Bull Pharm Sci Assiut University Vol 30 Part 2 2007 pp 241-258

ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــReceived in 10112007 Received in revised form in 29122007 amp Accepted in 30122007

Corresponding auther e-mail address ramzia_ismailyahoocom

SIMULTANEOUS DETERMINATION OF INTACTLOMEFLOXACIN AND CIPROFLOXACIN IN THEPRESENCE OF THEIR ACID DEGRADATIONPRODUCTS

Sonia T Hassib Ramzia I El-Bagary Hanaa M Hashem andMaha M El-Hakim

Department of Pharmaceutical Chemistry Faculty of Pharmacy CairoUniversity Egypt

١٨ ٣) ٢٠٨٠٠٦(بنسبة

مل ١٥٣٢٨٠٥

٦٢٠

لفصل ) ٢٠٨٠( ٢٨٨٢٧٩

١٦١٠

٠٢٥٢٥١٠

٢٩٢٢٦٠

٢٨١٠٥١٢

Sonia T Hassib et al

242

A reversed phase HPLC method was developed for thedetermination of lomefloxacin and its degradation product Inaddition two other methods have been developed for thedetermination of lomefloxacin hydrochloride (LFHCl) andciprofloxacin hydrochloride (CFHCl) in presence of their acidinduced degradation products

For the reversed phase HPLC method (determination ofLFHCl) the mobile phase used was a mixture of wateracetonitrile triethylamine (802006 vvv) adjusted to pH 30with o-phosphoric acid The flow rate was 15 mlmin and thedetection was carried out at 328 nm The linearity range was foundto be 05-6 microg 20 microl for LFHCl The limits of detection andquantification (LOD amp LOQ) were 022 microg 20 microl amp 074 microg 20microl respectively

The second method was densitometric method for thedetermination of both LFHCl and CFHCl the developing systemused was a mixture of methanol and ammonia buffer (8020 vv)Detection was carried out at 288 nm amp 279 nm for intact LFHCland CFHCl respectively The linearity ranges were found to be 1-6microg 10 microl amp 025-25 microg 10 microl for intact LFHCl and CFHClrespectively LOD amp LOQ were 01 034 microg 10 microl amp 005 018 microg 10 microl for both drugs respectively

The third method was derivative spectrophotometric method forthe determination of (LFHCl) amp (CFHCL) The linearity rangeswere found to be 2-8 microgml amp 5-12 microgml for LFHCl and CFHClrespectively LOD amp LOQ were 039 microg 129 microgml amp 103 345microgml for LFHCl and CFHCl respectively

Separation and identification of the acid degradation productsof lomefloxacin hydrochloride and ciprofloxacin hydrochloridewere carried out

The three described methods proved to be sensitive precise andapplicable to both dosage forms and laboratory prepared mixturesof the intact drugs and their acid degradation products

INTRODUCTION

Fluoroquinolone antibacterialagents which are relatively latearrivals on the antibacterial sceneproved to be very useful therapeuticagents They are particularly efficientin the treatment of complicated

urinary tract infections and also in thetreatment of infections resistant to thetraditional antibacterial agents1 Thedrugs proved to be effective in thetreatment of respiratory infectionsexternal otitis chronic gram negativebacillary osteomyelitis gonorrhoeacervicitis and bacterial prostatitis

243

They also helped greatly ineradication of Salmonella typhi incarriers and anthrax2 Thisinvestigation is concerned with theanalysis of LFHCl and CFHCleither alone or in the presence of theiracid degradation products Fig (1)

NHN

F

NF

O

OH

O

CH3

CH3

NHN

F

N

O

OH

O

Lomefloxacin Ciprofloxacin HCl HCl

NHN

F

NF

O

OH

O

CH3

CH3

NHN

F

N

O

OH

O

Lomefloxacin Ciprofloxacin HCl HCl

The chemical structure of lomefloxacinHCl and ciprofloxacin HCl

NN

NH

O

R1

F

X

R2

Compound X R1 R2

Ciprofloxacin H cyclopropyl HLomefloxacin F ethyl CH3

The identified chemical structures ofthe acid degradation products oflomefloxacin HCland ciprofloxacinHCl

Fig 1

Numerous analytical methods hadbeen reported for the determination ofCFHCl while few methods weredescribed for LFHCl hydrochlorideFor example volumetric3amp4 spectro-photometric5-15 fluorimetric16-23 andchromatographic methods24-41 werereported

These drugs are characterized bytheir susceptibility to photo-degradation into inactive productsThis fact encouraged the authors topropose stability-indicating proced-ures for the determination of theintact drug in the presence of its aciddegradation product New methodswere proposed in this work for theestimation of the components of theabove mentioned mixtures Theproposed methods were HPLCdensitometric TLC and derivativespectrophotometric methods

EXPERIMENTAL

Materials and reagentsAll chemicals and reagents used

were of analytical or pharmaceuticalgrade Distilled water was usedthroughout Solvents were of HPLCgrade or spectroscopic grade

Lomefloxacin hydrochloride(LFHCl) It was supplied by AlkanPharma SAE 6 October city EgyptIt was analyzed spectrophoto-metricaly42) and found to contain9917plusmn060 Lomefloxacin hydro-chloride

Ciprofloxacin hydrochloride It wassupplied by Biochemie Vienna

HCl

HCl

Sonia T Hassib et al

244

Austria It was analyzed by HPLC43

and found to contain 9943plusmn055CFHCl

ldquoAlkafloxrdquo tablets (batch no 001)nominally containing 400 mg ofLFHCl hydrochloride produced byAlkan Pharma SAE 6 OctoberCity Egypt

ldquoServifloxrdquotablets (batch no 030)nominally containing 2915 mg ofCFHCl hydrochloride (equivalent to250 mg of CFHCl) produced byBiochemie Vienna Austria

Methanol HPLC grade 2MHydrochloric acid ammoniabuffer 10 vv ammonim hydroxidein 1605 mv aqueous ammoniumchloride solution diethyl ether(BDH) 2 M sodium hydroxideacetonitrile HPLC grade triethyl-amine o-phosphoric acid doubledistilled water

Mobile phase for HPLCA mixture of water acetonitrile tri-

ethylamine (802006 vvv) adjustedto pH 30 with o-phosphoric acid

Developing system for densitometryA mixture of methanol and

ammonia buffer (8020 vv) wasfreshly prepared prior to analysis

Preparation of stock solutionsPreparation of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride stock solutions

A solution of LFHCl (05 mg ml)or CFHCL (1 mgml) was preparedin the specified solvent

Preparation of degraded lome-floxacin hydrochloride andciprofloxacin hydrochloride stocksolutions

An accurate weight of LFHCl orCFHCl (250 mg) was refluxed in 2M hydrochloric acid (40 ml) for 48hrs44amp45 After cooling the solutionwas neutralized with 2M sodiumhydroxide and then concentrated to asmall volume The concentratedsolution was extracted with diethylether (10 ml x 3) The ether extractwas evaporated to dryness on a warmwater bath The obtained residue wasused for preparing the degradedLFHCl solution (05 mgml) orCFHCl solution (1 mgml) in thespecified solvent

Preparation of internal standardstock solution

A solution of enrofloxacin (05mgml) was prepared in the mobilephase

ApparatusFor HPLC

HPLC apparatus consisted ofWaters autosampler injector Watersisocratic 610 pump Waters 486 uvdetector adjusted at 328 nm and microBondapack C18 reversed-phasecolumn (25 cm x 46 mm id)

For densitometryCS-9301 PC Dual wavelength

Flying Spot Scanning Shimadzudensitometer Japan

For spectrophotometryShimadzu uv-visible recording

spectrophotometer UV-265 Japan

245

For identification of degradedstructurea- For infra red spectra Shimadzu

Infra red spectrometer (IR- 435)Japan

b- For 1H- NMR spectra Joel FX90QNMR spectrometer

c- For mass spectra Finnigan SSQ7000 Gas chromatograph- mass

ProcedureHPLC method for the determinat-ion of LomefloxacinGeneral procedure and linearity

A solution containing intactLFHCl (05 mgml) and degradedLFHCl (04 mgml) was prepared inthe mobile phase Different aliquots(05-6 ml) of this solution wereintroduced into a series of 10 mlvolumetric flasks followed byinternal standard solution (1 ml) toeach flask The volume of each flaskwas completed with the mobile phaseTwenty-micro liters of each solutionwere injected onto the column Thechromatograms were recorded usingflow rate of 15 mlmin andwavelength of detection at 328 nm

The resulting chromatograms (Fig2) retention times (tR) of the peaksand the areas under the peaks (AUPs)were recorded The ratios (R) of therecorded AUPs of intact LFHCl or ofits degradation product to that of theinternal standard were plotted versusthe concentration (microg20 microl) and thecalibration graphs for intact anddegraded LFHCl were obtained

The following regressionequations (12) were computed

Concentration of intact LFHCl(microg20 microl) =05496 R + 00859 Eq (1)r2 = 09989 orR= 18195 X C (microg20microl) -01563

Concentration of degraded LFHCl(microg20 microl) =

04499 R + 01102 Eq(2)r2 = 09983 orR= 22227 X C (microg20 microl) -02449

Where C is the concentration in microg20microl R is the ratio of AUP ofdrugAUP of IS and r2 is thecorrelation coefficient

Retention time (tR)

Fig 2 HPLC chromatogram of degraded LFHCl (tR= 216 min) intactLFHCl (tR= 2451 min) and enrofloxacin (tR= 2832 min)

Sonia T Hassib et al

246

Simultaneous determination ofintact and degraded lomefloxacinhydrochloride in laboratoryprepared mixtures by RP-HPLC

Two solutions of intact LFHCland degraded LFHCl (06 mgml)each in the mobile phase wereprepared Different aliquots (15-45ml) and (05-35 ml) of both intactand degraded LFHCl solutionsrespectively were introduced into aseries of 10 ml volumetric flasksOne- ml aliquot of internal standardsolution was added to each flask andthe volumes were completed with themobile phase Twenty-micro liters ofeach mixture solution was injectedonto the column and thechromatograms were recorded usingthe same parameters mentioned undergeneral procedure and linearity Theconcentrations of intact and degradedLFHCl were calculated using theregression equations (1amp2)

Determination of intact lome-floxacin hydrochloride inldquoAlkafloxrdquo tablets by RP-HPLC

An accurate weight of thepowdered twenty tablets equivalent to(35 mg) of intact LFHCl wasintroduced into a 100 ml volumetricflask The mobile phase (80 ml) wasadded and the solution was shaken(using a mechanical shaker) for 10minutes and the volume wascompleted with the mobile phase Thesolution was filtered through a dryfilter paper The first 10 ml of thefiltrate was rejected and the filtratewas refiltered through 045 micrommillipore teflon filter Differentaliquots (2-4 ml) of this solution were

transferred into a series of 10 mlvolumetric flasks one ml aliquot ofthe internal standard solution wasadded to each flask and the volumewas completed with the mobile phaseTwenty-micro liters of each solutionwere injected onto the column and thechromatograms were recorded usingthe same parameters used undergeneral procedure and linearity onpage The procedure was repeatedusing the standard addition techniqueThe ratio (R) of AUP of the drug tothe AUP of the internal standard wasused in calculating the recoveredconcentrations of labeled and addedLFHCl

Densitometric simultaneous deter-mination of intact lomefloxacinhydrochloride and ciprofloxacinhydrochloride and their aciddegradation products

General procedure and linearityDifferent aliquots of intact LFHCl

stock solution (1-6 ml) or CFHClstock solution (05-5 ml) weretransferred into two series of 10 mlvolumetric flasks Also severalaliquots of degraded LF HCl stocksolution (05-6 ml) or degradedCFHCl stock solution (05-5 ml)were transferred into two series of 10ml volumetric flasks Each flask wascompleted to volume with methanol

A ten-microliter aliquot of eachsolution was applied on thin-layerchromatographic plates (20x20 cm)using a micropipette Spots werespaced 25 cm apart and 2 cm fromthe bottom edge of the plate Theplates were developed for at least one

247

hour in a chromatographic tankpreviously saturated with thespecified developing system byascending chromatography to adistance of 15 cm The plates wereleft to dry at room temperature Thespots were detected under a uv lamp(254 nm) and the two drugs werescanned under the followingconditionsPhoto mode Reflection scan modeZigzag swing width 12 mmWavelengthi) 288 and 328 nm for intact and

degraded LFHCl respectivelyii) 279 and 323 nm for intact and

degraded CFHCl respectively

Chromatograms result outputsand the areas under the peaks (AUPs)were recorded The recorded AUPswere plotted versus the concentrationused (microg10 microl) and the calibrationcurves for both intact and degradedLFHCl or CFHCl were obtainedThe following regression equations(345 and 6) were computed forcalculating the concentrations ofintact LFHCl degraded LFHClintact CFHCl and degraded CFHClrespectively

Concentration of intact LFHCl(microg10 microl) =00287 AUP ndash 00022 Eq (3)r2 = 09998 OrAUP= 34843 X C (microg10microl) +00767

Concentration of degraded LFHCl(microg10 microl) =00388 AUPndash 01587 Eq (4)r2 = 09982 OrAUP= 25773 X C (microg10microl) +40902

Concentration of intact CFHCl (microg10 microl) =00194 AUPndash 00398 Eq (5)r2 = 09991 OrAUP= 51546 X C (microg10microl) +20515

Concentration of degraded CFHCl(microg10 microl) =00246 AUP ndash00758 Eq (6)r2 = 09980 OrAUP= 40650 X C (microg10microl) +30813

Where C is the concentration inmicrog10microl AUP is the area under thepeak and r2 is the correlationcoefficient

Densitometric determination ofintact and degraded lomefloxacinhydrochloride and ciprofloxacinhydrochloride in laboratoryprepared mixtures

Four solutions of intact anddegraded LFHCl (12 mgml) ofintact and degraded CFHCl (04 mgml) were prepared in methanol

Different aliquots of both intactLFHCl (1-45 ml) or of intactCFHCl (15-4 ml) were transferredinto two series of 10 ml volumetricflasks Different aliquots of degradedLFHCl (05-4 ml) or degradedCFHCl (1-35 ml) were added tointact LFHCl series or to intactCFHCl series respectively [series (1)and (2)] for LFHCl and CFHClrespectively Each volumetric flaskwas completed to volume withmethanol Ten-microlitre aliquots ofeach mixture solution were applied onthin-layer chromatographic platesusing a micropipette and theprocedure was completed using the

Sonia T Hassib et al

248

same parameters mentioned undergeneral procedure and linearity

The recorded AUPs were used forcalculating the concentrations of thetwo components (intact and degradeddrug) in each mixture solution usingthe regression equations (345 and 6)for intact and degraded LFHCl andCFHCl respectively

Densitometric determination oflomefloxacin hydrochloride andciprofloxacin hydrochloride inldquoAlkafloxrdquo and ldquoServifloxrdquo tabletsrespectively

An accurate weight of thepowdered twenty tablets equivalent toLFHCl (35 mg) or CFHCl (30 mg)was extracted with methanol (10 ml x3) Each extract was filtered into a50ml volumetric flask The residuewas washed with methanol (5 ml x 3)the volume was completed withmethanol Aliquots (2-4 ml) ofLFHCl solution or (1-2 ml) ofCFHCl solution were transferredinto two series of 10 ml volumetricflasks and completed to volume withmethanol Ten-microlitre aliquot ofeach solution was applied on thin-chromartographgic plates and theprocedure was completed using thesame parameters mentioned undergeneral procedure for linearity Thisexperiment was repeated applying thestandard addition technique Therecorded AUPs for the spots wereused for calculating the concentrationof labelled and added intact LFHClor CFHCl from regression equations(3 and 5)

Simultaneous derivative spectro-photometric determin-ation ofintact lomefloxacin hydrochlorideor ciprofloxa-cin hydrochlorideand their acid degradationproducts

General procedure and linearityAn aqueous solution of intact LF

HCl (05 mgml) or CF HCl (1mgml) was prepared A ten-mlaliquot of this solution containingLFHCl or CFHCl was introducedinto a 100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid

Accurately measured aliquots (2-8ml) or (25-6 ml) of intact solution ofLFHCl or CF HCl respectivelywere transferred into two series of 50ml volumetric flasks followed by theaddition of accurately measuredvolumes (2-8 ml) or (4-8 ml) ofdegraded solution of LF HCl or CFHCl respectively Each flask wascompleted to volume with the samesolvent

The first and second derivativespectra for each solution wererecorded against a solvent blankusing the following parameters

Real time date Δλ2 speed fastslit 1 cycT(min) 0 ordinate rangelimit - 03 to + 03 or ndash 04 to + 04for LFHCl or CFHCl respectively

Wavelength range 200-400 nm or200-350 nm for LFHCl or CFHClrespectively

Also the second derivativespectrum for each flask solution wasrecorded against a solvent blankusing the same above parametersexcept for the following

249

1- Ordinate range limit - 009 to +006 or -012 to + 008 fordegraded LFHCl or CFHClrespectively

2- Wavelength range 250-350 nmor 225-325 nm for degradedLFHCl or CF HCl respectively

The heights (mm) of the firstderivative spectra at 292 nm forLFHCl or at 260 nm for CFHClwere measured and the calibrationgraph for each drug was plotted Theheights (mm) of the second derivativespectra at 272 or at 262 nm fordegraded LFHCl or CFHClrespectively were measured and thecalibration graphs for the degradeddrugs were constructed

The following regressionequations 789 and 10 werecomputed for calculating theconcentration of intact and degradedlomefloxacin and ciprofloxacinrespectively

Concentration of intact LFHCl(microgml) =01631 H292+ 00394 Eq(7)r2= 09998 OrH292= 61312 X C (microgml) -02416

Concentration of intact CFHCl(microgml) =01916H 260 + 02480 Eq(8)r2= 09984 OrH 260 = 52192 XC (microgml) -12944

Concentration of degraded LFHCl(microgml) =01534H272 + 00359 Eq(9)r2= 09998 OrH272 = 65189 X C (microg ml) -02340

Concentration of degraded CFHCl(microgml) =02123H 262 + 05092 Eq (10)r2= 09963 OrH 262 = 47103 X C (microg ml) -23985

Where C is the concentration in microgml H is height of the peak and r2 isthe correlation coefficient

Derivative spectrophotometricdetermination of lomefloxacinhydrochloride and ciprofloxacinhydrochloride in ldquoAlkafloxrdquo andldquoServifloxrdquo tablets respectively

An accurate weight of thepowdered tablets equivalent toLFHCl or CFHCl (20 mg) wasextracted with methanol (15 ml x 3)then filtered into a 100 ml volumetricflask After complete washing of theresidue with methanol (10 ml x 3) thesolution was diluted to volume withmethanol A twenty-ml aliquot of thissolution equivalent to LF HCl orCFHCl (4 mg) was evaporated on awarm water bath The residue wasdissolved in 2 M hydrochloric acidand quantitatively transferred into a100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid Accuratelymeasured aliquots of the abovesolution (2-6 ml) or (6-9 ml) wereintroduced into two series of 50 mlvolumetric flasks Each flask wascompleted to volume with 2Mhydrochloric acid

The first derivative spectrum ofeach drug was recorded against ablank using the same parametersmentioned under general procedureand linearity This procedure was

Sonia T Hassib et al

250

repeated using the standard additiontechnique

The heights (mm) at 292 and 260nm were measured and used forcalculating the corresponding labeledand added concentrations of intactLFHCl and CFHCl respectively

RESULTS AND DISCUSSION

HPLC methodIn this study different mobile

phases were attempted for theseparation of LFHCl in presence ofits degradation product(s) HoweverThoppil amp Amin38 reported astability-indicating reversed-phaseliquid chromatographic procedure forthe determination of CFHCl as bulkdrug in pharmaceutical preparationsand in the presence of its acid andalkaline degraded solutions Themobile phase consisted of water-acetonitrile-triethylamine (802006vvv) adjusted with o-phosphoricacid to pH 3

The above mobile systempreviously applied for CFHCl wassuccessfully used for the separationand determination of intact anddegraded LFHCl since it showedgood resolution between the elutedpeaks Detection was carried out at328 nm The polarity of the systemwas calculated46 and found to be(109) The flow rate was set at 15mlmin Enrofloxacin was used as aninternal standard with the elutionorder degraded LFHCl (tR= 2160)intact LFHCl (tR= 2451) andenrofloxacin (tR= 2832) Fig (2) Thechromatogram of the mixture shows

complete separation and goodresolution

A linear relationship wasobserved when the ratios of the AUPsof each component to the AUP of theinternal standard were plotted versusthe concentration of each componentThe concentration range for eachcomponent in the mixture regressionequations mean percentagerecoveries limit of detection (LOD)and limit of quantification (LOQ)when using the proposed methods aredisplayed in Table (1)

Densitometric methodFew developing systems were

used for TLC separation and solventpolarity optimization of LFH|Cl47 andCFHCl27amp48 in the presence of eitherrelated impurities or photo-degradation products in irradiatedsolutions Seija Tammilehto et al48

studied the photodegradation ofCFHCl irradiated in aqueoussolutions through using a mixture ofacetonitrile and ammonia buffer (pH106-111) as a developing system(6535 vv) for the separation of thecompound and its photodegradationproducts

Using methanol instead ofacetonitrile in a different proportionin the present work offered betterseparation of the drug and its aciddegradation product The polarity ofthe used system was calculated andfound to be (515)46

The TLC chromatogram of bothintact and degraded LFHCl showedcomplete separation with (Rf= 066)for the intact drug and (Rf= 083) for

251

Table 1 Results obtained in the chromatographic methods for the determinationof intact and degraded lomefloxacin hydrochloride and ciprofloxacinhydrochloride

TLC method HPLC methodLFHCl CFHCl LFHClItem

Intact Deg Intact Deg Intact DegConcentrationrange

1-6microg10 microl

05-6microg10 microl

025-25microg10 microl

025-25microg10 microl

05-6microg20 microl

04-48microg20 microl

Mean percentagerecoveries

1-Laboratorypreparedmixtures

9960plusmn 055

9951plusmn 067

9955plusmn072

9922plusmn069

9968plusmn077

10030plusmn089

2-Added standardto lsquoAlkafloxrsquoor lsquoServifloxrsquo

tablets

9925plusmn080

9963plusmn 064

9938plusmn070

LOD010

(microg 10microl)005

(microg 10microl)022

(microg 20microl)

LOQ034

(microg 10microl)018

(microg 10microl)074

(microg 20microl)

the degraded product The separatedspots of both components werescanned on the same plate at 288 and328 nm for the intact and degradedLFHCl respectively

Upon applying this modifieddeveloping system to the analysis ofboth intact and degraded CFHCl theTLC chromatogram showed completeresolution with (Rf= 053) for theintact drug and (Rf= 075) for thedegraded drug The separated spots ofboth components were scanned on thesame plate at 279 and 323 nm for theintact and degraded CFHClrespectively

The concentration range for eachcomponent in the two mixturesregression equations meanpercentage recoveries limit ofdetection and limit of quantificationwhen using the proposed methods aredisplayed in Table (1)

The results obtained in thedetermination of intact LFHCl andCFHCl in the presence of their aciddegradation products using HPLCand densitometric methods werestatistically compared to the reportedmethods (spectrophotometric42 andHPLC43 respectively) showing nosignificant difference concerningaccuracy and precision Table (2)

Sonia T Hassib et al

252

Table 2 Statistical analysis for the chromatographic methods for thedetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochlorideCiprofloxacinhydrochlorideStatistcal Term

HPLCmethod

TLCmethod

Reference42

TLCmethod

Reference43

Mean 9968 9960 9917 9955 9943

SD plusmn077 plusmn055 plusmn060 plusmn072 plusmn055

RSDwithin day

077 055 072

RSDbetween days

117 116 119

SE plusmn031 plusmn022 plusmn024 plusmn029 plusmn023

n 6 6 6 6 6

V 059 030 036 052 030

LOD022

(microg 20microl)010

(microg 10microl)005

(microg 10microl)

LOQ074

(microg 20microl)034

(microg 10microl)018

(microg 10microl)

t 128 130 032

F 164 120 173

The corresponding theoretical values for t and F at p = 005 are 223 and 505respectively

Derivative spectrophotometricmethod

The zero-order spectra of intactand degraded LFHCl and CFHClFigs (3amp4) show completeoverlapping which prevents thedetermination of the intact anddegraded drugs in presence of eachother using this technique

In this work a derivativespectrophotometric procedure ispresented to solve this problem

through measurements in both thefirst and second derivative spectraThe first derivative spectra Figs(5amp6) show that intact LFHCldisplays a peak at 272 nm and avalley at 292 nm while the degradedproduct has zero crossing at both 292and 271 nm Thus the ordinate valueat 292 nm was chosen for thedetermination of the intact drug in thepresence of its degradation productIn addition the first derivative

253

Wavelength (nm)

Fig 3 Zero-order spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 4 Zero-order spectra of intactciprofloxacin hydrochloride (056mg50ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 5 First-derivative spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 6 First-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Abs

orba

nce

Abs

orba

nce

Sonia T Hassib et al

254

spectrum of intact CFHCl Fig (6)exhibits a peak at 262 nm and a valleyat 282 nm while its degradationproduct has zero crossing at both 260and 286 nm Consequently theordinate value at 260 nm waspreferred for calculating theconcentration of the intact drug in thepresence of its degradation product

Figures (5amp6) show that thedegradation product of LFHCl orCFHCl could not be determinedthrough measurements in the firstderivative spectra On the other handin the second derivative spectra Fig(7) the degraded LFHCl shows avalley at 272 nm at which the intactdrug has a zero crossing Thus theordinate value at 272 nm was used forcomputing the concentration of thedegradation product in the presenceof the intact drug

Similarly the second derivativespectra Fig (8) show that thedegraded CFHCl exhibits a valley at262 nm at which the intact drugshows no interference Therefore theordinate value at 262 nm was used forcalculating the concentration of thedegradation product in the presenceof the intact drug

Linearity was shown over theconcentration range (12-96 microgml)or (28-126 microgml) of intact LFH|Clor CFHCl respectively (HPLC andDensitometric) linearity was alsoobserved over the concentration range(12-96 microgml) or (64-16 microgml) ofdegraded LFH|Cl or CFHClrespectively

Wavelength (nm)

Fig 7 Second-derivative spectra of intactlomefloxacin hydrochloride (024mg50 ml) (mdashmdashmdash) and ofdegraded drug (024 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 8 Second-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

255

The mean percentage recoveriesof intact and degraded LFHCl werefound to be 9972plusmn056 and 9893plusmn085 respectively (Table 3) Also themean percentage recoveries of intactand degraded CFHCl were 9962plusmn047 and 9967plusmn039 respectively(Table 3)

The method was successfullyapplied for the quantitative estimationof LFHCl or CFHCl in ldquoAlkafloxrdquoor ldquoServifloxrdquo tablets respectivelywithout interference from additivesThe standard addition technique wasapplied The mean percentagerecoveries of labelled and addedLFHCl were found to be 9976plusmn075and 9958plusmn064 respectively Alsothe mean percentage recoveries oflabelled and added CFHCl were

9949plusmn066 and 9978plusmn034respectively The second derivativespectra of ldquoAlkafloxrdquo and ldquoServifloxrdquotablets showed complete absence ofthe corresponding acid degradationproduct

The proposed methods are validand applicable for the determinationof intact LFHCl or CFHCl inlaboratory prepared mixturescontaining (20-80) or (40-75) ofthe degraded LFHCl or degradedCFHCl respectively

Statistical comparison between theproposed and the reported methods(spectrophotometric42 and HPLC43for LFHCl and CFHCl respectively)showed no significant differencebetween them concerning accuracyand precision (Table 3)

Table 3 Statistical analysis for the first derivative spectrophotometricdetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochloride Ciprofloxacin hydrochlorideStatistical

TermFirst

Derivativemethod

Comparisonmethod13

FirstDerivative

method

Comparisonmethod12

Mean 9972 9917 9962 9943

SD plusmn056 plusmn060 plusmn047 plusmn055

RSD 056 047

SE plusmn021 plusmn024 plusmn018 plusmn022

n 7 6 7 6

V 031 036 022 030

LOD(microgml)

039 103

LOQ(microgml)

129 345

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

243

They also helped greatly ineradication of Salmonella typhi incarriers and anthrax2 Thisinvestigation is concerned with theanalysis of LFHCl and CFHCleither alone or in the presence of theiracid degradation products Fig (1)

NHN

F

NF

O

OH

O

CH3

CH3

NHN

F

N

O

OH

O

Lomefloxacin Ciprofloxacin HCl HCl

NHN

F

NF

O

OH

O

CH3

CH3

NHN

F

N

O

OH

O

Lomefloxacin Ciprofloxacin HCl HCl

The chemical structure of lomefloxacinHCl and ciprofloxacin HCl

NN

NH

O

R1

F

X

R2

Compound X R1 R2

Ciprofloxacin H cyclopropyl HLomefloxacin F ethyl CH3

The identified chemical structures ofthe acid degradation products oflomefloxacin HCland ciprofloxacinHCl

Fig 1

Numerous analytical methods hadbeen reported for the determination ofCFHCl while few methods weredescribed for LFHCl hydrochlorideFor example volumetric3amp4 spectro-photometric5-15 fluorimetric16-23 andchromatographic methods24-41 werereported

These drugs are characterized bytheir susceptibility to photo-degradation into inactive productsThis fact encouraged the authors topropose stability-indicating proced-ures for the determination of theintact drug in the presence of its aciddegradation product New methodswere proposed in this work for theestimation of the components of theabove mentioned mixtures Theproposed methods were HPLCdensitometric TLC and derivativespectrophotometric methods

EXPERIMENTAL

Materials and reagentsAll chemicals and reagents used

were of analytical or pharmaceuticalgrade Distilled water was usedthroughout Solvents were of HPLCgrade or spectroscopic grade

Lomefloxacin hydrochloride(LFHCl) It was supplied by AlkanPharma SAE 6 October city EgyptIt was analyzed spectrophoto-metricaly42) and found to contain9917plusmn060 Lomefloxacin hydro-chloride

Ciprofloxacin hydrochloride It wassupplied by Biochemie Vienna

HCl

HCl

Sonia T Hassib et al

244

Austria It was analyzed by HPLC43

and found to contain 9943plusmn055CFHCl

ldquoAlkafloxrdquo tablets (batch no 001)nominally containing 400 mg ofLFHCl hydrochloride produced byAlkan Pharma SAE 6 OctoberCity Egypt

ldquoServifloxrdquotablets (batch no 030)nominally containing 2915 mg ofCFHCl hydrochloride (equivalent to250 mg of CFHCl) produced byBiochemie Vienna Austria

Methanol HPLC grade 2MHydrochloric acid ammoniabuffer 10 vv ammonim hydroxidein 1605 mv aqueous ammoniumchloride solution diethyl ether(BDH) 2 M sodium hydroxideacetonitrile HPLC grade triethyl-amine o-phosphoric acid doubledistilled water

Mobile phase for HPLCA mixture of water acetonitrile tri-

ethylamine (802006 vvv) adjustedto pH 30 with o-phosphoric acid

Developing system for densitometryA mixture of methanol and

ammonia buffer (8020 vv) wasfreshly prepared prior to analysis

Preparation of stock solutionsPreparation of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride stock solutions

A solution of LFHCl (05 mg ml)or CFHCL (1 mgml) was preparedin the specified solvent

Preparation of degraded lome-floxacin hydrochloride andciprofloxacin hydrochloride stocksolutions

An accurate weight of LFHCl orCFHCl (250 mg) was refluxed in 2M hydrochloric acid (40 ml) for 48hrs44amp45 After cooling the solutionwas neutralized with 2M sodiumhydroxide and then concentrated to asmall volume The concentratedsolution was extracted with diethylether (10 ml x 3) The ether extractwas evaporated to dryness on a warmwater bath The obtained residue wasused for preparing the degradedLFHCl solution (05 mgml) orCFHCl solution (1 mgml) in thespecified solvent

Preparation of internal standardstock solution

A solution of enrofloxacin (05mgml) was prepared in the mobilephase

ApparatusFor HPLC

HPLC apparatus consisted ofWaters autosampler injector Watersisocratic 610 pump Waters 486 uvdetector adjusted at 328 nm and microBondapack C18 reversed-phasecolumn (25 cm x 46 mm id)

For densitometryCS-9301 PC Dual wavelength

Flying Spot Scanning Shimadzudensitometer Japan

For spectrophotometryShimadzu uv-visible recording

spectrophotometer UV-265 Japan

245

For identification of degradedstructurea- For infra red spectra Shimadzu

Infra red spectrometer (IR- 435)Japan

b- For 1H- NMR spectra Joel FX90QNMR spectrometer

c- For mass spectra Finnigan SSQ7000 Gas chromatograph- mass

ProcedureHPLC method for the determinat-ion of LomefloxacinGeneral procedure and linearity

A solution containing intactLFHCl (05 mgml) and degradedLFHCl (04 mgml) was prepared inthe mobile phase Different aliquots(05-6 ml) of this solution wereintroduced into a series of 10 mlvolumetric flasks followed byinternal standard solution (1 ml) toeach flask The volume of each flaskwas completed with the mobile phaseTwenty-micro liters of each solutionwere injected onto the column Thechromatograms were recorded usingflow rate of 15 mlmin andwavelength of detection at 328 nm

The resulting chromatograms (Fig2) retention times (tR) of the peaksand the areas under the peaks (AUPs)were recorded The ratios (R) of therecorded AUPs of intact LFHCl or ofits degradation product to that of theinternal standard were plotted versusthe concentration (microg20 microl) and thecalibration graphs for intact anddegraded LFHCl were obtained

The following regressionequations (12) were computed

Concentration of intact LFHCl(microg20 microl) =05496 R + 00859 Eq (1)r2 = 09989 orR= 18195 X C (microg20microl) -01563

Concentration of degraded LFHCl(microg20 microl) =

04499 R + 01102 Eq(2)r2 = 09983 orR= 22227 X C (microg20 microl) -02449

Where C is the concentration in microg20microl R is the ratio of AUP ofdrugAUP of IS and r2 is thecorrelation coefficient

Retention time (tR)

Fig 2 HPLC chromatogram of degraded LFHCl (tR= 216 min) intactLFHCl (tR= 2451 min) and enrofloxacin (tR= 2832 min)

Sonia T Hassib et al

246

Simultaneous determination ofintact and degraded lomefloxacinhydrochloride in laboratoryprepared mixtures by RP-HPLC

Two solutions of intact LFHCland degraded LFHCl (06 mgml)each in the mobile phase wereprepared Different aliquots (15-45ml) and (05-35 ml) of both intactand degraded LFHCl solutionsrespectively were introduced into aseries of 10 ml volumetric flasksOne- ml aliquot of internal standardsolution was added to each flask andthe volumes were completed with themobile phase Twenty-micro liters ofeach mixture solution was injectedonto the column and thechromatograms were recorded usingthe same parameters mentioned undergeneral procedure and linearity Theconcentrations of intact and degradedLFHCl were calculated using theregression equations (1amp2)

Determination of intact lome-floxacin hydrochloride inldquoAlkafloxrdquo tablets by RP-HPLC

An accurate weight of thepowdered twenty tablets equivalent to(35 mg) of intact LFHCl wasintroduced into a 100 ml volumetricflask The mobile phase (80 ml) wasadded and the solution was shaken(using a mechanical shaker) for 10minutes and the volume wascompleted with the mobile phase Thesolution was filtered through a dryfilter paper The first 10 ml of thefiltrate was rejected and the filtratewas refiltered through 045 micrommillipore teflon filter Differentaliquots (2-4 ml) of this solution were

transferred into a series of 10 mlvolumetric flasks one ml aliquot ofthe internal standard solution wasadded to each flask and the volumewas completed with the mobile phaseTwenty-micro liters of each solutionwere injected onto the column and thechromatograms were recorded usingthe same parameters used undergeneral procedure and linearity onpage The procedure was repeatedusing the standard addition techniqueThe ratio (R) of AUP of the drug tothe AUP of the internal standard wasused in calculating the recoveredconcentrations of labeled and addedLFHCl

Densitometric simultaneous deter-mination of intact lomefloxacinhydrochloride and ciprofloxacinhydrochloride and their aciddegradation products

General procedure and linearityDifferent aliquots of intact LFHCl

stock solution (1-6 ml) or CFHClstock solution (05-5 ml) weretransferred into two series of 10 mlvolumetric flasks Also severalaliquots of degraded LF HCl stocksolution (05-6 ml) or degradedCFHCl stock solution (05-5 ml)were transferred into two series of 10ml volumetric flasks Each flask wascompleted to volume with methanol

A ten-microliter aliquot of eachsolution was applied on thin-layerchromatographic plates (20x20 cm)using a micropipette Spots werespaced 25 cm apart and 2 cm fromthe bottom edge of the plate Theplates were developed for at least one

247

hour in a chromatographic tankpreviously saturated with thespecified developing system byascending chromatography to adistance of 15 cm The plates wereleft to dry at room temperature Thespots were detected under a uv lamp(254 nm) and the two drugs werescanned under the followingconditionsPhoto mode Reflection scan modeZigzag swing width 12 mmWavelengthi) 288 and 328 nm for intact and

degraded LFHCl respectivelyii) 279 and 323 nm for intact and

degraded CFHCl respectively

Chromatograms result outputsand the areas under the peaks (AUPs)were recorded The recorded AUPswere plotted versus the concentrationused (microg10 microl) and the calibrationcurves for both intact and degradedLFHCl or CFHCl were obtainedThe following regression equations(345 and 6) were computed forcalculating the concentrations ofintact LFHCl degraded LFHClintact CFHCl and degraded CFHClrespectively

Concentration of intact LFHCl(microg10 microl) =00287 AUP ndash 00022 Eq (3)r2 = 09998 OrAUP= 34843 X C (microg10microl) +00767

Concentration of degraded LFHCl(microg10 microl) =00388 AUPndash 01587 Eq (4)r2 = 09982 OrAUP= 25773 X C (microg10microl) +40902

Concentration of intact CFHCl (microg10 microl) =00194 AUPndash 00398 Eq (5)r2 = 09991 OrAUP= 51546 X C (microg10microl) +20515

Concentration of degraded CFHCl(microg10 microl) =00246 AUP ndash00758 Eq (6)r2 = 09980 OrAUP= 40650 X C (microg10microl) +30813

Where C is the concentration inmicrog10microl AUP is the area under thepeak and r2 is the correlationcoefficient

Densitometric determination ofintact and degraded lomefloxacinhydrochloride and ciprofloxacinhydrochloride in laboratoryprepared mixtures

Four solutions of intact anddegraded LFHCl (12 mgml) ofintact and degraded CFHCl (04 mgml) were prepared in methanol

Different aliquots of both intactLFHCl (1-45 ml) or of intactCFHCl (15-4 ml) were transferredinto two series of 10 ml volumetricflasks Different aliquots of degradedLFHCl (05-4 ml) or degradedCFHCl (1-35 ml) were added tointact LFHCl series or to intactCFHCl series respectively [series (1)and (2)] for LFHCl and CFHClrespectively Each volumetric flaskwas completed to volume withmethanol Ten-microlitre aliquots ofeach mixture solution were applied onthin-layer chromatographic platesusing a micropipette and theprocedure was completed using the

Sonia T Hassib et al

248

same parameters mentioned undergeneral procedure and linearity

The recorded AUPs were used forcalculating the concentrations of thetwo components (intact and degradeddrug) in each mixture solution usingthe regression equations (345 and 6)for intact and degraded LFHCl andCFHCl respectively

Densitometric determination oflomefloxacin hydrochloride andciprofloxacin hydrochloride inldquoAlkafloxrdquo and ldquoServifloxrdquo tabletsrespectively

An accurate weight of thepowdered twenty tablets equivalent toLFHCl (35 mg) or CFHCl (30 mg)was extracted with methanol (10 ml x3) Each extract was filtered into a50ml volumetric flask The residuewas washed with methanol (5 ml x 3)the volume was completed withmethanol Aliquots (2-4 ml) ofLFHCl solution or (1-2 ml) ofCFHCl solution were transferredinto two series of 10 ml volumetricflasks and completed to volume withmethanol Ten-microlitre aliquot ofeach solution was applied on thin-chromartographgic plates and theprocedure was completed using thesame parameters mentioned undergeneral procedure for linearity Thisexperiment was repeated applying thestandard addition technique Therecorded AUPs for the spots wereused for calculating the concentrationof labelled and added intact LFHClor CFHCl from regression equations(3 and 5)

Simultaneous derivative spectro-photometric determin-ation ofintact lomefloxacin hydrochlorideor ciprofloxa-cin hydrochlorideand their acid degradationproducts

General procedure and linearityAn aqueous solution of intact LF

HCl (05 mgml) or CF HCl (1mgml) was prepared A ten-mlaliquot of this solution containingLFHCl or CFHCl was introducedinto a 100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid

Accurately measured aliquots (2-8ml) or (25-6 ml) of intact solution ofLFHCl or CF HCl respectivelywere transferred into two series of 50ml volumetric flasks followed by theaddition of accurately measuredvolumes (2-8 ml) or (4-8 ml) ofdegraded solution of LF HCl or CFHCl respectively Each flask wascompleted to volume with the samesolvent

The first and second derivativespectra for each solution wererecorded against a solvent blankusing the following parameters

Real time date Δλ2 speed fastslit 1 cycT(min) 0 ordinate rangelimit - 03 to + 03 or ndash 04 to + 04for LFHCl or CFHCl respectively

Wavelength range 200-400 nm or200-350 nm for LFHCl or CFHClrespectively

Also the second derivativespectrum for each flask solution wasrecorded against a solvent blankusing the same above parametersexcept for the following

249

1- Ordinate range limit - 009 to +006 or -012 to + 008 fordegraded LFHCl or CFHClrespectively

2- Wavelength range 250-350 nmor 225-325 nm for degradedLFHCl or CF HCl respectively

The heights (mm) of the firstderivative spectra at 292 nm forLFHCl or at 260 nm for CFHClwere measured and the calibrationgraph for each drug was plotted Theheights (mm) of the second derivativespectra at 272 or at 262 nm fordegraded LFHCl or CFHClrespectively were measured and thecalibration graphs for the degradeddrugs were constructed

The following regressionequations 789 and 10 werecomputed for calculating theconcentration of intact and degradedlomefloxacin and ciprofloxacinrespectively

Concentration of intact LFHCl(microgml) =01631 H292+ 00394 Eq(7)r2= 09998 OrH292= 61312 X C (microgml) -02416

Concentration of intact CFHCl(microgml) =01916H 260 + 02480 Eq(8)r2= 09984 OrH 260 = 52192 XC (microgml) -12944

Concentration of degraded LFHCl(microgml) =01534H272 + 00359 Eq(9)r2= 09998 OrH272 = 65189 X C (microg ml) -02340

Concentration of degraded CFHCl(microgml) =02123H 262 + 05092 Eq (10)r2= 09963 OrH 262 = 47103 X C (microg ml) -23985

Where C is the concentration in microgml H is height of the peak and r2 isthe correlation coefficient

Derivative spectrophotometricdetermination of lomefloxacinhydrochloride and ciprofloxacinhydrochloride in ldquoAlkafloxrdquo andldquoServifloxrdquo tablets respectively

An accurate weight of thepowdered tablets equivalent toLFHCl or CFHCl (20 mg) wasextracted with methanol (15 ml x 3)then filtered into a 100 ml volumetricflask After complete washing of theresidue with methanol (10 ml x 3) thesolution was diluted to volume withmethanol A twenty-ml aliquot of thissolution equivalent to LF HCl orCFHCl (4 mg) was evaporated on awarm water bath The residue wasdissolved in 2 M hydrochloric acidand quantitatively transferred into a100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid Accuratelymeasured aliquots of the abovesolution (2-6 ml) or (6-9 ml) wereintroduced into two series of 50 mlvolumetric flasks Each flask wascompleted to volume with 2Mhydrochloric acid

The first derivative spectrum ofeach drug was recorded against ablank using the same parametersmentioned under general procedureand linearity This procedure was

Sonia T Hassib et al

250

repeated using the standard additiontechnique

The heights (mm) at 292 and 260nm were measured and used forcalculating the corresponding labeledand added concentrations of intactLFHCl and CFHCl respectively

RESULTS AND DISCUSSION

HPLC methodIn this study different mobile

phases were attempted for theseparation of LFHCl in presence ofits degradation product(s) HoweverThoppil amp Amin38 reported astability-indicating reversed-phaseliquid chromatographic procedure forthe determination of CFHCl as bulkdrug in pharmaceutical preparationsand in the presence of its acid andalkaline degraded solutions Themobile phase consisted of water-acetonitrile-triethylamine (802006vvv) adjusted with o-phosphoricacid to pH 3

The above mobile systempreviously applied for CFHCl wassuccessfully used for the separationand determination of intact anddegraded LFHCl since it showedgood resolution between the elutedpeaks Detection was carried out at328 nm The polarity of the systemwas calculated46 and found to be(109) The flow rate was set at 15mlmin Enrofloxacin was used as aninternal standard with the elutionorder degraded LFHCl (tR= 2160)intact LFHCl (tR= 2451) andenrofloxacin (tR= 2832) Fig (2) Thechromatogram of the mixture shows

complete separation and goodresolution

A linear relationship wasobserved when the ratios of the AUPsof each component to the AUP of theinternal standard were plotted versusthe concentration of each componentThe concentration range for eachcomponent in the mixture regressionequations mean percentagerecoveries limit of detection (LOD)and limit of quantification (LOQ)when using the proposed methods aredisplayed in Table (1)

Densitometric methodFew developing systems were

used for TLC separation and solventpolarity optimization of LFH|Cl47 andCFHCl27amp48 in the presence of eitherrelated impurities or photo-degradation products in irradiatedsolutions Seija Tammilehto et al48

studied the photodegradation ofCFHCl irradiated in aqueoussolutions through using a mixture ofacetonitrile and ammonia buffer (pH106-111) as a developing system(6535 vv) for the separation of thecompound and its photodegradationproducts

Using methanol instead ofacetonitrile in a different proportionin the present work offered betterseparation of the drug and its aciddegradation product The polarity ofthe used system was calculated andfound to be (515)46

The TLC chromatogram of bothintact and degraded LFHCl showedcomplete separation with (Rf= 066)for the intact drug and (Rf= 083) for

251

Table 1 Results obtained in the chromatographic methods for the determinationof intact and degraded lomefloxacin hydrochloride and ciprofloxacinhydrochloride

TLC method HPLC methodLFHCl CFHCl LFHClItem

Intact Deg Intact Deg Intact DegConcentrationrange

1-6microg10 microl

05-6microg10 microl

025-25microg10 microl

025-25microg10 microl

05-6microg20 microl

04-48microg20 microl

Mean percentagerecoveries

1-Laboratorypreparedmixtures

9960plusmn 055

9951plusmn 067

9955plusmn072

9922plusmn069

9968plusmn077

10030plusmn089

2-Added standardto lsquoAlkafloxrsquoor lsquoServifloxrsquo

tablets

9925plusmn080

9963plusmn 064

9938plusmn070

LOD010

(microg 10microl)005

(microg 10microl)022

(microg 20microl)

LOQ034

(microg 10microl)018

(microg 10microl)074

(microg 20microl)

the degraded product The separatedspots of both components werescanned on the same plate at 288 and328 nm for the intact and degradedLFHCl respectively

Upon applying this modifieddeveloping system to the analysis ofboth intact and degraded CFHCl theTLC chromatogram showed completeresolution with (Rf= 053) for theintact drug and (Rf= 075) for thedegraded drug The separated spots ofboth components were scanned on thesame plate at 279 and 323 nm for theintact and degraded CFHClrespectively

The concentration range for eachcomponent in the two mixturesregression equations meanpercentage recoveries limit ofdetection and limit of quantificationwhen using the proposed methods aredisplayed in Table (1)

The results obtained in thedetermination of intact LFHCl andCFHCl in the presence of their aciddegradation products using HPLCand densitometric methods werestatistically compared to the reportedmethods (spectrophotometric42 andHPLC43 respectively) showing nosignificant difference concerningaccuracy and precision Table (2)

Sonia T Hassib et al

252

Table 2 Statistical analysis for the chromatographic methods for thedetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochlorideCiprofloxacinhydrochlorideStatistcal Term

HPLCmethod

TLCmethod

Reference42

TLCmethod

Reference43

Mean 9968 9960 9917 9955 9943

SD plusmn077 plusmn055 plusmn060 plusmn072 plusmn055

RSDwithin day

077 055 072

RSDbetween days

117 116 119

SE plusmn031 plusmn022 plusmn024 plusmn029 plusmn023

n 6 6 6 6 6

V 059 030 036 052 030

LOD022

(microg 20microl)010

(microg 10microl)005

(microg 10microl)

LOQ074

(microg 20microl)034

(microg 10microl)018

(microg 10microl)

t 128 130 032

F 164 120 173

The corresponding theoretical values for t and F at p = 005 are 223 and 505respectively

Derivative spectrophotometricmethod

The zero-order spectra of intactand degraded LFHCl and CFHClFigs (3amp4) show completeoverlapping which prevents thedetermination of the intact anddegraded drugs in presence of eachother using this technique

In this work a derivativespectrophotometric procedure ispresented to solve this problem

through measurements in both thefirst and second derivative spectraThe first derivative spectra Figs(5amp6) show that intact LFHCldisplays a peak at 272 nm and avalley at 292 nm while the degradedproduct has zero crossing at both 292and 271 nm Thus the ordinate valueat 292 nm was chosen for thedetermination of the intact drug in thepresence of its degradation productIn addition the first derivative

253

Wavelength (nm)

Fig 3 Zero-order spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 4 Zero-order spectra of intactciprofloxacin hydrochloride (056mg50ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 5 First-derivative spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 6 First-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Abs

orba

nce

Abs

orba

nce

Sonia T Hassib et al

254

spectrum of intact CFHCl Fig (6)exhibits a peak at 262 nm and a valleyat 282 nm while its degradationproduct has zero crossing at both 260and 286 nm Consequently theordinate value at 260 nm waspreferred for calculating theconcentration of the intact drug in thepresence of its degradation product

Figures (5amp6) show that thedegradation product of LFHCl orCFHCl could not be determinedthrough measurements in the firstderivative spectra On the other handin the second derivative spectra Fig(7) the degraded LFHCl shows avalley at 272 nm at which the intactdrug has a zero crossing Thus theordinate value at 272 nm was used forcomputing the concentration of thedegradation product in the presenceof the intact drug

Similarly the second derivativespectra Fig (8) show that thedegraded CFHCl exhibits a valley at262 nm at which the intact drugshows no interference Therefore theordinate value at 262 nm was used forcalculating the concentration of thedegradation product in the presenceof the intact drug

Linearity was shown over theconcentration range (12-96 microgml)or (28-126 microgml) of intact LFH|Clor CFHCl respectively (HPLC andDensitometric) linearity was alsoobserved over the concentration range(12-96 microgml) or (64-16 microgml) ofdegraded LFH|Cl or CFHClrespectively

Wavelength (nm)

Fig 7 Second-derivative spectra of intactlomefloxacin hydrochloride (024mg50 ml) (mdashmdashmdash) and ofdegraded drug (024 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 8 Second-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

255

The mean percentage recoveriesof intact and degraded LFHCl werefound to be 9972plusmn056 and 9893plusmn085 respectively (Table 3) Also themean percentage recoveries of intactand degraded CFHCl were 9962plusmn047 and 9967plusmn039 respectively(Table 3)

The method was successfullyapplied for the quantitative estimationof LFHCl or CFHCl in ldquoAlkafloxrdquoor ldquoServifloxrdquo tablets respectivelywithout interference from additivesThe standard addition technique wasapplied The mean percentagerecoveries of labelled and addedLFHCl were found to be 9976plusmn075and 9958plusmn064 respectively Alsothe mean percentage recoveries oflabelled and added CFHCl were

9949plusmn066 and 9978plusmn034respectively The second derivativespectra of ldquoAlkafloxrdquo and ldquoServifloxrdquotablets showed complete absence ofthe corresponding acid degradationproduct

The proposed methods are validand applicable for the determinationof intact LFHCl or CFHCl inlaboratory prepared mixturescontaining (20-80) or (40-75) ofthe degraded LFHCl or degradedCFHCl respectively

Statistical comparison between theproposed and the reported methods(spectrophotometric42 and HPLC43for LFHCl and CFHCl respectively)showed no significant differencebetween them concerning accuracyand precision (Table 3)

Table 3 Statistical analysis for the first derivative spectrophotometricdetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochloride Ciprofloxacin hydrochlorideStatistical

TermFirst

Derivativemethod

Comparisonmethod13

FirstDerivative

method

Comparisonmethod12

Mean 9972 9917 9962 9943

SD plusmn056 plusmn060 plusmn047 plusmn055

RSD 056 047

SE plusmn021 plusmn024 plusmn018 plusmn022

n 7 6 7 6

V 031 036 022 030

LOD(microgml)

039 103

LOQ(microgml)

129 345

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

Sonia T Hassib et al

244

Austria It was analyzed by HPLC43

and found to contain 9943plusmn055CFHCl

ldquoAlkafloxrdquo tablets (batch no 001)nominally containing 400 mg ofLFHCl hydrochloride produced byAlkan Pharma SAE 6 OctoberCity Egypt

ldquoServifloxrdquotablets (batch no 030)nominally containing 2915 mg ofCFHCl hydrochloride (equivalent to250 mg of CFHCl) produced byBiochemie Vienna Austria

Methanol HPLC grade 2MHydrochloric acid ammoniabuffer 10 vv ammonim hydroxidein 1605 mv aqueous ammoniumchloride solution diethyl ether(BDH) 2 M sodium hydroxideacetonitrile HPLC grade triethyl-amine o-phosphoric acid doubledistilled water

Mobile phase for HPLCA mixture of water acetonitrile tri-

ethylamine (802006 vvv) adjustedto pH 30 with o-phosphoric acid

Developing system for densitometryA mixture of methanol and

ammonia buffer (8020 vv) wasfreshly prepared prior to analysis

Preparation of stock solutionsPreparation of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride stock solutions

A solution of LFHCl (05 mg ml)or CFHCL (1 mgml) was preparedin the specified solvent

Preparation of degraded lome-floxacin hydrochloride andciprofloxacin hydrochloride stocksolutions

An accurate weight of LFHCl orCFHCl (250 mg) was refluxed in 2M hydrochloric acid (40 ml) for 48hrs44amp45 After cooling the solutionwas neutralized with 2M sodiumhydroxide and then concentrated to asmall volume The concentratedsolution was extracted with diethylether (10 ml x 3) The ether extractwas evaporated to dryness on a warmwater bath The obtained residue wasused for preparing the degradedLFHCl solution (05 mgml) orCFHCl solution (1 mgml) in thespecified solvent

Preparation of internal standardstock solution

A solution of enrofloxacin (05mgml) was prepared in the mobilephase

ApparatusFor HPLC

HPLC apparatus consisted ofWaters autosampler injector Watersisocratic 610 pump Waters 486 uvdetector adjusted at 328 nm and microBondapack C18 reversed-phasecolumn (25 cm x 46 mm id)

For densitometryCS-9301 PC Dual wavelength

Flying Spot Scanning Shimadzudensitometer Japan

For spectrophotometryShimadzu uv-visible recording

spectrophotometer UV-265 Japan

245

For identification of degradedstructurea- For infra red spectra Shimadzu

Infra red spectrometer (IR- 435)Japan

b- For 1H- NMR spectra Joel FX90QNMR spectrometer

c- For mass spectra Finnigan SSQ7000 Gas chromatograph- mass

ProcedureHPLC method for the determinat-ion of LomefloxacinGeneral procedure and linearity

A solution containing intactLFHCl (05 mgml) and degradedLFHCl (04 mgml) was prepared inthe mobile phase Different aliquots(05-6 ml) of this solution wereintroduced into a series of 10 mlvolumetric flasks followed byinternal standard solution (1 ml) toeach flask The volume of each flaskwas completed with the mobile phaseTwenty-micro liters of each solutionwere injected onto the column Thechromatograms were recorded usingflow rate of 15 mlmin andwavelength of detection at 328 nm

The resulting chromatograms (Fig2) retention times (tR) of the peaksand the areas under the peaks (AUPs)were recorded The ratios (R) of therecorded AUPs of intact LFHCl or ofits degradation product to that of theinternal standard were plotted versusthe concentration (microg20 microl) and thecalibration graphs for intact anddegraded LFHCl were obtained

The following regressionequations (12) were computed

Concentration of intact LFHCl(microg20 microl) =05496 R + 00859 Eq (1)r2 = 09989 orR= 18195 X C (microg20microl) -01563

Concentration of degraded LFHCl(microg20 microl) =

04499 R + 01102 Eq(2)r2 = 09983 orR= 22227 X C (microg20 microl) -02449

Where C is the concentration in microg20microl R is the ratio of AUP ofdrugAUP of IS and r2 is thecorrelation coefficient

Retention time (tR)

Fig 2 HPLC chromatogram of degraded LFHCl (tR= 216 min) intactLFHCl (tR= 2451 min) and enrofloxacin (tR= 2832 min)

Sonia T Hassib et al

246

Simultaneous determination ofintact and degraded lomefloxacinhydrochloride in laboratoryprepared mixtures by RP-HPLC

Two solutions of intact LFHCland degraded LFHCl (06 mgml)each in the mobile phase wereprepared Different aliquots (15-45ml) and (05-35 ml) of both intactand degraded LFHCl solutionsrespectively were introduced into aseries of 10 ml volumetric flasksOne- ml aliquot of internal standardsolution was added to each flask andthe volumes were completed with themobile phase Twenty-micro liters ofeach mixture solution was injectedonto the column and thechromatograms were recorded usingthe same parameters mentioned undergeneral procedure and linearity Theconcentrations of intact and degradedLFHCl were calculated using theregression equations (1amp2)

Determination of intact lome-floxacin hydrochloride inldquoAlkafloxrdquo tablets by RP-HPLC

An accurate weight of thepowdered twenty tablets equivalent to(35 mg) of intact LFHCl wasintroduced into a 100 ml volumetricflask The mobile phase (80 ml) wasadded and the solution was shaken(using a mechanical shaker) for 10minutes and the volume wascompleted with the mobile phase Thesolution was filtered through a dryfilter paper The first 10 ml of thefiltrate was rejected and the filtratewas refiltered through 045 micrommillipore teflon filter Differentaliquots (2-4 ml) of this solution were

transferred into a series of 10 mlvolumetric flasks one ml aliquot ofthe internal standard solution wasadded to each flask and the volumewas completed with the mobile phaseTwenty-micro liters of each solutionwere injected onto the column and thechromatograms were recorded usingthe same parameters used undergeneral procedure and linearity onpage The procedure was repeatedusing the standard addition techniqueThe ratio (R) of AUP of the drug tothe AUP of the internal standard wasused in calculating the recoveredconcentrations of labeled and addedLFHCl

Densitometric simultaneous deter-mination of intact lomefloxacinhydrochloride and ciprofloxacinhydrochloride and their aciddegradation products

General procedure and linearityDifferent aliquots of intact LFHCl

stock solution (1-6 ml) or CFHClstock solution (05-5 ml) weretransferred into two series of 10 mlvolumetric flasks Also severalaliquots of degraded LF HCl stocksolution (05-6 ml) or degradedCFHCl stock solution (05-5 ml)were transferred into two series of 10ml volumetric flasks Each flask wascompleted to volume with methanol

A ten-microliter aliquot of eachsolution was applied on thin-layerchromatographic plates (20x20 cm)using a micropipette Spots werespaced 25 cm apart and 2 cm fromthe bottom edge of the plate Theplates were developed for at least one

247

hour in a chromatographic tankpreviously saturated with thespecified developing system byascending chromatography to adistance of 15 cm The plates wereleft to dry at room temperature Thespots were detected under a uv lamp(254 nm) and the two drugs werescanned under the followingconditionsPhoto mode Reflection scan modeZigzag swing width 12 mmWavelengthi) 288 and 328 nm for intact and

degraded LFHCl respectivelyii) 279 and 323 nm for intact and

degraded CFHCl respectively

Chromatograms result outputsand the areas under the peaks (AUPs)were recorded The recorded AUPswere plotted versus the concentrationused (microg10 microl) and the calibrationcurves for both intact and degradedLFHCl or CFHCl were obtainedThe following regression equations(345 and 6) were computed forcalculating the concentrations ofintact LFHCl degraded LFHClintact CFHCl and degraded CFHClrespectively

Concentration of intact LFHCl(microg10 microl) =00287 AUP ndash 00022 Eq (3)r2 = 09998 OrAUP= 34843 X C (microg10microl) +00767

Concentration of degraded LFHCl(microg10 microl) =00388 AUPndash 01587 Eq (4)r2 = 09982 OrAUP= 25773 X C (microg10microl) +40902

Concentration of intact CFHCl (microg10 microl) =00194 AUPndash 00398 Eq (5)r2 = 09991 OrAUP= 51546 X C (microg10microl) +20515

Concentration of degraded CFHCl(microg10 microl) =00246 AUP ndash00758 Eq (6)r2 = 09980 OrAUP= 40650 X C (microg10microl) +30813

Where C is the concentration inmicrog10microl AUP is the area under thepeak and r2 is the correlationcoefficient

Densitometric determination ofintact and degraded lomefloxacinhydrochloride and ciprofloxacinhydrochloride in laboratoryprepared mixtures

Four solutions of intact anddegraded LFHCl (12 mgml) ofintact and degraded CFHCl (04 mgml) were prepared in methanol

Different aliquots of both intactLFHCl (1-45 ml) or of intactCFHCl (15-4 ml) were transferredinto two series of 10 ml volumetricflasks Different aliquots of degradedLFHCl (05-4 ml) or degradedCFHCl (1-35 ml) were added tointact LFHCl series or to intactCFHCl series respectively [series (1)and (2)] for LFHCl and CFHClrespectively Each volumetric flaskwas completed to volume withmethanol Ten-microlitre aliquots ofeach mixture solution were applied onthin-layer chromatographic platesusing a micropipette and theprocedure was completed using the

Sonia T Hassib et al

248

same parameters mentioned undergeneral procedure and linearity

The recorded AUPs were used forcalculating the concentrations of thetwo components (intact and degradeddrug) in each mixture solution usingthe regression equations (345 and 6)for intact and degraded LFHCl andCFHCl respectively

Densitometric determination oflomefloxacin hydrochloride andciprofloxacin hydrochloride inldquoAlkafloxrdquo and ldquoServifloxrdquo tabletsrespectively

An accurate weight of thepowdered twenty tablets equivalent toLFHCl (35 mg) or CFHCl (30 mg)was extracted with methanol (10 ml x3) Each extract was filtered into a50ml volumetric flask The residuewas washed with methanol (5 ml x 3)the volume was completed withmethanol Aliquots (2-4 ml) ofLFHCl solution or (1-2 ml) ofCFHCl solution were transferredinto two series of 10 ml volumetricflasks and completed to volume withmethanol Ten-microlitre aliquot ofeach solution was applied on thin-chromartographgic plates and theprocedure was completed using thesame parameters mentioned undergeneral procedure for linearity Thisexperiment was repeated applying thestandard addition technique Therecorded AUPs for the spots wereused for calculating the concentrationof labelled and added intact LFHClor CFHCl from regression equations(3 and 5)

Simultaneous derivative spectro-photometric determin-ation ofintact lomefloxacin hydrochlorideor ciprofloxa-cin hydrochlorideand their acid degradationproducts

General procedure and linearityAn aqueous solution of intact LF

HCl (05 mgml) or CF HCl (1mgml) was prepared A ten-mlaliquot of this solution containingLFHCl or CFHCl was introducedinto a 100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid

Accurately measured aliquots (2-8ml) or (25-6 ml) of intact solution ofLFHCl or CF HCl respectivelywere transferred into two series of 50ml volumetric flasks followed by theaddition of accurately measuredvolumes (2-8 ml) or (4-8 ml) ofdegraded solution of LF HCl or CFHCl respectively Each flask wascompleted to volume with the samesolvent

The first and second derivativespectra for each solution wererecorded against a solvent blankusing the following parameters

Real time date Δλ2 speed fastslit 1 cycT(min) 0 ordinate rangelimit - 03 to + 03 or ndash 04 to + 04for LFHCl or CFHCl respectively

Wavelength range 200-400 nm or200-350 nm for LFHCl or CFHClrespectively

Also the second derivativespectrum for each flask solution wasrecorded against a solvent blankusing the same above parametersexcept for the following

249

1- Ordinate range limit - 009 to +006 or -012 to + 008 fordegraded LFHCl or CFHClrespectively

2- Wavelength range 250-350 nmor 225-325 nm for degradedLFHCl or CF HCl respectively

The heights (mm) of the firstderivative spectra at 292 nm forLFHCl or at 260 nm for CFHClwere measured and the calibrationgraph for each drug was plotted Theheights (mm) of the second derivativespectra at 272 or at 262 nm fordegraded LFHCl or CFHClrespectively were measured and thecalibration graphs for the degradeddrugs were constructed

The following regressionequations 789 and 10 werecomputed for calculating theconcentration of intact and degradedlomefloxacin and ciprofloxacinrespectively

Concentration of intact LFHCl(microgml) =01631 H292+ 00394 Eq(7)r2= 09998 OrH292= 61312 X C (microgml) -02416

Concentration of intact CFHCl(microgml) =01916H 260 + 02480 Eq(8)r2= 09984 OrH 260 = 52192 XC (microgml) -12944

Concentration of degraded LFHCl(microgml) =01534H272 + 00359 Eq(9)r2= 09998 OrH272 = 65189 X C (microg ml) -02340

Concentration of degraded CFHCl(microgml) =02123H 262 + 05092 Eq (10)r2= 09963 OrH 262 = 47103 X C (microg ml) -23985

Where C is the concentration in microgml H is height of the peak and r2 isthe correlation coefficient

Derivative spectrophotometricdetermination of lomefloxacinhydrochloride and ciprofloxacinhydrochloride in ldquoAlkafloxrdquo andldquoServifloxrdquo tablets respectively

An accurate weight of thepowdered tablets equivalent toLFHCl or CFHCl (20 mg) wasextracted with methanol (15 ml x 3)then filtered into a 100 ml volumetricflask After complete washing of theresidue with methanol (10 ml x 3) thesolution was diluted to volume withmethanol A twenty-ml aliquot of thissolution equivalent to LF HCl orCFHCl (4 mg) was evaporated on awarm water bath The residue wasdissolved in 2 M hydrochloric acidand quantitatively transferred into a100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid Accuratelymeasured aliquots of the abovesolution (2-6 ml) or (6-9 ml) wereintroduced into two series of 50 mlvolumetric flasks Each flask wascompleted to volume with 2Mhydrochloric acid

The first derivative spectrum ofeach drug was recorded against ablank using the same parametersmentioned under general procedureand linearity This procedure was

Sonia T Hassib et al

250

repeated using the standard additiontechnique

The heights (mm) at 292 and 260nm were measured and used forcalculating the corresponding labeledand added concentrations of intactLFHCl and CFHCl respectively

RESULTS AND DISCUSSION

HPLC methodIn this study different mobile

phases were attempted for theseparation of LFHCl in presence ofits degradation product(s) HoweverThoppil amp Amin38 reported astability-indicating reversed-phaseliquid chromatographic procedure forthe determination of CFHCl as bulkdrug in pharmaceutical preparationsand in the presence of its acid andalkaline degraded solutions Themobile phase consisted of water-acetonitrile-triethylamine (802006vvv) adjusted with o-phosphoricacid to pH 3

The above mobile systempreviously applied for CFHCl wassuccessfully used for the separationand determination of intact anddegraded LFHCl since it showedgood resolution between the elutedpeaks Detection was carried out at328 nm The polarity of the systemwas calculated46 and found to be(109) The flow rate was set at 15mlmin Enrofloxacin was used as aninternal standard with the elutionorder degraded LFHCl (tR= 2160)intact LFHCl (tR= 2451) andenrofloxacin (tR= 2832) Fig (2) Thechromatogram of the mixture shows

complete separation and goodresolution

A linear relationship wasobserved when the ratios of the AUPsof each component to the AUP of theinternal standard were plotted versusthe concentration of each componentThe concentration range for eachcomponent in the mixture regressionequations mean percentagerecoveries limit of detection (LOD)and limit of quantification (LOQ)when using the proposed methods aredisplayed in Table (1)

Densitometric methodFew developing systems were

used for TLC separation and solventpolarity optimization of LFH|Cl47 andCFHCl27amp48 in the presence of eitherrelated impurities or photo-degradation products in irradiatedsolutions Seija Tammilehto et al48

studied the photodegradation ofCFHCl irradiated in aqueoussolutions through using a mixture ofacetonitrile and ammonia buffer (pH106-111) as a developing system(6535 vv) for the separation of thecompound and its photodegradationproducts

Using methanol instead ofacetonitrile in a different proportionin the present work offered betterseparation of the drug and its aciddegradation product The polarity ofthe used system was calculated andfound to be (515)46

The TLC chromatogram of bothintact and degraded LFHCl showedcomplete separation with (Rf= 066)for the intact drug and (Rf= 083) for

251

Table 1 Results obtained in the chromatographic methods for the determinationof intact and degraded lomefloxacin hydrochloride and ciprofloxacinhydrochloride

TLC method HPLC methodLFHCl CFHCl LFHClItem

Intact Deg Intact Deg Intact DegConcentrationrange

1-6microg10 microl

05-6microg10 microl

025-25microg10 microl

025-25microg10 microl

05-6microg20 microl

04-48microg20 microl

Mean percentagerecoveries

1-Laboratorypreparedmixtures

9960plusmn 055

9951plusmn 067

9955plusmn072

9922plusmn069

9968plusmn077

10030plusmn089

2-Added standardto lsquoAlkafloxrsquoor lsquoServifloxrsquo

tablets

9925plusmn080

9963plusmn 064

9938plusmn070

LOD010

(microg 10microl)005

(microg 10microl)022

(microg 20microl)

LOQ034

(microg 10microl)018

(microg 10microl)074

(microg 20microl)

the degraded product The separatedspots of both components werescanned on the same plate at 288 and328 nm for the intact and degradedLFHCl respectively

Upon applying this modifieddeveloping system to the analysis ofboth intact and degraded CFHCl theTLC chromatogram showed completeresolution with (Rf= 053) for theintact drug and (Rf= 075) for thedegraded drug The separated spots ofboth components were scanned on thesame plate at 279 and 323 nm for theintact and degraded CFHClrespectively

The concentration range for eachcomponent in the two mixturesregression equations meanpercentage recoveries limit ofdetection and limit of quantificationwhen using the proposed methods aredisplayed in Table (1)

The results obtained in thedetermination of intact LFHCl andCFHCl in the presence of their aciddegradation products using HPLCand densitometric methods werestatistically compared to the reportedmethods (spectrophotometric42 andHPLC43 respectively) showing nosignificant difference concerningaccuracy and precision Table (2)

Sonia T Hassib et al

252

Table 2 Statistical analysis for the chromatographic methods for thedetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochlorideCiprofloxacinhydrochlorideStatistcal Term

HPLCmethod

TLCmethod

Reference42

TLCmethod

Reference43

Mean 9968 9960 9917 9955 9943

SD plusmn077 plusmn055 plusmn060 plusmn072 plusmn055

RSDwithin day

077 055 072

RSDbetween days

117 116 119

SE plusmn031 plusmn022 plusmn024 plusmn029 plusmn023

n 6 6 6 6 6

V 059 030 036 052 030

LOD022

(microg 20microl)010

(microg 10microl)005

(microg 10microl)

LOQ074

(microg 20microl)034

(microg 10microl)018

(microg 10microl)

t 128 130 032

F 164 120 173

The corresponding theoretical values for t and F at p = 005 are 223 and 505respectively

Derivative spectrophotometricmethod

The zero-order spectra of intactand degraded LFHCl and CFHClFigs (3amp4) show completeoverlapping which prevents thedetermination of the intact anddegraded drugs in presence of eachother using this technique

In this work a derivativespectrophotometric procedure ispresented to solve this problem

through measurements in both thefirst and second derivative spectraThe first derivative spectra Figs(5amp6) show that intact LFHCldisplays a peak at 272 nm and avalley at 292 nm while the degradedproduct has zero crossing at both 292and 271 nm Thus the ordinate valueat 292 nm was chosen for thedetermination of the intact drug in thepresence of its degradation productIn addition the first derivative

253

Wavelength (nm)

Fig 3 Zero-order spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 4 Zero-order spectra of intactciprofloxacin hydrochloride (056mg50ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 5 First-derivative spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 6 First-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Abs

orba

nce

Abs

orba

nce

Sonia T Hassib et al

254

spectrum of intact CFHCl Fig (6)exhibits a peak at 262 nm and a valleyat 282 nm while its degradationproduct has zero crossing at both 260and 286 nm Consequently theordinate value at 260 nm waspreferred for calculating theconcentration of the intact drug in thepresence of its degradation product

Figures (5amp6) show that thedegradation product of LFHCl orCFHCl could not be determinedthrough measurements in the firstderivative spectra On the other handin the second derivative spectra Fig(7) the degraded LFHCl shows avalley at 272 nm at which the intactdrug has a zero crossing Thus theordinate value at 272 nm was used forcomputing the concentration of thedegradation product in the presenceof the intact drug

Similarly the second derivativespectra Fig (8) show that thedegraded CFHCl exhibits a valley at262 nm at which the intact drugshows no interference Therefore theordinate value at 262 nm was used forcalculating the concentration of thedegradation product in the presenceof the intact drug

Linearity was shown over theconcentration range (12-96 microgml)or (28-126 microgml) of intact LFH|Clor CFHCl respectively (HPLC andDensitometric) linearity was alsoobserved over the concentration range(12-96 microgml) or (64-16 microgml) ofdegraded LFH|Cl or CFHClrespectively

Wavelength (nm)

Fig 7 Second-derivative spectra of intactlomefloxacin hydrochloride (024mg50 ml) (mdashmdashmdash) and ofdegraded drug (024 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 8 Second-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

255

The mean percentage recoveriesof intact and degraded LFHCl werefound to be 9972plusmn056 and 9893plusmn085 respectively (Table 3) Also themean percentage recoveries of intactand degraded CFHCl were 9962plusmn047 and 9967plusmn039 respectively(Table 3)

The method was successfullyapplied for the quantitative estimationof LFHCl or CFHCl in ldquoAlkafloxrdquoor ldquoServifloxrdquo tablets respectivelywithout interference from additivesThe standard addition technique wasapplied The mean percentagerecoveries of labelled and addedLFHCl were found to be 9976plusmn075and 9958plusmn064 respectively Alsothe mean percentage recoveries oflabelled and added CFHCl were

9949plusmn066 and 9978plusmn034respectively The second derivativespectra of ldquoAlkafloxrdquo and ldquoServifloxrdquotablets showed complete absence ofthe corresponding acid degradationproduct

The proposed methods are validand applicable for the determinationof intact LFHCl or CFHCl inlaboratory prepared mixturescontaining (20-80) or (40-75) ofthe degraded LFHCl or degradedCFHCl respectively

Statistical comparison between theproposed and the reported methods(spectrophotometric42 and HPLC43for LFHCl and CFHCl respectively)showed no significant differencebetween them concerning accuracyand precision (Table 3)

Table 3 Statistical analysis for the first derivative spectrophotometricdetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochloride Ciprofloxacin hydrochlorideStatistical

TermFirst

Derivativemethod

Comparisonmethod13

FirstDerivative

method

Comparisonmethod12

Mean 9972 9917 9962 9943

SD plusmn056 plusmn060 plusmn047 plusmn055

RSD 056 047

SE plusmn021 plusmn024 plusmn018 plusmn022

n 7 6 7 6

V 031 036 022 030

LOD(microgml)

039 103

LOQ(microgml)

129 345

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

245

For identification of degradedstructurea- For infra red spectra Shimadzu

Infra red spectrometer (IR- 435)Japan

b- For 1H- NMR spectra Joel FX90QNMR spectrometer

c- For mass spectra Finnigan SSQ7000 Gas chromatograph- mass

ProcedureHPLC method for the determinat-ion of LomefloxacinGeneral procedure and linearity

A solution containing intactLFHCl (05 mgml) and degradedLFHCl (04 mgml) was prepared inthe mobile phase Different aliquots(05-6 ml) of this solution wereintroduced into a series of 10 mlvolumetric flasks followed byinternal standard solution (1 ml) toeach flask The volume of each flaskwas completed with the mobile phaseTwenty-micro liters of each solutionwere injected onto the column Thechromatograms were recorded usingflow rate of 15 mlmin andwavelength of detection at 328 nm

The resulting chromatograms (Fig2) retention times (tR) of the peaksand the areas under the peaks (AUPs)were recorded The ratios (R) of therecorded AUPs of intact LFHCl or ofits degradation product to that of theinternal standard were plotted versusthe concentration (microg20 microl) and thecalibration graphs for intact anddegraded LFHCl were obtained

The following regressionequations (12) were computed

Concentration of intact LFHCl(microg20 microl) =05496 R + 00859 Eq (1)r2 = 09989 orR= 18195 X C (microg20microl) -01563

Concentration of degraded LFHCl(microg20 microl) =

04499 R + 01102 Eq(2)r2 = 09983 orR= 22227 X C (microg20 microl) -02449

Where C is the concentration in microg20microl R is the ratio of AUP ofdrugAUP of IS and r2 is thecorrelation coefficient

Retention time (tR)

Fig 2 HPLC chromatogram of degraded LFHCl (tR= 216 min) intactLFHCl (tR= 2451 min) and enrofloxacin (tR= 2832 min)

Sonia T Hassib et al

246

Simultaneous determination ofintact and degraded lomefloxacinhydrochloride in laboratoryprepared mixtures by RP-HPLC

Two solutions of intact LFHCland degraded LFHCl (06 mgml)each in the mobile phase wereprepared Different aliquots (15-45ml) and (05-35 ml) of both intactand degraded LFHCl solutionsrespectively were introduced into aseries of 10 ml volumetric flasksOne- ml aliquot of internal standardsolution was added to each flask andthe volumes were completed with themobile phase Twenty-micro liters ofeach mixture solution was injectedonto the column and thechromatograms were recorded usingthe same parameters mentioned undergeneral procedure and linearity Theconcentrations of intact and degradedLFHCl were calculated using theregression equations (1amp2)

Determination of intact lome-floxacin hydrochloride inldquoAlkafloxrdquo tablets by RP-HPLC

An accurate weight of thepowdered twenty tablets equivalent to(35 mg) of intact LFHCl wasintroduced into a 100 ml volumetricflask The mobile phase (80 ml) wasadded and the solution was shaken(using a mechanical shaker) for 10minutes and the volume wascompleted with the mobile phase Thesolution was filtered through a dryfilter paper The first 10 ml of thefiltrate was rejected and the filtratewas refiltered through 045 micrommillipore teflon filter Differentaliquots (2-4 ml) of this solution were

transferred into a series of 10 mlvolumetric flasks one ml aliquot ofthe internal standard solution wasadded to each flask and the volumewas completed with the mobile phaseTwenty-micro liters of each solutionwere injected onto the column and thechromatograms were recorded usingthe same parameters used undergeneral procedure and linearity onpage The procedure was repeatedusing the standard addition techniqueThe ratio (R) of AUP of the drug tothe AUP of the internal standard wasused in calculating the recoveredconcentrations of labeled and addedLFHCl

Densitometric simultaneous deter-mination of intact lomefloxacinhydrochloride and ciprofloxacinhydrochloride and their aciddegradation products

General procedure and linearityDifferent aliquots of intact LFHCl

stock solution (1-6 ml) or CFHClstock solution (05-5 ml) weretransferred into two series of 10 mlvolumetric flasks Also severalaliquots of degraded LF HCl stocksolution (05-6 ml) or degradedCFHCl stock solution (05-5 ml)were transferred into two series of 10ml volumetric flasks Each flask wascompleted to volume with methanol

A ten-microliter aliquot of eachsolution was applied on thin-layerchromatographic plates (20x20 cm)using a micropipette Spots werespaced 25 cm apart and 2 cm fromthe bottom edge of the plate Theplates were developed for at least one

247

hour in a chromatographic tankpreviously saturated with thespecified developing system byascending chromatography to adistance of 15 cm The plates wereleft to dry at room temperature Thespots were detected under a uv lamp(254 nm) and the two drugs werescanned under the followingconditionsPhoto mode Reflection scan modeZigzag swing width 12 mmWavelengthi) 288 and 328 nm for intact and

degraded LFHCl respectivelyii) 279 and 323 nm for intact and

degraded CFHCl respectively

Chromatograms result outputsand the areas under the peaks (AUPs)were recorded The recorded AUPswere plotted versus the concentrationused (microg10 microl) and the calibrationcurves for both intact and degradedLFHCl or CFHCl were obtainedThe following regression equations(345 and 6) were computed forcalculating the concentrations ofintact LFHCl degraded LFHClintact CFHCl and degraded CFHClrespectively

Concentration of intact LFHCl(microg10 microl) =00287 AUP ndash 00022 Eq (3)r2 = 09998 OrAUP= 34843 X C (microg10microl) +00767

Concentration of degraded LFHCl(microg10 microl) =00388 AUPndash 01587 Eq (4)r2 = 09982 OrAUP= 25773 X C (microg10microl) +40902

Concentration of intact CFHCl (microg10 microl) =00194 AUPndash 00398 Eq (5)r2 = 09991 OrAUP= 51546 X C (microg10microl) +20515

Concentration of degraded CFHCl(microg10 microl) =00246 AUP ndash00758 Eq (6)r2 = 09980 OrAUP= 40650 X C (microg10microl) +30813

Where C is the concentration inmicrog10microl AUP is the area under thepeak and r2 is the correlationcoefficient

Densitometric determination ofintact and degraded lomefloxacinhydrochloride and ciprofloxacinhydrochloride in laboratoryprepared mixtures

Four solutions of intact anddegraded LFHCl (12 mgml) ofintact and degraded CFHCl (04 mgml) were prepared in methanol

Different aliquots of both intactLFHCl (1-45 ml) or of intactCFHCl (15-4 ml) were transferredinto two series of 10 ml volumetricflasks Different aliquots of degradedLFHCl (05-4 ml) or degradedCFHCl (1-35 ml) were added tointact LFHCl series or to intactCFHCl series respectively [series (1)and (2)] for LFHCl and CFHClrespectively Each volumetric flaskwas completed to volume withmethanol Ten-microlitre aliquots ofeach mixture solution were applied onthin-layer chromatographic platesusing a micropipette and theprocedure was completed using the

Sonia T Hassib et al

248

same parameters mentioned undergeneral procedure and linearity

The recorded AUPs were used forcalculating the concentrations of thetwo components (intact and degradeddrug) in each mixture solution usingthe regression equations (345 and 6)for intact and degraded LFHCl andCFHCl respectively

Densitometric determination oflomefloxacin hydrochloride andciprofloxacin hydrochloride inldquoAlkafloxrdquo and ldquoServifloxrdquo tabletsrespectively

An accurate weight of thepowdered twenty tablets equivalent toLFHCl (35 mg) or CFHCl (30 mg)was extracted with methanol (10 ml x3) Each extract was filtered into a50ml volumetric flask The residuewas washed with methanol (5 ml x 3)the volume was completed withmethanol Aliquots (2-4 ml) ofLFHCl solution or (1-2 ml) ofCFHCl solution were transferredinto two series of 10 ml volumetricflasks and completed to volume withmethanol Ten-microlitre aliquot ofeach solution was applied on thin-chromartographgic plates and theprocedure was completed using thesame parameters mentioned undergeneral procedure for linearity Thisexperiment was repeated applying thestandard addition technique Therecorded AUPs for the spots wereused for calculating the concentrationof labelled and added intact LFHClor CFHCl from regression equations(3 and 5)

Simultaneous derivative spectro-photometric determin-ation ofintact lomefloxacin hydrochlorideor ciprofloxa-cin hydrochlorideand their acid degradationproducts

General procedure and linearityAn aqueous solution of intact LF

HCl (05 mgml) or CF HCl (1mgml) was prepared A ten-mlaliquot of this solution containingLFHCl or CFHCl was introducedinto a 100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid

Accurately measured aliquots (2-8ml) or (25-6 ml) of intact solution ofLFHCl or CF HCl respectivelywere transferred into two series of 50ml volumetric flasks followed by theaddition of accurately measuredvolumes (2-8 ml) or (4-8 ml) ofdegraded solution of LF HCl or CFHCl respectively Each flask wascompleted to volume with the samesolvent

The first and second derivativespectra for each solution wererecorded against a solvent blankusing the following parameters

Real time date Δλ2 speed fastslit 1 cycT(min) 0 ordinate rangelimit - 03 to + 03 or ndash 04 to + 04for LFHCl or CFHCl respectively

Wavelength range 200-400 nm or200-350 nm for LFHCl or CFHClrespectively

Also the second derivativespectrum for each flask solution wasrecorded against a solvent blankusing the same above parametersexcept for the following

249

1- Ordinate range limit - 009 to +006 or -012 to + 008 fordegraded LFHCl or CFHClrespectively

2- Wavelength range 250-350 nmor 225-325 nm for degradedLFHCl or CF HCl respectively

The heights (mm) of the firstderivative spectra at 292 nm forLFHCl or at 260 nm for CFHClwere measured and the calibrationgraph for each drug was plotted Theheights (mm) of the second derivativespectra at 272 or at 262 nm fordegraded LFHCl or CFHClrespectively were measured and thecalibration graphs for the degradeddrugs were constructed

The following regressionequations 789 and 10 werecomputed for calculating theconcentration of intact and degradedlomefloxacin and ciprofloxacinrespectively

Concentration of intact LFHCl(microgml) =01631 H292+ 00394 Eq(7)r2= 09998 OrH292= 61312 X C (microgml) -02416

Concentration of intact CFHCl(microgml) =01916H 260 + 02480 Eq(8)r2= 09984 OrH 260 = 52192 XC (microgml) -12944

Concentration of degraded LFHCl(microgml) =01534H272 + 00359 Eq(9)r2= 09998 OrH272 = 65189 X C (microg ml) -02340

Concentration of degraded CFHCl(microgml) =02123H 262 + 05092 Eq (10)r2= 09963 OrH 262 = 47103 X C (microg ml) -23985

Where C is the concentration in microgml H is height of the peak and r2 isthe correlation coefficient

Derivative spectrophotometricdetermination of lomefloxacinhydrochloride and ciprofloxacinhydrochloride in ldquoAlkafloxrdquo andldquoServifloxrdquo tablets respectively

An accurate weight of thepowdered tablets equivalent toLFHCl or CFHCl (20 mg) wasextracted with methanol (15 ml x 3)then filtered into a 100 ml volumetricflask After complete washing of theresidue with methanol (10 ml x 3) thesolution was diluted to volume withmethanol A twenty-ml aliquot of thissolution equivalent to LF HCl orCFHCl (4 mg) was evaporated on awarm water bath The residue wasdissolved in 2 M hydrochloric acidand quantitatively transferred into a100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid Accuratelymeasured aliquots of the abovesolution (2-6 ml) or (6-9 ml) wereintroduced into two series of 50 mlvolumetric flasks Each flask wascompleted to volume with 2Mhydrochloric acid

The first derivative spectrum ofeach drug was recorded against ablank using the same parametersmentioned under general procedureand linearity This procedure was

Sonia T Hassib et al

250

repeated using the standard additiontechnique

The heights (mm) at 292 and 260nm were measured and used forcalculating the corresponding labeledand added concentrations of intactLFHCl and CFHCl respectively

RESULTS AND DISCUSSION

HPLC methodIn this study different mobile

phases were attempted for theseparation of LFHCl in presence ofits degradation product(s) HoweverThoppil amp Amin38 reported astability-indicating reversed-phaseliquid chromatographic procedure forthe determination of CFHCl as bulkdrug in pharmaceutical preparationsand in the presence of its acid andalkaline degraded solutions Themobile phase consisted of water-acetonitrile-triethylamine (802006vvv) adjusted with o-phosphoricacid to pH 3

The above mobile systempreviously applied for CFHCl wassuccessfully used for the separationand determination of intact anddegraded LFHCl since it showedgood resolution between the elutedpeaks Detection was carried out at328 nm The polarity of the systemwas calculated46 and found to be(109) The flow rate was set at 15mlmin Enrofloxacin was used as aninternal standard with the elutionorder degraded LFHCl (tR= 2160)intact LFHCl (tR= 2451) andenrofloxacin (tR= 2832) Fig (2) Thechromatogram of the mixture shows

complete separation and goodresolution

A linear relationship wasobserved when the ratios of the AUPsof each component to the AUP of theinternal standard were plotted versusthe concentration of each componentThe concentration range for eachcomponent in the mixture regressionequations mean percentagerecoveries limit of detection (LOD)and limit of quantification (LOQ)when using the proposed methods aredisplayed in Table (1)

Densitometric methodFew developing systems were

used for TLC separation and solventpolarity optimization of LFH|Cl47 andCFHCl27amp48 in the presence of eitherrelated impurities or photo-degradation products in irradiatedsolutions Seija Tammilehto et al48

studied the photodegradation ofCFHCl irradiated in aqueoussolutions through using a mixture ofacetonitrile and ammonia buffer (pH106-111) as a developing system(6535 vv) for the separation of thecompound and its photodegradationproducts

Using methanol instead ofacetonitrile in a different proportionin the present work offered betterseparation of the drug and its aciddegradation product The polarity ofthe used system was calculated andfound to be (515)46

The TLC chromatogram of bothintact and degraded LFHCl showedcomplete separation with (Rf= 066)for the intact drug and (Rf= 083) for

251

Table 1 Results obtained in the chromatographic methods for the determinationof intact and degraded lomefloxacin hydrochloride and ciprofloxacinhydrochloride

TLC method HPLC methodLFHCl CFHCl LFHClItem

Intact Deg Intact Deg Intact DegConcentrationrange

1-6microg10 microl

05-6microg10 microl

025-25microg10 microl

025-25microg10 microl

05-6microg20 microl

04-48microg20 microl

Mean percentagerecoveries

1-Laboratorypreparedmixtures

9960plusmn 055

9951plusmn 067

9955plusmn072

9922plusmn069

9968plusmn077

10030plusmn089

2-Added standardto lsquoAlkafloxrsquoor lsquoServifloxrsquo

tablets

9925plusmn080

9963plusmn 064

9938plusmn070

LOD010

(microg 10microl)005

(microg 10microl)022

(microg 20microl)

LOQ034

(microg 10microl)018

(microg 10microl)074

(microg 20microl)

the degraded product The separatedspots of both components werescanned on the same plate at 288 and328 nm for the intact and degradedLFHCl respectively

Upon applying this modifieddeveloping system to the analysis ofboth intact and degraded CFHCl theTLC chromatogram showed completeresolution with (Rf= 053) for theintact drug and (Rf= 075) for thedegraded drug The separated spots ofboth components were scanned on thesame plate at 279 and 323 nm for theintact and degraded CFHClrespectively

The concentration range for eachcomponent in the two mixturesregression equations meanpercentage recoveries limit ofdetection and limit of quantificationwhen using the proposed methods aredisplayed in Table (1)

The results obtained in thedetermination of intact LFHCl andCFHCl in the presence of their aciddegradation products using HPLCand densitometric methods werestatistically compared to the reportedmethods (spectrophotometric42 andHPLC43 respectively) showing nosignificant difference concerningaccuracy and precision Table (2)

Sonia T Hassib et al

252

Table 2 Statistical analysis for the chromatographic methods for thedetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochlorideCiprofloxacinhydrochlorideStatistcal Term

HPLCmethod

TLCmethod

Reference42

TLCmethod

Reference43

Mean 9968 9960 9917 9955 9943

SD plusmn077 plusmn055 plusmn060 plusmn072 plusmn055

RSDwithin day

077 055 072

RSDbetween days

117 116 119

SE plusmn031 plusmn022 plusmn024 plusmn029 plusmn023

n 6 6 6 6 6

V 059 030 036 052 030

LOD022

(microg 20microl)010

(microg 10microl)005

(microg 10microl)

LOQ074

(microg 20microl)034

(microg 10microl)018

(microg 10microl)

t 128 130 032

F 164 120 173

The corresponding theoretical values for t and F at p = 005 are 223 and 505respectively

Derivative spectrophotometricmethod

The zero-order spectra of intactand degraded LFHCl and CFHClFigs (3amp4) show completeoverlapping which prevents thedetermination of the intact anddegraded drugs in presence of eachother using this technique

In this work a derivativespectrophotometric procedure ispresented to solve this problem

through measurements in both thefirst and second derivative spectraThe first derivative spectra Figs(5amp6) show that intact LFHCldisplays a peak at 272 nm and avalley at 292 nm while the degradedproduct has zero crossing at both 292and 271 nm Thus the ordinate valueat 292 nm was chosen for thedetermination of the intact drug in thepresence of its degradation productIn addition the first derivative

253

Wavelength (nm)

Fig 3 Zero-order spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 4 Zero-order spectra of intactciprofloxacin hydrochloride (056mg50ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 5 First-derivative spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 6 First-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Abs

orba

nce

Abs

orba

nce

Sonia T Hassib et al

254

spectrum of intact CFHCl Fig (6)exhibits a peak at 262 nm and a valleyat 282 nm while its degradationproduct has zero crossing at both 260and 286 nm Consequently theordinate value at 260 nm waspreferred for calculating theconcentration of the intact drug in thepresence of its degradation product

Figures (5amp6) show that thedegradation product of LFHCl orCFHCl could not be determinedthrough measurements in the firstderivative spectra On the other handin the second derivative spectra Fig(7) the degraded LFHCl shows avalley at 272 nm at which the intactdrug has a zero crossing Thus theordinate value at 272 nm was used forcomputing the concentration of thedegradation product in the presenceof the intact drug

Similarly the second derivativespectra Fig (8) show that thedegraded CFHCl exhibits a valley at262 nm at which the intact drugshows no interference Therefore theordinate value at 262 nm was used forcalculating the concentration of thedegradation product in the presenceof the intact drug

Linearity was shown over theconcentration range (12-96 microgml)or (28-126 microgml) of intact LFH|Clor CFHCl respectively (HPLC andDensitometric) linearity was alsoobserved over the concentration range(12-96 microgml) or (64-16 microgml) ofdegraded LFH|Cl or CFHClrespectively

Wavelength (nm)

Fig 7 Second-derivative spectra of intactlomefloxacin hydrochloride (024mg50 ml) (mdashmdashmdash) and ofdegraded drug (024 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 8 Second-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

255

The mean percentage recoveriesof intact and degraded LFHCl werefound to be 9972plusmn056 and 9893plusmn085 respectively (Table 3) Also themean percentage recoveries of intactand degraded CFHCl were 9962plusmn047 and 9967plusmn039 respectively(Table 3)

The method was successfullyapplied for the quantitative estimationof LFHCl or CFHCl in ldquoAlkafloxrdquoor ldquoServifloxrdquo tablets respectivelywithout interference from additivesThe standard addition technique wasapplied The mean percentagerecoveries of labelled and addedLFHCl were found to be 9976plusmn075and 9958plusmn064 respectively Alsothe mean percentage recoveries oflabelled and added CFHCl were

9949plusmn066 and 9978plusmn034respectively The second derivativespectra of ldquoAlkafloxrdquo and ldquoServifloxrdquotablets showed complete absence ofthe corresponding acid degradationproduct

The proposed methods are validand applicable for the determinationof intact LFHCl or CFHCl inlaboratory prepared mixturescontaining (20-80) or (40-75) ofthe degraded LFHCl or degradedCFHCl respectively

Statistical comparison between theproposed and the reported methods(spectrophotometric42 and HPLC43for LFHCl and CFHCl respectively)showed no significant differencebetween them concerning accuracyand precision (Table 3)

Table 3 Statistical analysis for the first derivative spectrophotometricdetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochloride Ciprofloxacin hydrochlorideStatistical

TermFirst

Derivativemethod

Comparisonmethod13

FirstDerivative

method

Comparisonmethod12

Mean 9972 9917 9962 9943

SD plusmn056 plusmn060 plusmn047 plusmn055

RSD 056 047

SE plusmn021 plusmn024 plusmn018 plusmn022

n 7 6 7 6

V 031 036 022 030

LOD(microgml)

039 103

LOQ(microgml)

129 345

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

Sonia T Hassib et al

246

Simultaneous determination ofintact and degraded lomefloxacinhydrochloride in laboratoryprepared mixtures by RP-HPLC

Two solutions of intact LFHCland degraded LFHCl (06 mgml)each in the mobile phase wereprepared Different aliquots (15-45ml) and (05-35 ml) of both intactand degraded LFHCl solutionsrespectively were introduced into aseries of 10 ml volumetric flasksOne- ml aliquot of internal standardsolution was added to each flask andthe volumes were completed with themobile phase Twenty-micro liters ofeach mixture solution was injectedonto the column and thechromatograms were recorded usingthe same parameters mentioned undergeneral procedure and linearity Theconcentrations of intact and degradedLFHCl were calculated using theregression equations (1amp2)

Determination of intact lome-floxacin hydrochloride inldquoAlkafloxrdquo tablets by RP-HPLC

An accurate weight of thepowdered twenty tablets equivalent to(35 mg) of intact LFHCl wasintroduced into a 100 ml volumetricflask The mobile phase (80 ml) wasadded and the solution was shaken(using a mechanical shaker) for 10minutes and the volume wascompleted with the mobile phase Thesolution was filtered through a dryfilter paper The first 10 ml of thefiltrate was rejected and the filtratewas refiltered through 045 micrommillipore teflon filter Differentaliquots (2-4 ml) of this solution were

transferred into a series of 10 mlvolumetric flasks one ml aliquot ofthe internal standard solution wasadded to each flask and the volumewas completed with the mobile phaseTwenty-micro liters of each solutionwere injected onto the column and thechromatograms were recorded usingthe same parameters used undergeneral procedure and linearity onpage The procedure was repeatedusing the standard addition techniqueThe ratio (R) of AUP of the drug tothe AUP of the internal standard wasused in calculating the recoveredconcentrations of labeled and addedLFHCl

Densitometric simultaneous deter-mination of intact lomefloxacinhydrochloride and ciprofloxacinhydrochloride and their aciddegradation products

General procedure and linearityDifferent aliquots of intact LFHCl

stock solution (1-6 ml) or CFHClstock solution (05-5 ml) weretransferred into two series of 10 mlvolumetric flasks Also severalaliquots of degraded LF HCl stocksolution (05-6 ml) or degradedCFHCl stock solution (05-5 ml)were transferred into two series of 10ml volumetric flasks Each flask wascompleted to volume with methanol

A ten-microliter aliquot of eachsolution was applied on thin-layerchromatographic plates (20x20 cm)using a micropipette Spots werespaced 25 cm apart and 2 cm fromthe bottom edge of the plate Theplates were developed for at least one

247

hour in a chromatographic tankpreviously saturated with thespecified developing system byascending chromatography to adistance of 15 cm The plates wereleft to dry at room temperature Thespots were detected under a uv lamp(254 nm) and the two drugs werescanned under the followingconditionsPhoto mode Reflection scan modeZigzag swing width 12 mmWavelengthi) 288 and 328 nm for intact and

degraded LFHCl respectivelyii) 279 and 323 nm for intact and

degraded CFHCl respectively

Chromatograms result outputsand the areas under the peaks (AUPs)were recorded The recorded AUPswere plotted versus the concentrationused (microg10 microl) and the calibrationcurves for both intact and degradedLFHCl or CFHCl were obtainedThe following regression equations(345 and 6) were computed forcalculating the concentrations ofintact LFHCl degraded LFHClintact CFHCl and degraded CFHClrespectively

Concentration of intact LFHCl(microg10 microl) =00287 AUP ndash 00022 Eq (3)r2 = 09998 OrAUP= 34843 X C (microg10microl) +00767

Concentration of degraded LFHCl(microg10 microl) =00388 AUPndash 01587 Eq (4)r2 = 09982 OrAUP= 25773 X C (microg10microl) +40902

Concentration of intact CFHCl (microg10 microl) =00194 AUPndash 00398 Eq (5)r2 = 09991 OrAUP= 51546 X C (microg10microl) +20515

Concentration of degraded CFHCl(microg10 microl) =00246 AUP ndash00758 Eq (6)r2 = 09980 OrAUP= 40650 X C (microg10microl) +30813

Where C is the concentration inmicrog10microl AUP is the area under thepeak and r2 is the correlationcoefficient

Densitometric determination ofintact and degraded lomefloxacinhydrochloride and ciprofloxacinhydrochloride in laboratoryprepared mixtures

Four solutions of intact anddegraded LFHCl (12 mgml) ofintact and degraded CFHCl (04 mgml) were prepared in methanol

Different aliquots of both intactLFHCl (1-45 ml) or of intactCFHCl (15-4 ml) were transferredinto two series of 10 ml volumetricflasks Different aliquots of degradedLFHCl (05-4 ml) or degradedCFHCl (1-35 ml) were added tointact LFHCl series or to intactCFHCl series respectively [series (1)and (2)] for LFHCl and CFHClrespectively Each volumetric flaskwas completed to volume withmethanol Ten-microlitre aliquots ofeach mixture solution were applied onthin-layer chromatographic platesusing a micropipette and theprocedure was completed using the

Sonia T Hassib et al

248

same parameters mentioned undergeneral procedure and linearity

The recorded AUPs were used forcalculating the concentrations of thetwo components (intact and degradeddrug) in each mixture solution usingthe regression equations (345 and 6)for intact and degraded LFHCl andCFHCl respectively

Densitometric determination oflomefloxacin hydrochloride andciprofloxacin hydrochloride inldquoAlkafloxrdquo and ldquoServifloxrdquo tabletsrespectively

An accurate weight of thepowdered twenty tablets equivalent toLFHCl (35 mg) or CFHCl (30 mg)was extracted with methanol (10 ml x3) Each extract was filtered into a50ml volumetric flask The residuewas washed with methanol (5 ml x 3)the volume was completed withmethanol Aliquots (2-4 ml) ofLFHCl solution or (1-2 ml) ofCFHCl solution were transferredinto two series of 10 ml volumetricflasks and completed to volume withmethanol Ten-microlitre aliquot ofeach solution was applied on thin-chromartographgic plates and theprocedure was completed using thesame parameters mentioned undergeneral procedure for linearity Thisexperiment was repeated applying thestandard addition technique Therecorded AUPs for the spots wereused for calculating the concentrationof labelled and added intact LFHClor CFHCl from regression equations(3 and 5)

Simultaneous derivative spectro-photometric determin-ation ofintact lomefloxacin hydrochlorideor ciprofloxa-cin hydrochlorideand their acid degradationproducts

General procedure and linearityAn aqueous solution of intact LF

HCl (05 mgml) or CF HCl (1mgml) was prepared A ten-mlaliquot of this solution containingLFHCl or CFHCl was introducedinto a 100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid

Accurately measured aliquots (2-8ml) or (25-6 ml) of intact solution ofLFHCl or CF HCl respectivelywere transferred into two series of 50ml volumetric flasks followed by theaddition of accurately measuredvolumes (2-8 ml) or (4-8 ml) ofdegraded solution of LF HCl or CFHCl respectively Each flask wascompleted to volume with the samesolvent

The first and second derivativespectra for each solution wererecorded against a solvent blankusing the following parameters

Real time date Δλ2 speed fastslit 1 cycT(min) 0 ordinate rangelimit - 03 to + 03 or ndash 04 to + 04for LFHCl or CFHCl respectively

Wavelength range 200-400 nm or200-350 nm for LFHCl or CFHClrespectively

Also the second derivativespectrum for each flask solution wasrecorded against a solvent blankusing the same above parametersexcept for the following

249

1- Ordinate range limit - 009 to +006 or -012 to + 008 fordegraded LFHCl or CFHClrespectively

2- Wavelength range 250-350 nmor 225-325 nm for degradedLFHCl or CF HCl respectively

The heights (mm) of the firstderivative spectra at 292 nm forLFHCl or at 260 nm for CFHClwere measured and the calibrationgraph for each drug was plotted Theheights (mm) of the second derivativespectra at 272 or at 262 nm fordegraded LFHCl or CFHClrespectively were measured and thecalibration graphs for the degradeddrugs were constructed

The following regressionequations 789 and 10 werecomputed for calculating theconcentration of intact and degradedlomefloxacin and ciprofloxacinrespectively

Concentration of intact LFHCl(microgml) =01631 H292+ 00394 Eq(7)r2= 09998 OrH292= 61312 X C (microgml) -02416

Concentration of intact CFHCl(microgml) =01916H 260 + 02480 Eq(8)r2= 09984 OrH 260 = 52192 XC (microgml) -12944

Concentration of degraded LFHCl(microgml) =01534H272 + 00359 Eq(9)r2= 09998 OrH272 = 65189 X C (microg ml) -02340

Concentration of degraded CFHCl(microgml) =02123H 262 + 05092 Eq (10)r2= 09963 OrH 262 = 47103 X C (microg ml) -23985

Where C is the concentration in microgml H is height of the peak and r2 isthe correlation coefficient

Derivative spectrophotometricdetermination of lomefloxacinhydrochloride and ciprofloxacinhydrochloride in ldquoAlkafloxrdquo andldquoServifloxrdquo tablets respectively

An accurate weight of thepowdered tablets equivalent toLFHCl or CFHCl (20 mg) wasextracted with methanol (15 ml x 3)then filtered into a 100 ml volumetricflask After complete washing of theresidue with methanol (10 ml x 3) thesolution was diluted to volume withmethanol A twenty-ml aliquot of thissolution equivalent to LF HCl orCFHCl (4 mg) was evaporated on awarm water bath The residue wasdissolved in 2 M hydrochloric acidand quantitatively transferred into a100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid Accuratelymeasured aliquots of the abovesolution (2-6 ml) or (6-9 ml) wereintroduced into two series of 50 mlvolumetric flasks Each flask wascompleted to volume with 2Mhydrochloric acid

The first derivative spectrum ofeach drug was recorded against ablank using the same parametersmentioned under general procedureand linearity This procedure was

Sonia T Hassib et al

250

repeated using the standard additiontechnique

The heights (mm) at 292 and 260nm were measured and used forcalculating the corresponding labeledand added concentrations of intactLFHCl and CFHCl respectively

RESULTS AND DISCUSSION

HPLC methodIn this study different mobile

phases were attempted for theseparation of LFHCl in presence ofits degradation product(s) HoweverThoppil amp Amin38 reported astability-indicating reversed-phaseliquid chromatographic procedure forthe determination of CFHCl as bulkdrug in pharmaceutical preparationsand in the presence of its acid andalkaline degraded solutions Themobile phase consisted of water-acetonitrile-triethylamine (802006vvv) adjusted with o-phosphoricacid to pH 3

The above mobile systempreviously applied for CFHCl wassuccessfully used for the separationand determination of intact anddegraded LFHCl since it showedgood resolution between the elutedpeaks Detection was carried out at328 nm The polarity of the systemwas calculated46 and found to be(109) The flow rate was set at 15mlmin Enrofloxacin was used as aninternal standard with the elutionorder degraded LFHCl (tR= 2160)intact LFHCl (tR= 2451) andenrofloxacin (tR= 2832) Fig (2) Thechromatogram of the mixture shows

complete separation and goodresolution

A linear relationship wasobserved when the ratios of the AUPsof each component to the AUP of theinternal standard were plotted versusthe concentration of each componentThe concentration range for eachcomponent in the mixture regressionequations mean percentagerecoveries limit of detection (LOD)and limit of quantification (LOQ)when using the proposed methods aredisplayed in Table (1)

Densitometric methodFew developing systems were

used for TLC separation and solventpolarity optimization of LFH|Cl47 andCFHCl27amp48 in the presence of eitherrelated impurities or photo-degradation products in irradiatedsolutions Seija Tammilehto et al48

studied the photodegradation ofCFHCl irradiated in aqueoussolutions through using a mixture ofacetonitrile and ammonia buffer (pH106-111) as a developing system(6535 vv) for the separation of thecompound and its photodegradationproducts

Using methanol instead ofacetonitrile in a different proportionin the present work offered betterseparation of the drug and its aciddegradation product The polarity ofthe used system was calculated andfound to be (515)46

The TLC chromatogram of bothintact and degraded LFHCl showedcomplete separation with (Rf= 066)for the intact drug and (Rf= 083) for

251

Table 1 Results obtained in the chromatographic methods for the determinationof intact and degraded lomefloxacin hydrochloride and ciprofloxacinhydrochloride

TLC method HPLC methodLFHCl CFHCl LFHClItem

Intact Deg Intact Deg Intact DegConcentrationrange

1-6microg10 microl

05-6microg10 microl

025-25microg10 microl

025-25microg10 microl

05-6microg20 microl

04-48microg20 microl

Mean percentagerecoveries

1-Laboratorypreparedmixtures

9960plusmn 055

9951plusmn 067

9955plusmn072

9922plusmn069

9968plusmn077

10030plusmn089

2-Added standardto lsquoAlkafloxrsquoor lsquoServifloxrsquo

tablets

9925plusmn080

9963plusmn 064

9938plusmn070

LOD010

(microg 10microl)005

(microg 10microl)022

(microg 20microl)

LOQ034

(microg 10microl)018

(microg 10microl)074

(microg 20microl)

the degraded product The separatedspots of both components werescanned on the same plate at 288 and328 nm for the intact and degradedLFHCl respectively

Upon applying this modifieddeveloping system to the analysis ofboth intact and degraded CFHCl theTLC chromatogram showed completeresolution with (Rf= 053) for theintact drug and (Rf= 075) for thedegraded drug The separated spots ofboth components were scanned on thesame plate at 279 and 323 nm for theintact and degraded CFHClrespectively

The concentration range for eachcomponent in the two mixturesregression equations meanpercentage recoveries limit ofdetection and limit of quantificationwhen using the proposed methods aredisplayed in Table (1)

The results obtained in thedetermination of intact LFHCl andCFHCl in the presence of their aciddegradation products using HPLCand densitometric methods werestatistically compared to the reportedmethods (spectrophotometric42 andHPLC43 respectively) showing nosignificant difference concerningaccuracy and precision Table (2)

Sonia T Hassib et al

252

Table 2 Statistical analysis for the chromatographic methods for thedetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochlorideCiprofloxacinhydrochlorideStatistcal Term

HPLCmethod

TLCmethod

Reference42

TLCmethod

Reference43

Mean 9968 9960 9917 9955 9943

SD plusmn077 plusmn055 plusmn060 plusmn072 plusmn055

RSDwithin day

077 055 072

RSDbetween days

117 116 119

SE plusmn031 plusmn022 plusmn024 plusmn029 plusmn023

n 6 6 6 6 6

V 059 030 036 052 030

LOD022

(microg 20microl)010

(microg 10microl)005

(microg 10microl)

LOQ074

(microg 20microl)034

(microg 10microl)018

(microg 10microl)

t 128 130 032

F 164 120 173

The corresponding theoretical values for t and F at p = 005 are 223 and 505respectively

Derivative spectrophotometricmethod

The zero-order spectra of intactand degraded LFHCl and CFHClFigs (3amp4) show completeoverlapping which prevents thedetermination of the intact anddegraded drugs in presence of eachother using this technique

In this work a derivativespectrophotometric procedure ispresented to solve this problem

through measurements in both thefirst and second derivative spectraThe first derivative spectra Figs(5amp6) show that intact LFHCldisplays a peak at 272 nm and avalley at 292 nm while the degradedproduct has zero crossing at both 292and 271 nm Thus the ordinate valueat 292 nm was chosen for thedetermination of the intact drug in thepresence of its degradation productIn addition the first derivative

253

Wavelength (nm)

Fig 3 Zero-order spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 4 Zero-order spectra of intactciprofloxacin hydrochloride (056mg50ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 5 First-derivative spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 6 First-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Abs

orba

nce

Abs

orba

nce

Sonia T Hassib et al

254

spectrum of intact CFHCl Fig (6)exhibits a peak at 262 nm and a valleyat 282 nm while its degradationproduct has zero crossing at both 260and 286 nm Consequently theordinate value at 260 nm waspreferred for calculating theconcentration of the intact drug in thepresence of its degradation product

Figures (5amp6) show that thedegradation product of LFHCl orCFHCl could not be determinedthrough measurements in the firstderivative spectra On the other handin the second derivative spectra Fig(7) the degraded LFHCl shows avalley at 272 nm at which the intactdrug has a zero crossing Thus theordinate value at 272 nm was used forcomputing the concentration of thedegradation product in the presenceof the intact drug

Similarly the second derivativespectra Fig (8) show that thedegraded CFHCl exhibits a valley at262 nm at which the intact drugshows no interference Therefore theordinate value at 262 nm was used forcalculating the concentration of thedegradation product in the presenceof the intact drug

Linearity was shown over theconcentration range (12-96 microgml)or (28-126 microgml) of intact LFH|Clor CFHCl respectively (HPLC andDensitometric) linearity was alsoobserved over the concentration range(12-96 microgml) or (64-16 microgml) ofdegraded LFH|Cl or CFHClrespectively

Wavelength (nm)

Fig 7 Second-derivative spectra of intactlomefloxacin hydrochloride (024mg50 ml) (mdashmdashmdash) and ofdegraded drug (024 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 8 Second-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

255

The mean percentage recoveriesof intact and degraded LFHCl werefound to be 9972plusmn056 and 9893plusmn085 respectively (Table 3) Also themean percentage recoveries of intactand degraded CFHCl were 9962plusmn047 and 9967plusmn039 respectively(Table 3)

The method was successfullyapplied for the quantitative estimationof LFHCl or CFHCl in ldquoAlkafloxrdquoor ldquoServifloxrdquo tablets respectivelywithout interference from additivesThe standard addition technique wasapplied The mean percentagerecoveries of labelled and addedLFHCl were found to be 9976plusmn075and 9958plusmn064 respectively Alsothe mean percentage recoveries oflabelled and added CFHCl were

9949plusmn066 and 9978plusmn034respectively The second derivativespectra of ldquoAlkafloxrdquo and ldquoServifloxrdquotablets showed complete absence ofthe corresponding acid degradationproduct

The proposed methods are validand applicable for the determinationof intact LFHCl or CFHCl inlaboratory prepared mixturescontaining (20-80) or (40-75) ofthe degraded LFHCl or degradedCFHCl respectively

Statistical comparison between theproposed and the reported methods(spectrophotometric42 and HPLC43for LFHCl and CFHCl respectively)showed no significant differencebetween them concerning accuracyand precision (Table 3)

Table 3 Statistical analysis for the first derivative spectrophotometricdetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochloride Ciprofloxacin hydrochlorideStatistical

TermFirst

Derivativemethod

Comparisonmethod13

FirstDerivative

method

Comparisonmethod12

Mean 9972 9917 9962 9943

SD plusmn056 plusmn060 plusmn047 plusmn055

RSD 056 047

SE plusmn021 plusmn024 plusmn018 plusmn022

n 7 6 7 6

V 031 036 022 030

LOD(microgml)

039 103

LOQ(microgml)

129 345

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

247

hour in a chromatographic tankpreviously saturated with thespecified developing system byascending chromatography to adistance of 15 cm The plates wereleft to dry at room temperature Thespots were detected under a uv lamp(254 nm) and the two drugs werescanned under the followingconditionsPhoto mode Reflection scan modeZigzag swing width 12 mmWavelengthi) 288 and 328 nm for intact and

degraded LFHCl respectivelyii) 279 and 323 nm for intact and

degraded CFHCl respectively

Chromatograms result outputsand the areas under the peaks (AUPs)were recorded The recorded AUPswere plotted versus the concentrationused (microg10 microl) and the calibrationcurves for both intact and degradedLFHCl or CFHCl were obtainedThe following regression equations(345 and 6) were computed forcalculating the concentrations ofintact LFHCl degraded LFHClintact CFHCl and degraded CFHClrespectively

Concentration of intact LFHCl(microg10 microl) =00287 AUP ndash 00022 Eq (3)r2 = 09998 OrAUP= 34843 X C (microg10microl) +00767

Concentration of degraded LFHCl(microg10 microl) =00388 AUPndash 01587 Eq (4)r2 = 09982 OrAUP= 25773 X C (microg10microl) +40902

Concentration of intact CFHCl (microg10 microl) =00194 AUPndash 00398 Eq (5)r2 = 09991 OrAUP= 51546 X C (microg10microl) +20515

Concentration of degraded CFHCl(microg10 microl) =00246 AUP ndash00758 Eq (6)r2 = 09980 OrAUP= 40650 X C (microg10microl) +30813

Where C is the concentration inmicrog10microl AUP is the area under thepeak and r2 is the correlationcoefficient

Densitometric determination ofintact and degraded lomefloxacinhydrochloride and ciprofloxacinhydrochloride in laboratoryprepared mixtures

Four solutions of intact anddegraded LFHCl (12 mgml) ofintact and degraded CFHCl (04 mgml) were prepared in methanol

Different aliquots of both intactLFHCl (1-45 ml) or of intactCFHCl (15-4 ml) were transferredinto two series of 10 ml volumetricflasks Different aliquots of degradedLFHCl (05-4 ml) or degradedCFHCl (1-35 ml) were added tointact LFHCl series or to intactCFHCl series respectively [series (1)and (2)] for LFHCl and CFHClrespectively Each volumetric flaskwas completed to volume withmethanol Ten-microlitre aliquots ofeach mixture solution were applied onthin-layer chromatographic platesusing a micropipette and theprocedure was completed using the

Sonia T Hassib et al

248

same parameters mentioned undergeneral procedure and linearity

The recorded AUPs were used forcalculating the concentrations of thetwo components (intact and degradeddrug) in each mixture solution usingthe regression equations (345 and 6)for intact and degraded LFHCl andCFHCl respectively

Densitometric determination oflomefloxacin hydrochloride andciprofloxacin hydrochloride inldquoAlkafloxrdquo and ldquoServifloxrdquo tabletsrespectively

An accurate weight of thepowdered twenty tablets equivalent toLFHCl (35 mg) or CFHCl (30 mg)was extracted with methanol (10 ml x3) Each extract was filtered into a50ml volumetric flask The residuewas washed with methanol (5 ml x 3)the volume was completed withmethanol Aliquots (2-4 ml) ofLFHCl solution or (1-2 ml) ofCFHCl solution were transferredinto two series of 10 ml volumetricflasks and completed to volume withmethanol Ten-microlitre aliquot ofeach solution was applied on thin-chromartographgic plates and theprocedure was completed using thesame parameters mentioned undergeneral procedure for linearity Thisexperiment was repeated applying thestandard addition technique Therecorded AUPs for the spots wereused for calculating the concentrationof labelled and added intact LFHClor CFHCl from regression equations(3 and 5)

Simultaneous derivative spectro-photometric determin-ation ofintact lomefloxacin hydrochlorideor ciprofloxa-cin hydrochlorideand their acid degradationproducts

General procedure and linearityAn aqueous solution of intact LF

HCl (05 mgml) or CF HCl (1mgml) was prepared A ten-mlaliquot of this solution containingLFHCl or CFHCl was introducedinto a 100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid

Accurately measured aliquots (2-8ml) or (25-6 ml) of intact solution ofLFHCl or CF HCl respectivelywere transferred into two series of 50ml volumetric flasks followed by theaddition of accurately measuredvolumes (2-8 ml) or (4-8 ml) ofdegraded solution of LF HCl or CFHCl respectively Each flask wascompleted to volume with the samesolvent

The first and second derivativespectra for each solution wererecorded against a solvent blankusing the following parameters

Real time date Δλ2 speed fastslit 1 cycT(min) 0 ordinate rangelimit - 03 to + 03 or ndash 04 to + 04for LFHCl or CFHCl respectively

Wavelength range 200-400 nm or200-350 nm for LFHCl or CFHClrespectively

Also the second derivativespectrum for each flask solution wasrecorded against a solvent blankusing the same above parametersexcept for the following

249

1- Ordinate range limit - 009 to +006 or -012 to + 008 fordegraded LFHCl or CFHClrespectively

2- Wavelength range 250-350 nmor 225-325 nm for degradedLFHCl or CF HCl respectively

The heights (mm) of the firstderivative spectra at 292 nm forLFHCl or at 260 nm for CFHClwere measured and the calibrationgraph for each drug was plotted Theheights (mm) of the second derivativespectra at 272 or at 262 nm fordegraded LFHCl or CFHClrespectively were measured and thecalibration graphs for the degradeddrugs were constructed

The following regressionequations 789 and 10 werecomputed for calculating theconcentration of intact and degradedlomefloxacin and ciprofloxacinrespectively

Concentration of intact LFHCl(microgml) =01631 H292+ 00394 Eq(7)r2= 09998 OrH292= 61312 X C (microgml) -02416

Concentration of intact CFHCl(microgml) =01916H 260 + 02480 Eq(8)r2= 09984 OrH 260 = 52192 XC (microgml) -12944

Concentration of degraded LFHCl(microgml) =01534H272 + 00359 Eq(9)r2= 09998 OrH272 = 65189 X C (microg ml) -02340

Concentration of degraded CFHCl(microgml) =02123H 262 + 05092 Eq (10)r2= 09963 OrH 262 = 47103 X C (microg ml) -23985

Where C is the concentration in microgml H is height of the peak and r2 isthe correlation coefficient

Derivative spectrophotometricdetermination of lomefloxacinhydrochloride and ciprofloxacinhydrochloride in ldquoAlkafloxrdquo andldquoServifloxrdquo tablets respectively

An accurate weight of thepowdered tablets equivalent toLFHCl or CFHCl (20 mg) wasextracted with methanol (15 ml x 3)then filtered into a 100 ml volumetricflask After complete washing of theresidue with methanol (10 ml x 3) thesolution was diluted to volume withmethanol A twenty-ml aliquot of thissolution equivalent to LF HCl orCFHCl (4 mg) was evaporated on awarm water bath The residue wasdissolved in 2 M hydrochloric acidand quantitatively transferred into a100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid Accuratelymeasured aliquots of the abovesolution (2-6 ml) or (6-9 ml) wereintroduced into two series of 50 mlvolumetric flasks Each flask wascompleted to volume with 2Mhydrochloric acid

The first derivative spectrum ofeach drug was recorded against ablank using the same parametersmentioned under general procedureand linearity This procedure was

Sonia T Hassib et al

250

repeated using the standard additiontechnique

The heights (mm) at 292 and 260nm were measured and used forcalculating the corresponding labeledand added concentrations of intactLFHCl and CFHCl respectively

RESULTS AND DISCUSSION

HPLC methodIn this study different mobile

phases were attempted for theseparation of LFHCl in presence ofits degradation product(s) HoweverThoppil amp Amin38 reported astability-indicating reversed-phaseliquid chromatographic procedure forthe determination of CFHCl as bulkdrug in pharmaceutical preparationsand in the presence of its acid andalkaline degraded solutions Themobile phase consisted of water-acetonitrile-triethylamine (802006vvv) adjusted with o-phosphoricacid to pH 3

The above mobile systempreviously applied for CFHCl wassuccessfully used for the separationand determination of intact anddegraded LFHCl since it showedgood resolution between the elutedpeaks Detection was carried out at328 nm The polarity of the systemwas calculated46 and found to be(109) The flow rate was set at 15mlmin Enrofloxacin was used as aninternal standard with the elutionorder degraded LFHCl (tR= 2160)intact LFHCl (tR= 2451) andenrofloxacin (tR= 2832) Fig (2) Thechromatogram of the mixture shows

complete separation and goodresolution

A linear relationship wasobserved when the ratios of the AUPsof each component to the AUP of theinternal standard were plotted versusthe concentration of each componentThe concentration range for eachcomponent in the mixture regressionequations mean percentagerecoveries limit of detection (LOD)and limit of quantification (LOQ)when using the proposed methods aredisplayed in Table (1)

Densitometric methodFew developing systems were

used for TLC separation and solventpolarity optimization of LFH|Cl47 andCFHCl27amp48 in the presence of eitherrelated impurities or photo-degradation products in irradiatedsolutions Seija Tammilehto et al48

studied the photodegradation ofCFHCl irradiated in aqueoussolutions through using a mixture ofacetonitrile and ammonia buffer (pH106-111) as a developing system(6535 vv) for the separation of thecompound and its photodegradationproducts

Using methanol instead ofacetonitrile in a different proportionin the present work offered betterseparation of the drug and its aciddegradation product The polarity ofthe used system was calculated andfound to be (515)46

The TLC chromatogram of bothintact and degraded LFHCl showedcomplete separation with (Rf= 066)for the intact drug and (Rf= 083) for

251

Table 1 Results obtained in the chromatographic methods for the determinationof intact and degraded lomefloxacin hydrochloride and ciprofloxacinhydrochloride

TLC method HPLC methodLFHCl CFHCl LFHClItem

Intact Deg Intact Deg Intact DegConcentrationrange

1-6microg10 microl

05-6microg10 microl

025-25microg10 microl

025-25microg10 microl

05-6microg20 microl

04-48microg20 microl

Mean percentagerecoveries

1-Laboratorypreparedmixtures

9960plusmn 055

9951plusmn 067

9955plusmn072

9922plusmn069

9968plusmn077

10030plusmn089

2-Added standardto lsquoAlkafloxrsquoor lsquoServifloxrsquo

tablets

9925plusmn080

9963plusmn 064

9938plusmn070

LOD010

(microg 10microl)005

(microg 10microl)022

(microg 20microl)

LOQ034

(microg 10microl)018

(microg 10microl)074

(microg 20microl)

the degraded product The separatedspots of both components werescanned on the same plate at 288 and328 nm for the intact and degradedLFHCl respectively

Upon applying this modifieddeveloping system to the analysis ofboth intact and degraded CFHCl theTLC chromatogram showed completeresolution with (Rf= 053) for theintact drug and (Rf= 075) for thedegraded drug The separated spots ofboth components were scanned on thesame plate at 279 and 323 nm for theintact and degraded CFHClrespectively

The concentration range for eachcomponent in the two mixturesregression equations meanpercentage recoveries limit ofdetection and limit of quantificationwhen using the proposed methods aredisplayed in Table (1)

The results obtained in thedetermination of intact LFHCl andCFHCl in the presence of their aciddegradation products using HPLCand densitometric methods werestatistically compared to the reportedmethods (spectrophotometric42 andHPLC43 respectively) showing nosignificant difference concerningaccuracy and precision Table (2)

Sonia T Hassib et al

252

Table 2 Statistical analysis for the chromatographic methods for thedetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochlorideCiprofloxacinhydrochlorideStatistcal Term

HPLCmethod

TLCmethod

Reference42

TLCmethod

Reference43

Mean 9968 9960 9917 9955 9943

SD plusmn077 plusmn055 plusmn060 plusmn072 plusmn055

RSDwithin day

077 055 072

RSDbetween days

117 116 119

SE plusmn031 plusmn022 plusmn024 plusmn029 plusmn023

n 6 6 6 6 6

V 059 030 036 052 030

LOD022

(microg 20microl)010

(microg 10microl)005

(microg 10microl)

LOQ074

(microg 20microl)034

(microg 10microl)018

(microg 10microl)

t 128 130 032

F 164 120 173

The corresponding theoretical values for t and F at p = 005 are 223 and 505respectively

Derivative spectrophotometricmethod

The zero-order spectra of intactand degraded LFHCl and CFHClFigs (3amp4) show completeoverlapping which prevents thedetermination of the intact anddegraded drugs in presence of eachother using this technique

In this work a derivativespectrophotometric procedure ispresented to solve this problem

through measurements in both thefirst and second derivative spectraThe first derivative spectra Figs(5amp6) show that intact LFHCldisplays a peak at 272 nm and avalley at 292 nm while the degradedproduct has zero crossing at both 292and 271 nm Thus the ordinate valueat 292 nm was chosen for thedetermination of the intact drug in thepresence of its degradation productIn addition the first derivative

253

Wavelength (nm)

Fig 3 Zero-order spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 4 Zero-order spectra of intactciprofloxacin hydrochloride (056mg50ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 5 First-derivative spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 6 First-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Abs

orba

nce

Abs

orba

nce

Sonia T Hassib et al

254

spectrum of intact CFHCl Fig (6)exhibits a peak at 262 nm and a valleyat 282 nm while its degradationproduct has zero crossing at both 260and 286 nm Consequently theordinate value at 260 nm waspreferred for calculating theconcentration of the intact drug in thepresence of its degradation product

Figures (5amp6) show that thedegradation product of LFHCl orCFHCl could not be determinedthrough measurements in the firstderivative spectra On the other handin the second derivative spectra Fig(7) the degraded LFHCl shows avalley at 272 nm at which the intactdrug has a zero crossing Thus theordinate value at 272 nm was used forcomputing the concentration of thedegradation product in the presenceof the intact drug

Similarly the second derivativespectra Fig (8) show that thedegraded CFHCl exhibits a valley at262 nm at which the intact drugshows no interference Therefore theordinate value at 262 nm was used forcalculating the concentration of thedegradation product in the presenceof the intact drug

Linearity was shown over theconcentration range (12-96 microgml)or (28-126 microgml) of intact LFH|Clor CFHCl respectively (HPLC andDensitometric) linearity was alsoobserved over the concentration range(12-96 microgml) or (64-16 microgml) ofdegraded LFH|Cl or CFHClrespectively

Wavelength (nm)

Fig 7 Second-derivative spectra of intactlomefloxacin hydrochloride (024mg50 ml) (mdashmdashmdash) and ofdegraded drug (024 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 8 Second-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

255

The mean percentage recoveriesof intact and degraded LFHCl werefound to be 9972plusmn056 and 9893plusmn085 respectively (Table 3) Also themean percentage recoveries of intactand degraded CFHCl were 9962plusmn047 and 9967plusmn039 respectively(Table 3)

The method was successfullyapplied for the quantitative estimationof LFHCl or CFHCl in ldquoAlkafloxrdquoor ldquoServifloxrdquo tablets respectivelywithout interference from additivesThe standard addition technique wasapplied The mean percentagerecoveries of labelled and addedLFHCl were found to be 9976plusmn075and 9958plusmn064 respectively Alsothe mean percentage recoveries oflabelled and added CFHCl were

9949plusmn066 and 9978plusmn034respectively The second derivativespectra of ldquoAlkafloxrdquo and ldquoServifloxrdquotablets showed complete absence ofthe corresponding acid degradationproduct

The proposed methods are validand applicable for the determinationof intact LFHCl or CFHCl inlaboratory prepared mixturescontaining (20-80) or (40-75) ofthe degraded LFHCl or degradedCFHCl respectively

Statistical comparison between theproposed and the reported methods(spectrophotometric42 and HPLC43for LFHCl and CFHCl respectively)showed no significant differencebetween them concerning accuracyand precision (Table 3)

Table 3 Statistical analysis for the first derivative spectrophotometricdetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochloride Ciprofloxacin hydrochlorideStatistical

TermFirst

Derivativemethod

Comparisonmethod13

FirstDerivative

method

Comparisonmethod12

Mean 9972 9917 9962 9943

SD plusmn056 plusmn060 plusmn047 plusmn055

RSD 056 047

SE plusmn021 plusmn024 plusmn018 plusmn022

n 7 6 7 6

V 031 036 022 030

LOD(microgml)

039 103

LOQ(microgml)

129 345

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

Sonia T Hassib et al

248

same parameters mentioned undergeneral procedure and linearity

The recorded AUPs were used forcalculating the concentrations of thetwo components (intact and degradeddrug) in each mixture solution usingthe regression equations (345 and 6)for intact and degraded LFHCl andCFHCl respectively

Densitometric determination oflomefloxacin hydrochloride andciprofloxacin hydrochloride inldquoAlkafloxrdquo and ldquoServifloxrdquo tabletsrespectively

An accurate weight of thepowdered twenty tablets equivalent toLFHCl (35 mg) or CFHCl (30 mg)was extracted with methanol (10 ml x3) Each extract was filtered into a50ml volumetric flask The residuewas washed with methanol (5 ml x 3)the volume was completed withmethanol Aliquots (2-4 ml) ofLFHCl solution or (1-2 ml) ofCFHCl solution were transferredinto two series of 10 ml volumetricflasks and completed to volume withmethanol Ten-microlitre aliquot ofeach solution was applied on thin-chromartographgic plates and theprocedure was completed using thesame parameters mentioned undergeneral procedure for linearity Thisexperiment was repeated applying thestandard addition technique Therecorded AUPs for the spots wereused for calculating the concentrationof labelled and added intact LFHClor CFHCl from regression equations(3 and 5)

Simultaneous derivative spectro-photometric determin-ation ofintact lomefloxacin hydrochlorideor ciprofloxa-cin hydrochlorideand their acid degradationproducts

General procedure and linearityAn aqueous solution of intact LF

HCl (05 mgml) or CF HCl (1mgml) was prepared A ten-mlaliquot of this solution containingLFHCl or CFHCl was introducedinto a 100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid

Accurately measured aliquots (2-8ml) or (25-6 ml) of intact solution ofLFHCl or CF HCl respectivelywere transferred into two series of 50ml volumetric flasks followed by theaddition of accurately measuredvolumes (2-8 ml) or (4-8 ml) ofdegraded solution of LF HCl or CFHCl respectively Each flask wascompleted to volume with the samesolvent

The first and second derivativespectra for each solution wererecorded against a solvent blankusing the following parameters

Real time date Δλ2 speed fastslit 1 cycT(min) 0 ordinate rangelimit - 03 to + 03 or ndash 04 to + 04for LFHCl or CFHCl respectively

Wavelength range 200-400 nm or200-350 nm for LFHCl or CFHClrespectively

Also the second derivativespectrum for each flask solution wasrecorded against a solvent blankusing the same above parametersexcept for the following

249

1- Ordinate range limit - 009 to +006 or -012 to + 008 fordegraded LFHCl or CFHClrespectively

2- Wavelength range 250-350 nmor 225-325 nm for degradedLFHCl or CF HCl respectively

The heights (mm) of the firstderivative spectra at 292 nm forLFHCl or at 260 nm for CFHClwere measured and the calibrationgraph for each drug was plotted Theheights (mm) of the second derivativespectra at 272 or at 262 nm fordegraded LFHCl or CFHClrespectively were measured and thecalibration graphs for the degradeddrugs were constructed

The following regressionequations 789 and 10 werecomputed for calculating theconcentration of intact and degradedlomefloxacin and ciprofloxacinrespectively

Concentration of intact LFHCl(microgml) =01631 H292+ 00394 Eq(7)r2= 09998 OrH292= 61312 X C (microgml) -02416

Concentration of intact CFHCl(microgml) =01916H 260 + 02480 Eq(8)r2= 09984 OrH 260 = 52192 XC (microgml) -12944

Concentration of degraded LFHCl(microgml) =01534H272 + 00359 Eq(9)r2= 09998 OrH272 = 65189 X C (microg ml) -02340

Concentration of degraded CFHCl(microgml) =02123H 262 + 05092 Eq (10)r2= 09963 OrH 262 = 47103 X C (microg ml) -23985

Where C is the concentration in microgml H is height of the peak and r2 isthe correlation coefficient

Derivative spectrophotometricdetermination of lomefloxacinhydrochloride and ciprofloxacinhydrochloride in ldquoAlkafloxrdquo andldquoServifloxrdquo tablets respectively

An accurate weight of thepowdered tablets equivalent toLFHCl or CFHCl (20 mg) wasextracted with methanol (15 ml x 3)then filtered into a 100 ml volumetricflask After complete washing of theresidue with methanol (10 ml x 3) thesolution was diluted to volume withmethanol A twenty-ml aliquot of thissolution equivalent to LF HCl orCFHCl (4 mg) was evaporated on awarm water bath The residue wasdissolved in 2 M hydrochloric acidand quantitatively transferred into a100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid Accuratelymeasured aliquots of the abovesolution (2-6 ml) or (6-9 ml) wereintroduced into two series of 50 mlvolumetric flasks Each flask wascompleted to volume with 2Mhydrochloric acid

The first derivative spectrum ofeach drug was recorded against ablank using the same parametersmentioned under general procedureand linearity This procedure was

Sonia T Hassib et al

250

repeated using the standard additiontechnique

The heights (mm) at 292 and 260nm were measured and used forcalculating the corresponding labeledand added concentrations of intactLFHCl and CFHCl respectively

RESULTS AND DISCUSSION

HPLC methodIn this study different mobile

phases were attempted for theseparation of LFHCl in presence ofits degradation product(s) HoweverThoppil amp Amin38 reported astability-indicating reversed-phaseliquid chromatographic procedure forthe determination of CFHCl as bulkdrug in pharmaceutical preparationsand in the presence of its acid andalkaline degraded solutions Themobile phase consisted of water-acetonitrile-triethylamine (802006vvv) adjusted with o-phosphoricacid to pH 3

The above mobile systempreviously applied for CFHCl wassuccessfully used for the separationand determination of intact anddegraded LFHCl since it showedgood resolution between the elutedpeaks Detection was carried out at328 nm The polarity of the systemwas calculated46 and found to be(109) The flow rate was set at 15mlmin Enrofloxacin was used as aninternal standard with the elutionorder degraded LFHCl (tR= 2160)intact LFHCl (tR= 2451) andenrofloxacin (tR= 2832) Fig (2) Thechromatogram of the mixture shows

complete separation and goodresolution

A linear relationship wasobserved when the ratios of the AUPsof each component to the AUP of theinternal standard were plotted versusthe concentration of each componentThe concentration range for eachcomponent in the mixture regressionequations mean percentagerecoveries limit of detection (LOD)and limit of quantification (LOQ)when using the proposed methods aredisplayed in Table (1)

Densitometric methodFew developing systems were

used for TLC separation and solventpolarity optimization of LFH|Cl47 andCFHCl27amp48 in the presence of eitherrelated impurities or photo-degradation products in irradiatedsolutions Seija Tammilehto et al48

studied the photodegradation ofCFHCl irradiated in aqueoussolutions through using a mixture ofacetonitrile and ammonia buffer (pH106-111) as a developing system(6535 vv) for the separation of thecompound and its photodegradationproducts

Using methanol instead ofacetonitrile in a different proportionin the present work offered betterseparation of the drug and its aciddegradation product The polarity ofthe used system was calculated andfound to be (515)46

The TLC chromatogram of bothintact and degraded LFHCl showedcomplete separation with (Rf= 066)for the intact drug and (Rf= 083) for

251

Table 1 Results obtained in the chromatographic methods for the determinationof intact and degraded lomefloxacin hydrochloride and ciprofloxacinhydrochloride

TLC method HPLC methodLFHCl CFHCl LFHClItem

Intact Deg Intact Deg Intact DegConcentrationrange

1-6microg10 microl

05-6microg10 microl

025-25microg10 microl

025-25microg10 microl

05-6microg20 microl

04-48microg20 microl

Mean percentagerecoveries

1-Laboratorypreparedmixtures

9960plusmn 055

9951plusmn 067

9955plusmn072

9922plusmn069

9968plusmn077

10030plusmn089

2-Added standardto lsquoAlkafloxrsquoor lsquoServifloxrsquo

tablets

9925plusmn080

9963plusmn 064

9938plusmn070

LOD010

(microg 10microl)005

(microg 10microl)022

(microg 20microl)

LOQ034

(microg 10microl)018

(microg 10microl)074

(microg 20microl)

the degraded product The separatedspots of both components werescanned on the same plate at 288 and328 nm for the intact and degradedLFHCl respectively

Upon applying this modifieddeveloping system to the analysis ofboth intact and degraded CFHCl theTLC chromatogram showed completeresolution with (Rf= 053) for theintact drug and (Rf= 075) for thedegraded drug The separated spots ofboth components were scanned on thesame plate at 279 and 323 nm for theintact and degraded CFHClrespectively

The concentration range for eachcomponent in the two mixturesregression equations meanpercentage recoveries limit ofdetection and limit of quantificationwhen using the proposed methods aredisplayed in Table (1)

The results obtained in thedetermination of intact LFHCl andCFHCl in the presence of their aciddegradation products using HPLCand densitometric methods werestatistically compared to the reportedmethods (spectrophotometric42 andHPLC43 respectively) showing nosignificant difference concerningaccuracy and precision Table (2)

Sonia T Hassib et al

252

Table 2 Statistical analysis for the chromatographic methods for thedetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochlorideCiprofloxacinhydrochlorideStatistcal Term

HPLCmethod

TLCmethod

Reference42

TLCmethod

Reference43

Mean 9968 9960 9917 9955 9943

SD plusmn077 plusmn055 plusmn060 plusmn072 plusmn055

RSDwithin day

077 055 072

RSDbetween days

117 116 119

SE plusmn031 plusmn022 plusmn024 plusmn029 plusmn023

n 6 6 6 6 6

V 059 030 036 052 030

LOD022

(microg 20microl)010

(microg 10microl)005

(microg 10microl)

LOQ074

(microg 20microl)034

(microg 10microl)018

(microg 10microl)

t 128 130 032

F 164 120 173

The corresponding theoretical values for t and F at p = 005 are 223 and 505respectively

Derivative spectrophotometricmethod

The zero-order spectra of intactand degraded LFHCl and CFHClFigs (3amp4) show completeoverlapping which prevents thedetermination of the intact anddegraded drugs in presence of eachother using this technique

In this work a derivativespectrophotometric procedure ispresented to solve this problem

through measurements in both thefirst and second derivative spectraThe first derivative spectra Figs(5amp6) show that intact LFHCldisplays a peak at 272 nm and avalley at 292 nm while the degradedproduct has zero crossing at both 292and 271 nm Thus the ordinate valueat 292 nm was chosen for thedetermination of the intact drug in thepresence of its degradation productIn addition the first derivative

253

Wavelength (nm)

Fig 3 Zero-order spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 4 Zero-order spectra of intactciprofloxacin hydrochloride (056mg50ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 5 First-derivative spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 6 First-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Abs

orba

nce

Abs

orba

nce

Sonia T Hassib et al

254

spectrum of intact CFHCl Fig (6)exhibits a peak at 262 nm and a valleyat 282 nm while its degradationproduct has zero crossing at both 260and 286 nm Consequently theordinate value at 260 nm waspreferred for calculating theconcentration of the intact drug in thepresence of its degradation product

Figures (5amp6) show that thedegradation product of LFHCl orCFHCl could not be determinedthrough measurements in the firstderivative spectra On the other handin the second derivative spectra Fig(7) the degraded LFHCl shows avalley at 272 nm at which the intactdrug has a zero crossing Thus theordinate value at 272 nm was used forcomputing the concentration of thedegradation product in the presenceof the intact drug

Similarly the second derivativespectra Fig (8) show that thedegraded CFHCl exhibits a valley at262 nm at which the intact drugshows no interference Therefore theordinate value at 262 nm was used forcalculating the concentration of thedegradation product in the presenceof the intact drug

Linearity was shown over theconcentration range (12-96 microgml)or (28-126 microgml) of intact LFH|Clor CFHCl respectively (HPLC andDensitometric) linearity was alsoobserved over the concentration range(12-96 microgml) or (64-16 microgml) ofdegraded LFH|Cl or CFHClrespectively

Wavelength (nm)

Fig 7 Second-derivative spectra of intactlomefloxacin hydrochloride (024mg50 ml) (mdashmdashmdash) and ofdegraded drug (024 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 8 Second-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

255

The mean percentage recoveriesof intact and degraded LFHCl werefound to be 9972plusmn056 and 9893plusmn085 respectively (Table 3) Also themean percentage recoveries of intactand degraded CFHCl were 9962plusmn047 and 9967plusmn039 respectively(Table 3)

The method was successfullyapplied for the quantitative estimationof LFHCl or CFHCl in ldquoAlkafloxrdquoor ldquoServifloxrdquo tablets respectivelywithout interference from additivesThe standard addition technique wasapplied The mean percentagerecoveries of labelled and addedLFHCl were found to be 9976plusmn075and 9958plusmn064 respectively Alsothe mean percentage recoveries oflabelled and added CFHCl were

9949plusmn066 and 9978plusmn034respectively The second derivativespectra of ldquoAlkafloxrdquo and ldquoServifloxrdquotablets showed complete absence ofthe corresponding acid degradationproduct

The proposed methods are validand applicable for the determinationof intact LFHCl or CFHCl inlaboratory prepared mixturescontaining (20-80) or (40-75) ofthe degraded LFHCl or degradedCFHCl respectively

Statistical comparison between theproposed and the reported methods(spectrophotometric42 and HPLC43for LFHCl and CFHCl respectively)showed no significant differencebetween them concerning accuracyand precision (Table 3)

Table 3 Statistical analysis for the first derivative spectrophotometricdetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochloride Ciprofloxacin hydrochlorideStatistical

TermFirst

Derivativemethod

Comparisonmethod13

FirstDerivative

method

Comparisonmethod12

Mean 9972 9917 9962 9943

SD plusmn056 plusmn060 plusmn047 plusmn055

RSD 056 047

SE plusmn021 plusmn024 plusmn018 plusmn022

n 7 6 7 6

V 031 036 022 030

LOD(microgml)

039 103

LOQ(microgml)

129 345

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

249

1- Ordinate range limit - 009 to +006 or -012 to + 008 fordegraded LFHCl or CFHClrespectively

2- Wavelength range 250-350 nmor 225-325 nm for degradedLFHCl or CF HCl respectively

The heights (mm) of the firstderivative spectra at 292 nm forLFHCl or at 260 nm for CFHClwere measured and the calibrationgraph for each drug was plotted Theheights (mm) of the second derivativespectra at 272 or at 262 nm fordegraded LFHCl or CFHClrespectively were measured and thecalibration graphs for the degradeddrugs were constructed

The following regressionequations 789 and 10 werecomputed for calculating theconcentration of intact and degradedlomefloxacin and ciprofloxacinrespectively

Concentration of intact LFHCl(microgml) =01631 H292+ 00394 Eq(7)r2= 09998 OrH292= 61312 X C (microgml) -02416

Concentration of intact CFHCl(microgml) =01916H 260 + 02480 Eq(8)r2= 09984 OrH 260 = 52192 XC (microgml) -12944

Concentration of degraded LFHCl(microgml) =01534H272 + 00359 Eq(9)r2= 09998 OrH272 = 65189 X C (microg ml) -02340

Concentration of degraded CFHCl(microgml) =02123H 262 + 05092 Eq (10)r2= 09963 OrH 262 = 47103 X C (microg ml) -23985

Where C is the concentration in microgml H is height of the peak and r2 isthe correlation coefficient

Derivative spectrophotometricdetermination of lomefloxacinhydrochloride and ciprofloxacinhydrochloride in ldquoAlkafloxrdquo andldquoServifloxrdquo tablets respectively

An accurate weight of thepowdered tablets equivalent toLFHCl or CFHCl (20 mg) wasextracted with methanol (15 ml x 3)then filtered into a 100 ml volumetricflask After complete washing of theresidue with methanol (10 ml x 3) thesolution was diluted to volume withmethanol A twenty-ml aliquot of thissolution equivalent to LF HCl orCFHCl (4 mg) was evaporated on awarm water bath The residue wasdissolved in 2 M hydrochloric acidand quantitatively transferred into a100 ml volumetric flask and thevolume was completed with 2 Mhydrochloric acid Accuratelymeasured aliquots of the abovesolution (2-6 ml) or (6-9 ml) wereintroduced into two series of 50 mlvolumetric flasks Each flask wascompleted to volume with 2Mhydrochloric acid

The first derivative spectrum ofeach drug was recorded against ablank using the same parametersmentioned under general procedureand linearity This procedure was

Sonia T Hassib et al

250

repeated using the standard additiontechnique

The heights (mm) at 292 and 260nm were measured and used forcalculating the corresponding labeledand added concentrations of intactLFHCl and CFHCl respectively

RESULTS AND DISCUSSION

HPLC methodIn this study different mobile

phases were attempted for theseparation of LFHCl in presence ofits degradation product(s) HoweverThoppil amp Amin38 reported astability-indicating reversed-phaseliquid chromatographic procedure forthe determination of CFHCl as bulkdrug in pharmaceutical preparationsand in the presence of its acid andalkaline degraded solutions Themobile phase consisted of water-acetonitrile-triethylamine (802006vvv) adjusted with o-phosphoricacid to pH 3

The above mobile systempreviously applied for CFHCl wassuccessfully used for the separationand determination of intact anddegraded LFHCl since it showedgood resolution between the elutedpeaks Detection was carried out at328 nm The polarity of the systemwas calculated46 and found to be(109) The flow rate was set at 15mlmin Enrofloxacin was used as aninternal standard with the elutionorder degraded LFHCl (tR= 2160)intact LFHCl (tR= 2451) andenrofloxacin (tR= 2832) Fig (2) Thechromatogram of the mixture shows

complete separation and goodresolution

A linear relationship wasobserved when the ratios of the AUPsof each component to the AUP of theinternal standard were plotted versusthe concentration of each componentThe concentration range for eachcomponent in the mixture regressionequations mean percentagerecoveries limit of detection (LOD)and limit of quantification (LOQ)when using the proposed methods aredisplayed in Table (1)

Densitometric methodFew developing systems were

used for TLC separation and solventpolarity optimization of LFH|Cl47 andCFHCl27amp48 in the presence of eitherrelated impurities or photo-degradation products in irradiatedsolutions Seija Tammilehto et al48

studied the photodegradation ofCFHCl irradiated in aqueoussolutions through using a mixture ofacetonitrile and ammonia buffer (pH106-111) as a developing system(6535 vv) for the separation of thecompound and its photodegradationproducts

Using methanol instead ofacetonitrile in a different proportionin the present work offered betterseparation of the drug and its aciddegradation product The polarity ofthe used system was calculated andfound to be (515)46

The TLC chromatogram of bothintact and degraded LFHCl showedcomplete separation with (Rf= 066)for the intact drug and (Rf= 083) for

251

Table 1 Results obtained in the chromatographic methods for the determinationof intact and degraded lomefloxacin hydrochloride and ciprofloxacinhydrochloride

TLC method HPLC methodLFHCl CFHCl LFHClItem

Intact Deg Intact Deg Intact DegConcentrationrange

1-6microg10 microl

05-6microg10 microl

025-25microg10 microl

025-25microg10 microl

05-6microg20 microl

04-48microg20 microl

Mean percentagerecoveries

1-Laboratorypreparedmixtures

9960plusmn 055

9951plusmn 067

9955plusmn072

9922plusmn069

9968plusmn077

10030plusmn089

2-Added standardto lsquoAlkafloxrsquoor lsquoServifloxrsquo

tablets

9925plusmn080

9963plusmn 064

9938plusmn070

LOD010

(microg 10microl)005

(microg 10microl)022

(microg 20microl)

LOQ034

(microg 10microl)018

(microg 10microl)074

(microg 20microl)

the degraded product The separatedspots of both components werescanned on the same plate at 288 and328 nm for the intact and degradedLFHCl respectively

Upon applying this modifieddeveloping system to the analysis ofboth intact and degraded CFHCl theTLC chromatogram showed completeresolution with (Rf= 053) for theintact drug and (Rf= 075) for thedegraded drug The separated spots ofboth components were scanned on thesame plate at 279 and 323 nm for theintact and degraded CFHClrespectively

The concentration range for eachcomponent in the two mixturesregression equations meanpercentage recoveries limit ofdetection and limit of quantificationwhen using the proposed methods aredisplayed in Table (1)

The results obtained in thedetermination of intact LFHCl andCFHCl in the presence of their aciddegradation products using HPLCand densitometric methods werestatistically compared to the reportedmethods (spectrophotometric42 andHPLC43 respectively) showing nosignificant difference concerningaccuracy and precision Table (2)

Sonia T Hassib et al

252

Table 2 Statistical analysis for the chromatographic methods for thedetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochlorideCiprofloxacinhydrochlorideStatistcal Term

HPLCmethod

TLCmethod

Reference42

TLCmethod

Reference43

Mean 9968 9960 9917 9955 9943

SD plusmn077 plusmn055 plusmn060 plusmn072 plusmn055

RSDwithin day

077 055 072

RSDbetween days

117 116 119

SE plusmn031 plusmn022 plusmn024 plusmn029 plusmn023

n 6 6 6 6 6

V 059 030 036 052 030

LOD022

(microg 20microl)010

(microg 10microl)005

(microg 10microl)

LOQ074

(microg 20microl)034

(microg 10microl)018

(microg 10microl)

t 128 130 032

F 164 120 173

The corresponding theoretical values for t and F at p = 005 are 223 and 505respectively

Derivative spectrophotometricmethod

The zero-order spectra of intactand degraded LFHCl and CFHClFigs (3amp4) show completeoverlapping which prevents thedetermination of the intact anddegraded drugs in presence of eachother using this technique

In this work a derivativespectrophotometric procedure ispresented to solve this problem

through measurements in both thefirst and second derivative spectraThe first derivative spectra Figs(5amp6) show that intact LFHCldisplays a peak at 272 nm and avalley at 292 nm while the degradedproduct has zero crossing at both 292and 271 nm Thus the ordinate valueat 292 nm was chosen for thedetermination of the intact drug in thepresence of its degradation productIn addition the first derivative

253

Wavelength (nm)

Fig 3 Zero-order spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 4 Zero-order spectra of intactciprofloxacin hydrochloride (056mg50ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 5 First-derivative spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 6 First-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Abs

orba

nce

Abs

orba

nce

Sonia T Hassib et al

254

spectrum of intact CFHCl Fig (6)exhibits a peak at 262 nm and a valleyat 282 nm while its degradationproduct has zero crossing at both 260and 286 nm Consequently theordinate value at 260 nm waspreferred for calculating theconcentration of the intact drug in thepresence of its degradation product

Figures (5amp6) show that thedegradation product of LFHCl orCFHCl could not be determinedthrough measurements in the firstderivative spectra On the other handin the second derivative spectra Fig(7) the degraded LFHCl shows avalley at 272 nm at which the intactdrug has a zero crossing Thus theordinate value at 272 nm was used forcomputing the concentration of thedegradation product in the presenceof the intact drug

Similarly the second derivativespectra Fig (8) show that thedegraded CFHCl exhibits a valley at262 nm at which the intact drugshows no interference Therefore theordinate value at 262 nm was used forcalculating the concentration of thedegradation product in the presenceof the intact drug

Linearity was shown over theconcentration range (12-96 microgml)or (28-126 microgml) of intact LFH|Clor CFHCl respectively (HPLC andDensitometric) linearity was alsoobserved over the concentration range(12-96 microgml) or (64-16 microgml) ofdegraded LFH|Cl or CFHClrespectively

Wavelength (nm)

Fig 7 Second-derivative spectra of intactlomefloxacin hydrochloride (024mg50 ml) (mdashmdashmdash) and ofdegraded drug (024 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 8 Second-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

255

The mean percentage recoveriesof intact and degraded LFHCl werefound to be 9972plusmn056 and 9893plusmn085 respectively (Table 3) Also themean percentage recoveries of intactand degraded CFHCl were 9962plusmn047 and 9967plusmn039 respectively(Table 3)

The method was successfullyapplied for the quantitative estimationof LFHCl or CFHCl in ldquoAlkafloxrdquoor ldquoServifloxrdquo tablets respectivelywithout interference from additivesThe standard addition technique wasapplied The mean percentagerecoveries of labelled and addedLFHCl were found to be 9976plusmn075and 9958plusmn064 respectively Alsothe mean percentage recoveries oflabelled and added CFHCl were

9949plusmn066 and 9978plusmn034respectively The second derivativespectra of ldquoAlkafloxrdquo and ldquoServifloxrdquotablets showed complete absence ofthe corresponding acid degradationproduct

The proposed methods are validand applicable for the determinationof intact LFHCl or CFHCl inlaboratory prepared mixturescontaining (20-80) or (40-75) ofthe degraded LFHCl or degradedCFHCl respectively

Statistical comparison between theproposed and the reported methods(spectrophotometric42 and HPLC43for LFHCl and CFHCl respectively)showed no significant differencebetween them concerning accuracyand precision (Table 3)

Table 3 Statistical analysis for the first derivative spectrophotometricdetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochloride Ciprofloxacin hydrochlorideStatistical

TermFirst

Derivativemethod

Comparisonmethod13

FirstDerivative

method

Comparisonmethod12

Mean 9972 9917 9962 9943

SD plusmn056 plusmn060 plusmn047 plusmn055

RSD 056 047

SE plusmn021 plusmn024 plusmn018 plusmn022

n 7 6 7 6

V 031 036 022 030

LOD(microgml)

039 103

LOQ(microgml)

129 345

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

Sonia T Hassib et al

250

repeated using the standard additiontechnique

The heights (mm) at 292 and 260nm were measured and used forcalculating the corresponding labeledand added concentrations of intactLFHCl and CFHCl respectively

RESULTS AND DISCUSSION

HPLC methodIn this study different mobile

phases were attempted for theseparation of LFHCl in presence ofits degradation product(s) HoweverThoppil amp Amin38 reported astability-indicating reversed-phaseliquid chromatographic procedure forthe determination of CFHCl as bulkdrug in pharmaceutical preparationsand in the presence of its acid andalkaline degraded solutions Themobile phase consisted of water-acetonitrile-triethylamine (802006vvv) adjusted with o-phosphoricacid to pH 3

The above mobile systempreviously applied for CFHCl wassuccessfully used for the separationand determination of intact anddegraded LFHCl since it showedgood resolution between the elutedpeaks Detection was carried out at328 nm The polarity of the systemwas calculated46 and found to be(109) The flow rate was set at 15mlmin Enrofloxacin was used as aninternal standard with the elutionorder degraded LFHCl (tR= 2160)intact LFHCl (tR= 2451) andenrofloxacin (tR= 2832) Fig (2) Thechromatogram of the mixture shows

complete separation and goodresolution

A linear relationship wasobserved when the ratios of the AUPsof each component to the AUP of theinternal standard were plotted versusthe concentration of each componentThe concentration range for eachcomponent in the mixture regressionequations mean percentagerecoveries limit of detection (LOD)and limit of quantification (LOQ)when using the proposed methods aredisplayed in Table (1)

Densitometric methodFew developing systems were

used for TLC separation and solventpolarity optimization of LFH|Cl47 andCFHCl27amp48 in the presence of eitherrelated impurities or photo-degradation products in irradiatedsolutions Seija Tammilehto et al48

studied the photodegradation ofCFHCl irradiated in aqueoussolutions through using a mixture ofacetonitrile and ammonia buffer (pH106-111) as a developing system(6535 vv) for the separation of thecompound and its photodegradationproducts

Using methanol instead ofacetonitrile in a different proportionin the present work offered betterseparation of the drug and its aciddegradation product The polarity ofthe used system was calculated andfound to be (515)46

The TLC chromatogram of bothintact and degraded LFHCl showedcomplete separation with (Rf= 066)for the intact drug and (Rf= 083) for

251

Table 1 Results obtained in the chromatographic methods for the determinationof intact and degraded lomefloxacin hydrochloride and ciprofloxacinhydrochloride

TLC method HPLC methodLFHCl CFHCl LFHClItem

Intact Deg Intact Deg Intact DegConcentrationrange

1-6microg10 microl

05-6microg10 microl

025-25microg10 microl

025-25microg10 microl

05-6microg20 microl

04-48microg20 microl

Mean percentagerecoveries

1-Laboratorypreparedmixtures

9960plusmn 055

9951plusmn 067

9955plusmn072

9922plusmn069

9968plusmn077

10030plusmn089

2-Added standardto lsquoAlkafloxrsquoor lsquoServifloxrsquo

tablets

9925plusmn080

9963plusmn 064

9938plusmn070

LOD010

(microg 10microl)005

(microg 10microl)022

(microg 20microl)

LOQ034

(microg 10microl)018

(microg 10microl)074

(microg 20microl)

the degraded product The separatedspots of both components werescanned on the same plate at 288 and328 nm for the intact and degradedLFHCl respectively

Upon applying this modifieddeveloping system to the analysis ofboth intact and degraded CFHCl theTLC chromatogram showed completeresolution with (Rf= 053) for theintact drug and (Rf= 075) for thedegraded drug The separated spots ofboth components were scanned on thesame plate at 279 and 323 nm for theintact and degraded CFHClrespectively

The concentration range for eachcomponent in the two mixturesregression equations meanpercentage recoveries limit ofdetection and limit of quantificationwhen using the proposed methods aredisplayed in Table (1)

The results obtained in thedetermination of intact LFHCl andCFHCl in the presence of their aciddegradation products using HPLCand densitometric methods werestatistically compared to the reportedmethods (spectrophotometric42 andHPLC43 respectively) showing nosignificant difference concerningaccuracy and precision Table (2)

Sonia T Hassib et al

252

Table 2 Statistical analysis for the chromatographic methods for thedetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochlorideCiprofloxacinhydrochlorideStatistcal Term

HPLCmethod

TLCmethod

Reference42

TLCmethod

Reference43

Mean 9968 9960 9917 9955 9943

SD plusmn077 plusmn055 plusmn060 plusmn072 plusmn055

RSDwithin day

077 055 072

RSDbetween days

117 116 119

SE plusmn031 plusmn022 plusmn024 plusmn029 plusmn023

n 6 6 6 6 6

V 059 030 036 052 030

LOD022

(microg 20microl)010

(microg 10microl)005

(microg 10microl)

LOQ074

(microg 20microl)034

(microg 10microl)018

(microg 10microl)

t 128 130 032

F 164 120 173

The corresponding theoretical values for t and F at p = 005 are 223 and 505respectively

Derivative spectrophotometricmethod

The zero-order spectra of intactand degraded LFHCl and CFHClFigs (3amp4) show completeoverlapping which prevents thedetermination of the intact anddegraded drugs in presence of eachother using this technique

In this work a derivativespectrophotometric procedure ispresented to solve this problem

through measurements in both thefirst and second derivative spectraThe first derivative spectra Figs(5amp6) show that intact LFHCldisplays a peak at 272 nm and avalley at 292 nm while the degradedproduct has zero crossing at both 292and 271 nm Thus the ordinate valueat 292 nm was chosen for thedetermination of the intact drug in thepresence of its degradation productIn addition the first derivative

253

Wavelength (nm)

Fig 3 Zero-order spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 4 Zero-order spectra of intactciprofloxacin hydrochloride (056mg50ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 5 First-derivative spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 6 First-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Abs

orba

nce

Abs

orba

nce

Sonia T Hassib et al

254

spectrum of intact CFHCl Fig (6)exhibits a peak at 262 nm and a valleyat 282 nm while its degradationproduct has zero crossing at both 260and 286 nm Consequently theordinate value at 260 nm waspreferred for calculating theconcentration of the intact drug in thepresence of its degradation product

Figures (5amp6) show that thedegradation product of LFHCl orCFHCl could not be determinedthrough measurements in the firstderivative spectra On the other handin the second derivative spectra Fig(7) the degraded LFHCl shows avalley at 272 nm at which the intactdrug has a zero crossing Thus theordinate value at 272 nm was used forcomputing the concentration of thedegradation product in the presenceof the intact drug

Similarly the second derivativespectra Fig (8) show that thedegraded CFHCl exhibits a valley at262 nm at which the intact drugshows no interference Therefore theordinate value at 262 nm was used forcalculating the concentration of thedegradation product in the presenceof the intact drug

Linearity was shown over theconcentration range (12-96 microgml)or (28-126 microgml) of intact LFH|Clor CFHCl respectively (HPLC andDensitometric) linearity was alsoobserved over the concentration range(12-96 microgml) or (64-16 microgml) ofdegraded LFH|Cl or CFHClrespectively

Wavelength (nm)

Fig 7 Second-derivative spectra of intactlomefloxacin hydrochloride (024mg50 ml) (mdashmdashmdash) and ofdegraded drug (024 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 8 Second-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

255

The mean percentage recoveriesof intact and degraded LFHCl werefound to be 9972plusmn056 and 9893plusmn085 respectively (Table 3) Also themean percentage recoveries of intactand degraded CFHCl were 9962plusmn047 and 9967plusmn039 respectively(Table 3)

The method was successfullyapplied for the quantitative estimationof LFHCl or CFHCl in ldquoAlkafloxrdquoor ldquoServifloxrdquo tablets respectivelywithout interference from additivesThe standard addition technique wasapplied The mean percentagerecoveries of labelled and addedLFHCl were found to be 9976plusmn075and 9958plusmn064 respectively Alsothe mean percentage recoveries oflabelled and added CFHCl were

9949plusmn066 and 9978plusmn034respectively The second derivativespectra of ldquoAlkafloxrdquo and ldquoServifloxrdquotablets showed complete absence ofthe corresponding acid degradationproduct

The proposed methods are validand applicable for the determinationof intact LFHCl or CFHCl inlaboratory prepared mixturescontaining (20-80) or (40-75) ofthe degraded LFHCl or degradedCFHCl respectively

Statistical comparison between theproposed and the reported methods(spectrophotometric42 and HPLC43for LFHCl and CFHCl respectively)showed no significant differencebetween them concerning accuracyand precision (Table 3)

Table 3 Statistical analysis for the first derivative spectrophotometricdetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochloride Ciprofloxacin hydrochlorideStatistical

TermFirst

Derivativemethod

Comparisonmethod13

FirstDerivative

method

Comparisonmethod12

Mean 9972 9917 9962 9943

SD plusmn056 plusmn060 plusmn047 plusmn055

RSD 056 047

SE plusmn021 plusmn024 plusmn018 plusmn022

n 7 6 7 6

V 031 036 022 030

LOD(microgml)

039 103

LOQ(microgml)

129 345

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

251

Table 1 Results obtained in the chromatographic methods for the determinationof intact and degraded lomefloxacin hydrochloride and ciprofloxacinhydrochloride

TLC method HPLC methodLFHCl CFHCl LFHClItem

Intact Deg Intact Deg Intact DegConcentrationrange

1-6microg10 microl

05-6microg10 microl

025-25microg10 microl

025-25microg10 microl

05-6microg20 microl

04-48microg20 microl

Mean percentagerecoveries

1-Laboratorypreparedmixtures

9960plusmn 055

9951plusmn 067

9955plusmn072

9922plusmn069

9968plusmn077

10030plusmn089

2-Added standardto lsquoAlkafloxrsquoor lsquoServifloxrsquo

tablets

9925plusmn080

9963plusmn 064

9938plusmn070

LOD010

(microg 10microl)005

(microg 10microl)022

(microg 20microl)

LOQ034

(microg 10microl)018

(microg 10microl)074

(microg 20microl)

the degraded product The separatedspots of both components werescanned on the same plate at 288 and328 nm for the intact and degradedLFHCl respectively

Upon applying this modifieddeveloping system to the analysis ofboth intact and degraded CFHCl theTLC chromatogram showed completeresolution with (Rf= 053) for theintact drug and (Rf= 075) for thedegraded drug The separated spots ofboth components were scanned on thesame plate at 279 and 323 nm for theintact and degraded CFHClrespectively

The concentration range for eachcomponent in the two mixturesregression equations meanpercentage recoveries limit ofdetection and limit of quantificationwhen using the proposed methods aredisplayed in Table (1)

The results obtained in thedetermination of intact LFHCl andCFHCl in the presence of their aciddegradation products using HPLCand densitometric methods werestatistically compared to the reportedmethods (spectrophotometric42 andHPLC43 respectively) showing nosignificant difference concerningaccuracy and precision Table (2)

Sonia T Hassib et al

252

Table 2 Statistical analysis for the chromatographic methods for thedetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochlorideCiprofloxacinhydrochlorideStatistcal Term

HPLCmethod

TLCmethod

Reference42

TLCmethod

Reference43

Mean 9968 9960 9917 9955 9943

SD plusmn077 plusmn055 plusmn060 plusmn072 plusmn055

RSDwithin day

077 055 072

RSDbetween days

117 116 119

SE plusmn031 plusmn022 plusmn024 plusmn029 plusmn023

n 6 6 6 6 6

V 059 030 036 052 030

LOD022

(microg 20microl)010

(microg 10microl)005

(microg 10microl)

LOQ074

(microg 20microl)034

(microg 10microl)018

(microg 10microl)

t 128 130 032

F 164 120 173

The corresponding theoretical values for t and F at p = 005 are 223 and 505respectively

Derivative spectrophotometricmethod

The zero-order spectra of intactand degraded LFHCl and CFHClFigs (3amp4) show completeoverlapping which prevents thedetermination of the intact anddegraded drugs in presence of eachother using this technique

In this work a derivativespectrophotometric procedure ispresented to solve this problem

through measurements in both thefirst and second derivative spectraThe first derivative spectra Figs(5amp6) show that intact LFHCldisplays a peak at 272 nm and avalley at 292 nm while the degradedproduct has zero crossing at both 292and 271 nm Thus the ordinate valueat 292 nm was chosen for thedetermination of the intact drug in thepresence of its degradation productIn addition the first derivative

253

Wavelength (nm)

Fig 3 Zero-order spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 4 Zero-order spectra of intactciprofloxacin hydrochloride (056mg50ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 5 First-derivative spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 6 First-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Abs

orba

nce

Abs

orba

nce

Sonia T Hassib et al

254

spectrum of intact CFHCl Fig (6)exhibits a peak at 262 nm and a valleyat 282 nm while its degradationproduct has zero crossing at both 260and 286 nm Consequently theordinate value at 260 nm waspreferred for calculating theconcentration of the intact drug in thepresence of its degradation product

Figures (5amp6) show that thedegradation product of LFHCl orCFHCl could not be determinedthrough measurements in the firstderivative spectra On the other handin the second derivative spectra Fig(7) the degraded LFHCl shows avalley at 272 nm at which the intactdrug has a zero crossing Thus theordinate value at 272 nm was used forcomputing the concentration of thedegradation product in the presenceof the intact drug

Similarly the second derivativespectra Fig (8) show that thedegraded CFHCl exhibits a valley at262 nm at which the intact drugshows no interference Therefore theordinate value at 262 nm was used forcalculating the concentration of thedegradation product in the presenceof the intact drug

Linearity was shown over theconcentration range (12-96 microgml)or (28-126 microgml) of intact LFH|Clor CFHCl respectively (HPLC andDensitometric) linearity was alsoobserved over the concentration range(12-96 microgml) or (64-16 microgml) ofdegraded LFH|Cl or CFHClrespectively

Wavelength (nm)

Fig 7 Second-derivative spectra of intactlomefloxacin hydrochloride (024mg50 ml) (mdashmdashmdash) and ofdegraded drug (024 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 8 Second-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

255

The mean percentage recoveriesof intact and degraded LFHCl werefound to be 9972plusmn056 and 9893plusmn085 respectively (Table 3) Also themean percentage recoveries of intactand degraded CFHCl were 9962plusmn047 and 9967plusmn039 respectively(Table 3)

The method was successfullyapplied for the quantitative estimationof LFHCl or CFHCl in ldquoAlkafloxrdquoor ldquoServifloxrdquo tablets respectivelywithout interference from additivesThe standard addition technique wasapplied The mean percentagerecoveries of labelled and addedLFHCl were found to be 9976plusmn075and 9958plusmn064 respectively Alsothe mean percentage recoveries oflabelled and added CFHCl were

9949plusmn066 and 9978plusmn034respectively The second derivativespectra of ldquoAlkafloxrdquo and ldquoServifloxrdquotablets showed complete absence ofthe corresponding acid degradationproduct

The proposed methods are validand applicable for the determinationof intact LFHCl or CFHCl inlaboratory prepared mixturescontaining (20-80) or (40-75) ofthe degraded LFHCl or degradedCFHCl respectively

Statistical comparison between theproposed and the reported methods(spectrophotometric42 and HPLC43for LFHCl and CFHCl respectively)showed no significant differencebetween them concerning accuracyand precision (Table 3)

Table 3 Statistical analysis for the first derivative spectrophotometricdetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochloride Ciprofloxacin hydrochlorideStatistical

TermFirst

Derivativemethod

Comparisonmethod13

FirstDerivative

method

Comparisonmethod12

Mean 9972 9917 9962 9943

SD plusmn056 plusmn060 plusmn047 plusmn055

RSD 056 047

SE plusmn021 plusmn024 plusmn018 plusmn022

n 7 6 7 6

V 031 036 022 030

LOD(microgml)

039 103

LOQ(microgml)

129 345

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

Sonia T Hassib et al

252

Table 2 Statistical analysis for the chromatographic methods for thedetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochlorideCiprofloxacinhydrochlorideStatistcal Term

HPLCmethod

TLCmethod

Reference42

TLCmethod

Reference43

Mean 9968 9960 9917 9955 9943

SD plusmn077 plusmn055 plusmn060 plusmn072 plusmn055

RSDwithin day

077 055 072

RSDbetween days

117 116 119

SE plusmn031 plusmn022 plusmn024 plusmn029 plusmn023

n 6 6 6 6 6

V 059 030 036 052 030

LOD022

(microg 20microl)010

(microg 10microl)005

(microg 10microl)

LOQ074

(microg 20microl)034

(microg 10microl)018

(microg 10microl)

t 128 130 032

F 164 120 173

The corresponding theoretical values for t and F at p = 005 are 223 and 505respectively

Derivative spectrophotometricmethod

The zero-order spectra of intactand degraded LFHCl and CFHClFigs (3amp4) show completeoverlapping which prevents thedetermination of the intact anddegraded drugs in presence of eachother using this technique

In this work a derivativespectrophotometric procedure ispresented to solve this problem

through measurements in both thefirst and second derivative spectraThe first derivative spectra Figs(5amp6) show that intact LFHCldisplays a peak at 272 nm and avalley at 292 nm while the degradedproduct has zero crossing at both 292and 271 nm Thus the ordinate valueat 292 nm was chosen for thedetermination of the intact drug in thepresence of its degradation productIn addition the first derivative

253

Wavelength (nm)

Fig 3 Zero-order spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 4 Zero-order spectra of intactciprofloxacin hydrochloride (056mg50ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 5 First-derivative spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 6 First-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Abs

orba

nce

Abs

orba

nce

Sonia T Hassib et al

254

spectrum of intact CFHCl Fig (6)exhibits a peak at 262 nm and a valleyat 282 nm while its degradationproduct has zero crossing at both 260and 286 nm Consequently theordinate value at 260 nm waspreferred for calculating theconcentration of the intact drug in thepresence of its degradation product

Figures (5amp6) show that thedegradation product of LFHCl orCFHCl could not be determinedthrough measurements in the firstderivative spectra On the other handin the second derivative spectra Fig(7) the degraded LFHCl shows avalley at 272 nm at which the intactdrug has a zero crossing Thus theordinate value at 272 nm was used forcomputing the concentration of thedegradation product in the presenceof the intact drug

Similarly the second derivativespectra Fig (8) show that thedegraded CFHCl exhibits a valley at262 nm at which the intact drugshows no interference Therefore theordinate value at 262 nm was used forcalculating the concentration of thedegradation product in the presenceof the intact drug

Linearity was shown over theconcentration range (12-96 microgml)or (28-126 microgml) of intact LFH|Clor CFHCl respectively (HPLC andDensitometric) linearity was alsoobserved over the concentration range(12-96 microgml) or (64-16 microgml) ofdegraded LFH|Cl or CFHClrespectively

Wavelength (nm)

Fig 7 Second-derivative spectra of intactlomefloxacin hydrochloride (024mg50 ml) (mdashmdashmdash) and ofdegraded drug (024 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 8 Second-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

255

The mean percentage recoveriesof intact and degraded LFHCl werefound to be 9972plusmn056 and 9893plusmn085 respectively (Table 3) Also themean percentage recoveries of intactand degraded CFHCl were 9962plusmn047 and 9967plusmn039 respectively(Table 3)

The method was successfullyapplied for the quantitative estimationof LFHCl or CFHCl in ldquoAlkafloxrdquoor ldquoServifloxrdquo tablets respectivelywithout interference from additivesThe standard addition technique wasapplied The mean percentagerecoveries of labelled and addedLFHCl were found to be 9976plusmn075and 9958plusmn064 respectively Alsothe mean percentage recoveries oflabelled and added CFHCl were

9949plusmn066 and 9978plusmn034respectively The second derivativespectra of ldquoAlkafloxrdquo and ldquoServifloxrdquotablets showed complete absence ofthe corresponding acid degradationproduct

The proposed methods are validand applicable for the determinationof intact LFHCl or CFHCl inlaboratory prepared mixturescontaining (20-80) or (40-75) ofthe degraded LFHCl or degradedCFHCl respectively

Statistical comparison between theproposed and the reported methods(spectrophotometric42 and HPLC43for LFHCl and CFHCl respectively)showed no significant differencebetween them concerning accuracyand precision (Table 3)

Table 3 Statistical analysis for the first derivative spectrophotometricdetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochloride Ciprofloxacin hydrochlorideStatistical

TermFirst

Derivativemethod

Comparisonmethod13

FirstDerivative

method

Comparisonmethod12

Mean 9972 9917 9962 9943

SD plusmn056 plusmn060 plusmn047 plusmn055

RSD 056 047

SE plusmn021 plusmn024 plusmn018 plusmn022

n 7 6 7 6

V 031 036 022 030

LOD(microgml)

039 103

LOQ(microgml)

129 345

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

253

Wavelength (nm)

Fig 3 Zero-order spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 4 Zero-order spectra of intactciprofloxacin hydrochloride (056mg50ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 5 First-derivative spectra of intactlomefloxacin hydrochloride (024mg50ml) (mdashmdash) and of degradeddrug (024 mg50 ml) ( ndash ndash ndash ) in2N HCl

Wavelength (nm)

Fig 6 First-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Abs

orba

nce

Abs

orba

nce

Sonia T Hassib et al

254

spectrum of intact CFHCl Fig (6)exhibits a peak at 262 nm and a valleyat 282 nm while its degradationproduct has zero crossing at both 260and 286 nm Consequently theordinate value at 260 nm waspreferred for calculating theconcentration of the intact drug in thepresence of its degradation product

Figures (5amp6) show that thedegradation product of LFHCl orCFHCl could not be determinedthrough measurements in the firstderivative spectra On the other handin the second derivative spectra Fig(7) the degraded LFHCl shows avalley at 272 nm at which the intactdrug has a zero crossing Thus theordinate value at 272 nm was used forcomputing the concentration of thedegradation product in the presenceof the intact drug

Similarly the second derivativespectra Fig (8) show that thedegraded CFHCl exhibits a valley at262 nm at which the intact drugshows no interference Therefore theordinate value at 262 nm was used forcalculating the concentration of thedegradation product in the presenceof the intact drug

Linearity was shown over theconcentration range (12-96 microgml)or (28-126 microgml) of intact LFH|Clor CFHCl respectively (HPLC andDensitometric) linearity was alsoobserved over the concentration range(12-96 microgml) or (64-16 microgml) ofdegraded LFH|Cl or CFHClrespectively

Wavelength (nm)

Fig 7 Second-derivative spectra of intactlomefloxacin hydrochloride (024mg50 ml) (mdashmdashmdash) and ofdegraded drug (024 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 8 Second-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

255

The mean percentage recoveriesof intact and degraded LFHCl werefound to be 9972plusmn056 and 9893plusmn085 respectively (Table 3) Also themean percentage recoveries of intactand degraded CFHCl were 9962plusmn047 and 9967plusmn039 respectively(Table 3)

The method was successfullyapplied for the quantitative estimationof LFHCl or CFHCl in ldquoAlkafloxrdquoor ldquoServifloxrdquo tablets respectivelywithout interference from additivesThe standard addition technique wasapplied The mean percentagerecoveries of labelled and addedLFHCl were found to be 9976plusmn075and 9958plusmn064 respectively Alsothe mean percentage recoveries oflabelled and added CFHCl were

9949plusmn066 and 9978plusmn034respectively The second derivativespectra of ldquoAlkafloxrdquo and ldquoServifloxrdquotablets showed complete absence ofthe corresponding acid degradationproduct

The proposed methods are validand applicable for the determinationof intact LFHCl or CFHCl inlaboratory prepared mixturescontaining (20-80) or (40-75) ofthe degraded LFHCl or degradedCFHCl respectively

Statistical comparison between theproposed and the reported methods(spectrophotometric42 and HPLC43for LFHCl and CFHCl respectively)showed no significant differencebetween them concerning accuracyand precision (Table 3)

Table 3 Statistical analysis for the first derivative spectrophotometricdetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochloride Ciprofloxacin hydrochlorideStatistical

TermFirst

Derivativemethod

Comparisonmethod13

FirstDerivative

method

Comparisonmethod12

Mean 9972 9917 9962 9943

SD plusmn056 plusmn060 plusmn047 plusmn055

RSD 056 047

SE plusmn021 plusmn024 plusmn018 plusmn022

n 7 6 7 6

V 031 036 022 030

LOD(microgml)

039 103

LOQ(microgml)

129 345

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

Sonia T Hassib et al

254

spectrum of intact CFHCl Fig (6)exhibits a peak at 262 nm and a valleyat 282 nm while its degradationproduct has zero crossing at both 260and 286 nm Consequently theordinate value at 260 nm waspreferred for calculating theconcentration of the intact drug in thepresence of its degradation product

Figures (5amp6) show that thedegradation product of LFHCl orCFHCl could not be determinedthrough measurements in the firstderivative spectra On the other handin the second derivative spectra Fig(7) the degraded LFHCl shows avalley at 272 nm at which the intactdrug has a zero crossing Thus theordinate value at 272 nm was used forcomputing the concentration of thedegradation product in the presenceof the intact drug

Similarly the second derivativespectra Fig (8) show that thedegraded CFHCl exhibits a valley at262 nm at which the intact drugshows no interference Therefore theordinate value at 262 nm was used forcalculating the concentration of thedegradation product in the presenceof the intact drug

Linearity was shown over theconcentration range (12-96 microgml)or (28-126 microgml) of intact LFH|Clor CFHCl respectively (HPLC andDensitometric) linearity was alsoobserved over the concentration range(12-96 microgml) or (64-16 microgml) ofdegraded LFH|Cl or CFHClrespectively

Wavelength (nm)

Fig 7 Second-derivative spectra of intactlomefloxacin hydrochloride (024mg50 ml) (mdashmdashmdash) and ofdegraded drug (024 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

Wavelength (nm)

Fig 8 Second-derivative spectra of intactciprofloxacin hydrochloride (056mg50 ml) (mdashmdashmdash) and ofdegraded drug (056 mg50 ml)( ndash ndash ndash ndash ) in 2N HCl

255

The mean percentage recoveriesof intact and degraded LFHCl werefound to be 9972plusmn056 and 9893plusmn085 respectively (Table 3) Also themean percentage recoveries of intactand degraded CFHCl were 9962plusmn047 and 9967plusmn039 respectively(Table 3)

The method was successfullyapplied for the quantitative estimationof LFHCl or CFHCl in ldquoAlkafloxrdquoor ldquoServifloxrdquo tablets respectivelywithout interference from additivesThe standard addition technique wasapplied The mean percentagerecoveries of labelled and addedLFHCl were found to be 9976plusmn075and 9958plusmn064 respectively Alsothe mean percentage recoveries oflabelled and added CFHCl were

9949plusmn066 and 9978plusmn034respectively The second derivativespectra of ldquoAlkafloxrdquo and ldquoServifloxrdquotablets showed complete absence ofthe corresponding acid degradationproduct

The proposed methods are validand applicable for the determinationof intact LFHCl or CFHCl inlaboratory prepared mixturescontaining (20-80) or (40-75) ofthe degraded LFHCl or degradedCFHCl respectively

Statistical comparison between theproposed and the reported methods(spectrophotometric42 and HPLC43for LFHCl and CFHCl respectively)showed no significant differencebetween them concerning accuracyand precision (Table 3)

Table 3 Statistical analysis for the first derivative spectrophotometricdetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochloride Ciprofloxacin hydrochlorideStatistical

TermFirst

Derivativemethod

Comparisonmethod13

FirstDerivative

method

Comparisonmethod12

Mean 9972 9917 9962 9943

SD plusmn056 plusmn060 plusmn047 plusmn055

RSD 056 047

SE plusmn021 plusmn024 plusmn018 plusmn022

n 7 6 7 6

V 031 036 022 030

LOD(microgml)

039 103

LOQ(microgml)

129 345

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

255

The mean percentage recoveriesof intact and degraded LFHCl werefound to be 9972plusmn056 and 9893plusmn085 respectively (Table 3) Also themean percentage recoveries of intactand degraded CFHCl were 9962plusmn047 and 9967plusmn039 respectively(Table 3)

The method was successfullyapplied for the quantitative estimationof LFHCl or CFHCl in ldquoAlkafloxrdquoor ldquoServifloxrdquo tablets respectivelywithout interference from additivesThe standard addition technique wasapplied The mean percentagerecoveries of labelled and addedLFHCl were found to be 9976plusmn075and 9958plusmn064 respectively Alsothe mean percentage recoveries oflabelled and added CFHCl were

9949plusmn066 and 9978plusmn034respectively The second derivativespectra of ldquoAlkafloxrdquo and ldquoServifloxrdquotablets showed complete absence ofthe corresponding acid degradationproduct

The proposed methods are validand applicable for the determinationof intact LFHCl or CFHCl inlaboratory prepared mixturescontaining (20-80) or (40-75) ofthe degraded LFHCl or degradedCFHCl respectively

Statistical comparison between theproposed and the reported methods(spectrophotometric42 and HPLC43for LFHCl and CFHCl respectively)showed no significant differencebetween them concerning accuracyand precision (Table 3)

Table 3 Statistical analysis for the first derivative spectrophotometricdetermination of lomefloxacin hydrochloride and ciprofloxacinhydrochloride

Lomefloxacin hydrochloride Ciprofloxacin hydrochlorideStatistical

TermFirst

Derivativemethod

Comparisonmethod13

FirstDerivative

method

Comparisonmethod12

Mean 9972 9917 9962 9943

SD plusmn056 plusmn060 plusmn047 plusmn055

RSD 056 047

SE plusmn021 plusmn024 plusmn018 plusmn022

n 7 6 7 6

V 031 036 022 030

LOD(microgml)

039 103

LOQ(microgml)

129 345

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

Sonia T Hassib et al

256

Identification of the degradationproducts of lomefloxacin hydro-chloride and ciprofloxacin hydro-chloride

The degradation product of eachof LFHCl and CFHCl was separatedand crystallized from diethylether andhas a melting point at 100degC or192degC respectively

The structure for each degradationproduct was confirmed by spectralanalysis (IR KBr disc) 1H NMR(DMSO) and MS

Degradation product of LFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ 09 (d 3H)

13 (t 3H) (27- 31) (m 7H) 42 (q2H) 6 (d 1H) 76 (d 1H) 79 (d1H) M S (M+ 307)

Degradation product of CFHClIR 1620 cm-1 (C=O of pyridone)1H NMR (DMSO) δ(1-12) (m

4H) (28-31) (m 8H) 34 (t 1H)58 (d 1H) 73 (d 1H) 76 (d 1H)78 (d 1H) MS (M+ 287)

The previous data revealed theabsence of carboxylic acid group inboth degraded LFHCl and CFHCl(Fig 1) which played a vital role inthe antibacterial activity of the drugs

The following structures for thedegradation products were suggested(Fig 1)

ConclusionA reversed phase HPLC method

has been developed for thedetermination of lomefloxacin and itsacid degradation product In additiontwo other methods have been

developed for the determination oflomefloxacin hydrochloride (LFHCl)and ciprofloxacin hydrochloride(CFHCl) in presence of their acidinduced degradation products Thedeveloped methods are rapid simpleand accurate The developed methodsare recommended for the routineanalysis of cited drugs in dosageforms and in drug controllaboratories

REFERENCES

1- Graham L Patrick AnIntroduction to MedicinalChemistry 2nd Edition OxfordUniversity Press (2001)

2- H P Rang M M Dale J MRitter and P K MoorePharmacology ElsevierScience Limited 2003 pp 620-653

3- B K Dubey Renu UpadhyayA K Tiwari and I C Shukla Jof the Indian Chemical Society81 511 (2004)

4- E Kilic F Koseoglu and M AAkay J Pharm Biomed Anal12 347 (1994)

5- M I Pascual-Reguera G PerezParras and A Molina DiazMicrochim J 77 79 (2004)

6- M I Pascual-Reguera G PerezParras and A Molina Diaz JPharm Biomed Anal 35 689(2004)

7- D N Tipre and A V KastureIndian Drugs 37 148 (2000)

8- A Bungalowala D K Jain andP Trivedi ibid 35 348 (1998)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

257

9- C S Xuan Z Y Wang and J LSong Anal Lett 31 1185(1998)

10- A S Amin Mikrochim Acta134 89 (2000)

11- B G Gowda and JSeetharamappa Anal Sci 19461 (2003)

12- P Djurdjevic M Todorovic MJ Stankov and J Odovic AnalLett 33 657 (2000)

13- B S Nagaralli JSeetharamappa and M BMelwanki J Pharm BiomedAnal 29 859 (2002)

14- M Rizk F Belal F Ibrahim SAhmed and Z A Sheribah JAOAC Int 84 368 (2001)

15- Li Ming Du Ya Qin Yang andQing Mei Wang Anal ChimActa 516 237 (2004)

16- H He H Y Ye L Dai Q CJiao and P H ChuongGuangpuxue-Yu-Guangpu-Fenxi 26 480 (2006)

17- A Navalon O Ballesteros RBlanc and J L Vilchez Talanta52 845 (2000)

18- Yi Rao Yan Tong XiunrongZhang Guoan Luo and Willy RG Baeyens Anal Lett 33 1117(2000)

19- F A Aly A A Alwarthan andS A Al-Tamimi Talanta 53885 (2001)

20- Y D Liang J F Song and X FYang Anal Chim Acta 510 21(2004)

21- R C Rodriguez-Diaz M PAguilar-Caballos and A Gomez-Hens ibid 494 55 (2003)

22- M E El-Kommos G A SalehS M El-Gizawi and M AAbou-Elwafa Talanta 60 1033(2003)

23- R M Cuenca-Trujillo M JAyora-Canada and A Molina-Diaz J AOAC Int 85 1268(2002)

24- M A Garcia C Solans ACalvo M Royo E HernandezR Rey and M A BreganteChromatographia 54 577(2001)

25- G C G Tozzo and H R NSalagado J AOAC Int 891305 (2006)

26- G Carlucci Mazzeo VetuschiC J Liq Chromatogr RelatTechnol 26 2053 (2003)

27- J Novakovic K Nesmerak HNova and K Filka J PharmBiomed Anal 25 957 (2001)

28- P-L Wang and L Chen JAOAC Int 84 684 (2001)

29- I M Choma J LiqChromatogr Relat Technol 262673 (2003)

30- S Imre M T Dogaru C EVari T Muntean and LKelemen J Pharm BiomedAnal 33 125 (2003)

31- M T Maya N J Goncalves NB Silva and J A Morais JChromatogr B Biomed Appl755 305 (2001)

32- A Shafiee M Amini and MKhanavi Indian Drugs 39 110(2002)

33- A Espinosa-Mansilla AMunoz-de-la-Pena D Gonzalea-Gomez and F Canada-Canada J

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)

Sonia T Hassib et al

258

of Separation Science 29 1969(2006)

34- A Zotou and N Miltiadou ibid28 559 (2002)

35- H R Liang M B Kays and KM Sowinski J Chromatogr BAnalyt Technol Biomed LifeSci 772 53 (2002)

36- V F Samanidou C EDemetriou and I NPapadoyanis Anal BioanalChem 375 623 (2003)

37- M I R M Santoro N MKassab A K Singh and E RM Kedor-Hackmam J PharmBiomed Anal 40 179 (2006)

38- S O Thoppil and P D Aminibid 22 699 (2000)

39- F Hudrea C Grosset J Alaryand M Bojita BiomedChromatogr 14 17 (2000)

40- S S Zarapkar A V Rane andS H Rane Indian Drugs 36408 (1999)

41- A P Argekar and S G Powaribid 36 399 (1999)

42- Y D Sanzgiri and S R KnaubAnalytical Profiles of DrugSubstances and ExcipientsEditor K Flowery Vol 23Academic Press Inc (1994)

43- The British Pharmacopoeia HerMajestyrsquos Stationary Office(2001)

44- S Z El-Khateeb S A AbdelRazek and M M Amer JPharm Biomed Anal 17 829(1998)

45- H Gu L Sun X Nu Z Taoand S Zhao Yaowu FenxiZazhi 17 89 (1997) ThroughChem Abst 127 336731z(1998)

46- H-Z Xie C Dong Y-L Fen andC-S Liu Anal Lett 30 79(1997)

47- R A Sodhi J L Chawla and RT Sane Indian Drugs 34 512(1997)

48- S Tammilehto H Salomies andK Torniainen J Planar Chrom7 368 (1994)