Hindawi Publishing CorporationChromatography Research InternationalVolume 2013 Article ID 315145 12 pageshttpdxdoiorg1011552013315145

Research ArticleDevelopment and Validation of a Stability-Indicating RP-HPLCMethod for the Simultaneous Estimation of Guaifenesin andDextromethorphan Impurities in Pharmaceutical Formulations

Thummala V Raghava Raju12 Noru Anil Kumar1 Seshadri Raja Kumar1

Annarapu Malleswara Reddy1 Nittala Someswara Rao2 and Ivaturi Mrutyunjaya Rao2

1 Analytical Research and Development Integrated Product Development Dr Reddyrsquos Laboratories Limited BachupallyHyderabad-500072 India

2 School of Chemistry Andhra University Visakhapatnam Andhra Pradesh 530003 India

Correspondence should be addressed toThummala V Raghava Raju raghavartvdrreddyscom

Received 8 June 2013 Revised 9 August 2013 Accepted 9 August 2013

Academic Editor Osama Y Aldirbashi

Copyright copy 2013 Thummala V Raghava Raju et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

A sensitive stability-indicating gradient RP-HPLC method has been developed for the simultaneous estimation of impurities ofGuaifenesin and Dextromethorphan in pharmaceutical formulations Efficient chromatographic separation was achieved on aSunfire C18 250times 46mm 5 120583m column with mobile phase containing a gradient mixture of solvents A and BThe flow rate of themobile phase was 08mLminminus1 with column temperature of 50∘C and detection wavelength at 224 nm Regression analysis showedan r value (correlation coefficient) greater than 0999 for Guaifenesin Dextromethorphan and their impurities Guaifenesin andDextromethorphan formulation sample was subjected to the stress conditions of oxidative acid base hydrolytic thermal andphotolytic degradation Guaifenesin was found stable and Dextromethorphan was found to degrade significantly in peroxide stresscondition The degradation products were well resolved from Guaifenesin Dextromethorphan and their impurities The peakpurity test results confirmed that the Guaifenesin and Dextromethorphan peak was homogenous and pure in all stress samples andthemass balance was found to bemore than 98 thus proving the stability-indicating power of themethodThe developedmethodwas validated according to ICH guidelines with respect to specificity linearity limits of detection and quantification accuracyprecision and robustness

1 Introduction

Guaifenesin (GN) (+)-3-(2-methoxyphenoxy)-propane-12-diol is a widely used expectorant useful for the symptomaticrelief of respiratory conditions Its empirical formula isC10H14O4 which corresponds to a molecular weight of19821 It is a white or slightly gray crystalline substance witha slightly bitter aromatic taste Its solid oral dosage form isavailable as extended release tablets for oral administration[1]

Dextromethorphan (DN) [2ndash4] is the dextrorotatoryenantiomer of themethyl ether of levorphanol and stereoiso-mer of levomethorphan DN is an antitussive (cough sup-pressant) drug and used for pain relief and psychological

applications [5ndash7] Its empirical formula is C8H25NO whichcorresponds to a molecular weight of 2714 It is a whitepowder The combination of GN and DN is used to treatcough and chest congestion caused by the common coldinfections or allergies The chemical structures of GN andDN are shown in Table 1

Impurity profiling of active pharmaceutical ingredients(API) in both bulk material and finalized formulations is oneof the most challenging tasks for pharmaceutical analyticalchemists under industrial environment [8] The presenceof unwanted or in certain cases unknown chemicals evenin small amounts may influence not only the therapeuticefficacy but also the safety of the pharmaceutical products [9]For these reasons all major international pharmacopoeias

2 Chromatography Research International

have established maximum allowed limits for related com-pounds for both bulk and formulated APIs As per therequirements of various regulatory authorities the impurityprofile study of drug substances and drug products has tobe carried out using a suitable analytical method in the finalproduct [10 11]

GN andDNare official in theUnited States pharmacopeiaand European pharmacopeia but its combination is notofficial in any of the pharmacopeias In the literature surveythere were several LC assay methods that have been reportedfor the determination of GN and DN in pharmaceuticalpreparation either individually or in combination with otherdrugs [12ndash20] and in human plasma by LC-MS [21 22] Fewmethods were available for the determination of impuritiesindividually for GN and DN [27ndash29]

There is no single method reported for the simultaneousdetermination of the impurities in pharmaceuticals formula-tions of GN andDN It is felt to develop a stability-indicatingmethod for simultaneous determination of GN and DNrelated impurities in pharmaceutical formulation

Hence an attempt has been made to develop an acc-urate rapid specific and reproducible method for the deter-mination of GN and DN impurities (Tables 2(a) and 2(b)) inpharmaceutical dosage forms along with method validationas per ICH norms [23 24] The stability tests were alsoperformed on both drug substances and drug products as perICH norms [25 26]

2 Experimental

21 Chemicals and Reagents GN + DN tablets were receivedfrom the formulation research and development laboratoryof Dr Reddyrsquos Laboratories Ltd IPDO Hyderabad IndiaGN API and impurities were procured from SynthochemLab India DN API and impurities were procured fromWochardt Laboratories Ltd India Sodiumdihydrogen phos-phatemonohydrate 1-octane sulfonic acid sodium saltmono-hydrate HPLC grade acetonitrile methanol and orthophos-phoric acid were purchased from Merck Germany RegisTechnologies Inc USA and highly pure water was preparedby using Millipore MilliQ plus purification system

22 Equipment The LC system used for method devel-opment and method validation was Waters with a diodearray detector (model 2998 detector and e2695 separationmodule) The output signal was monitored and processedusing Waters Empower software Weighing was performedwith a Mettler XS 205 Dual Range (Mettler-Toledo GmbHGreifensee Switzerland) Photo stability studies were carriedout in a photo stability chamber (SUN TEST XLS+ AtlasUSA) Thermal stability studies were performed in a dry airoven (Merck Pharmatech Hyderabad India)

23 Chromatographic Conditions HPLCmeasurements werecarried out using a reversed phase Sunfire C18 250times 46mm5 120583m particle size column (Waters India Pvt Ltd) operatedat 50∘C with gradient elution at 08mLminminus1 using a mobilephase buffer as a mixture of 001M sodium dihydrogen

phosphate monohydrate and 00046M 1-octane sulfonic acidsodium saltmonohydrate of pH 30 (pH adjustedwith dilutedorthophosphoric acid) UV absorbance at 224 nm injectionvolume 20120583LThemobile phase A consisted of pH 30 bufferand acetonitrile (90 10 vv) mobile phase B consisted of pH30 buffer acetonitrile and methanol (10 10 80 vvv) TheLC gradient program was set as and time (min) mobilephase B 00115 155 2030 3050 6085 6515 and 7515Pressure Range was 1700 psi to 2200 psi Mixture of wateracetonitrile and methanol (60 20 20 vvv) was used asdiluent for sample preparation

24 Preparation of Standard Solution and System SuitabilitySolution We prepared individual stock solutions for GNDN and their impurities (each 500120583gmL) Working solu-tion was prepared from the above stock solutions for relatedsubstances determination (48120583gmL of GN and 24120583gmL ofDN) Amixture of all impurities (48120583gmL of GN impuritiesand 24120583gmL of DN impurities) along with GN and DN(24000 120583gmL of GN and 1200120583gmL of DN) was alsoprepared in diluent Stock solutions were used for methoddevelopment and method validation

25 Preparation of Test Solution Twenty tablets (1200mg ofGN + 60mg of DN) were weighed and the average weightwas calculated The tablets were crushed into fine powderand powder equivalent to 6000mg of GN (or equivalent to300mg of DN) was transferred into a 250mL volumetricflask Approximately 170mL of diluent was added shakedto disperse the material and sonicated for 20 minuteswith intermediate shaking The solution was then dilutedto 250mL and centrifuged at 3000 rpm for 10min Thesupernatant (24000120583gmL of GN and 1200120583gmL of DN)was collected and filtered through a 045120583m pore size nylon66-membrane filter (make Rankem)The filtrate was used assample solution

3 Method Validation

The proposed method was validated as per ICH guidelines[24]

31 System Suitability System suitability parameters wereevaluated to verify the system performance System preci-sion was determined on six replicate injections of standardpreparations All the important characteristics including therelative standard deviation peak tailing and theoretical platenumber were measured Resolution between impurities wasmeasured by injecting system suitability solution All thesesystem suitability parameters covered the system methodand column performance

32 Specificity Stress studies were performed at an initialconcentration of 24000 120583gmLminus1 of GN and 1200120583gmLminus1 ofDN in active pharmaceutical ingredients (API) and formu-lated sample to provide the stability-indicating property andspecificity of the proposed method Intentional degradation

Chromatography Research International 3

Table 1 Structures of Guaifenesin and Dextromethorphan

Molecule name Chemical name Chemical structure

Guaifenesin (+)-3-(2-Methoxyphenoxy)-propane-12-diolO

O

OH

OH

Dextromethorphan Ent-3-methoxy-17-methylmorphinan

N

H

O

was attempted by the stress conditions of exposure to pho-tolytic stress (12million lux hours followed by 200Watt hoursmminus2) heat (exposed at 105∘C for 15 h) acid (05N HCl for2 hours at 60∘C) base (05N NaOH for 2 hours at 60∘C)oxidation (10 peroxide for 30min at 60∘C) water (refluxedfor 12 hours at 60∘C) and humidity (exposed to 90 RH for7 days)

33 Precision The precision of the determination of theimpurities was checked by injecting six individual prepa-rations of (24000 120583gmL of GN and 1200120583gmL of DN)test preparation spiked with 48120583gmLminus1 of GN impuritiesand 24 120583gmLminus1 of DN impurities (02 of the target testconcentration) and calculating the RSD of impurity foreach compound The intermediate precision of the methodwas also evaluated using different analysts and a differentinstrument in the same laboratory

34 Limits of Detection (LOD) and Quantification (LOQ)LOD and LOQ for all impurities including GN and DNwere determined at a signal-to-noise ratio of 3 1 and 10 1respectively by injecting a series of dilute solutions withknown concentrations Precision study was also carried outat LOQ level by injecting six individual preparations ofimpurities and RSD was calculated

35 Linearity Linearity test solutions for the method wereprepared by diluting stock solution to the required concentra-tions The solutions were prepared at six concentration levelsfrom LOQ to the target test concentration The peak areaversus concentration in 120583gmLminus1 data was subjected to least-squares linear regression analysis

36 Accuracy Accuracy of the method was evaluated byusing concentration levels LOQ 01 02 04 08 and10 on GN + DN tablets Six preparations were performedat LOQ and 10 level and three preparations were per-formed at different levels Standard addition and recoveryexperiments were conducted on real sample to determine

accuracy of the related substance methodThe percentages ofrecoveries for all impurities GN and DN were calculated

37 Robustness To determine the robustness of the devel-oped method experimental conditions were deliberatelychanged and the resolution between GN DN and theirimpurities tailing factor and theoretical plates of GN andDN peaks were evaluated Also relative retention times for allthe impurities and column pressure throughout the run weremonitored

To study the effect of the flow rate on the developedmethod it was changed from 08mLminminus1 to 06 and10mLminminus1 The effect of column temperature on thedeveloped method was studied at 45 and 55∘C (instead of50∘C) The effect of pH was studied by varying plusmn02 pH units(ie 28 and 32) and the mobile phase composition waschanged plusmn10 from the initial composition In all the abovevaried conditions the component of the mobile phase washeld constant

38 Stability in Solution and in the Mobile Phase GN andDN spiked samples (impurities spiked at 02 of the targettest concentration ie 48 120583gmLminus1 of GN impurities and24 120583gmLminus1 of DN impurities) were prepared in the diluentleaving the test solutions at room temperature The spikedsamples were injected at 0 24 and 48 hrs time intervals Theimpurity content was calculated and the consistency in the area of the principal peak at each interval was checkedThe prepared mobile phase was kept constant during thestudy period The mobile phase study was demonstrated byinjecting the freshly prepared sample solution at differenttime intervals (0ndash2 days)

4 Results and Discussion

41 Optimization of Chromatographic Conditions The maincriterion was developing an RP-HPLC method for thesimultaneous determination of impurities in GN and DNpharmaceutical dosage form in a single run with emphasis

4 Chromatography Research International

Table 2 (a) Structures of Guaifenesin impurities (b) Structures of Dextromethorphan impurities

(a)

Impurity name Chemical name Chemical structure

Impurity A (degradant) 2-Methoxyphenol

OH

O

Impurity B (degradant) 2-(2-Methoxyphenoxy)propane-13-diolHO

HO

O

O

Impurity C (degradant) 111015840-Oxybis[3-(2-methoxyphenoxy)propan-2-ol]OHOH

O

OOO

O

Impurity D (process related impurity) 13-Bis(2-methoxyphenoxy)propan-2-ol

OHOO

O O

(b)

Impurity name Chemical name Chemical structure

Impurity A (degradant) Ent-3-methoxymorphinan

NH

H

O

Impurity B (degradant) Ent-17-methylmorphinan-3-ol

HO

H

N

Impurity C (degradant) Ent-3-methoxy-17-methylmorphinan-10-one H

N

O

O

N-Oxide (degradant) 3-Methoxy-N-methylmorphinan N-oxide H

O

Ominus

N+

N-Formyl Morphine (NFM)(process related impurity)

3-Methoxy-6788a910-hexahydro-5H-94b-(epiminoethano)phenanthrene-11-carbaldehyde

N

CHO

H3CO

N-Formyl octabase (NFO)(process related impurity)

(S)-1-(4-Methoxybenzyl)-345678-hexahydroisoquinoline-2(1H)-carbaldehyde

H

NCHO

H3CO

Chromatography Research International 5

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

23833 Guaifenesin imp A15533 Guaifenesin imp B 43267 Guaifenesin imp C

45500 Guaifenesin imp D

(a)

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

21400 Guaifenesin40733 Dextromethorphan

(b)

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

34017 Dextromethorphan imp B40117 Dextromethorphan imp C41450 Dextromethorphan imp N-Oxide42033 Dextromethorphan imp A51983 Dextromethorphan imp NFM55967 Dextromethorphan imp NFO

(c)

Figure 1 Spectra of GN DN and their impurities

on themethod being accurate reproducible robust stability-indicating linear free of interference fromother formulationexcipients and convenient enough for routine use in qualitycontrol laboratories

Individual stock solutions of GN DN and their impu-rities were injected and the spectra were checked of eachcomponent (Figure 1) From the spectra all the impuritieswere having absorbance maximum at about 224 Hence224 nm was selected for the estimation of GN and DNimpurities

A spiked solution of impurities (48120583gmLminus1 of GNimpurities and 24 120583gmLminus1 of DN impurities) GN + DN(24000120583gmLminus1 + 1200120583gmLminus1) and placebo peaks weresubjected to separation by RP-HPLC Initially the separationwas tried with the existing methods (USP Pharma Europeand the literature method) [27ndash29] It was observed thatplacebo peaks and GN impurity peaks were merging with

each other and two DN known impurities (NFM and NFO)were not eluting in DN API Pharma Europe method In USPand Pharma Europe GN API method DN impurities werenot separated from DN peak and two DN known impuritieswere eluting at longer retention with broad peak shapes Incase of the literature method [29] DN impurities were notseparated fromDN peak and twoDN known impurities wereeluting at longer retention times with better peak shapes

Method development was initiated by changing dif-ferent gradient programmes different pH values of themobile phase buffer different phosphate buffers and differentcolumns with the literature method [29] Sharp peak shapeswere observed with Sunfire column and sodium dihydrogenphosphate monohydrate buffer at pH 30 but separation wasnot up to the mark for DN DN-N-oxide and DN-impurityC Sharp peak shapes were due to the properties of the Sunfirecolumn (high mass loading capability excellent low pH

6 Chromatography Research International

020

018

016

014

012

010

008

006

004

002

000000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 2 Overlaid chromatogram of blank and system suitabilitypreparation

020

018

016

014

012

010

008

006

004

002

000

000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 3 Overlaid chromatogram of placebo and spiked testpreparation

stability superior peak shapes and high efficiency) Since wewere using a very high concentration ofGN (24000120583gmLminus1)it was decided to use Sunfire column for further developmenttrails by using ion pair reagent in the mobile phase for betterseparation between DN DN-Impurity A and DN-ImpurityB Separation was achieved between all the pairs of peaks butpeak shapes of DN-NFM Impurity and DN-NFO Impurityare not sharp Acetonitrile was added in the mobile phase inaddition to methanol to get sharp peaks

The chromatographic separation was achieved by areversed phase Sunfire C18 250 times 46mm 5120583m particlesize column operated at 50∘C with gradient elution at

020

018

016

014

012

010

008

006

004

002

000

000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 4 Typical chromatogram of peroxide stressed sample

08mLminminus1 using a mobile phase buffer as a mixtureof 001M sodium dihydrogen phosphate monohydrate and00046M 1-octane sulfonic acid sodium salt monohydrateof pH 30 (pH adjusted with diluted orthophosphoric acid)UV absorbance at 224 nm and injection volume 20 120583L Themobile phase A consisted of pH 30 buffer and acetonitrile(90 10 vv) mobile phase B consisted of pH 30 bufferacetonitrile andmethanol (10 10 80 vvv)The LC gradientprogram was set as time (min) mobile phase B 00115155 2030 3050 6085 6515 and 7515 All the impuritieswere well separated with a resolution greater than 2 Nochromatographic interference due to the blank (diluent)and other excipients (placebo) at the retention time of GNDN and their impurities was observed The typical overlaychromatogram of blank and system suitability solution andspiked test is shown in Figures 2 and 3

42MethodValidation After the development of themethodit was subject to method validation as per ICH guidelines[24] The method was validated to demonstrate that it issuitable for its intended purpose by the standard procedureto evaluate adequate validation characteristics (system suit-ability specificity accuracy precision linearity robustnessruggedness solution stability LOD and LOQ and stability-indicating capability)

421 System Suitability The percentage relative standarddeviation (RSD) of area from six replicate injections wasbelow 50 (diluted standard solution 48 120583gmLminus1 of GNand 24 120583gmLminus1 of DN) Low values of RSD for replicateinjections indicate that the system is precise The results ofother system suitability parameters such as resolution peaktailing and theoretical plates are presented in Table 3 As seenfrom this data the acceptable system suitability parameterswould be as follows the relative standard deviation of

Chromatography Research International 7

000200000000400000000600000000800000000

1000000000120000000014000000001600000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesiny = 56119662x + 118692128

R2 = 0999

(a)

02000000400000060000008000000

10000000120000001400000016000000

0000 50000 100000 150000 200000 250000 300000

Linearity of detector response of Guaifenesin impurity Ay = 62278038x + 138787601

R2 = 0998

Are

a

(b)

02000000400000060000008000000

100000001200000014000000

0000 50000 100000 150000 200000 250000 300000

Linearity of detector response of Guaifenesin impurity By = 46707746x + 109527559

R2 = 0998

Are

a

(c)

02000000400000060000008000000

100000001200000014000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesin impurity Cy = 49 606000x +

R2 = 0999

78596824

(d)

0

5000000

10000000

15000000

20000000

25000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesin impurity Dy = 79962049x + 156337857

R2 = 0999

(e)

050000

100000150000200000250000300000350000400000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphany = 30824867x + 4487696

R2 = 1000

(f)

050000

100000150000200000250000300000350000400000450000500000

0000 2000 4000 6000 8000 10000 12000 14000 16000

Are

a

Linearity of detector response of Dextromethorphan impurity A

y = 32920087x + 331349

R2 = 1000

(g)

050000

100000150000200000250000300000

0000 2000 4000 6000 8000 10000 12000

Are

a

Linearity of detector response of Dextromethorphan impurity B

y = 25035731x minus 57593

R2 = 1000

(h)

050000

100000150000200000250000300000350000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphan impurity C

y = 24213547x + 450726

R2 = 1000

(i)

050000

100000150000200000250000300000350000400000450000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphan N-oxide

y = 33 682946x + 523536

R2 = 1000

(j)

0100000200000300000400000500000600000700000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Concentration (ppm)

Linearity of detector response of Dextromethorphan impurity NFM

y = 49617284x + 22697199

R2 = 0996

(k)

0100000200000300000400000500000600000700000800000900000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Concentration (ppm)

Linearity of detector response of Dextromethorphan impurity NFO

y = 66581365x minus 1352313

R2 = 1000

(l)

Figure 5 (a) Linearity graph of Guaifenesin (b) Linearity graph of Guaifenesin impurity A (c) Linearity graph of Guaifenesin impurity B(d) Linearity graph of Guaifenesin impurity C (e) Linearity graph of Guaifenesin impurity D (f) Linearity graph of Dextromethorphan(g) Linearity graph of Dextromethorphan impurity A (h) Linearity graph of Dextromethorphan impurity B (i) Linearity graph ofDextromethorphan impurity C (j) Linearity graph of DextromethorphanN-oxide (k) Linearity graph of Dextromethorphan impurity NFM(l) Linearity graph of Dextromethorphan impurity NFO

8 Chromatography Research International

Table 3 System suitability results

Parameter RSDlowast of standard Theoretical plateslowast Tailing factorlowast Resolution 1 Resolution 2GN DN GN DN GN DN

As such method 05 07 45320 498659 10 10 27 23At 06mLmin flow rate 12 06 41091 495497 10 10 29 25At 10mLmin flow rate 09 04 45828 515551 10 10 25 22At 50∘C column temperature 15 08 45321 508966 10 10 26 23At 60∘C column temperature 21 23 40929 515807 10 10 29 26At pH 28 (buffer pH) 11 05 43970 463987 10 10 24 23At pH 32 (buffer pH) 07 15 44619 532891 10 10 26 27lowastAverage of six replicate standard injections

Table 4 (a) Forced degradation data for Guaifenesin (b) Forced degradation data for Dextromethorphan

(a)

Degradation conditions Guaifenesin degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 million lux hours followed by 200Watt hours) 006 3585 4142 1002Exposed to humidity at 25∘C and 90 RH for about 7 days 007 3881 4241 998Refluxed with purified water for about 12 hours at 60∘C 005 3285 3845 986Refluxed with 05 N HCL solution for about 2 hours at 60∘C 010 5293 7073 992Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 006 3067 3489 989Refluxed with 10 H2O2 solution for about 30min at 60∘C 013 2755 3607 983Exposed to dry heat for about 15 hours at 105∘C 007 3346 4331 994

(b)

Degradation conditions Dextromethorphan degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 Million lux hours followed by 200Watt hours) 001 0115 0242 998Exposed to humidity at 25∘C and 90 RH for about 7 days 001 0124 0261 995Refluxed with purified water for about 12 hours at 60∘C 001 0101 0261 989Refluxed with 05 N HCL solution for about 2 hours at 60∘C 002 0154 0262 981Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 001 0103 0256 992Refluxed with 10 H2O2 solution for about 30min at 60∘C 105 0102 0250 997Exposed to dry heat for about 15 hours at 105∘C 001 0099 0261 987

Table 5 Results of precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0198 0201 0189 0213 0205 0209 0203 42GN-Impurity B 0209 0195 0192 0201 0206 0194 0200 35GN-Impurity C 0189 0195 0186 0192 0198 0184 0191 28GN-Impurity D 0208 0190 0192 0193 0188 0186 0193 41DN-Impurity A 0215 0214 0215 0209 0212 0205 0212 19DN-Impurity B 0204 0200 0202 0203 0205 0191 0201 25DN-Impurity C 0195 0186 0198 0199 0184 0194 0193 32DN-N-oxide 0216 0214 0218 0219 0211 0210 0215 17DN-NFM Impurity 0216 0218 0215 0215 0216 0212 0215 09DN-NFO Impurity 0198 0195 0213 0192 0214 0215 0205 52GN 0203 0212 0204 0206 0205 0210 0207 17DN 0182 0189 0194 0188 0187 0201 0190 34

Chromatography Research International 9

Table 6 Results of intermediate precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0191 0204 0201 0199 0210 0212 0203 38GN-Impurity B 0212 0204 0210 0209 0211 0204 0208 17GN-Impurity C 0192 0198 0191 0194 0202 0209 0198 35GN-Impurity D 0185 0192 0191 0194 0193 0192 0192 15DN-Impurity A 0202 0202 0203 0202 0202 0204 0203 04DN-Impurity B 0191 0188 0204 0209 0189 0202 0197 45DN-Impurity C 0183 0189 0191 0186 0205 0206 0193 51DN-N-oxide 0197 0210 0213 0202 0198 0212 0205 35DN-NFM Impurity 0183 0187 0179 0184 0185 0188 0184 17DN-NFO Impurity 0206 0197 0209 0212 0194 0208 0204 35GN 0199 0205 0201 0204 0207 0208 0204 17DN 0187 0185 0192 0186 0194 0191 0189 19

Table 7 LOD LOQ and precision data

Compound LOD () LOQ () SN ratio RSD at LOQ Level precisionLOD LOQ

GN-Impurity A 0004 0001 27 95 42GN-Impurity B 0008 0002 29 98 25GN-Impurity C 0005 0002 30 98 31GN-Impurity D 0003 0001 29 97 25DN-Impurity A 0014 0004 28 101 11DN-Impurity B 0021 0006 27 104 34DN-Impurity C 0023 0007 28 99 49DN-N-oxide 0016 0005 31 98 22DN-NFM Impurity 0016 0005 27 101 15DN-NFO Impurity 0041 0012 29 106 42GN 0006 0002 31 98 21DN 0015 0005 28 96 28

replicate injections is notmore than 50 resolution betweenimpurities 20 the tailing factor for GN and DN is not morethan 15 and the theoretical plates are not less than 5000

422 Specificity All forced degradation samples were ana-lyzed with the aforementioned HPLC conditions using aPDA detector to monitor the homogeneity and purity of theGN DN and their related impurities Individual impuritiesplacebo GN and DN were verified and proved to be nonin-terfering with each other thus proving the specificity of themethod

Figure 3 shows that there is no interference at the RT(retention time) of GN DN and all known impurities fromthe other excipients Degradation was not observed in pho-tolytic stress humidity acid hydrolysis base hydrolysis waterhydrolysis and thermal stress studies Significant degradationwas observed in oxidative conditions The typical oxidativestressed chromatogram was shown in Figure 4 It was inter-esting to note that all the peaks due to degradation were well

resolved from the peaks of GN DN and their impuritiesFurther the peak purity of GN DN and their impuritieswas found to be homogeneous based on the evaluationparameters such as purity angle and purity threshold usingWaters Empower Networking Software The verification ofpeak purity indicates that there is no interference fromdegradants facilitating error-free quantification of GN andDN impurities Also themass balance of stressed samples wasfound to be more than 98 Thus the method is consideredto be ldquostability-indicatingrdquo The specificity results were shownin Tables 4(a) and 4(b)

423 Precision The RSD for the individual of allimpurities in impurities method precision study was within34 The results obtained in the intermediated precisionstudy for the RSD of the individual of all impurities werewell within 41 conforming high precision of the methodThe results are shown in Tables 5 and 6

10 Chromatography Research International

Table 8 Accuracy of the method

Compound Recovery at each levela

LOQ 01 02 04 08 10GN-Impurity A 985 995 1012 967 999 1035GN-Impurity B 1056 1027 997 1051 985 975GN-Impurity C 1026 1056 1019 1061 1035 1021GN-Impurity D 956 972 965 958 965 985DN-Impurity A 935 965 952 942 985 967DN-Impurity B 985 967 999 989 975 965DN-Impurity C 1011 1023 1025 1037 1025 1018DN-N-oxide 1056 1065 1051 1045 1029 1037DN-NFM Impurity 1015 985 1025 1012 995 986DN-NFO Impurity 1071 1056 1069 1058 1065 1058GN 985 965 972 968 974 987DN 992 985 971 995 1005 1012aAverage of six determinations at LOQ level amp 10 level and three determinations at remaining levels

Table 9 Regression statistics

Substance Linearity range (120583gmLminus1) Correlation coefficient (119877) 119884-intercept bias in GN-Impurity A 093 to 23961 0999 09GN-Impurity B 201 to 24278 0999 10GN-Impurity C 126 to 24192 1000 07GN-Impurity D 079 to 24255 0999 08DN-Impurity A 017 to 1410 1000 04DN-Impurity B 025 to 1010 1000 01DN-Impurity C 027 to 1223 1000 08DN-N-oxide 019 to 1249 1000 06DN-NFM Impurity 019 to 1219 0998 15DN-NFO Impurity 048 to 1222 1000 09GN 153 to 24478 0999 16DN 018 to 1215 1000 01

Table 10 Results of robustness study

S no Impurity name

RRTrsquos of impurities

As suchmethod

At06mLminflow rate

At10mLminflow rate

At 50∘Ccolumn

temperature

At 60∘Ccolumn

temperature

At pH 28(buffer pH)

At pH 32(buffer pH)

1 GN-Impurity A 074 076 073 072 071 074 073

2 GN-Impurity B 112 111 113 111 110 112 113

3 GN-Impurity C 204 206 205 205 202 204 205

4 GN-Impurity D 214 212 215 213 211 213 214

5 DN-Impurity B 083 082 081 082 082 083 082

6 DN-Impurity C 098 097 098 098 097 097 098

7 DN-N-oxide 101 102 103 102 103 102 104

8 DN-Impurity A 103 104 105 104 105 104 106

9 DN-NFM Impurity 128 131 130 131 129 128 119

10 DN-NFO Impurity 138 141 142 139 139 139 141Note GN known impurities RRTs were calculated against GN main peak and DN known impurities RRTs were calculated against DN main peak

Chromatography Research International 11

424 Limit of Detection (LOD) and Limit of Quantification(LOQ) The determined limit of detection limit of quantifi-cation and precision at LOQ values for GN DN and theirimpurities were reported inTable 7TheRSD for peak areas ofGNDN and their related impurities at limit of quantificationlevel was within 100

425 Accuracy The recovery of all the impurities fromfinished pharmaceutical dosage form ranged from 850 to1150 The summary of recovery for individual impuritywas mentioned in Table 8

426 Linearity Linear calibration plot for the related sub-stance method was obtained over the calibration rangestested that is LOQ to 10 of the target test concentrationThe correlation coefficient obtained was greater than 0997for all the componentsThe slope and y-intercept values werealso provided in Table 9 which confirmed good linearitybetween peak areas and concentration The linearity graphswere shown in Figure 5(a) to Figure 5(l)

427 Robustness No significant effect was observed onsystem suitability parameters such as resolution RSD tailingfactor RRTs of impurities or the theoretical plates of GNand DN when small but deliberate changes were made tochromatographic conditions The results were presented inTables 3 and 10 along with the system suitability parametersof normal conditions Thus the method was found to berobust with respect to variability in applied conditions

428 Stability in Solution and in the Mobile Phase Nosignificant changeswere observed in the content of impuritiesduring solution stability and mobile phase stability exper-iments when performed using the impurities method Thesolution stability and mobile phase stability experiment dataconfirms that the sample solution and mobile phases usedduring the impurity determination were stable for at least48 h

5 Conclusions

The gradient HPLC method developed for the simultaneousdetermination of GN and DN impurities in pharmaceuticaldosage form was precise accurate and specific The methodis validated as per ICH guidelines and found to be specificprecise linear accurate rugged and robust The developedmethod can be used for the stability analysis of GN and DNeither individually or in their combination dosage forms

Acknowledgments

The authors wish to thank the management of Dr Reddyrsquosgroup for supporting this workThe authors wish to acknowl-edge the formulation development group for providing thesamples for their research They would also like to thankcolleagues in bulkmanufacturers for providing chemicals andimpurity standards for their research work

References

[1] httpwwwrxlistcomorganidin-nr-drughtm[2] W Zhang TWang L Qin et al ldquoNeuroprotective effect of dex-

tromethorphan in the MPTP Parkinsonrsquos disease model role ofNADPHoxidaserdquoTheFASEB Journal vol 18 no 3 pp 589ndash5912004

[3] Dextromethorphan NHTSA[4] ldquoChild deaths lead to FDA hearing on cough cold medsrdquo 2007

httpwwwcnncom[5] B KuKanich and M G Papich ldquoPlasma profile and pharmac-

okinetics of dextromethorphan after intravenous and oraladministration in healthy dogsrdquo Journal of Veterinary Pharma-cology andTherapeutics vol 27 no 5 pp 337ndash341 2004

[6] ldquoKidsrsquo cough medicine no better than placebordquo San FranciscoChronicle 2004

[7] I M Paul K E Yoder K R Crowell et al ldquoEffect of dextro-methorphan diphenhydramine and placebo on nocturnalcough and sleep quality for coughing children and their par-entsrdquo Pediatrics vol 114 no 1 pp e85ndashe90 2004

[8] S Gorog M Babjak G Balogh et al ldquoDrug impurity profilingstrategiesrdquo Talanta vol 44 no 9 pp 1517ndash1526 1997

[9] B B Sanjay R K Bharati S J Yogini and A S Atul ldquoImpurityprofile significance in active pharmaceutical ingredientrdquo Eura-sian Journal of Analytical Chemistry vol 2 pp 32ndash53 2007

[10] ICH ldquoImpurities in new drug substances Q3A (R2)rdquo 2006[11] ICH ldquoImpurities in new drug products Q3B (R2)rdquo 2006[12] G Grosa E D Grosso R Russo and G Allegrone ldquoSimulta-

neous stability indicating HPLC-DAD determination ofguaifenesin and methyl and propyl-parabens in cough syruprdquoJournal of Pharmaceutical and Biomedical Analysis vol 41 no3 pp 798ndash803 2006

[13] M Senthilraja and P Giriraj ldquoReverse phase hplc method forthe simultaneous estimation of terbutanile sulphate bromhex-ine HCl and guaifenesin in cough syruprdquo Asian Journal ofPharmaceutical and Clinical Research vol 4 no 2 pp 13ndash152011

[14] M L Wilcox and J T Stewart ldquoHPLC determination of guaife-nesin with selected medications on underivatized silica with anaqueous-organic mobile phaserdquo Journal of Pharmaceutical andBiomedical Analysis vol 23 no 5 pp 909ndash916 2000

[15] A Ozdemir H Aksoy E Dinc D Baleanu and S Dermis ldquoDe-termination of guaifenesin and dextromethorphan in a coughsyrup by HPLC with fluorometric detectionrdquo Revue Roumainede Chimie vol 51 no 2 pp 117ndash122 2006

[16] I Demian ldquoHigh-performance liquid chromatography (HPLC)chiral separations of guaifenesin methocarbamol and race-morphanrdquo Chirality vol 5 no 4 pp 238ndash240 1993

[17] S Suzen C Akay and S Cevheroglu ldquoSimultaneous determi-nation of guaiphenesin and codeine phosphate in tablets byhigh-performance liquid chromatographyrdquo Farmaco vol 54no 10 pp 705ndash709 1999

[18] S M Amer S S Abbas M A Shehata and NM Ali ldquoSimulta-neous determination of phenylephrine hydrochloride guaifen-esin and chlorpheniraminemaleate in cough syrup by gradientliquid chromatographyrdquo Journal of AOAC International vol 91no 2 pp 276ndash284 2008

[19] V Galli andC Barbas ldquoHigh-performance liquid chromatogra-phic analysis of dextromethorphan guaifenesin and benzoate ina cough syrup for stability testingrdquo Journal of ChromatographyA vol 1048 no 2 pp 207ndash211 2004

12 Chromatography Research International

[20] X Chen J Huang Z Kong and D Zhong ldquoSensitive liquidchromatography-tandem mass spectrometry method for thesimultaneous determination of paracetamol and guaifenesin inhuman plasmardquo Journal of Chromatography B vol 817 no 2 pp263ndash269 2005

[21] R M Gudipati J E Wallace and S A Stavchansky ldquoHigh per-formance liquid chromatography determination of guaifenesinin dog plasmardquo Analytical Letters vol 24 pp 265ndash274 1991

[22] Q-H Ge Z Zhou X-J Zhi L-L Ma and C-G Ding ldquoSim-ultaneous determination of guaifenesin bromhexine andambroxol in human plasma by LC-MSMS and its pharmacoki-netic studiesrdquo Chinese Pharmaceutical Journal vol 44 no 13pp 1025ndash1028 2009

[23] R Rajagopalan ldquoReview of regulatory guidance on impuritiesrdquoSeparation Science and Technology vol 5 pp 27ndash37 2004

[24] ICH Harmonized Tripartite Guideline Validation of AnalyticalProcedures Text and Methodology Q2(R1) 2005

[25] ICH ldquoStability testing of new drug substances and productsQ1A (R2)rdquo 2003

[26] ICH ldquoPhotostability testing of new drug substances and prod-ucts Q1Brdquo 1996

[27] The United States Pharmacopeia USP35-NF30 3829ndash3837[28] European Pharmacopeia 70 1821-1822 amp 2128-2129[29] P Sunil Reddy K Sudhakar Babu N Kumar and Y V V Sasi

Sekhar ldquoDevelopment and validation of stability indicating theRP-HPLC method for the estimation of related compounds ofguaifenesin in pharmaceutical dosage formsrdquo PharmaceuticalMethods vol 2 pp 229ndash234 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

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Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

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Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

2 Chromatography Research International

have established maximum allowed limits for related com-pounds for both bulk and formulated APIs As per therequirements of various regulatory authorities the impurityprofile study of drug substances and drug products has tobe carried out using a suitable analytical method in the finalproduct [10 11]

GN andDNare official in theUnited States pharmacopeiaand European pharmacopeia but its combination is notofficial in any of the pharmacopeias In the literature surveythere were several LC assay methods that have been reportedfor the determination of GN and DN in pharmaceuticalpreparation either individually or in combination with otherdrugs [12ndash20] and in human plasma by LC-MS [21 22] Fewmethods were available for the determination of impuritiesindividually for GN and DN [27ndash29]

There is no single method reported for the simultaneousdetermination of the impurities in pharmaceuticals formula-tions of GN andDN It is felt to develop a stability-indicatingmethod for simultaneous determination of GN and DNrelated impurities in pharmaceutical formulation

Hence an attempt has been made to develop an acc-urate rapid specific and reproducible method for the deter-mination of GN and DN impurities (Tables 2(a) and 2(b)) inpharmaceutical dosage forms along with method validationas per ICH norms [23 24] The stability tests were alsoperformed on both drug substances and drug products as perICH norms [25 26]

2 Experimental

21 Chemicals and Reagents GN + DN tablets were receivedfrom the formulation research and development laboratoryof Dr Reddyrsquos Laboratories Ltd IPDO Hyderabad IndiaGN API and impurities were procured from SynthochemLab India DN API and impurities were procured fromWochardt Laboratories Ltd India Sodiumdihydrogen phos-phatemonohydrate 1-octane sulfonic acid sodium saltmono-hydrate HPLC grade acetonitrile methanol and orthophos-phoric acid were purchased from Merck Germany RegisTechnologies Inc USA and highly pure water was preparedby using Millipore MilliQ plus purification system

22 Equipment The LC system used for method devel-opment and method validation was Waters with a diodearray detector (model 2998 detector and e2695 separationmodule) The output signal was monitored and processedusing Waters Empower software Weighing was performedwith a Mettler XS 205 Dual Range (Mettler-Toledo GmbHGreifensee Switzerland) Photo stability studies were carriedout in a photo stability chamber (SUN TEST XLS+ AtlasUSA) Thermal stability studies were performed in a dry airoven (Merck Pharmatech Hyderabad India)

23 Chromatographic Conditions HPLCmeasurements werecarried out using a reversed phase Sunfire C18 250times 46mm5 120583m particle size column (Waters India Pvt Ltd) operatedat 50∘C with gradient elution at 08mLminminus1 using a mobilephase buffer as a mixture of 001M sodium dihydrogen

phosphate monohydrate and 00046M 1-octane sulfonic acidsodium saltmonohydrate of pH 30 (pH adjustedwith dilutedorthophosphoric acid) UV absorbance at 224 nm injectionvolume 20120583LThemobile phase A consisted of pH 30 bufferand acetonitrile (90 10 vv) mobile phase B consisted of pH30 buffer acetonitrile and methanol (10 10 80 vvv) TheLC gradient program was set as and time (min) mobilephase B 00115 155 2030 3050 6085 6515 and 7515Pressure Range was 1700 psi to 2200 psi Mixture of wateracetonitrile and methanol (60 20 20 vvv) was used asdiluent for sample preparation

24 Preparation of Standard Solution and System SuitabilitySolution We prepared individual stock solutions for GNDN and their impurities (each 500120583gmL) Working solu-tion was prepared from the above stock solutions for relatedsubstances determination (48120583gmL of GN and 24120583gmL ofDN) Amixture of all impurities (48120583gmL of GN impuritiesand 24120583gmL of DN impurities) along with GN and DN(24000 120583gmL of GN and 1200120583gmL of DN) was alsoprepared in diluent Stock solutions were used for methoddevelopment and method validation

25 Preparation of Test Solution Twenty tablets (1200mg ofGN + 60mg of DN) were weighed and the average weightwas calculated The tablets were crushed into fine powderand powder equivalent to 6000mg of GN (or equivalent to300mg of DN) was transferred into a 250mL volumetricflask Approximately 170mL of diluent was added shakedto disperse the material and sonicated for 20 minuteswith intermediate shaking The solution was then dilutedto 250mL and centrifuged at 3000 rpm for 10min Thesupernatant (24000120583gmL of GN and 1200120583gmL of DN)was collected and filtered through a 045120583m pore size nylon66-membrane filter (make Rankem)The filtrate was used assample solution

3 Method Validation

The proposed method was validated as per ICH guidelines[24]

31 System Suitability System suitability parameters wereevaluated to verify the system performance System preci-sion was determined on six replicate injections of standardpreparations All the important characteristics including therelative standard deviation peak tailing and theoretical platenumber were measured Resolution between impurities wasmeasured by injecting system suitability solution All thesesystem suitability parameters covered the system methodand column performance

32 Specificity Stress studies were performed at an initialconcentration of 24000 120583gmLminus1 of GN and 1200120583gmLminus1 ofDN in active pharmaceutical ingredients (API) and formu-lated sample to provide the stability-indicating property andspecificity of the proposed method Intentional degradation

Chromatography Research International 3

Table 1 Structures of Guaifenesin and Dextromethorphan

Molecule name Chemical name Chemical structure

Guaifenesin (+)-3-(2-Methoxyphenoxy)-propane-12-diolO

O

OH

OH

Dextromethorphan Ent-3-methoxy-17-methylmorphinan

N

H

O

was attempted by the stress conditions of exposure to pho-tolytic stress (12million lux hours followed by 200Watt hoursmminus2) heat (exposed at 105∘C for 15 h) acid (05N HCl for2 hours at 60∘C) base (05N NaOH for 2 hours at 60∘C)oxidation (10 peroxide for 30min at 60∘C) water (refluxedfor 12 hours at 60∘C) and humidity (exposed to 90 RH for7 days)

33 Precision The precision of the determination of theimpurities was checked by injecting six individual prepa-rations of (24000 120583gmL of GN and 1200120583gmL of DN)test preparation spiked with 48120583gmLminus1 of GN impuritiesand 24 120583gmLminus1 of DN impurities (02 of the target testconcentration) and calculating the RSD of impurity foreach compound The intermediate precision of the methodwas also evaluated using different analysts and a differentinstrument in the same laboratory

34 Limits of Detection (LOD) and Quantification (LOQ)LOD and LOQ for all impurities including GN and DNwere determined at a signal-to-noise ratio of 3 1 and 10 1respectively by injecting a series of dilute solutions withknown concentrations Precision study was also carried outat LOQ level by injecting six individual preparations ofimpurities and RSD was calculated

35 Linearity Linearity test solutions for the method wereprepared by diluting stock solution to the required concentra-tions The solutions were prepared at six concentration levelsfrom LOQ to the target test concentration The peak areaversus concentration in 120583gmLminus1 data was subjected to least-squares linear regression analysis

36 Accuracy Accuracy of the method was evaluated byusing concentration levels LOQ 01 02 04 08 and10 on GN + DN tablets Six preparations were performedat LOQ and 10 level and three preparations were per-formed at different levels Standard addition and recoveryexperiments were conducted on real sample to determine

accuracy of the related substance methodThe percentages ofrecoveries for all impurities GN and DN were calculated

37 Robustness To determine the robustness of the devel-oped method experimental conditions were deliberatelychanged and the resolution between GN DN and theirimpurities tailing factor and theoretical plates of GN andDN peaks were evaluated Also relative retention times for allthe impurities and column pressure throughout the run weremonitored

To study the effect of the flow rate on the developedmethod it was changed from 08mLminminus1 to 06 and10mLminminus1 The effect of column temperature on thedeveloped method was studied at 45 and 55∘C (instead of50∘C) The effect of pH was studied by varying plusmn02 pH units(ie 28 and 32) and the mobile phase composition waschanged plusmn10 from the initial composition In all the abovevaried conditions the component of the mobile phase washeld constant

38 Stability in Solution and in the Mobile Phase GN andDN spiked samples (impurities spiked at 02 of the targettest concentration ie 48 120583gmLminus1 of GN impurities and24 120583gmLminus1 of DN impurities) were prepared in the diluentleaving the test solutions at room temperature The spikedsamples were injected at 0 24 and 48 hrs time intervals Theimpurity content was calculated and the consistency in the area of the principal peak at each interval was checkedThe prepared mobile phase was kept constant during thestudy period The mobile phase study was demonstrated byinjecting the freshly prepared sample solution at differenttime intervals (0ndash2 days)

4 Results and Discussion

41 Optimization of Chromatographic Conditions The maincriterion was developing an RP-HPLC method for thesimultaneous determination of impurities in GN and DNpharmaceutical dosage form in a single run with emphasis

4 Chromatography Research International

Table 2 (a) Structures of Guaifenesin impurities (b) Structures of Dextromethorphan impurities

(a)

Impurity name Chemical name Chemical structure

Impurity A (degradant) 2-Methoxyphenol

OH

O

Impurity B (degradant) 2-(2-Methoxyphenoxy)propane-13-diolHO

HO

O

O

Impurity C (degradant) 111015840-Oxybis[3-(2-methoxyphenoxy)propan-2-ol]OHOH

O

OOO

O

Impurity D (process related impurity) 13-Bis(2-methoxyphenoxy)propan-2-ol

OHOO

O O

(b)

Impurity name Chemical name Chemical structure

Impurity A (degradant) Ent-3-methoxymorphinan

NH

H

O

Impurity B (degradant) Ent-17-methylmorphinan-3-ol

HO

H

N

Impurity C (degradant) Ent-3-methoxy-17-methylmorphinan-10-one H

N

O

O

N-Oxide (degradant) 3-Methoxy-N-methylmorphinan N-oxide H

O

Ominus

N+

N-Formyl Morphine (NFM)(process related impurity)

3-Methoxy-6788a910-hexahydro-5H-94b-(epiminoethano)phenanthrene-11-carbaldehyde

N

CHO

H3CO

N-Formyl octabase (NFO)(process related impurity)

(S)-1-(4-Methoxybenzyl)-345678-hexahydroisoquinoline-2(1H)-carbaldehyde

H

NCHO

H3CO

Chromatography Research International 5

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

23833 Guaifenesin imp A15533 Guaifenesin imp B 43267 Guaifenesin imp C

45500 Guaifenesin imp D

(a)

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

21400 Guaifenesin40733 Dextromethorphan

(b)

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

34017 Dextromethorphan imp B40117 Dextromethorphan imp C41450 Dextromethorphan imp N-Oxide42033 Dextromethorphan imp A51983 Dextromethorphan imp NFM55967 Dextromethorphan imp NFO

(c)

Figure 1 Spectra of GN DN and their impurities

on themethod being accurate reproducible robust stability-indicating linear free of interference fromother formulationexcipients and convenient enough for routine use in qualitycontrol laboratories

Individual stock solutions of GN DN and their impu-rities were injected and the spectra were checked of eachcomponent (Figure 1) From the spectra all the impuritieswere having absorbance maximum at about 224 Hence224 nm was selected for the estimation of GN and DNimpurities

A spiked solution of impurities (48120583gmLminus1 of GNimpurities and 24 120583gmLminus1 of DN impurities) GN + DN(24000120583gmLminus1 + 1200120583gmLminus1) and placebo peaks weresubjected to separation by RP-HPLC Initially the separationwas tried with the existing methods (USP Pharma Europeand the literature method) [27ndash29] It was observed thatplacebo peaks and GN impurity peaks were merging with

each other and two DN known impurities (NFM and NFO)were not eluting in DN API Pharma Europe method In USPand Pharma Europe GN API method DN impurities werenot separated from DN peak and two DN known impuritieswere eluting at longer retention with broad peak shapes Incase of the literature method [29] DN impurities were notseparated fromDN peak and twoDN known impurities wereeluting at longer retention times with better peak shapes

Method development was initiated by changing dif-ferent gradient programmes different pH values of themobile phase buffer different phosphate buffers and differentcolumns with the literature method [29] Sharp peak shapeswere observed with Sunfire column and sodium dihydrogenphosphate monohydrate buffer at pH 30 but separation wasnot up to the mark for DN DN-N-oxide and DN-impurityC Sharp peak shapes were due to the properties of the Sunfirecolumn (high mass loading capability excellent low pH

6 Chromatography Research International

020

018

016

014

012

010

008

006

004

002

000000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 2 Overlaid chromatogram of blank and system suitabilitypreparation

020

018

016

014

012

010

008

006

004

002

000

000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 3 Overlaid chromatogram of placebo and spiked testpreparation

stability superior peak shapes and high efficiency) Since wewere using a very high concentration ofGN (24000120583gmLminus1)it was decided to use Sunfire column for further developmenttrails by using ion pair reagent in the mobile phase for betterseparation between DN DN-Impurity A and DN-ImpurityB Separation was achieved between all the pairs of peaks butpeak shapes of DN-NFM Impurity and DN-NFO Impurityare not sharp Acetonitrile was added in the mobile phase inaddition to methanol to get sharp peaks

The chromatographic separation was achieved by areversed phase Sunfire C18 250 times 46mm 5120583m particlesize column operated at 50∘C with gradient elution at

020

018

016

014

012

010

008

006

004

002

000

000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 4 Typical chromatogram of peroxide stressed sample

08mLminminus1 using a mobile phase buffer as a mixtureof 001M sodium dihydrogen phosphate monohydrate and00046M 1-octane sulfonic acid sodium salt monohydrateof pH 30 (pH adjusted with diluted orthophosphoric acid)UV absorbance at 224 nm and injection volume 20 120583L Themobile phase A consisted of pH 30 buffer and acetonitrile(90 10 vv) mobile phase B consisted of pH 30 bufferacetonitrile andmethanol (10 10 80 vvv)The LC gradientprogram was set as time (min) mobile phase B 00115155 2030 3050 6085 6515 and 7515 All the impuritieswere well separated with a resolution greater than 2 Nochromatographic interference due to the blank (diluent)and other excipients (placebo) at the retention time of GNDN and their impurities was observed The typical overlaychromatogram of blank and system suitability solution andspiked test is shown in Figures 2 and 3

42MethodValidation After the development of themethodit was subject to method validation as per ICH guidelines[24] The method was validated to demonstrate that it issuitable for its intended purpose by the standard procedureto evaluate adequate validation characteristics (system suit-ability specificity accuracy precision linearity robustnessruggedness solution stability LOD and LOQ and stability-indicating capability)

421 System Suitability The percentage relative standarddeviation (RSD) of area from six replicate injections wasbelow 50 (diluted standard solution 48 120583gmLminus1 of GNand 24 120583gmLminus1 of DN) Low values of RSD for replicateinjections indicate that the system is precise The results ofother system suitability parameters such as resolution peaktailing and theoretical plates are presented in Table 3 As seenfrom this data the acceptable system suitability parameterswould be as follows the relative standard deviation of

Chromatography Research International 7

000200000000400000000600000000800000000

1000000000120000000014000000001600000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesiny = 56119662x + 118692128

R2 = 0999

(a)

02000000400000060000008000000

10000000120000001400000016000000

0000 50000 100000 150000 200000 250000 300000

Linearity of detector response of Guaifenesin impurity Ay = 62278038x + 138787601

R2 = 0998

Are

a

(b)

02000000400000060000008000000

100000001200000014000000

0000 50000 100000 150000 200000 250000 300000

Linearity of detector response of Guaifenesin impurity By = 46707746x + 109527559

R2 = 0998

Are

a

(c)

02000000400000060000008000000

100000001200000014000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesin impurity Cy = 49 606000x +

R2 = 0999

78596824

(d)

0

5000000

10000000

15000000

20000000

25000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesin impurity Dy = 79962049x + 156337857

R2 = 0999

(e)

050000

100000150000200000250000300000350000400000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphany = 30824867x + 4487696

R2 = 1000

(f)

050000

100000150000200000250000300000350000400000450000500000

0000 2000 4000 6000 8000 10000 12000 14000 16000

Are

a

Linearity of detector response of Dextromethorphan impurity A

y = 32920087x + 331349

R2 = 1000

(g)

050000

100000150000200000250000300000

0000 2000 4000 6000 8000 10000 12000

Are

a

Linearity of detector response of Dextromethorphan impurity B

y = 25035731x minus 57593

R2 = 1000

(h)

050000

100000150000200000250000300000350000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphan impurity C

y = 24213547x + 450726

R2 = 1000

(i)

050000

100000150000200000250000300000350000400000450000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphan N-oxide

y = 33 682946x + 523536

R2 = 1000

(j)

0100000200000300000400000500000600000700000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Concentration (ppm)

Linearity of detector response of Dextromethorphan impurity NFM

y = 49617284x + 22697199

R2 = 0996

(k)

0100000200000300000400000500000600000700000800000900000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Concentration (ppm)

Linearity of detector response of Dextromethorphan impurity NFO

y = 66581365x minus 1352313

R2 = 1000

(l)

Figure 5 (a) Linearity graph of Guaifenesin (b) Linearity graph of Guaifenesin impurity A (c) Linearity graph of Guaifenesin impurity B(d) Linearity graph of Guaifenesin impurity C (e) Linearity graph of Guaifenesin impurity D (f) Linearity graph of Dextromethorphan(g) Linearity graph of Dextromethorphan impurity A (h) Linearity graph of Dextromethorphan impurity B (i) Linearity graph ofDextromethorphan impurity C (j) Linearity graph of DextromethorphanN-oxide (k) Linearity graph of Dextromethorphan impurity NFM(l) Linearity graph of Dextromethorphan impurity NFO

8 Chromatography Research International

Table 3 System suitability results

Parameter RSDlowast of standard Theoretical plateslowast Tailing factorlowast Resolution 1 Resolution 2GN DN GN DN GN DN

As such method 05 07 45320 498659 10 10 27 23At 06mLmin flow rate 12 06 41091 495497 10 10 29 25At 10mLmin flow rate 09 04 45828 515551 10 10 25 22At 50∘C column temperature 15 08 45321 508966 10 10 26 23At 60∘C column temperature 21 23 40929 515807 10 10 29 26At pH 28 (buffer pH) 11 05 43970 463987 10 10 24 23At pH 32 (buffer pH) 07 15 44619 532891 10 10 26 27lowastAverage of six replicate standard injections

Table 4 (a) Forced degradation data for Guaifenesin (b) Forced degradation data for Dextromethorphan

(a)

Degradation conditions Guaifenesin degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 million lux hours followed by 200Watt hours) 006 3585 4142 1002Exposed to humidity at 25∘C and 90 RH for about 7 days 007 3881 4241 998Refluxed with purified water for about 12 hours at 60∘C 005 3285 3845 986Refluxed with 05 N HCL solution for about 2 hours at 60∘C 010 5293 7073 992Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 006 3067 3489 989Refluxed with 10 H2O2 solution for about 30min at 60∘C 013 2755 3607 983Exposed to dry heat for about 15 hours at 105∘C 007 3346 4331 994

(b)

Degradation conditions Dextromethorphan degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 Million lux hours followed by 200Watt hours) 001 0115 0242 998Exposed to humidity at 25∘C and 90 RH for about 7 days 001 0124 0261 995Refluxed with purified water for about 12 hours at 60∘C 001 0101 0261 989Refluxed with 05 N HCL solution for about 2 hours at 60∘C 002 0154 0262 981Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 001 0103 0256 992Refluxed with 10 H2O2 solution for about 30min at 60∘C 105 0102 0250 997Exposed to dry heat for about 15 hours at 105∘C 001 0099 0261 987

Table 5 Results of precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0198 0201 0189 0213 0205 0209 0203 42GN-Impurity B 0209 0195 0192 0201 0206 0194 0200 35GN-Impurity C 0189 0195 0186 0192 0198 0184 0191 28GN-Impurity D 0208 0190 0192 0193 0188 0186 0193 41DN-Impurity A 0215 0214 0215 0209 0212 0205 0212 19DN-Impurity B 0204 0200 0202 0203 0205 0191 0201 25DN-Impurity C 0195 0186 0198 0199 0184 0194 0193 32DN-N-oxide 0216 0214 0218 0219 0211 0210 0215 17DN-NFM Impurity 0216 0218 0215 0215 0216 0212 0215 09DN-NFO Impurity 0198 0195 0213 0192 0214 0215 0205 52GN 0203 0212 0204 0206 0205 0210 0207 17DN 0182 0189 0194 0188 0187 0201 0190 34

Chromatography Research International 9

Table 6 Results of intermediate precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0191 0204 0201 0199 0210 0212 0203 38GN-Impurity B 0212 0204 0210 0209 0211 0204 0208 17GN-Impurity C 0192 0198 0191 0194 0202 0209 0198 35GN-Impurity D 0185 0192 0191 0194 0193 0192 0192 15DN-Impurity A 0202 0202 0203 0202 0202 0204 0203 04DN-Impurity B 0191 0188 0204 0209 0189 0202 0197 45DN-Impurity C 0183 0189 0191 0186 0205 0206 0193 51DN-N-oxide 0197 0210 0213 0202 0198 0212 0205 35DN-NFM Impurity 0183 0187 0179 0184 0185 0188 0184 17DN-NFO Impurity 0206 0197 0209 0212 0194 0208 0204 35GN 0199 0205 0201 0204 0207 0208 0204 17DN 0187 0185 0192 0186 0194 0191 0189 19

Table 7 LOD LOQ and precision data

Compound LOD () LOQ () SN ratio RSD at LOQ Level precisionLOD LOQ

GN-Impurity A 0004 0001 27 95 42GN-Impurity B 0008 0002 29 98 25GN-Impurity C 0005 0002 30 98 31GN-Impurity D 0003 0001 29 97 25DN-Impurity A 0014 0004 28 101 11DN-Impurity B 0021 0006 27 104 34DN-Impurity C 0023 0007 28 99 49DN-N-oxide 0016 0005 31 98 22DN-NFM Impurity 0016 0005 27 101 15DN-NFO Impurity 0041 0012 29 106 42GN 0006 0002 31 98 21DN 0015 0005 28 96 28

replicate injections is notmore than 50 resolution betweenimpurities 20 the tailing factor for GN and DN is not morethan 15 and the theoretical plates are not less than 5000

422 Specificity All forced degradation samples were ana-lyzed with the aforementioned HPLC conditions using aPDA detector to monitor the homogeneity and purity of theGN DN and their related impurities Individual impuritiesplacebo GN and DN were verified and proved to be nonin-terfering with each other thus proving the specificity of themethod

Figure 3 shows that there is no interference at the RT(retention time) of GN DN and all known impurities fromthe other excipients Degradation was not observed in pho-tolytic stress humidity acid hydrolysis base hydrolysis waterhydrolysis and thermal stress studies Significant degradationwas observed in oxidative conditions The typical oxidativestressed chromatogram was shown in Figure 4 It was inter-esting to note that all the peaks due to degradation were well

resolved from the peaks of GN DN and their impuritiesFurther the peak purity of GN DN and their impuritieswas found to be homogeneous based on the evaluationparameters such as purity angle and purity threshold usingWaters Empower Networking Software The verification ofpeak purity indicates that there is no interference fromdegradants facilitating error-free quantification of GN andDN impurities Also themass balance of stressed samples wasfound to be more than 98 Thus the method is consideredto be ldquostability-indicatingrdquo The specificity results were shownin Tables 4(a) and 4(b)

423 Precision The RSD for the individual of allimpurities in impurities method precision study was within34 The results obtained in the intermediated precisionstudy for the RSD of the individual of all impurities werewell within 41 conforming high precision of the methodThe results are shown in Tables 5 and 6

10 Chromatography Research International

Table 8 Accuracy of the method

Compound Recovery at each levela

LOQ 01 02 04 08 10GN-Impurity A 985 995 1012 967 999 1035GN-Impurity B 1056 1027 997 1051 985 975GN-Impurity C 1026 1056 1019 1061 1035 1021GN-Impurity D 956 972 965 958 965 985DN-Impurity A 935 965 952 942 985 967DN-Impurity B 985 967 999 989 975 965DN-Impurity C 1011 1023 1025 1037 1025 1018DN-N-oxide 1056 1065 1051 1045 1029 1037DN-NFM Impurity 1015 985 1025 1012 995 986DN-NFO Impurity 1071 1056 1069 1058 1065 1058GN 985 965 972 968 974 987DN 992 985 971 995 1005 1012aAverage of six determinations at LOQ level amp 10 level and three determinations at remaining levels

Table 9 Regression statistics

Substance Linearity range (120583gmLminus1) Correlation coefficient (119877) 119884-intercept bias in GN-Impurity A 093 to 23961 0999 09GN-Impurity B 201 to 24278 0999 10GN-Impurity C 126 to 24192 1000 07GN-Impurity D 079 to 24255 0999 08DN-Impurity A 017 to 1410 1000 04DN-Impurity B 025 to 1010 1000 01DN-Impurity C 027 to 1223 1000 08DN-N-oxide 019 to 1249 1000 06DN-NFM Impurity 019 to 1219 0998 15DN-NFO Impurity 048 to 1222 1000 09GN 153 to 24478 0999 16DN 018 to 1215 1000 01

Table 10 Results of robustness study

S no Impurity name

RRTrsquos of impurities

As suchmethod

At06mLminflow rate

At10mLminflow rate

At 50∘Ccolumn

temperature

At 60∘Ccolumn

temperature

At pH 28(buffer pH)

At pH 32(buffer pH)

1 GN-Impurity A 074 076 073 072 071 074 073

2 GN-Impurity B 112 111 113 111 110 112 113

3 GN-Impurity C 204 206 205 205 202 204 205

4 GN-Impurity D 214 212 215 213 211 213 214

5 DN-Impurity B 083 082 081 082 082 083 082

6 DN-Impurity C 098 097 098 098 097 097 098

7 DN-N-oxide 101 102 103 102 103 102 104

8 DN-Impurity A 103 104 105 104 105 104 106

9 DN-NFM Impurity 128 131 130 131 129 128 119

10 DN-NFO Impurity 138 141 142 139 139 139 141Note GN known impurities RRTs were calculated against GN main peak and DN known impurities RRTs were calculated against DN main peak

Chromatography Research International 11

424 Limit of Detection (LOD) and Limit of Quantification(LOQ) The determined limit of detection limit of quantifi-cation and precision at LOQ values for GN DN and theirimpurities were reported inTable 7TheRSD for peak areas ofGNDN and their related impurities at limit of quantificationlevel was within 100

425 Accuracy The recovery of all the impurities fromfinished pharmaceutical dosage form ranged from 850 to1150 The summary of recovery for individual impuritywas mentioned in Table 8

426 Linearity Linear calibration plot for the related sub-stance method was obtained over the calibration rangestested that is LOQ to 10 of the target test concentrationThe correlation coefficient obtained was greater than 0997for all the componentsThe slope and y-intercept values werealso provided in Table 9 which confirmed good linearitybetween peak areas and concentration The linearity graphswere shown in Figure 5(a) to Figure 5(l)

427 Robustness No significant effect was observed onsystem suitability parameters such as resolution RSD tailingfactor RRTs of impurities or the theoretical plates of GNand DN when small but deliberate changes were made tochromatographic conditions The results were presented inTables 3 and 10 along with the system suitability parametersof normal conditions Thus the method was found to berobust with respect to variability in applied conditions

428 Stability in Solution and in the Mobile Phase Nosignificant changeswere observed in the content of impuritiesduring solution stability and mobile phase stability exper-iments when performed using the impurities method Thesolution stability and mobile phase stability experiment dataconfirms that the sample solution and mobile phases usedduring the impurity determination were stable for at least48 h

5 Conclusions

The gradient HPLC method developed for the simultaneousdetermination of GN and DN impurities in pharmaceuticaldosage form was precise accurate and specific The methodis validated as per ICH guidelines and found to be specificprecise linear accurate rugged and robust The developedmethod can be used for the stability analysis of GN and DNeither individually or in their combination dosage forms

Acknowledgments

The authors wish to thank the management of Dr Reddyrsquosgroup for supporting this workThe authors wish to acknowl-edge the formulation development group for providing thesamples for their research They would also like to thankcolleagues in bulkmanufacturers for providing chemicals andimpurity standards for their research work

References

[1] httpwwwrxlistcomorganidin-nr-drughtm[2] W Zhang TWang L Qin et al ldquoNeuroprotective effect of dex-

tromethorphan in the MPTP Parkinsonrsquos disease model role ofNADPHoxidaserdquoTheFASEB Journal vol 18 no 3 pp 589ndash5912004

[3] Dextromethorphan NHTSA[4] ldquoChild deaths lead to FDA hearing on cough cold medsrdquo 2007

httpwwwcnncom[5] B KuKanich and M G Papich ldquoPlasma profile and pharmac-

okinetics of dextromethorphan after intravenous and oraladministration in healthy dogsrdquo Journal of Veterinary Pharma-cology andTherapeutics vol 27 no 5 pp 337ndash341 2004

[6] ldquoKidsrsquo cough medicine no better than placebordquo San FranciscoChronicle 2004

[7] I M Paul K E Yoder K R Crowell et al ldquoEffect of dextro-methorphan diphenhydramine and placebo on nocturnalcough and sleep quality for coughing children and their par-entsrdquo Pediatrics vol 114 no 1 pp e85ndashe90 2004

[8] S Gorog M Babjak G Balogh et al ldquoDrug impurity profilingstrategiesrdquo Talanta vol 44 no 9 pp 1517ndash1526 1997

[9] B B Sanjay R K Bharati S J Yogini and A S Atul ldquoImpurityprofile significance in active pharmaceutical ingredientrdquo Eura-sian Journal of Analytical Chemistry vol 2 pp 32ndash53 2007

[10] ICH ldquoImpurities in new drug substances Q3A (R2)rdquo 2006[11] ICH ldquoImpurities in new drug products Q3B (R2)rdquo 2006[12] G Grosa E D Grosso R Russo and G Allegrone ldquoSimulta-

neous stability indicating HPLC-DAD determination ofguaifenesin and methyl and propyl-parabens in cough syruprdquoJournal of Pharmaceutical and Biomedical Analysis vol 41 no3 pp 798ndash803 2006

[13] M Senthilraja and P Giriraj ldquoReverse phase hplc method forthe simultaneous estimation of terbutanile sulphate bromhex-ine HCl and guaifenesin in cough syruprdquo Asian Journal ofPharmaceutical and Clinical Research vol 4 no 2 pp 13ndash152011

[14] M L Wilcox and J T Stewart ldquoHPLC determination of guaife-nesin with selected medications on underivatized silica with anaqueous-organic mobile phaserdquo Journal of Pharmaceutical andBiomedical Analysis vol 23 no 5 pp 909ndash916 2000

[15] A Ozdemir H Aksoy E Dinc D Baleanu and S Dermis ldquoDe-termination of guaifenesin and dextromethorphan in a coughsyrup by HPLC with fluorometric detectionrdquo Revue Roumainede Chimie vol 51 no 2 pp 117ndash122 2006

[16] I Demian ldquoHigh-performance liquid chromatography (HPLC)chiral separations of guaifenesin methocarbamol and race-morphanrdquo Chirality vol 5 no 4 pp 238ndash240 1993

[17] S Suzen C Akay and S Cevheroglu ldquoSimultaneous determi-nation of guaiphenesin and codeine phosphate in tablets byhigh-performance liquid chromatographyrdquo Farmaco vol 54no 10 pp 705ndash709 1999

[18] S M Amer S S Abbas M A Shehata and NM Ali ldquoSimulta-neous determination of phenylephrine hydrochloride guaifen-esin and chlorpheniraminemaleate in cough syrup by gradientliquid chromatographyrdquo Journal of AOAC International vol 91no 2 pp 276ndash284 2008

[19] V Galli andC Barbas ldquoHigh-performance liquid chromatogra-phic analysis of dextromethorphan guaifenesin and benzoate ina cough syrup for stability testingrdquo Journal of ChromatographyA vol 1048 no 2 pp 207ndash211 2004

12 Chromatography Research International

[20] X Chen J Huang Z Kong and D Zhong ldquoSensitive liquidchromatography-tandem mass spectrometry method for thesimultaneous determination of paracetamol and guaifenesin inhuman plasmardquo Journal of Chromatography B vol 817 no 2 pp263ndash269 2005

[21] R M Gudipati J E Wallace and S A Stavchansky ldquoHigh per-formance liquid chromatography determination of guaifenesinin dog plasmardquo Analytical Letters vol 24 pp 265ndash274 1991

[22] Q-H Ge Z Zhou X-J Zhi L-L Ma and C-G Ding ldquoSim-ultaneous determination of guaifenesin bromhexine andambroxol in human plasma by LC-MSMS and its pharmacoki-netic studiesrdquo Chinese Pharmaceutical Journal vol 44 no 13pp 1025ndash1028 2009

[23] R Rajagopalan ldquoReview of regulatory guidance on impuritiesrdquoSeparation Science and Technology vol 5 pp 27ndash37 2004

[24] ICH Harmonized Tripartite Guideline Validation of AnalyticalProcedures Text and Methodology Q2(R1) 2005

[25] ICH ldquoStability testing of new drug substances and productsQ1A (R2)rdquo 2003

[26] ICH ldquoPhotostability testing of new drug substances and prod-ucts Q1Brdquo 1996

[27] The United States Pharmacopeia USP35-NF30 3829ndash3837[28] European Pharmacopeia 70 1821-1822 amp 2128-2129[29] P Sunil Reddy K Sudhakar Babu N Kumar and Y V V Sasi

Sekhar ldquoDevelopment and validation of stability indicating theRP-HPLC method for the estimation of related compounds ofguaifenesin in pharmaceutical dosage formsrdquo PharmaceuticalMethods vol 2 pp 229ndash234 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Chromatography Research International 3

Table 1 Structures of Guaifenesin and Dextromethorphan

Molecule name Chemical name Chemical structure

Guaifenesin (+)-3-(2-Methoxyphenoxy)-propane-12-diolO

O

OH

OH

Dextromethorphan Ent-3-methoxy-17-methylmorphinan

N

H

O

was attempted by the stress conditions of exposure to pho-tolytic stress (12million lux hours followed by 200Watt hoursmminus2) heat (exposed at 105∘C for 15 h) acid (05N HCl for2 hours at 60∘C) base (05N NaOH for 2 hours at 60∘C)oxidation (10 peroxide for 30min at 60∘C) water (refluxedfor 12 hours at 60∘C) and humidity (exposed to 90 RH for7 days)

33 Precision The precision of the determination of theimpurities was checked by injecting six individual prepa-rations of (24000 120583gmL of GN and 1200120583gmL of DN)test preparation spiked with 48120583gmLminus1 of GN impuritiesand 24 120583gmLminus1 of DN impurities (02 of the target testconcentration) and calculating the RSD of impurity foreach compound The intermediate precision of the methodwas also evaluated using different analysts and a differentinstrument in the same laboratory

34 Limits of Detection (LOD) and Quantification (LOQ)LOD and LOQ for all impurities including GN and DNwere determined at a signal-to-noise ratio of 3 1 and 10 1respectively by injecting a series of dilute solutions withknown concentrations Precision study was also carried outat LOQ level by injecting six individual preparations ofimpurities and RSD was calculated

35 Linearity Linearity test solutions for the method wereprepared by diluting stock solution to the required concentra-tions The solutions were prepared at six concentration levelsfrom LOQ to the target test concentration The peak areaversus concentration in 120583gmLminus1 data was subjected to least-squares linear regression analysis

36 Accuracy Accuracy of the method was evaluated byusing concentration levels LOQ 01 02 04 08 and10 on GN + DN tablets Six preparations were performedat LOQ and 10 level and three preparations were per-formed at different levels Standard addition and recoveryexperiments were conducted on real sample to determine

accuracy of the related substance methodThe percentages ofrecoveries for all impurities GN and DN were calculated

37 Robustness To determine the robustness of the devel-oped method experimental conditions were deliberatelychanged and the resolution between GN DN and theirimpurities tailing factor and theoretical plates of GN andDN peaks were evaluated Also relative retention times for allthe impurities and column pressure throughout the run weremonitored

To study the effect of the flow rate on the developedmethod it was changed from 08mLminminus1 to 06 and10mLminminus1 The effect of column temperature on thedeveloped method was studied at 45 and 55∘C (instead of50∘C) The effect of pH was studied by varying plusmn02 pH units(ie 28 and 32) and the mobile phase composition waschanged plusmn10 from the initial composition In all the abovevaried conditions the component of the mobile phase washeld constant

38 Stability in Solution and in the Mobile Phase GN andDN spiked samples (impurities spiked at 02 of the targettest concentration ie 48 120583gmLminus1 of GN impurities and24 120583gmLminus1 of DN impurities) were prepared in the diluentleaving the test solutions at room temperature The spikedsamples were injected at 0 24 and 48 hrs time intervals Theimpurity content was calculated and the consistency in the area of the principal peak at each interval was checkedThe prepared mobile phase was kept constant during thestudy period The mobile phase study was demonstrated byinjecting the freshly prepared sample solution at differenttime intervals (0ndash2 days)

4 Results and Discussion

41 Optimization of Chromatographic Conditions The maincriterion was developing an RP-HPLC method for thesimultaneous determination of impurities in GN and DNpharmaceutical dosage form in a single run with emphasis

4 Chromatography Research International

Table 2 (a) Structures of Guaifenesin impurities (b) Structures of Dextromethorphan impurities

(a)

Impurity name Chemical name Chemical structure

Impurity A (degradant) 2-Methoxyphenol

OH

O

Impurity B (degradant) 2-(2-Methoxyphenoxy)propane-13-diolHO

HO

O

O

Impurity C (degradant) 111015840-Oxybis[3-(2-methoxyphenoxy)propan-2-ol]OHOH

O

OOO

O

Impurity D (process related impurity) 13-Bis(2-methoxyphenoxy)propan-2-ol

OHOO

O O

(b)

Impurity name Chemical name Chemical structure

Impurity A (degradant) Ent-3-methoxymorphinan

NH

H

O

Impurity B (degradant) Ent-17-methylmorphinan-3-ol

HO

H

N

Impurity C (degradant) Ent-3-methoxy-17-methylmorphinan-10-one H

N

O

O

N-Oxide (degradant) 3-Methoxy-N-methylmorphinan N-oxide H

O

Ominus

N+

N-Formyl Morphine (NFM)(process related impurity)

3-Methoxy-6788a910-hexahydro-5H-94b-(epiminoethano)phenanthrene-11-carbaldehyde

N

CHO

H3CO

N-Formyl octabase (NFO)(process related impurity)

(S)-1-(4-Methoxybenzyl)-345678-hexahydroisoquinoline-2(1H)-carbaldehyde

H

NCHO

H3CO

Chromatography Research International 5

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

23833 Guaifenesin imp A15533 Guaifenesin imp B 43267 Guaifenesin imp C

45500 Guaifenesin imp D

(a)

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

21400 Guaifenesin40733 Dextromethorphan

(b)

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

34017 Dextromethorphan imp B40117 Dextromethorphan imp C41450 Dextromethorphan imp N-Oxide42033 Dextromethorphan imp A51983 Dextromethorphan imp NFM55967 Dextromethorphan imp NFO

(c)

Figure 1 Spectra of GN DN and their impurities

on themethod being accurate reproducible robust stability-indicating linear free of interference fromother formulationexcipients and convenient enough for routine use in qualitycontrol laboratories

Individual stock solutions of GN DN and their impu-rities were injected and the spectra were checked of eachcomponent (Figure 1) From the spectra all the impuritieswere having absorbance maximum at about 224 Hence224 nm was selected for the estimation of GN and DNimpurities

A spiked solution of impurities (48120583gmLminus1 of GNimpurities and 24 120583gmLminus1 of DN impurities) GN + DN(24000120583gmLminus1 + 1200120583gmLminus1) and placebo peaks weresubjected to separation by RP-HPLC Initially the separationwas tried with the existing methods (USP Pharma Europeand the literature method) [27ndash29] It was observed thatplacebo peaks and GN impurity peaks were merging with

each other and two DN known impurities (NFM and NFO)were not eluting in DN API Pharma Europe method In USPand Pharma Europe GN API method DN impurities werenot separated from DN peak and two DN known impuritieswere eluting at longer retention with broad peak shapes Incase of the literature method [29] DN impurities were notseparated fromDN peak and twoDN known impurities wereeluting at longer retention times with better peak shapes

Method development was initiated by changing dif-ferent gradient programmes different pH values of themobile phase buffer different phosphate buffers and differentcolumns with the literature method [29] Sharp peak shapeswere observed with Sunfire column and sodium dihydrogenphosphate monohydrate buffer at pH 30 but separation wasnot up to the mark for DN DN-N-oxide and DN-impurityC Sharp peak shapes were due to the properties of the Sunfirecolumn (high mass loading capability excellent low pH

6 Chromatography Research International

020

018

016

014

012

010

008

006

004

002

000000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 2 Overlaid chromatogram of blank and system suitabilitypreparation

020

018

016

014

012

010

008

006

004

002

000

000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 3 Overlaid chromatogram of placebo and spiked testpreparation

stability superior peak shapes and high efficiency) Since wewere using a very high concentration ofGN (24000120583gmLminus1)it was decided to use Sunfire column for further developmenttrails by using ion pair reagent in the mobile phase for betterseparation between DN DN-Impurity A and DN-ImpurityB Separation was achieved between all the pairs of peaks butpeak shapes of DN-NFM Impurity and DN-NFO Impurityare not sharp Acetonitrile was added in the mobile phase inaddition to methanol to get sharp peaks

The chromatographic separation was achieved by areversed phase Sunfire C18 250 times 46mm 5120583m particlesize column operated at 50∘C with gradient elution at

020

018

016

014

012

010

008

006

004

002

000

000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 4 Typical chromatogram of peroxide stressed sample

08mLminminus1 using a mobile phase buffer as a mixtureof 001M sodium dihydrogen phosphate monohydrate and00046M 1-octane sulfonic acid sodium salt monohydrateof pH 30 (pH adjusted with diluted orthophosphoric acid)UV absorbance at 224 nm and injection volume 20 120583L Themobile phase A consisted of pH 30 buffer and acetonitrile(90 10 vv) mobile phase B consisted of pH 30 bufferacetonitrile andmethanol (10 10 80 vvv)The LC gradientprogram was set as time (min) mobile phase B 00115155 2030 3050 6085 6515 and 7515 All the impuritieswere well separated with a resolution greater than 2 Nochromatographic interference due to the blank (diluent)and other excipients (placebo) at the retention time of GNDN and their impurities was observed The typical overlaychromatogram of blank and system suitability solution andspiked test is shown in Figures 2 and 3

42MethodValidation After the development of themethodit was subject to method validation as per ICH guidelines[24] The method was validated to demonstrate that it issuitable for its intended purpose by the standard procedureto evaluate adequate validation characteristics (system suit-ability specificity accuracy precision linearity robustnessruggedness solution stability LOD and LOQ and stability-indicating capability)

421 System Suitability The percentage relative standarddeviation (RSD) of area from six replicate injections wasbelow 50 (diluted standard solution 48 120583gmLminus1 of GNand 24 120583gmLminus1 of DN) Low values of RSD for replicateinjections indicate that the system is precise The results ofother system suitability parameters such as resolution peaktailing and theoretical plates are presented in Table 3 As seenfrom this data the acceptable system suitability parameterswould be as follows the relative standard deviation of

Chromatography Research International 7

000200000000400000000600000000800000000

1000000000120000000014000000001600000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesiny = 56119662x + 118692128

R2 = 0999

(a)

02000000400000060000008000000

10000000120000001400000016000000

0000 50000 100000 150000 200000 250000 300000

Linearity of detector response of Guaifenesin impurity Ay = 62278038x + 138787601

R2 = 0998

Are

a

(b)

02000000400000060000008000000

100000001200000014000000

0000 50000 100000 150000 200000 250000 300000

Linearity of detector response of Guaifenesin impurity By = 46707746x + 109527559

R2 = 0998

Are

a

(c)

02000000400000060000008000000

100000001200000014000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesin impurity Cy = 49 606000x +

R2 = 0999

78596824

(d)

0

5000000

10000000

15000000

20000000

25000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesin impurity Dy = 79962049x + 156337857

R2 = 0999

(e)

050000

100000150000200000250000300000350000400000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphany = 30824867x + 4487696

R2 = 1000

(f)

050000

100000150000200000250000300000350000400000450000500000

0000 2000 4000 6000 8000 10000 12000 14000 16000

Are

a

Linearity of detector response of Dextromethorphan impurity A

y = 32920087x + 331349

R2 = 1000

(g)

050000

100000150000200000250000300000

0000 2000 4000 6000 8000 10000 12000

Are

a

Linearity of detector response of Dextromethorphan impurity B

y = 25035731x minus 57593

R2 = 1000

(h)

050000

100000150000200000250000300000350000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphan impurity C

y = 24213547x + 450726

R2 = 1000

(i)

050000

100000150000200000250000300000350000400000450000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphan N-oxide

y = 33 682946x + 523536

R2 = 1000

(j)

0100000200000300000400000500000600000700000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Concentration (ppm)

Linearity of detector response of Dextromethorphan impurity NFM

y = 49617284x + 22697199

R2 = 0996

(k)

0100000200000300000400000500000600000700000800000900000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Concentration (ppm)

Linearity of detector response of Dextromethorphan impurity NFO

y = 66581365x minus 1352313

R2 = 1000

(l)

Figure 5 (a) Linearity graph of Guaifenesin (b) Linearity graph of Guaifenesin impurity A (c) Linearity graph of Guaifenesin impurity B(d) Linearity graph of Guaifenesin impurity C (e) Linearity graph of Guaifenesin impurity D (f) Linearity graph of Dextromethorphan(g) Linearity graph of Dextromethorphan impurity A (h) Linearity graph of Dextromethorphan impurity B (i) Linearity graph ofDextromethorphan impurity C (j) Linearity graph of DextromethorphanN-oxide (k) Linearity graph of Dextromethorphan impurity NFM(l) Linearity graph of Dextromethorphan impurity NFO

8 Chromatography Research International

Table 3 System suitability results

Parameter RSDlowast of standard Theoretical plateslowast Tailing factorlowast Resolution 1 Resolution 2GN DN GN DN GN DN

As such method 05 07 45320 498659 10 10 27 23At 06mLmin flow rate 12 06 41091 495497 10 10 29 25At 10mLmin flow rate 09 04 45828 515551 10 10 25 22At 50∘C column temperature 15 08 45321 508966 10 10 26 23At 60∘C column temperature 21 23 40929 515807 10 10 29 26At pH 28 (buffer pH) 11 05 43970 463987 10 10 24 23At pH 32 (buffer pH) 07 15 44619 532891 10 10 26 27lowastAverage of six replicate standard injections

Table 4 (a) Forced degradation data for Guaifenesin (b) Forced degradation data for Dextromethorphan

(a)

Degradation conditions Guaifenesin degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 million lux hours followed by 200Watt hours) 006 3585 4142 1002Exposed to humidity at 25∘C and 90 RH for about 7 days 007 3881 4241 998Refluxed with purified water for about 12 hours at 60∘C 005 3285 3845 986Refluxed with 05 N HCL solution for about 2 hours at 60∘C 010 5293 7073 992Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 006 3067 3489 989Refluxed with 10 H2O2 solution for about 30min at 60∘C 013 2755 3607 983Exposed to dry heat for about 15 hours at 105∘C 007 3346 4331 994

(b)

Degradation conditions Dextromethorphan degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 Million lux hours followed by 200Watt hours) 001 0115 0242 998Exposed to humidity at 25∘C and 90 RH for about 7 days 001 0124 0261 995Refluxed with purified water for about 12 hours at 60∘C 001 0101 0261 989Refluxed with 05 N HCL solution for about 2 hours at 60∘C 002 0154 0262 981Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 001 0103 0256 992Refluxed with 10 H2O2 solution for about 30min at 60∘C 105 0102 0250 997Exposed to dry heat for about 15 hours at 105∘C 001 0099 0261 987

Table 5 Results of precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0198 0201 0189 0213 0205 0209 0203 42GN-Impurity B 0209 0195 0192 0201 0206 0194 0200 35GN-Impurity C 0189 0195 0186 0192 0198 0184 0191 28GN-Impurity D 0208 0190 0192 0193 0188 0186 0193 41DN-Impurity A 0215 0214 0215 0209 0212 0205 0212 19DN-Impurity B 0204 0200 0202 0203 0205 0191 0201 25DN-Impurity C 0195 0186 0198 0199 0184 0194 0193 32DN-N-oxide 0216 0214 0218 0219 0211 0210 0215 17DN-NFM Impurity 0216 0218 0215 0215 0216 0212 0215 09DN-NFO Impurity 0198 0195 0213 0192 0214 0215 0205 52GN 0203 0212 0204 0206 0205 0210 0207 17DN 0182 0189 0194 0188 0187 0201 0190 34

Chromatography Research International 9

Table 6 Results of intermediate precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0191 0204 0201 0199 0210 0212 0203 38GN-Impurity B 0212 0204 0210 0209 0211 0204 0208 17GN-Impurity C 0192 0198 0191 0194 0202 0209 0198 35GN-Impurity D 0185 0192 0191 0194 0193 0192 0192 15DN-Impurity A 0202 0202 0203 0202 0202 0204 0203 04DN-Impurity B 0191 0188 0204 0209 0189 0202 0197 45DN-Impurity C 0183 0189 0191 0186 0205 0206 0193 51DN-N-oxide 0197 0210 0213 0202 0198 0212 0205 35DN-NFM Impurity 0183 0187 0179 0184 0185 0188 0184 17DN-NFO Impurity 0206 0197 0209 0212 0194 0208 0204 35GN 0199 0205 0201 0204 0207 0208 0204 17DN 0187 0185 0192 0186 0194 0191 0189 19

Table 7 LOD LOQ and precision data

Compound LOD () LOQ () SN ratio RSD at LOQ Level precisionLOD LOQ

GN-Impurity A 0004 0001 27 95 42GN-Impurity B 0008 0002 29 98 25GN-Impurity C 0005 0002 30 98 31GN-Impurity D 0003 0001 29 97 25DN-Impurity A 0014 0004 28 101 11DN-Impurity B 0021 0006 27 104 34DN-Impurity C 0023 0007 28 99 49DN-N-oxide 0016 0005 31 98 22DN-NFM Impurity 0016 0005 27 101 15DN-NFO Impurity 0041 0012 29 106 42GN 0006 0002 31 98 21DN 0015 0005 28 96 28

replicate injections is notmore than 50 resolution betweenimpurities 20 the tailing factor for GN and DN is not morethan 15 and the theoretical plates are not less than 5000

422 Specificity All forced degradation samples were ana-lyzed with the aforementioned HPLC conditions using aPDA detector to monitor the homogeneity and purity of theGN DN and their related impurities Individual impuritiesplacebo GN and DN were verified and proved to be nonin-terfering with each other thus proving the specificity of themethod

Figure 3 shows that there is no interference at the RT(retention time) of GN DN and all known impurities fromthe other excipients Degradation was not observed in pho-tolytic stress humidity acid hydrolysis base hydrolysis waterhydrolysis and thermal stress studies Significant degradationwas observed in oxidative conditions The typical oxidativestressed chromatogram was shown in Figure 4 It was inter-esting to note that all the peaks due to degradation were well

resolved from the peaks of GN DN and their impuritiesFurther the peak purity of GN DN and their impuritieswas found to be homogeneous based on the evaluationparameters such as purity angle and purity threshold usingWaters Empower Networking Software The verification ofpeak purity indicates that there is no interference fromdegradants facilitating error-free quantification of GN andDN impurities Also themass balance of stressed samples wasfound to be more than 98 Thus the method is consideredto be ldquostability-indicatingrdquo The specificity results were shownin Tables 4(a) and 4(b)

423 Precision The RSD for the individual of allimpurities in impurities method precision study was within34 The results obtained in the intermediated precisionstudy for the RSD of the individual of all impurities werewell within 41 conforming high precision of the methodThe results are shown in Tables 5 and 6

10 Chromatography Research International

Table 8 Accuracy of the method

Compound Recovery at each levela

LOQ 01 02 04 08 10GN-Impurity A 985 995 1012 967 999 1035GN-Impurity B 1056 1027 997 1051 985 975GN-Impurity C 1026 1056 1019 1061 1035 1021GN-Impurity D 956 972 965 958 965 985DN-Impurity A 935 965 952 942 985 967DN-Impurity B 985 967 999 989 975 965DN-Impurity C 1011 1023 1025 1037 1025 1018DN-N-oxide 1056 1065 1051 1045 1029 1037DN-NFM Impurity 1015 985 1025 1012 995 986DN-NFO Impurity 1071 1056 1069 1058 1065 1058GN 985 965 972 968 974 987DN 992 985 971 995 1005 1012aAverage of six determinations at LOQ level amp 10 level and three determinations at remaining levels

Table 9 Regression statistics

Substance Linearity range (120583gmLminus1) Correlation coefficient (119877) 119884-intercept bias in GN-Impurity A 093 to 23961 0999 09GN-Impurity B 201 to 24278 0999 10GN-Impurity C 126 to 24192 1000 07GN-Impurity D 079 to 24255 0999 08DN-Impurity A 017 to 1410 1000 04DN-Impurity B 025 to 1010 1000 01DN-Impurity C 027 to 1223 1000 08DN-N-oxide 019 to 1249 1000 06DN-NFM Impurity 019 to 1219 0998 15DN-NFO Impurity 048 to 1222 1000 09GN 153 to 24478 0999 16DN 018 to 1215 1000 01

Table 10 Results of robustness study

S no Impurity name

RRTrsquos of impurities

As suchmethod

At06mLminflow rate

At10mLminflow rate

At 50∘Ccolumn

temperature

At 60∘Ccolumn

temperature

At pH 28(buffer pH)

At pH 32(buffer pH)

1 GN-Impurity A 074 076 073 072 071 074 073

2 GN-Impurity B 112 111 113 111 110 112 113

3 GN-Impurity C 204 206 205 205 202 204 205

4 GN-Impurity D 214 212 215 213 211 213 214

5 DN-Impurity B 083 082 081 082 082 083 082

6 DN-Impurity C 098 097 098 098 097 097 098

7 DN-N-oxide 101 102 103 102 103 102 104

8 DN-Impurity A 103 104 105 104 105 104 106

9 DN-NFM Impurity 128 131 130 131 129 128 119

10 DN-NFO Impurity 138 141 142 139 139 139 141Note GN known impurities RRTs were calculated against GN main peak and DN known impurities RRTs were calculated against DN main peak

Chromatography Research International 11

424 Limit of Detection (LOD) and Limit of Quantification(LOQ) The determined limit of detection limit of quantifi-cation and precision at LOQ values for GN DN and theirimpurities were reported inTable 7TheRSD for peak areas ofGNDN and their related impurities at limit of quantificationlevel was within 100

425 Accuracy The recovery of all the impurities fromfinished pharmaceutical dosage form ranged from 850 to1150 The summary of recovery for individual impuritywas mentioned in Table 8

426 Linearity Linear calibration plot for the related sub-stance method was obtained over the calibration rangestested that is LOQ to 10 of the target test concentrationThe correlation coefficient obtained was greater than 0997for all the componentsThe slope and y-intercept values werealso provided in Table 9 which confirmed good linearitybetween peak areas and concentration The linearity graphswere shown in Figure 5(a) to Figure 5(l)

427 Robustness No significant effect was observed onsystem suitability parameters such as resolution RSD tailingfactor RRTs of impurities or the theoretical plates of GNand DN when small but deliberate changes were made tochromatographic conditions The results were presented inTables 3 and 10 along with the system suitability parametersof normal conditions Thus the method was found to berobust with respect to variability in applied conditions

428 Stability in Solution and in the Mobile Phase Nosignificant changeswere observed in the content of impuritiesduring solution stability and mobile phase stability exper-iments when performed using the impurities method Thesolution stability and mobile phase stability experiment dataconfirms that the sample solution and mobile phases usedduring the impurity determination were stable for at least48 h

5 Conclusions

The gradient HPLC method developed for the simultaneousdetermination of GN and DN impurities in pharmaceuticaldosage form was precise accurate and specific The methodis validated as per ICH guidelines and found to be specificprecise linear accurate rugged and robust The developedmethod can be used for the stability analysis of GN and DNeither individually or in their combination dosage forms

Acknowledgments

The authors wish to thank the management of Dr Reddyrsquosgroup for supporting this workThe authors wish to acknowl-edge the formulation development group for providing thesamples for their research They would also like to thankcolleagues in bulkmanufacturers for providing chemicals andimpurity standards for their research work

References

[1] httpwwwrxlistcomorganidin-nr-drughtm[2] W Zhang TWang L Qin et al ldquoNeuroprotective effect of dex-

tromethorphan in the MPTP Parkinsonrsquos disease model role ofNADPHoxidaserdquoTheFASEB Journal vol 18 no 3 pp 589ndash5912004

[3] Dextromethorphan NHTSA[4] ldquoChild deaths lead to FDA hearing on cough cold medsrdquo 2007

httpwwwcnncom[5] B KuKanich and M G Papich ldquoPlasma profile and pharmac-

okinetics of dextromethorphan after intravenous and oraladministration in healthy dogsrdquo Journal of Veterinary Pharma-cology andTherapeutics vol 27 no 5 pp 337ndash341 2004

[6] ldquoKidsrsquo cough medicine no better than placebordquo San FranciscoChronicle 2004

[7] I M Paul K E Yoder K R Crowell et al ldquoEffect of dextro-methorphan diphenhydramine and placebo on nocturnalcough and sleep quality for coughing children and their par-entsrdquo Pediatrics vol 114 no 1 pp e85ndashe90 2004

[8] S Gorog M Babjak G Balogh et al ldquoDrug impurity profilingstrategiesrdquo Talanta vol 44 no 9 pp 1517ndash1526 1997

[9] B B Sanjay R K Bharati S J Yogini and A S Atul ldquoImpurityprofile significance in active pharmaceutical ingredientrdquo Eura-sian Journal of Analytical Chemistry vol 2 pp 32ndash53 2007

[10] ICH ldquoImpurities in new drug substances Q3A (R2)rdquo 2006[11] ICH ldquoImpurities in new drug products Q3B (R2)rdquo 2006[12] G Grosa E D Grosso R Russo and G Allegrone ldquoSimulta-

neous stability indicating HPLC-DAD determination ofguaifenesin and methyl and propyl-parabens in cough syruprdquoJournal of Pharmaceutical and Biomedical Analysis vol 41 no3 pp 798ndash803 2006

[13] M Senthilraja and P Giriraj ldquoReverse phase hplc method forthe simultaneous estimation of terbutanile sulphate bromhex-ine HCl and guaifenesin in cough syruprdquo Asian Journal ofPharmaceutical and Clinical Research vol 4 no 2 pp 13ndash152011

[14] M L Wilcox and J T Stewart ldquoHPLC determination of guaife-nesin with selected medications on underivatized silica with anaqueous-organic mobile phaserdquo Journal of Pharmaceutical andBiomedical Analysis vol 23 no 5 pp 909ndash916 2000

[15] A Ozdemir H Aksoy E Dinc D Baleanu and S Dermis ldquoDe-termination of guaifenesin and dextromethorphan in a coughsyrup by HPLC with fluorometric detectionrdquo Revue Roumainede Chimie vol 51 no 2 pp 117ndash122 2006

[16] I Demian ldquoHigh-performance liquid chromatography (HPLC)chiral separations of guaifenesin methocarbamol and race-morphanrdquo Chirality vol 5 no 4 pp 238ndash240 1993

[17] S Suzen C Akay and S Cevheroglu ldquoSimultaneous determi-nation of guaiphenesin and codeine phosphate in tablets byhigh-performance liquid chromatographyrdquo Farmaco vol 54no 10 pp 705ndash709 1999

[18] S M Amer S S Abbas M A Shehata and NM Ali ldquoSimulta-neous determination of phenylephrine hydrochloride guaifen-esin and chlorpheniraminemaleate in cough syrup by gradientliquid chromatographyrdquo Journal of AOAC International vol 91no 2 pp 276ndash284 2008

[19] V Galli andC Barbas ldquoHigh-performance liquid chromatogra-phic analysis of dextromethorphan guaifenesin and benzoate ina cough syrup for stability testingrdquo Journal of ChromatographyA vol 1048 no 2 pp 207ndash211 2004

12 Chromatography Research International

[20] X Chen J Huang Z Kong and D Zhong ldquoSensitive liquidchromatography-tandem mass spectrometry method for thesimultaneous determination of paracetamol and guaifenesin inhuman plasmardquo Journal of Chromatography B vol 817 no 2 pp263ndash269 2005

[21] R M Gudipati J E Wallace and S A Stavchansky ldquoHigh per-formance liquid chromatography determination of guaifenesinin dog plasmardquo Analytical Letters vol 24 pp 265ndash274 1991

[22] Q-H Ge Z Zhou X-J Zhi L-L Ma and C-G Ding ldquoSim-ultaneous determination of guaifenesin bromhexine andambroxol in human plasma by LC-MSMS and its pharmacoki-netic studiesrdquo Chinese Pharmaceutical Journal vol 44 no 13pp 1025ndash1028 2009

[23] R Rajagopalan ldquoReview of regulatory guidance on impuritiesrdquoSeparation Science and Technology vol 5 pp 27ndash37 2004

[24] ICH Harmonized Tripartite Guideline Validation of AnalyticalProcedures Text and Methodology Q2(R1) 2005

[25] ICH ldquoStability testing of new drug substances and productsQ1A (R2)rdquo 2003

[26] ICH ldquoPhotostability testing of new drug substances and prod-ucts Q1Brdquo 1996

[27] The United States Pharmacopeia USP35-NF30 3829ndash3837[28] European Pharmacopeia 70 1821-1822 amp 2128-2129[29] P Sunil Reddy K Sudhakar Babu N Kumar and Y V V Sasi

Sekhar ldquoDevelopment and validation of stability indicating theRP-HPLC method for the estimation of related compounds ofguaifenesin in pharmaceutical dosage formsrdquo PharmaceuticalMethods vol 2 pp 229ndash234 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

4 Chromatography Research International

Table 2 (a) Structures of Guaifenesin impurities (b) Structures of Dextromethorphan impurities

(a)

Impurity name Chemical name Chemical structure

Impurity A (degradant) 2-Methoxyphenol

OH

O

Impurity B (degradant) 2-(2-Methoxyphenoxy)propane-13-diolHO

HO

O

O

Impurity C (degradant) 111015840-Oxybis[3-(2-methoxyphenoxy)propan-2-ol]OHOH

O

OOO

O

Impurity D (process related impurity) 13-Bis(2-methoxyphenoxy)propan-2-ol

OHOO

O O

(b)

Impurity name Chemical name Chemical structure

Impurity A (degradant) Ent-3-methoxymorphinan

NH

H

O

Impurity B (degradant) Ent-17-methylmorphinan-3-ol

HO

H

N

Impurity C (degradant) Ent-3-methoxy-17-methylmorphinan-10-one H

N

O

O

N-Oxide (degradant) 3-Methoxy-N-methylmorphinan N-oxide H

O

Ominus

N+

N-Formyl Morphine (NFM)(process related impurity)

3-Methoxy-6788a910-hexahydro-5H-94b-(epiminoethano)phenanthrene-11-carbaldehyde

N

CHO

H3CO

N-Formyl octabase (NFO)(process related impurity)

(S)-1-(4-Methoxybenzyl)-345678-hexahydroisoquinoline-2(1H)-carbaldehyde

H

NCHO

H3CO

Chromatography Research International 5

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

23833 Guaifenesin imp A15533 Guaifenesin imp B 43267 Guaifenesin imp C

45500 Guaifenesin imp D

(a)

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

21400 Guaifenesin40733 Dextromethorphan

(b)

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

34017 Dextromethorphan imp B40117 Dextromethorphan imp C41450 Dextromethorphan imp N-Oxide42033 Dextromethorphan imp A51983 Dextromethorphan imp NFM55967 Dextromethorphan imp NFO

(c)

Figure 1 Spectra of GN DN and their impurities

on themethod being accurate reproducible robust stability-indicating linear free of interference fromother formulationexcipients and convenient enough for routine use in qualitycontrol laboratories

Individual stock solutions of GN DN and their impu-rities were injected and the spectra were checked of eachcomponent (Figure 1) From the spectra all the impuritieswere having absorbance maximum at about 224 Hence224 nm was selected for the estimation of GN and DNimpurities

A spiked solution of impurities (48120583gmLminus1 of GNimpurities and 24 120583gmLminus1 of DN impurities) GN + DN(24000120583gmLminus1 + 1200120583gmLminus1) and placebo peaks weresubjected to separation by RP-HPLC Initially the separationwas tried with the existing methods (USP Pharma Europeand the literature method) [27ndash29] It was observed thatplacebo peaks and GN impurity peaks were merging with

each other and two DN known impurities (NFM and NFO)were not eluting in DN API Pharma Europe method In USPand Pharma Europe GN API method DN impurities werenot separated from DN peak and two DN known impuritieswere eluting at longer retention with broad peak shapes Incase of the literature method [29] DN impurities were notseparated fromDN peak and twoDN known impurities wereeluting at longer retention times with better peak shapes

Method development was initiated by changing dif-ferent gradient programmes different pH values of themobile phase buffer different phosphate buffers and differentcolumns with the literature method [29] Sharp peak shapeswere observed with Sunfire column and sodium dihydrogenphosphate monohydrate buffer at pH 30 but separation wasnot up to the mark for DN DN-N-oxide and DN-impurityC Sharp peak shapes were due to the properties of the Sunfirecolumn (high mass loading capability excellent low pH

6 Chromatography Research International

020

018

016

014

012

010

008

006

004

002

000000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 2 Overlaid chromatogram of blank and system suitabilitypreparation

020

018

016

014

012

010

008

006

004

002

000

000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 3 Overlaid chromatogram of placebo and spiked testpreparation

stability superior peak shapes and high efficiency) Since wewere using a very high concentration ofGN (24000120583gmLminus1)it was decided to use Sunfire column for further developmenttrails by using ion pair reagent in the mobile phase for betterseparation between DN DN-Impurity A and DN-ImpurityB Separation was achieved between all the pairs of peaks butpeak shapes of DN-NFM Impurity and DN-NFO Impurityare not sharp Acetonitrile was added in the mobile phase inaddition to methanol to get sharp peaks

The chromatographic separation was achieved by areversed phase Sunfire C18 250 times 46mm 5120583m particlesize column operated at 50∘C with gradient elution at

020

018

016

014

012

010

008

006

004

002

000

000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 4 Typical chromatogram of peroxide stressed sample

08mLminminus1 using a mobile phase buffer as a mixtureof 001M sodium dihydrogen phosphate monohydrate and00046M 1-octane sulfonic acid sodium salt monohydrateof pH 30 (pH adjusted with diluted orthophosphoric acid)UV absorbance at 224 nm and injection volume 20 120583L Themobile phase A consisted of pH 30 buffer and acetonitrile(90 10 vv) mobile phase B consisted of pH 30 bufferacetonitrile andmethanol (10 10 80 vvv)The LC gradientprogram was set as time (min) mobile phase B 00115155 2030 3050 6085 6515 and 7515 All the impuritieswere well separated with a resolution greater than 2 Nochromatographic interference due to the blank (diluent)and other excipients (placebo) at the retention time of GNDN and their impurities was observed The typical overlaychromatogram of blank and system suitability solution andspiked test is shown in Figures 2 and 3

42MethodValidation After the development of themethodit was subject to method validation as per ICH guidelines[24] The method was validated to demonstrate that it issuitable for its intended purpose by the standard procedureto evaluate adequate validation characteristics (system suit-ability specificity accuracy precision linearity robustnessruggedness solution stability LOD and LOQ and stability-indicating capability)

421 System Suitability The percentage relative standarddeviation (RSD) of area from six replicate injections wasbelow 50 (diluted standard solution 48 120583gmLminus1 of GNand 24 120583gmLminus1 of DN) Low values of RSD for replicateinjections indicate that the system is precise The results ofother system suitability parameters such as resolution peaktailing and theoretical plates are presented in Table 3 As seenfrom this data the acceptable system suitability parameterswould be as follows the relative standard deviation of

Chromatography Research International 7

000200000000400000000600000000800000000

1000000000120000000014000000001600000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesiny = 56119662x + 118692128

R2 = 0999

(a)

02000000400000060000008000000

10000000120000001400000016000000

0000 50000 100000 150000 200000 250000 300000

Linearity of detector response of Guaifenesin impurity Ay = 62278038x + 138787601

R2 = 0998

Are

a

(b)

02000000400000060000008000000

100000001200000014000000

0000 50000 100000 150000 200000 250000 300000

Linearity of detector response of Guaifenesin impurity By = 46707746x + 109527559

R2 = 0998

Are

a

(c)

02000000400000060000008000000

100000001200000014000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesin impurity Cy = 49 606000x +

R2 = 0999

78596824

(d)

0

5000000

10000000

15000000

20000000

25000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesin impurity Dy = 79962049x + 156337857

R2 = 0999

(e)

050000

100000150000200000250000300000350000400000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphany = 30824867x + 4487696

R2 = 1000

(f)

050000

100000150000200000250000300000350000400000450000500000

0000 2000 4000 6000 8000 10000 12000 14000 16000

Are

a

Linearity of detector response of Dextromethorphan impurity A

y = 32920087x + 331349

R2 = 1000

(g)

050000

100000150000200000250000300000

0000 2000 4000 6000 8000 10000 12000

Are

a

Linearity of detector response of Dextromethorphan impurity B

y = 25035731x minus 57593

R2 = 1000

(h)

050000

100000150000200000250000300000350000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphan impurity C

y = 24213547x + 450726

R2 = 1000

(i)

050000

100000150000200000250000300000350000400000450000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphan N-oxide

y = 33 682946x + 523536

R2 = 1000

(j)

0100000200000300000400000500000600000700000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Concentration (ppm)

Linearity of detector response of Dextromethorphan impurity NFM

y = 49617284x + 22697199

R2 = 0996

(k)

0100000200000300000400000500000600000700000800000900000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Concentration (ppm)

Linearity of detector response of Dextromethorphan impurity NFO

y = 66581365x minus 1352313

R2 = 1000

(l)

Figure 5 (a) Linearity graph of Guaifenesin (b) Linearity graph of Guaifenesin impurity A (c) Linearity graph of Guaifenesin impurity B(d) Linearity graph of Guaifenesin impurity C (e) Linearity graph of Guaifenesin impurity D (f) Linearity graph of Dextromethorphan(g) Linearity graph of Dextromethorphan impurity A (h) Linearity graph of Dextromethorphan impurity B (i) Linearity graph ofDextromethorphan impurity C (j) Linearity graph of DextromethorphanN-oxide (k) Linearity graph of Dextromethorphan impurity NFM(l) Linearity graph of Dextromethorphan impurity NFO

8 Chromatography Research International

Table 3 System suitability results

Parameter RSDlowast of standard Theoretical plateslowast Tailing factorlowast Resolution 1 Resolution 2GN DN GN DN GN DN

As such method 05 07 45320 498659 10 10 27 23At 06mLmin flow rate 12 06 41091 495497 10 10 29 25At 10mLmin flow rate 09 04 45828 515551 10 10 25 22At 50∘C column temperature 15 08 45321 508966 10 10 26 23At 60∘C column temperature 21 23 40929 515807 10 10 29 26At pH 28 (buffer pH) 11 05 43970 463987 10 10 24 23At pH 32 (buffer pH) 07 15 44619 532891 10 10 26 27lowastAverage of six replicate standard injections

Table 4 (a) Forced degradation data for Guaifenesin (b) Forced degradation data for Dextromethorphan

(a)

Degradation conditions Guaifenesin degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 million lux hours followed by 200Watt hours) 006 3585 4142 1002Exposed to humidity at 25∘C and 90 RH for about 7 days 007 3881 4241 998Refluxed with purified water for about 12 hours at 60∘C 005 3285 3845 986Refluxed with 05 N HCL solution for about 2 hours at 60∘C 010 5293 7073 992Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 006 3067 3489 989Refluxed with 10 H2O2 solution for about 30min at 60∘C 013 2755 3607 983Exposed to dry heat for about 15 hours at 105∘C 007 3346 4331 994

(b)

Degradation conditions Dextromethorphan degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 Million lux hours followed by 200Watt hours) 001 0115 0242 998Exposed to humidity at 25∘C and 90 RH for about 7 days 001 0124 0261 995Refluxed with purified water for about 12 hours at 60∘C 001 0101 0261 989Refluxed with 05 N HCL solution for about 2 hours at 60∘C 002 0154 0262 981Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 001 0103 0256 992Refluxed with 10 H2O2 solution for about 30min at 60∘C 105 0102 0250 997Exposed to dry heat for about 15 hours at 105∘C 001 0099 0261 987

Table 5 Results of precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0198 0201 0189 0213 0205 0209 0203 42GN-Impurity B 0209 0195 0192 0201 0206 0194 0200 35GN-Impurity C 0189 0195 0186 0192 0198 0184 0191 28GN-Impurity D 0208 0190 0192 0193 0188 0186 0193 41DN-Impurity A 0215 0214 0215 0209 0212 0205 0212 19DN-Impurity B 0204 0200 0202 0203 0205 0191 0201 25DN-Impurity C 0195 0186 0198 0199 0184 0194 0193 32DN-N-oxide 0216 0214 0218 0219 0211 0210 0215 17DN-NFM Impurity 0216 0218 0215 0215 0216 0212 0215 09DN-NFO Impurity 0198 0195 0213 0192 0214 0215 0205 52GN 0203 0212 0204 0206 0205 0210 0207 17DN 0182 0189 0194 0188 0187 0201 0190 34

Chromatography Research International 9

Table 6 Results of intermediate precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0191 0204 0201 0199 0210 0212 0203 38GN-Impurity B 0212 0204 0210 0209 0211 0204 0208 17GN-Impurity C 0192 0198 0191 0194 0202 0209 0198 35GN-Impurity D 0185 0192 0191 0194 0193 0192 0192 15DN-Impurity A 0202 0202 0203 0202 0202 0204 0203 04DN-Impurity B 0191 0188 0204 0209 0189 0202 0197 45DN-Impurity C 0183 0189 0191 0186 0205 0206 0193 51DN-N-oxide 0197 0210 0213 0202 0198 0212 0205 35DN-NFM Impurity 0183 0187 0179 0184 0185 0188 0184 17DN-NFO Impurity 0206 0197 0209 0212 0194 0208 0204 35GN 0199 0205 0201 0204 0207 0208 0204 17DN 0187 0185 0192 0186 0194 0191 0189 19

Table 7 LOD LOQ and precision data

Compound LOD () LOQ () SN ratio RSD at LOQ Level precisionLOD LOQ

GN-Impurity A 0004 0001 27 95 42GN-Impurity B 0008 0002 29 98 25GN-Impurity C 0005 0002 30 98 31GN-Impurity D 0003 0001 29 97 25DN-Impurity A 0014 0004 28 101 11DN-Impurity B 0021 0006 27 104 34DN-Impurity C 0023 0007 28 99 49DN-N-oxide 0016 0005 31 98 22DN-NFM Impurity 0016 0005 27 101 15DN-NFO Impurity 0041 0012 29 106 42GN 0006 0002 31 98 21DN 0015 0005 28 96 28

replicate injections is notmore than 50 resolution betweenimpurities 20 the tailing factor for GN and DN is not morethan 15 and the theoretical plates are not less than 5000

422 Specificity All forced degradation samples were ana-lyzed with the aforementioned HPLC conditions using aPDA detector to monitor the homogeneity and purity of theGN DN and their related impurities Individual impuritiesplacebo GN and DN were verified and proved to be nonin-terfering with each other thus proving the specificity of themethod

Figure 3 shows that there is no interference at the RT(retention time) of GN DN and all known impurities fromthe other excipients Degradation was not observed in pho-tolytic stress humidity acid hydrolysis base hydrolysis waterhydrolysis and thermal stress studies Significant degradationwas observed in oxidative conditions The typical oxidativestressed chromatogram was shown in Figure 4 It was inter-esting to note that all the peaks due to degradation were well

resolved from the peaks of GN DN and their impuritiesFurther the peak purity of GN DN and their impuritieswas found to be homogeneous based on the evaluationparameters such as purity angle and purity threshold usingWaters Empower Networking Software The verification ofpeak purity indicates that there is no interference fromdegradants facilitating error-free quantification of GN andDN impurities Also themass balance of stressed samples wasfound to be more than 98 Thus the method is consideredto be ldquostability-indicatingrdquo The specificity results were shownin Tables 4(a) and 4(b)

423 Precision The RSD for the individual of allimpurities in impurities method precision study was within34 The results obtained in the intermediated precisionstudy for the RSD of the individual of all impurities werewell within 41 conforming high precision of the methodThe results are shown in Tables 5 and 6

10 Chromatography Research International

Table 8 Accuracy of the method

Compound Recovery at each levela

LOQ 01 02 04 08 10GN-Impurity A 985 995 1012 967 999 1035GN-Impurity B 1056 1027 997 1051 985 975GN-Impurity C 1026 1056 1019 1061 1035 1021GN-Impurity D 956 972 965 958 965 985DN-Impurity A 935 965 952 942 985 967DN-Impurity B 985 967 999 989 975 965DN-Impurity C 1011 1023 1025 1037 1025 1018DN-N-oxide 1056 1065 1051 1045 1029 1037DN-NFM Impurity 1015 985 1025 1012 995 986DN-NFO Impurity 1071 1056 1069 1058 1065 1058GN 985 965 972 968 974 987DN 992 985 971 995 1005 1012aAverage of six determinations at LOQ level amp 10 level and three determinations at remaining levels

Table 9 Regression statistics

Substance Linearity range (120583gmLminus1) Correlation coefficient (119877) 119884-intercept bias in GN-Impurity A 093 to 23961 0999 09GN-Impurity B 201 to 24278 0999 10GN-Impurity C 126 to 24192 1000 07GN-Impurity D 079 to 24255 0999 08DN-Impurity A 017 to 1410 1000 04DN-Impurity B 025 to 1010 1000 01DN-Impurity C 027 to 1223 1000 08DN-N-oxide 019 to 1249 1000 06DN-NFM Impurity 019 to 1219 0998 15DN-NFO Impurity 048 to 1222 1000 09GN 153 to 24478 0999 16DN 018 to 1215 1000 01

Table 10 Results of robustness study

S no Impurity name

RRTrsquos of impurities

As suchmethod

At06mLminflow rate

At10mLminflow rate

At 50∘Ccolumn

temperature

At 60∘Ccolumn

temperature

At pH 28(buffer pH)

At pH 32(buffer pH)

1 GN-Impurity A 074 076 073 072 071 074 073

2 GN-Impurity B 112 111 113 111 110 112 113

3 GN-Impurity C 204 206 205 205 202 204 205

4 GN-Impurity D 214 212 215 213 211 213 214

5 DN-Impurity B 083 082 081 082 082 083 082

6 DN-Impurity C 098 097 098 098 097 097 098

7 DN-N-oxide 101 102 103 102 103 102 104

8 DN-Impurity A 103 104 105 104 105 104 106

9 DN-NFM Impurity 128 131 130 131 129 128 119

10 DN-NFO Impurity 138 141 142 139 139 139 141Note GN known impurities RRTs were calculated against GN main peak and DN known impurities RRTs were calculated against DN main peak

Chromatography Research International 11

424 Limit of Detection (LOD) and Limit of Quantification(LOQ) The determined limit of detection limit of quantifi-cation and precision at LOQ values for GN DN and theirimpurities were reported inTable 7TheRSD for peak areas ofGNDN and their related impurities at limit of quantificationlevel was within 100

425 Accuracy The recovery of all the impurities fromfinished pharmaceutical dosage form ranged from 850 to1150 The summary of recovery for individual impuritywas mentioned in Table 8

426 Linearity Linear calibration plot for the related sub-stance method was obtained over the calibration rangestested that is LOQ to 10 of the target test concentrationThe correlation coefficient obtained was greater than 0997for all the componentsThe slope and y-intercept values werealso provided in Table 9 which confirmed good linearitybetween peak areas and concentration The linearity graphswere shown in Figure 5(a) to Figure 5(l)

427 Robustness No significant effect was observed onsystem suitability parameters such as resolution RSD tailingfactor RRTs of impurities or the theoretical plates of GNand DN when small but deliberate changes were made tochromatographic conditions The results were presented inTables 3 and 10 along with the system suitability parametersof normal conditions Thus the method was found to berobust with respect to variability in applied conditions

428 Stability in Solution and in the Mobile Phase Nosignificant changeswere observed in the content of impuritiesduring solution stability and mobile phase stability exper-iments when performed using the impurities method Thesolution stability and mobile phase stability experiment dataconfirms that the sample solution and mobile phases usedduring the impurity determination were stable for at least48 h

5 Conclusions

The gradient HPLC method developed for the simultaneousdetermination of GN and DN impurities in pharmaceuticaldosage form was precise accurate and specific The methodis validated as per ICH guidelines and found to be specificprecise linear accurate rugged and robust The developedmethod can be used for the stability analysis of GN and DNeither individually or in their combination dosage forms

Acknowledgments

The authors wish to thank the management of Dr Reddyrsquosgroup for supporting this workThe authors wish to acknowl-edge the formulation development group for providing thesamples for their research They would also like to thankcolleagues in bulkmanufacturers for providing chemicals andimpurity standards for their research work

References

[1] httpwwwrxlistcomorganidin-nr-drughtm[2] W Zhang TWang L Qin et al ldquoNeuroprotective effect of dex-

tromethorphan in the MPTP Parkinsonrsquos disease model role ofNADPHoxidaserdquoTheFASEB Journal vol 18 no 3 pp 589ndash5912004

[3] Dextromethorphan NHTSA[4] ldquoChild deaths lead to FDA hearing on cough cold medsrdquo 2007

httpwwwcnncom[5] B KuKanich and M G Papich ldquoPlasma profile and pharmac-

okinetics of dextromethorphan after intravenous and oraladministration in healthy dogsrdquo Journal of Veterinary Pharma-cology andTherapeutics vol 27 no 5 pp 337ndash341 2004

[6] ldquoKidsrsquo cough medicine no better than placebordquo San FranciscoChronicle 2004

[7] I M Paul K E Yoder K R Crowell et al ldquoEffect of dextro-methorphan diphenhydramine and placebo on nocturnalcough and sleep quality for coughing children and their par-entsrdquo Pediatrics vol 114 no 1 pp e85ndashe90 2004

[8] S Gorog M Babjak G Balogh et al ldquoDrug impurity profilingstrategiesrdquo Talanta vol 44 no 9 pp 1517ndash1526 1997

[9] B B Sanjay R K Bharati S J Yogini and A S Atul ldquoImpurityprofile significance in active pharmaceutical ingredientrdquo Eura-sian Journal of Analytical Chemistry vol 2 pp 32ndash53 2007

[10] ICH ldquoImpurities in new drug substances Q3A (R2)rdquo 2006[11] ICH ldquoImpurities in new drug products Q3B (R2)rdquo 2006[12] G Grosa E D Grosso R Russo and G Allegrone ldquoSimulta-

neous stability indicating HPLC-DAD determination ofguaifenesin and methyl and propyl-parabens in cough syruprdquoJournal of Pharmaceutical and Biomedical Analysis vol 41 no3 pp 798ndash803 2006

[13] M Senthilraja and P Giriraj ldquoReverse phase hplc method forthe simultaneous estimation of terbutanile sulphate bromhex-ine HCl and guaifenesin in cough syruprdquo Asian Journal ofPharmaceutical and Clinical Research vol 4 no 2 pp 13ndash152011

[14] M L Wilcox and J T Stewart ldquoHPLC determination of guaife-nesin with selected medications on underivatized silica with anaqueous-organic mobile phaserdquo Journal of Pharmaceutical andBiomedical Analysis vol 23 no 5 pp 909ndash916 2000

[15] A Ozdemir H Aksoy E Dinc D Baleanu and S Dermis ldquoDe-termination of guaifenesin and dextromethorphan in a coughsyrup by HPLC with fluorometric detectionrdquo Revue Roumainede Chimie vol 51 no 2 pp 117ndash122 2006

[16] I Demian ldquoHigh-performance liquid chromatography (HPLC)chiral separations of guaifenesin methocarbamol and race-morphanrdquo Chirality vol 5 no 4 pp 238ndash240 1993

[17] S Suzen C Akay and S Cevheroglu ldquoSimultaneous determi-nation of guaiphenesin and codeine phosphate in tablets byhigh-performance liquid chromatographyrdquo Farmaco vol 54no 10 pp 705ndash709 1999

[18] S M Amer S S Abbas M A Shehata and NM Ali ldquoSimulta-neous determination of phenylephrine hydrochloride guaifen-esin and chlorpheniraminemaleate in cough syrup by gradientliquid chromatographyrdquo Journal of AOAC International vol 91no 2 pp 276ndash284 2008

[19] V Galli andC Barbas ldquoHigh-performance liquid chromatogra-phic analysis of dextromethorphan guaifenesin and benzoate ina cough syrup for stability testingrdquo Journal of ChromatographyA vol 1048 no 2 pp 207ndash211 2004

12 Chromatography Research International

[20] X Chen J Huang Z Kong and D Zhong ldquoSensitive liquidchromatography-tandem mass spectrometry method for thesimultaneous determination of paracetamol and guaifenesin inhuman plasmardquo Journal of Chromatography B vol 817 no 2 pp263ndash269 2005

[21] R M Gudipati J E Wallace and S A Stavchansky ldquoHigh per-formance liquid chromatography determination of guaifenesinin dog plasmardquo Analytical Letters vol 24 pp 265ndash274 1991

[22] Q-H Ge Z Zhou X-J Zhi L-L Ma and C-G Ding ldquoSim-ultaneous determination of guaifenesin bromhexine andambroxol in human plasma by LC-MSMS and its pharmacoki-netic studiesrdquo Chinese Pharmaceutical Journal vol 44 no 13pp 1025ndash1028 2009

[23] R Rajagopalan ldquoReview of regulatory guidance on impuritiesrdquoSeparation Science and Technology vol 5 pp 27ndash37 2004

[24] ICH Harmonized Tripartite Guideline Validation of AnalyticalProcedures Text and Methodology Q2(R1) 2005

[25] ICH ldquoStability testing of new drug substances and productsQ1A (R2)rdquo 2003

[26] ICH ldquoPhotostability testing of new drug substances and prod-ucts Q1Brdquo 1996

[27] The United States Pharmacopeia USP35-NF30 3829ndash3837[28] European Pharmacopeia 70 1821-1822 amp 2128-2129[29] P Sunil Reddy K Sudhakar Babu N Kumar and Y V V Sasi

Sekhar ldquoDevelopment and validation of stability indicating theRP-HPLC method for the estimation of related compounds ofguaifenesin in pharmaceutical dosage formsrdquo PharmaceuticalMethods vol 2 pp 229ndash234 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Chromatography Research International 5

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

23833 Guaifenesin imp A15533 Guaifenesin imp B 43267 Guaifenesin imp C

45500 Guaifenesin imp D

(a)

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

21400 Guaifenesin40733 Dextromethorphan

(b)

22000 24000 26000 28000 30000 32000 34000 36000 38000(nm)

34017 Dextromethorphan imp B40117 Dextromethorphan imp C41450 Dextromethorphan imp N-Oxide42033 Dextromethorphan imp A51983 Dextromethorphan imp NFM55967 Dextromethorphan imp NFO

(c)

Figure 1 Spectra of GN DN and their impurities

on themethod being accurate reproducible robust stability-indicating linear free of interference fromother formulationexcipients and convenient enough for routine use in qualitycontrol laboratories

Individual stock solutions of GN DN and their impu-rities were injected and the spectra were checked of eachcomponent (Figure 1) From the spectra all the impuritieswere having absorbance maximum at about 224 Hence224 nm was selected for the estimation of GN and DNimpurities

A spiked solution of impurities (48120583gmLminus1 of GNimpurities and 24 120583gmLminus1 of DN impurities) GN + DN(24000120583gmLminus1 + 1200120583gmLminus1) and placebo peaks weresubjected to separation by RP-HPLC Initially the separationwas tried with the existing methods (USP Pharma Europeand the literature method) [27ndash29] It was observed thatplacebo peaks and GN impurity peaks were merging with

each other and two DN known impurities (NFM and NFO)were not eluting in DN API Pharma Europe method In USPand Pharma Europe GN API method DN impurities werenot separated from DN peak and two DN known impuritieswere eluting at longer retention with broad peak shapes Incase of the literature method [29] DN impurities were notseparated fromDN peak and twoDN known impurities wereeluting at longer retention times with better peak shapes

Method development was initiated by changing dif-ferent gradient programmes different pH values of themobile phase buffer different phosphate buffers and differentcolumns with the literature method [29] Sharp peak shapeswere observed with Sunfire column and sodium dihydrogenphosphate monohydrate buffer at pH 30 but separation wasnot up to the mark for DN DN-N-oxide and DN-impurityC Sharp peak shapes were due to the properties of the Sunfirecolumn (high mass loading capability excellent low pH

6 Chromatography Research International

020

018

016

014

012

010

008

006

004

002

000000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 2 Overlaid chromatogram of blank and system suitabilitypreparation

020

018

016

014

012

010

008

006

004

002

000

000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 3 Overlaid chromatogram of placebo and spiked testpreparation

stability superior peak shapes and high efficiency) Since wewere using a very high concentration ofGN (24000120583gmLminus1)it was decided to use Sunfire column for further developmenttrails by using ion pair reagent in the mobile phase for betterseparation between DN DN-Impurity A and DN-ImpurityB Separation was achieved between all the pairs of peaks butpeak shapes of DN-NFM Impurity and DN-NFO Impurityare not sharp Acetonitrile was added in the mobile phase inaddition to methanol to get sharp peaks

The chromatographic separation was achieved by areversed phase Sunfire C18 250 times 46mm 5120583m particlesize column operated at 50∘C with gradient elution at

020

018

016

014

012

010

008

006

004

002

000

000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 4 Typical chromatogram of peroxide stressed sample

08mLminminus1 using a mobile phase buffer as a mixtureof 001M sodium dihydrogen phosphate monohydrate and00046M 1-octane sulfonic acid sodium salt monohydrateof pH 30 (pH adjusted with diluted orthophosphoric acid)UV absorbance at 224 nm and injection volume 20 120583L Themobile phase A consisted of pH 30 buffer and acetonitrile(90 10 vv) mobile phase B consisted of pH 30 bufferacetonitrile andmethanol (10 10 80 vvv)The LC gradientprogram was set as time (min) mobile phase B 00115155 2030 3050 6085 6515 and 7515 All the impuritieswere well separated with a resolution greater than 2 Nochromatographic interference due to the blank (diluent)and other excipients (placebo) at the retention time of GNDN and their impurities was observed The typical overlaychromatogram of blank and system suitability solution andspiked test is shown in Figures 2 and 3

42MethodValidation After the development of themethodit was subject to method validation as per ICH guidelines[24] The method was validated to demonstrate that it issuitable for its intended purpose by the standard procedureto evaluate adequate validation characteristics (system suit-ability specificity accuracy precision linearity robustnessruggedness solution stability LOD and LOQ and stability-indicating capability)

421 System Suitability The percentage relative standarddeviation (RSD) of area from six replicate injections wasbelow 50 (diluted standard solution 48 120583gmLminus1 of GNand 24 120583gmLminus1 of DN) Low values of RSD for replicateinjections indicate that the system is precise The results ofother system suitability parameters such as resolution peaktailing and theoretical plates are presented in Table 3 As seenfrom this data the acceptable system suitability parameterswould be as follows the relative standard deviation of

Chromatography Research International 7

000200000000400000000600000000800000000

1000000000120000000014000000001600000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesiny = 56119662x + 118692128

R2 = 0999

(a)

02000000400000060000008000000

10000000120000001400000016000000

0000 50000 100000 150000 200000 250000 300000

Linearity of detector response of Guaifenesin impurity Ay = 62278038x + 138787601

R2 = 0998

Are

a

(b)

02000000400000060000008000000

100000001200000014000000

0000 50000 100000 150000 200000 250000 300000

Linearity of detector response of Guaifenesin impurity By = 46707746x + 109527559

R2 = 0998

Are

a

(c)

02000000400000060000008000000

100000001200000014000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesin impurity Cy = 49 606000x +

R2 = 0999

78596824

(d)

0

5000000

10000000

15000000

20000000

25000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesin impurity Dy = 79962049x + 156337857

R2 = 0999

(e)

050000

100000150000200000250000300000350000400000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphany = 30824867x + 4487696

R2 = 1000

(f)

050000

100000150000200000250000300000350000400000450000500000

0000 2000 4000 6000 8000 10000 12000 14000 16000

Are

a

Linearity of detector response of Dextromethorphan impurity A

y = 32920087x + 331349

R2 = 1000

(g)

050000

100000150000200000250000300000

0000 2000 4000 6000 8000 10000 12000

Are

a

Linearity of detector response of Dextromethorphan impurity B

y = 25035731x minus 57593

R2 = 1000

(h)

050000

100000150000200000250000300000350000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphan impurity C

y = 24213547x + 450726

R2 = 1000

(i)

050000

100000150000200000250000300000350000400000450000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphan N-oxide

y = 33 682946x + 523536

R2 = 1000

(j)

0100000200000300000400000500000600000700000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Concentration (ppm)

Linearity of detector response of Dextromethorphan impurity NFM

y = 49617284x + 22697199

R2 = 0996

(k)

0100000200000300000400000500000600000700000800000900000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Concentration (ppm)

Linearity of detector response of Dextromethorphan impurity NFO

y = 66581365x minus 1352313

R2 = 1000

(l)

Figure 5 (a) Linearity graph of Guaifenesin (b) Linearity graph of Guaifenesin impurity A (c) Linearity graph of Guaifenesin impurity B(d) Linearity graph of Guaifenesin impurity C (e) Linearity graph of Guaifenesin impurity D (f) Linearity graph of Dextromethorphan(g) Linearity graph of Dextromethorphan impurity A (h) Linearity graph of Dextromethorphan impurity B (i) Linearity graph ofDextromethorphan impurity C (j) Linearity graph of DextromethorphanN-oxide (k) Linearity graph of Dextromethorphan impurity NFM(l) Linearity graph of Dextromethorphan impurity NFO

8 Chromatography Research International

Table 3 System suitability results

Parameter RSDlowast of standard Theoretical plateslowast Tailing factorlowast Resolution 1 Resolution 2GN DN GN DN GN DN

As such method 05 07 45320 498659 10 10 27 23At 06mLmin flow rate 12 06 41091 495497 10 10 29 25At 10mLmin flow rate 09 04 45828 515551 10 10 25 22At 50∘C column temperature 15 08 45321 508966 10 10 26 23At 60∘C column temperature 21 23 40929 515807 10 10 29 26At pH 28 (buffer pH) 11 05 43970 463987 10 10 24 23At pH 32 (buffer pH) 07 15 44619 532891 10 10 26 27lowastAverage of six replicate standard injections

Table 4 (a) Forced degradation data for Guaifenesin (b) Forced degradation data for Dextromethorphan

(a)

Degradation conditions Guaifenesin degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 million lux hours followed by 200Watt hours) 006 3585 4142 1002Exposed to humidity at 25∘C and 90 RH for about 7 days 007 3881 4241 998Refluxed with purified water for about 12 hours at 60∘C 005 3285 3845 986Refluxed with 05 N HCL solution for about 2 hours at 60∘C 010 5293 7073 992Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 006 3067 3489 989Refluxed with 10 H2O2 solution for about 30min at 60∘C 013 2755 3607 983Exposed to dry heat for about 15 hours at 105∘C 007 3346 4331 994

(b)

Degradation conditions Dextromethorphan degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 Million lux hours followed by 200Watt hours) 001 0115 0242 998Exposed to humidity at 25∘C and 90 RH for about 7 days 001 0124 0261 995Refluxed with purified water for about 12 hours at 60∘C 001 0101 0261 989Refluxed with 05 N HCL solution for about 2 hours at 60∘C 002 0154 0262 981Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 001 0103 0256 992Refluxed with 10 H2O2 solution for about 30min at 60∘C 105 0102 0250 997Exposed to dry heat for about 15 hours at 105∘C 001 0099 0261 987

Table 5 Results of precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0198 0201 0189 0213 0205 0209 0203 42GN-Impurity B 0209 0195 0192 0201 0206 0194 0200 35GN-Impurity C 0189 0195 0186 0192 0198 0184 0191 28GN-Impurity D 0208 0190 0192 0193 0188 0186 0193 41DN-Impurity A 0215 0214 0215 0209 0212 0205 0212 19DN-Impurity B 0204 0200 0202 0203 0205 0191 0201 25DN-Impurity C 0195 0186 0198 0199 0184 0194 0193 32DN-N-oxide 0216 0214 0218 0219 0211 0210 0215 17DN-NFM Impurity 0216 0218 0215 0215 0216 0212 0215 09DN-NFO Impurity 0198 0195 0213 0192 0214 0215 0205 52GN 0203 0212 0204 0206 0205 0210 0207 17DN 0182 0189 0194 0188 0187 0201 0190 34

Chromatography Research International 9

Table 6 Results of intermediate precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0191 0204 0201 0199 0210 0212 0203 38GN-Impurity B 0212 0204 0210 0209 0211 0204 0208 17GN-Impurity C 0192 0198 0191 0194 0202 0209 0198 35GN-Impurity D 0185 0192 0191 0194 0193 0192 0192 15DN-Impurity A 0202 0202 0203 0202 0202 0204 0203 04DN-Impurity B 0191 0188 0204 0209 0189 0202 0197 45DN-Impurity C 0183 0189 0191 0186 0205 0206 0193 51DN-N-oxide 0197 0210 0213 0202 0198 0212 0205 35DN-NFM Impurity 0183 0187 0179 0184 0185 0188 0184 17DN-NFO Impurity 0206 0197 0209 0212 0194 0208 0204 35GN 0199 0205 0201 0204 0207 0208 0204 17DN 0187 0185 0192 0186 0194 0191 0189 19

Table 7 LOD LOQ and precision data

Compound LOD () LOQ () SN ratio RSD at LOQ Level precisionLOD LOQ

GN-Impurity A 0004 0001 27 95 42GN-Impurity B 0008 0002 29 98 25GN-Impurity C 0005 0002 30 98 31GN-Impurity D 0003 0001 29 97 25DN-Impurity A 0014 0004 28 101 11DN-Impurity B 0021 0006 27 104 34DN-Impurity C 0023 0007 28 99 49DN-N-oxide 0016 0005 31 98 22DN-NFM Impurity 0016 0005 27 101 15DN-NFO Impurity 0041 0012 29 106 42GN 0006 0002 31 98 21DN 0015 0005 28 96 28

replicate injections is notmore than 50 resolution betweenimpurities 20 the tailing factor for GN and DN is not morethan 15 and the theoretical plates are not less than 5000

422 Specificity All forced degradation samples were ana-lyzed with the aforementioned HPLC conditions using aPDA detector to monitor the homogeneity and purity of theGN DN and their related impurities Individual impuritiesplacebo GN and DN were verified and proved to be nonin-terfering with each other thus proving the specificity of themethod

Figure 3 shows that there is no interference at the RT(retention time) of GN DN and all known impurities fromthe other excipients Degradation was not observed in pho-tolytic stress humidity acid hydrolysis base hydrolysis waterhydrolysis and thermal stress studies Significant degradationwas observed in oxidative conditions The typical oxidativestressed chromatogram was shown in Figure 4 It was inter-esting to note that all the peaks due to degradation were well

resolved from the peaks of GN DN and their impuritiesFurther the peak purity of GN DN and their impuritieswas found to be homogeneous based on the evaluationparameters such as purity angle and purity threshold usingWaters Empower Networking Software The verification ofpeak purity indicates that there is no interference fromdegradants facilitating error-free quantification of GN andDN impurities Also themass balance of stressed samples wasfound to be more than 98 Thus the method is consideredto be ldquostability-indicatingrdquo The specificity results were shownin Tables 4(a) and 4(b)

423 Precision The RSD for the individual of allimpurities in impurities method precision study was within34 The results obtained in the intermediated precisionstudy for the RSD of the individual of all impurities werewell within 41 conforming high precision of the methodThe results are shown in Tables 5 and 6

10 Chromatography Research International

Table 8 Accuracy of the method

Compound Recovery at each levela

LOQ 01 02 04 08 10GN-Impurity A 985 995 1012 967 999 1035GN-Impurity B 1056 1027 997 1051 985 975GN-Impurity C 1026 1056 1019 1061 1035 1021GN-Impurity D 956 972 965 958 965 985DN-Impurity A 935 965 952 942 985 967DN-Impurity B 985 967 999 989 975 965DN-Impurity C 1011 1023 1025 1037 1025 1018DN-N-oxide 1056 1065 1051 1045 1029 1037DN-NFM Impurity 1015 985 1025 1012 995 986DN-NFO Impurity 1071 1056 1069 1058 1065 1058GN 985 965 972 968 974 987DN 992 985 971 995 1005 1012aAverage of six determinations at LOQ level amp 10 level and three determinations at remaining levels

Table 9 Regression statistics

Substance Linearity range (120583gmLminus1) Correlation coefficient (119877) 119884-intercept bias in GN-Impurity A 093 to 23961 0999 09GN-Impurity B 201 to 24278 0999 10GN-Impurity C 126 to 24192 1000 07GN-Impurity D 079 to 24255 0999 08DN-Impurity A 017 to 1410 1000 04DN-Impurity B 025 to 1010 1000 01DN-Impurity C 027 to 1223 1000 08DN-N-oxide 019 to 1249 1000 06DN-NFM Impurity 019 to 1219 0998 15DN-NFO Impurity 048 to 1222 1000 09GN 153 to 24478 0999 16DN 018 to 1215 1000 01

Table 10 Results of robustness study

S no Impurity name

RRTrsquos of impurities

As suchmethod

At06mLminflow rate

At10mLminflow rate

At 50∘Ccolumn

temperature

At 60∘Ccolumn

temperature

At pH 28(buffer pH)

At pH 32(buffer pH)

1 GN-Impurity A 074 076 073 072 071 074 073

2 GN-Impurity B 112 111 113 111 110 112 113

3 GN-Impurity C 204 206 205 205 202 204 205

4 GN-Impurity D 214 212 215 213 211 213 214

5 DN-Impurity B 083 082 081 082 082 083 082

6 DN-Impurity C 098 097 098 098 097 097 098

7 DN-N-oxide 101 102 103 102 103 102 104

8 DN-Impurity A 103 104 105 104 105 104 106

9 DN-NFM Impurity 128 131 130 131 129 128 119

10 DN-NFO Impurity 138 141 142 139 139 139 141Note GN known impurities RRTs were calculated against GN main peak and DN known impurities RRTs were calculated against DN main peak

Chromatography Research International 11

424 Limit of Detection (LOD) and Limit of Quantification(LOQ) The determined limit of detection limit of quantifi-cation and precision at LOQ values for GN DN and theirimpurities were reported inTable 7TheRSD for peak areas ofGNDN and their related impurities at limit of quantificationlevel was within 100

425 Accuracy The recovery of all the impurities fromfinished pharmaceutical dosage form ranged from 850 to1150 The summary of recovery for individual impuritywas mentioned in Table 8

426 Linearity Linear calibration plot for the related sub-stance method was obtained over the calibration rangestested that is LOQ to 10 of the target test concentrationThe correlation coefficient obtained was greater than 0997for all the componentsThe slope and y-intercept values werealso provided in Table 9 which confirmed good linearitybetween peak areas and concentration The linearity graphswere shown in Figure 5(a) to Figure 5(l)

427 Robustness No significant effect was observed onsystem suitability parameters such as resolution RSD tailingfactor RRTs of impurities or the theoretical plates of GNand DN when small but deliberate changes were made tochromatographic conditions The results were presented inTables 3 and 10 along with the system suitability parametersof normal conditions Thus the method was found to berobust with respect to variability in applied conditions

428 Stability in Solution and in the Mobile Phase Nosignificant changeswere observed in the content of impuritiesduring solution stability and mobile phase stability exper-iments when performed using the impurities method Thesolution stability and mobile phase stability experiment dataconfirms that the sample solution and mobile phases usedduring the impurity determination were stable for at least48 h

5 Conclusions

The gradient HPLC method developed for the simultaneousdetermination of GN and DN impurities in pharmaceuticaldosage form was precise accurate and specific The methodis validated as per ICH guidelines and found to be specificprecise linear accurate rugged and robust The developedmethod can be used for the stability analysis of GN and DNeither individually or in their combination dosage forms

Acknowledgments

The authors wish to thank the management of Dr Reddyrsquosgroup for supporting this workThe authors wish to acknowl-edge the formulation development group for providing thesamples for their research They would also like to thankcolleagues in bulkmanufacturers for providing chemicals andimpurity standards for their research work

References

[1] httpwwwrxlistcomorganidin-nr-drughtm[2] W Zhang TWang L Qin et al ldquoNeuroprotective effect of dex-

tromethorphan in the MPTP Parkinsonrsquos disease model role ofNADPHoxidaserdquoTheFASEB Journal vol 18 no 3 pp 589ndash5912004

[3] Dextromethorphan NHTSA[4] ldquoChild deaths lead to FDA hearing on cough cold medsrdquo 2007

httpwwwcnncom[5] B KuKanich and M G Papich ldquoPlasma profile and pharmac-

okinetics of dextromethorphan after intravenous and oraladministration in healthy dogsrdquo Journal of Veterinary Pharma-cology andTherapeutics vol 27 no 5 pp 337ndash341 2004

[6] ldquoKidsrsquo cough medicine no better than placebordquo San FranciscoChronicle 2004

[7] I M Paul K E Yoder K R Crowell et al ldquoEffect of dextro-methorphan diphenhydramine and placebo on nocturnalcough and sleep quality for coughing children and their par-entsrdquo Pediatrics vol 114 no 1 pp e85ndashe90 2004

[8] S Gorog M Babjak G Balogh et al ldquoDrug impurity profilingstrategiesrdquo Talanta vol 44 no 9 pp 1517ndash1526 1997

[9] B B Sanjay R K Bharati S J Yogini and A S Atul ldquoImpurityprofile significance in active pharmaceutical ingredientrdquo Eura-sian Journal of Analytical Chemistry vol 2 pp 32ndash53 2007

[10] ICH ldquoImpurities in new drug substances Q3A (R2)rdquo 2006[11] ICH ldquoImpurities in new drug products Q3B (R2)rdquo 2006[12] G Grosa E D Grosso R Russo and G Allegrone ldquoSimulta-

neous stability indicating HPLC-DAD determination ofguaifenesin and methyl and propyl-parabens in cough syruprdquoJournal of Pharmaceutical and Biomedical Analysis vol 41 no3 pp 798ndash803 2006

[13] M Senthilraja and P Giriraj ldquoReverse phase hplc method forthe simultaneous estimation of terbutanile sulphate bromhex-ine HCl and guaifenesin in cough syruprdquo Asian Journal ofPharmaceutical and Clinical Research vol 4 no 2 pp 13ndash152011

[14] M L Wilcox and J T Stewart ldquoHPLC determination of guaife-nesin with selected medications on underivatized silica with anaqueous-organic mobile phaserdquo Journal of Pharmaceutical andBiomedical Analysis vol 23 no 5 pp 909ndash916 2000

[15] A Ozdemir H Aksoy E Dinc D Baleanu and S Dermis ldquoDe-termination of guaifenesin and dextromethorphan in a coughsyrup by HPLC with fluorometric detectionrdquo Revue Roumainede Chimie vol 51 no 2 pp 117ndash122 2006

[16] I Demian ldquoHigh-performance liquid chromatography (HPLC)chiral separations of guaifenesin methocarbamol and race-morphanrdquo Chirality vol 5 no 4 pp 238ndash240 1993

[17] S Suzen C Akay and S Cevheroglu ldquoSimultaneous determi-nation of guaiphenesin and codeine phosphate in tablets byhigh-performance liquid chromatographyrdquo Farmaco vol 54no 10 pp 705ndash709 1999

[18] S M Amer S S Abbas M A Shehata and NM Ali ldquoSimulta-neous determination of phenylephrine hydrochloride guaifen-esin and chlorpheniraminemaleate in cough syrup by gradientliquid chromatographyrdquo Journal of AOAC International vol 91no 2 pp 276ndash284 2008

[19] V Galli andC Barbas ldquoHigh-performance liquid chromatogra-phic analysis of dextromethorphan guaifenesin and benzoate ina cough syrup for stability testingrdquo Journal of ChromatographyA vol 1048 no 2 pp 207ndash211 2004

12 Chromatography Research International

[20] X Chen J Huang Z Kong and D Zhong ldquoSensitive liquidchromatography-tandem mass spectrometry method for thesimultaneous determination of paracetamol and guaifenesin inhuman plasmardquo Journal of Chromatography B vol 817 no 2 pp263ndash269 2005

[21] R M Gudipati J E Wallace and S A Stavchansky ldquoHigh per-formance liquid chromatography determination of guaifenesinin dog plasmardquo Analytical Letters vol 24 pp 265ndash274 1991

[22] Q-H Ge Z Zhou X-J Zhi L-L Ma and C-G Ding ldquoSim-ultaneous determination of guaifenesin bromhexine andambroxol in human plasma by LC-MSMS and its pharmacoki-netic studiesrdquo Chinese Pharmaceutical Journal vol 44 no 13pp 1025ndash1028 2009

[23] R Rajagopalan ldquoReview of regulatory guidance on impuritiesrdquoSeparation Science and Technology vol 5 pp 27ndash37 2004

[24] ICH Harmonized Tripartite Guideline Validation of AnalyticalProcedures Text and Methodology Q2(R1) 2005

[25] ICH ldquoStability testing of new drug substances and productsQ1A (R2)rdquo 2003

[26] ICH ldquoPhotostability testing of new drug substances and prod-ucts Q1Brdquo 1996

[27] The United States Pharmacopeia USP35-NF30 3829ndash3837[28] European Pharmacopeia 70 1821-1822 amp 2128-2129[29] P Sunil Reddy K Sudhakar Babu N Kumar and Y V V Sasi

Sekhar ldquoDevelopment and validation of stability indicating theRP-HPLC method for the estimation of related compounds ofguaifenesin in pharmaceutical dosage formsrdquo PharmaceuticalMethods vol 2 pp 229ndash234 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

6 Chromatography Research International

020

018

016

014

012

010

008

006

004

002

000000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 2 Overlaid chromatogram of blank and system suitabilitypreparation

020

018

016

014

012

010

008

006

004

002

000

000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 3 Overlaid chromatogram of placebo and spiked testpreparation

stability superior peak shapes and high efficiency) Since wewere using a very high concentration ofGN (24000120583gmLminus1)it was decided to use Sunfire column for further developmenttrails by using ion pair reagent in the mobile phase for betterseparation between DN DN-Impurity A and DN-ImpurityB Separation was achieved between all the pairs of peaks butpeak shapes of DN-NFM Impurity and DN-NFO Impurityare not sharp Acetonitrile was added in the mobile phase inaddition to methanol to get sharp peaks

The chromatographic separation was achieved by areversed phase Sunfire C18 250 times 46mm 5120583m particlesize column operated at 50∘C with gradient elution at

020

018

016

014

012

010

008

006

004

002

000

000 1000 2000 3000 4000 5000 6000 7000

(AU

)

(minutes)

Figure 4 Typical chromatogram of peroxide stressed sample

08mLminminus1 using a mobile phase buffer as a mixtureof 001M sodium dihydrogen phosphate monohydrate and00046M 1-octane sulfonic acid sodium salt monohydrateof pH 30 (pH adjusted with diluted orthophosphoric acid)UV absorbance at 224 nm and injection volume 20 120583L Themobile phase A consisted of pH 30 buffer and acetonitrile(90 10 vv) mobile phase B consisted of pH 30 bufferacetonitrile andmethanol (10 10 80 vvv)The LC gradientprogram was set as time (min) mobile phase B 00115155 2030 3050 6085 6515 and 7515 All the impuritieswere well separated with a resolution greater than 2 Nochromatographic interference due to the blank (diluent)and other excipients (placebo) at the retention time of GNDN and their impurities was observed The typical overlaychromatogram of blank and system suitability solution andspiked test is shown in Figures 2 and 3

42MethodValidation After the development of themethodit was subject to method validation as per ICH guidelines[24] The method was validated to demonstrate that it issuitable for its intended purpose by the standard procedureto evaluate adequate validation characteristics (system suit-ability specificity accuracy precision linearity robustnessruggedness solution stability LOD and LOQ and stability-indicating capability)

421 System Suitability The percentage relative standarddeviation (RSD) of area from six replicate injections wasbelow 50 (diluted standard solution 48 120583gmLminus1 of GNand 24 120583gmLminus1 of DN) Low values of RSD for replicateinjections indicate that the system is precise The results ofother system suitability parameters such as resolution peaktailing and theoretical plates are presented in Table 3 As seenfrom this data the acceptable system suitability parameterswould be as follows the relative standard deviation of

Chromatography Research International 7

000200000000400000000600000000800000000

1000000000120000000014000000001600000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesiny = 56119662x + 118692128

R2 = 0999

(a)

02000000400000060000008000000

10000000120000001400000016000000

0000 50000 100000 150000 200000 250000 300000

Linearity of detector response of Guaifenesin impurity Ay = 62278038x + 138787601

R2 = 0998

Are

a

(b)

02000000400000060000008000000

100000001200000014000000

0000 50000 100000 150000 200000 250000 300000

Linearity of detector response of Guaifenesin impurity By = 46707746x + 109527559

R2 = 0998

Are

a

(c)

02000000400000060000008000000

100000001200000014000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesin impurity Cy = 49 606000x +

R2 = 0999

78596824

(d)

0

5000000

10000000

15000000

20000000

25000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesin impurity Dy = 79962049x + 156337857

R2 = 0999

(e)

050000

100000150000200000250000300000350000400000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphany = 30824867x + 4487696

R2 = 1000

(f)

050000

100000150000200000250000300000350000400000450000500000

0000 2000 4000 6000 8000 10000 12000 14000 16000

Are

a

Linearity of detector response of Dextromethorphan impurity A

y = 32920087x + 331349

R2 = 1000

(g)

050000

100000150000200000250000300000

0000 2000 4000 6000 8000 10000 12000

Are

a

Linearity of detector response of Dextromethorphan impurity B

y = 25035731x minus 57593

R2 = 1000

(h)

050000

100000150000200000250000300000350000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphan impurity C

y = 24213547x + 450726

R2 = 1000

(i)

050000

100000150000200000250000300000350000400000450000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphan N-oxide

y = 33 682946x + 523536

R2 = 1000

(j)

0100000200000300000400000500000600000700000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Concentration (ppm)

Linearity of detector response of Dextromethorphan impurity NFM

y = 49617284x + 22697199

R2 = 0996

(k)

0100000200000300000400000500000600000700000800000900000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Concentration (ppm)

Linearity of detector response of Dextromethorphan impurity NFO

y = 66581365x minus 1352313

R2 = 1000

(l)

Figure 5 (a) Linearity graph of Guaifenesin (b) Linearity graph of Guaifenesin impurity A (c) Linearity graph of Guaifenesin impurity B(d) Linearity graph of Guaifenesin impurity C (e) Linearity graph of Guaifenesin impurity D (f) Linearity graph of Dextromethorphan(g) Linearity graph of Dextromethorphan impurity A (h) Linearity graph of Dextromethorphan impurity B (i) Linearity graph ofDextromethorphan impurity C (j) Linearity graph of DextromethorphanN-oxide (k) Linearity graph of Dextromethorphan impurity NFM(l) Linearity graph of Dextromethorphan impurity NFO

8 Chromatography Research International

Table 3 System suitability results

Parameter RSDlowast of standard Theoretical plateslowast Tailing factorlowast Resolution 1 Resolution 2GN DN GN DN GN DN

As such method 05 07 45320 498659 10 10 27 23At 06mLmin flow rate 12 06 41091 495497 10 10 29 25At 10mLmin flow rate 09 04 45828 515551 10 10 25 22At 50∘C column temperature 15 08 45321 508966 10 10 26 23At 60∘C column temperature 21 23 40929 515807 10 10 29 26At pH 28 (buffer pH) 11 05 43970 463987 10 10 24 23At pH 32 (buffer pH) 07 15 44619 532891 10 10 26 27lowastAverage of six replicate standard injections

Table 4 (a) Forced degradation data for Guaifenesin (b) Forced degradation data for Dextromethorphan

(a)

Degradation conditions Guaifenesin degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 million lux hours followed by 200Watt hours) 006 3585 4142 1002Exposed to humidity at 25∘C and 90 RH for about 7 days 007 3881 4241 998Refluxed with purified water for about 12 hours at 60∘C 005 3285 3845 986Refluxed with 05 N HCL solution for about 2 hours at 60∘C 010 5293 7073 992Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 006 3067 3489 989Refluxed with 10 H2O2 solution for about 30min at 60∘C 013 2755 3607 983Exposed to dry heat for about 15 hours at 105∘C 007 3346 4331 994

(b)

Degradation conditions Dextromethorphan degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 Million lux hours followed by 200Watt hours) 001 0115 0242 998Exposed to humidity at 25∘C and 90 RH for about 7 days 001 0124 0261 995Refluxed with purified water for about 12 hours at 60∘C 001 0101 0261 989Refluxed with 05 N HCL solution for about 2 hours at 60∘C 002 0154 0262 981Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 001 0103 0256 992Refluxed with 10 H2O2 solution for about 30min at 60∘C 105 0102 0250 997Exposed to dry heat for about 15 hours at 105∘C 001 0099 0261 987

Table 5 Results of precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0198 0201 0189 0213 0205 0209 0203 42GN-Impurity B 0209 0195 0192 0201 0206 0194 0200 35GN-Impurity C 0189 0195 0186 0192 0198 0184 0191 28GN-Impurity D 0208 0190 0192 0193 0188 0186 0193 41DN-Impurity A 0215 0214 0215 0209 0212 0205 0212 19DN-Impurity B 0204 0200 0202 0203 0205 0191 0201 25DN-Impurity C 0195 0186 0198 0199 0184 0194 0193 32DN-N-oxide 0216 0214 0218 0219 0211 0210 0215 17DN-NFM Impurity 0216 0218 0215 0215 0216 0212 0215 09DN-NFO Impurity 0198 0195 0213 0192 0214 0215 0205 52GN 0203 0212 0204 0206 0205 0210 0207 17DN 0182 0189 0194 0188 0187 0201 0190 34

Chromatography Research International 9

Table 6 Results of intermediate precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0191 0204 0201 0199 0210 0212 0203 38GN-Impurity B 0212 0204 0210 0209 0211 0204 0208 17GN-Impurity C 0192 0198 0191 0194 0202 0209 0198 35GN-Impurity D 0185 0192 0191 0194 0193 0192 0192 15DN-Impurity A 0202 0202 0203 0202 0202 0204 0203 04DN-Impurity B 0191 0188 0204 0209 0189 0202 0197 45DN-Impurity C 0183 0189 0191 0186 0205 0206 0193 51DN-N-oxide 0197 0210 0213 0202 0198 0212 0205 35DN-NFM Impurity 0183 0187 0179 0184 0185 0188 0184 17DN-NFO Impurity 0206 0197 0209 0212 0194 0208 0204 35GN 0199 0205 0201 0204 0207 0208 0204 17DN 0187 0185 0192 0186 0194 0191 0189 19

Table 7 LOD LOQ and precision data

Compound LOD () LOQ () SN ratio RSD at LOQ Level precisionLOD LOQ

GN-Impurity A 0004 0001 27 95 42GN-Impurity B 0008 0002 29 98 25GN-Impurity C 0005 0002 30 98 31GN-Impurity D 0003 0001 29 97 25DN-Impurity A 0014 0004 28 101 11DN-Impurity B 0021 0006 27 104 34DN-Impurity C 0023 0007 28 99 49DN-N-oxide 0016 0005 31 98 22DN-NFM Impurity 0016 0005 27 101 15DN-NFO Impurity 0041 0012 29 106 42GN 0006 0002 31 98 21DN 0015 0005 28 96 28

replicate injections is notmore than 50 resolution betweenimpurities 20 the tailing factor for GN and DN is not morethan 15 and the theoretical plates are not less than 5000

422 Specificity All forced degradation samples were ana-lyzed with the aforementioned HPLC conditions using aPDA detector to monitor the homogeneity and purity of theGN DN and their related impurities Individual impuritiesplacebo GN and DN were verified and proved to be nonin-terfering with each other thus proving the specificity of themethod

Figure 3 shows that there is no interference at the RT(retention time) of GN DN and all known impurities fromthe other excipients Degradation was not observed in pho-tolytic stress humidity acid hydrolysis base hydrolysis waterhydrolysis and thermal stress studies Significant degradationwas observed in oxidative conditions The typical oxidativestressed chromatogram was shown in Figure 4 It was inter-esting to note that all the peaks due to degradation were well

resolved from the peaks of GN DN and their impuritiesFurther the peak purity of GN DN and their impuritieswas found to be homogeneous based on the evaluationparameters such as purity angle and purity threshold usingWaters Empower Networking Software The verification ofpeak purity indicates that there is no interference fromdegradants facilitating error-free quantification of GN andDN impurities Also themass balance of stressed samples wasfound to be more than 98 Thus the method is consideredto be ldquostability-indicatingrdquo The specificity results were shownin Tables 4(a) and 4(b)

423 Precision The RSD for the individual of allimpurities in impurities method precision study was within34 The results obtained in the intermediated precisionstudy for the RSD of the individual of all impurities werewell within 41 conforming high precision of the methodThe results are shown in Tables 5 and 6

10 Chromatography Research International

Table 8 Accuracy of the method

Compound Recovery at each levela

LOQ 01 02 04 08 10GN-Impurity A 985 995 1012 967 999 1035GN-Impurity B 1056 1027 997 1051 985 975GN-Impurity C 1026 1056 1019 1061 1035 1021GN-Impurity D 956 972 965 958 965 985DN-Impurity A 935 965 952 942 985 967DN-Impurity B 985 967 999 989 975 965DN-Impurity C 1011 1023 1025 1037 1025 1018DN-N-oxide 1056 1065 1051 1045 1029 1037DN-NFM Impurity 1015 985 1025 1012 995 986DN-NFO Impurity 1071 1056 1069 1058 1065 1058GN 985 965 972 968 974 987DN 992 985 971 995 1005 1012aAverage of six determinations at LOQ level amp 10 level and three determinations at remaining levels

Table 9 Regression statistics

Substance Linearity range (120583gmLminus1) Correlation coefficient (119877) 119884-intercept bias in GN-Impurity A 093 to 23961 0999 09GN-Impurity B 201 to 24278 0999 10GN-Impurity C 126 to 24192 1000 07GN-Impurity D 079 to 24255 0999 08DN-Impurity A 017 to 1410 1000 04DN-Impurity B 025 to 1010 1000 01DN-Impurity C 027 to 1223 1000 08DN-N-oxide 019 to 1249 1000 06DN-NFM Impurity 019 to 1219 0998 15DN-NFO Impurity 048 to 1222 1000 09GN 153 to 24478 0999 16DN 018 to 1215 1000 01

Table 10 Results of robustness study

S no Impurity name

RRTrsquos of impurities

As suchmethod

At06mLminflow rate

At10mLminflow rate

At 50∘Ccolumn

temperature

At 60∘Ccolumn

temperature

At pH 28(buffer pH)

At pH 32(buffer pH)

1 GN-Impurity A 074 076 073 072 071 074 073

2 GN-Impurity B 112 111 113 111 110 112 113

3 GN-Impurity C 204 206 205 205 202 204 205

4 GN-Impurity D 214 212 215 213 211 213 214

5 DN-Impurity B 083 082 081 082 082 083 082

6 DN-Impurity C 098 097 098 098 097 097 098

7 DN-N-oxide 101 102 103 102 103 102 104

8 DN-Impurity A 103 104 105 104 105 104 106

9 DN-NFM Impurity 128 131 130 131 129 128 119

10 DN-NFO Impurity 138 141 142 139 139 139 141Note GN known impurities RRTs were calculated against GN main peak and DN known impurities RRTs were calculated against DN main peak

Chromatography Research International 11

424 Limit of Detection (LOD) and Limit of Quantification(LOQ) The determined limit of detection limit of quantifi-cation and precision at LOQ values for GN DN and theirimpurities were reported inTable 7TheRSD for peak areas ofGNDN and their related impurities at limit of quantificationlevel was within 100

425 Accuracy The recovery of all the impurities fromfinished pharmaceutical dosage form ranged from 850 to1150 The summary of recovery for individual impuritywas mentioned in Table 8

426 Linearity Linear calibration plot for the related sub-stance method was obtained over the calibration rangestested that is LOQ to 10 of the target test concentrationThe correlation coefficient obtained was greater than 0997for all the componentsThe slope and y-intercept values werealso provided in Table 9 which confirmed good linearitybetween peak areas and concentration The linearity graphswere shown in Figure 5(a) to Figure 5(l)

427 Robustness No significant effect was observed onsystem suitability parameters such as resolution RSD tailingfactor RRTs of impurities or the theoretical plates of GNand DN when small but deliberate changes were made tochromatographic conditions The results were presented inTables 3 and 10 along with the system suitability parametersof normal conditions Thus the method was found to berobust with respect to variability in applied conditions

428 Stability in Solution and in the Mobile Phase Nosignificant changeswere observed in the content of impuritiesduring solution stability and mobile phase stability exper-iments when performed using the impurities method Thesolution stability and mobile phase stability experiment dataconfirms that the sample solution and mobile phases usedduring the impurity determination were stable for at least48 h

5 Conclusions

The gradient HPLC method developed for the simultaneousdetermination of GN and DN impurities in pharmaceuticaldosage form was precise accurate and specific The methodis validated as per ICH guidelines and found to be specificprecise linear accurate rugged and robust The developedmethod can be used for the stability analysis of GN and DNeither individually or in their combination dosage forms

Acknowledgments

The authors wish to thank the management of Dr Reddyrsquosgroup for supporting this workThe authors wish to acknowl-edge the formulation development group for providing thesamples for their research They would also like to thankcolleagues in bulkmanufacturers for providing chemicals andimpurity standards for their research work

References

[1] httpwwwrxlistcomorganidin-nr-drughtm[2] W Zhang TWang L Qin et al ldquoNeuroprotective effect of dex-

tromethorphan in the MPTP Parkinsonrsquos disease model role ofNADPHoxidaserdquoTheFASEB Journal vol 18 no 3 pp 589ndash5912004

[3] Dextromethorphan NHTSA[4] ldquoChild deaths lead to FDA hearing on cough cold medsrdquo 2007

httpwwwcnncom[5] B KuKanich and M G Papich ldquoPlasma profile and pharmac-

okinetics of dextromethorphan after intravenous and oraladministration in healthy dogsrdquo Journal of Veterinary Pharma-cology andTherapeutics vol 27 no 5 pp 337ndash341 2004

[6] ldquoKidsrsquo cough medicine no better than placebordquo San FranciscoChronicle 2004

[7] I M Paul K E Yoder K R Crowell et al ldquoEffect of dextro-methorphan diphenhydramine and placebo on nocturnalcough and sleep quality for coughing children and their par-entsrdquo Pediatrics vol 114 no 1 pp e85ndashe90 2004

[8] S Gorog M Babjak G Balogh et al ldquoDrug impurity profilingstrategiesrdquo Talanta vol 44 no 9 pp 1517ndash1526 1997

[9] B B Sanjay R K Bharati S J Yogini and A S Atul ldquoImpurityprofile significance in active pharmaceutical ingredientrdquo Eura-sian Journal of Analytical Chemistry vol 2 pp 32ndash53 2007

[10] ICH ldquoImpurities in new drug substances Q3A (R2)rdquo 2006[11] ICH ldquoImpurities in new drug products Q3B (R2)rdquo 2006[12] G Grosa E D Grosso R Russo and G Allegrone ldquoSimulta-

neous stability indicating HPLC-DAD determination ofguaifenesin and methyl and propyl-parabens in cough syruprdquoJournal of Pharmaceutical and Biomedical Analysis vol 41 no3 pp 798ndash803 2006

[13] M Senthilraja and P Giriraj ldquoReverse phase hplc method forthe simultaneous estimation of terbutanile sulphate bromhex-ine HCl and guaifenesin in cough syruprdquo Asian Journal ofPharmaceutical and Clinical Research vol 4 no 2 pp 13ndash152011

[14] M L Wilcox and J T Stewart ldquoHPLC determination of guaife-nesin with selected medications on underivatized silica with anaqueous-organic mobile phaserdquo Journal of Pharmaceutical andBiomedical Analysis vol 23 no 5 pp 909ndash916 2000

[15] A Ozdemir H Aksoy E Dinc D Baleanu and S Dermis ldquoDe-termination of guaifenesin and dextromethorphan in a coughsyrup by HPLC with fluorometric detectionrdquo Revue Roumainede Chimie vol 51 no 2 pp 117ndash122 2006

[16] I Demian ldquoHigh-performance liquid chromatography (HPLC)chiral separations of guaifenesin methocarbamol and race-morphanrdquo Chirality vol 5 no 4 pp 238ndash240 1993

[17] S Suzen C Akay and S Cevheroglu ldquoSimultaneous determi-nation of guaiphenesin and codeine phosphate in tablets byhigh-performance liquid chromatographyrdquo Farmaco vol 54no 10 pp 705ndash709 1999

[18] S M Amer S S Abbas M A Shehata and NM Ali ldquoSimulta-neous determination of phenylephrine hydrochloride guaifen-esin and chlorpheniraminemaleate in cough syrup by gradientliquid chromatographyrdquo Journal of AOAC International vol 91no 2 pp 276ndash284 2008

[19] V Galli andC Barbas ldquoHigh-performance liquid chromatogra-phic analysis of dextromethorphan guaifenesin and benzoate ina cough syrup for stability testingrdquo Journal of ChromatographyA vol 1048 no 2 pp 207ndash211 2004

12 Chromatography Research International

[20] X Chen J Huang Z Kong and D Zhong ldquoSensitive liquidchromatography-tandem mass spectrometry method for thesimultaneous determination of paracetamol and guaifenesin inhuman plasmardquo Journal of Chromatography B vol 817 no 2 pp263ndash269 2005

[21] R M Gudipati J E Wallace and S A Stavchansky ldquoHigh per-formance liquid chromatography determination of guaifenesinin dog plasmardquo Analytical Letters vol 24 pp 265ndash274 1991

[22] Q-H Ge Z Zhou X-J Zhi L-L Ma and C-G Ding ldquoSim-ultaneous determination of guaifenesin bromhexine andambroxol in human plasma by LC-MSMS and its pharmacoki-netic studiesrdquo Chinese Pharmaceutical Journal vol 44 no 13pp 1025ndash1028 2009

[23] R Rajagopalan ldquoReview of regulatory guidance on impuritiesrdquoSeparation Science and Technology vol 5 pp 27ndash37 2004

[24] ICH Harmonized Tripartite Guideline Validation of AnalyticalProcedures Text and Methodology Q2(R1) 2005

[25] ICH ldquoStability testing of new drug substances and productsQ1A (R2)rdquo 2003

[26] ICH ldquoPhotostability testing of new drug substances and prod-ucts Q1Brdquo 1996

[27] The United States Pharmacopeia USP35-NF30 3829ndash3837[28] European Pharmacopeia 70 1821-1822 amp 2128-2129[29] P Sunil Reddy K Sudhakar Babu N Kumar and Y V V Sasi

Sekhar ldquoDevelopment and validation of stability indicating theRP-HPLC method for the estimation of related compounds ofguaifenesin in pharmaceutical dosage formsrdquo PharmaceuticalMethods vol 2 pp 229ndash234 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Chromatography Research International 7

000200000000400000000600000000800000000

1000000000120000000014000000001600000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesiny = 56119662x + 118692128

R2 = 0999

(a)

02000000400000060000008000000

10000000120000001400000016000000

0000 50000 100000 150000 200000 250000 300000

Linearity of detector response of Guaifenesin impurity Ay = 62278038x + 138787601

R2 = 0998

Are

a

(b)

02000000400000060000008000000

100000001200000014000000

0000 50000 100000 150000 200000 250000 300000

Linearity of detector response of Guaifenesin impurity By = 46707746x + 109527559

R2 = 0998

Are

a

(c)

02000000400000060000008000000

100000001200000014000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesin impurity Cy = 49 606000x +

R2 = 0999

78596824

(d)

0

5000000

10000000

15000000

20000000

25000000

0000 50000 100000 150000 200000 250000 300000

Are

a

Linearity of detector response of Guaifenesin impurity Dy = 79962049x + 156337857

R2 = 0999

(e)

050000

100000150000200000250000300000350000400000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphany = 30824867x + 4487696

R2 = 1000

(f)

050000

100000150000200000250000300000350000400000450000500000

0000 2000 4000 6000 8000 10000 12000 14000 16000

Are

a

Linearity of detector response of Dextromethorphan impurity A

y = 32920087x + 331349

R2 = 1000

(g)

050000

100000150000200000250000300000

0000 2000 4000 6000 8000 10000 12000

Are

a

Linearity of detector response of Dextromethorphan impurity B

y = 25035731x minus 57593

R2 = 1000

(h)

050000

100000150000200000250000300000350000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphan impurity C

y = 24213547x + 450726

R2 = 1000

(i)

050000

100000150000200000250000300000350000400000450000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Linearity of detector response of Dextromethorphan N-oxide

y = 33 682946x + 523536

R2 = 1000

(j)

0100000200000300000400000500000600000700000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Concentration (ppm)

Linearity of detector response of Dextromethorphan impurity NFM

y = 49617284x + 22697199

R2 = 0996

(k)

0100000200000300000400000500000600000700000800000900000

0000 2000 4000 6000 8000 10000 12000 14000

Are

a

Concentration (ppm)

Linearity of detector response of Dextromethorphan impurity NFO

y = 66581365x minus 1352313

R2 = 1000

(l)

Figure 5 (a) Linearity graph of Guaifenesin (b) Linearity graph of Guaifenesin impurity A (c) Linearity graph of Guaifenesin impurity B(d) Linearity graph of Guaifenesin impurity C (e) Linearity graph of Guaifenesin impurity D (f) Linearity graph of Dextromethorphan(g) Linearity graph of Dextromethorphan impurity A (h) Linearity graph of Dextromethorphan impurity B (i) Linearity graph ofDextromethorphan impurity C (j) Linearity graph of DextromethorphanN-oxide (k) Linearity graph of Dextromethorphan impurity NFM(l) Linearity graph of Dextromethorphan impurity NFO

8 Chromatography Research International

Table 3 System suitability results

Parameter RSDlowast of standard Theoretical plateslowast Tailing factorlowast Resolution 1 Resolution 2GN DN GN DN GN DN

As such method 05 07 45320 498659 10 10 27 23At 06mLmin flow rate 12 06 41091 495497 10 10 29 25At 10mLmin flow rate 09 04 45828 515551 10 10 25 22At 50∘C column temperature 15 08 45321 508966 10 10 26 23At 60∘C column temperature 21 23 40929 515807 10 10 29 26At pH 28 (buffer pH) 11 05 43970 463987 10 10 24 23At pH 32 (buffer pH) 07 15 44619 532891 10 10 26 27lowastAverage of six replicate standard injections

Table 4 (a) Forced degradation data for Guaifenesin (b) Forced degradation data for Dextromethorphan

(a)

Degradation conditions Guaifenesin degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 million lux hours followed by 200Watt hours) 006 3585 4142 1002Exposed to humidity at 25∘C and 90 RH for about 7 days 007 3881 4241 998Refluxed with purified water for about 12 hours at 60∘C 005 3285 3845 986Refluxed with 05 N HCL solution for about 2 hours at 60∘C 010 5293 7073 992Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 006 3067 3489 989Refluxed with 10 H2O2 solution for about 30min at 60∘C 013 2755 3607 983Exposed to dry heat for about 15 hours at 105∘C 007 3346 4331 994

(b)

Degradation conditions Dextromethorphan degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 Million lux hours followed by 200Watt hours) 001 0115 0242 998Exposed to humidity at 25∘C and 90 RH for about 7 days 001 0124 0261 995Refluxed with purified water for about 12 hours at 60∘C 001 0101 0261 989Refluxed with 05 N HCL solution for about 2 hours at 60∘C 002 0154 0262 981Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 001 0103 0256 992Refluxed with 10 H2O2 solution for about 30min at 60∘C 105 0102 0250 997Exposed to dry heat for about 15 hours at 105∘C 001 0099 0261 987

Table 5 Results of precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0198 0201 0189 0213 0205 0209 0203 42GN-Impurity B 0209 0195 0192 0201 0206 0194 0200 35GN-Impurity C 0189 0195 0186 0192 0198 0184 0191 28GN-Impurity D 0208 0190 0192 0193 0188 0186 0193 41DN-Impurity A 0215 0214 0215 0209 0212 0205 0212 19DN-Impurity B 0204 0200 0202 0203 0205 0191 0201 25DN-Impurity C 0195 0186 0198 0199 0184 0194 0193 32DN-N-oxide 0216 0214 0218 0219 0211 0210 0215 17DN-NFM Impurity 0216 0218 0215 0215 0216 0212 0215 09DN-NFO Impurity 0198 0195 0213 0192 0214 0215 0205 52GN 0203 0212 0204 0206 0205 0210 0207 17DN 0182 0189 0194 0188 0187 0201 0190 34

Chromatography Research International 9

Table 6 Results of intermediate precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0191 0204 0201 0199 0210 0212 0203 38GN-Impurity B 0212 0204 0210 0209 0211 0204 0208 17GN-Impurity C 0192 0198 0191 0194 0202 0209 0198 35GN-Impurity D 0185 0192 0191 0194 0193 0192 0192 15DN-Impurity A 0202 0202 0203 0202 0202 0204 0203 04DN-Impurity B 0191 0188 0204 0209 0189 0202 0197 45DN-Impurity C 0183 0189 0191 0186 0205 0206 0193 51DN-N-oxide 0197 0210 0213 0202 0198 0212 0205 35DN-NFM Impurity 0183 0187 0179 0184 0185 0188 0184 17DN-NFO Impurity 0206 0197 0209 0212 0194 0208 0204 35GN 0199 0205 0201 0204 0207 0208 0204 17DN 0187 0185 0192 0186 0194 0191 0189 19

Table 7 LOD LOQ and precision data

Compound LOD () LOQ () SN ratio RSD at LOQ Level precisionLOD LOQ

GN-Impurity A 0004 0001 27 95 42GN-Impurity B 0008 0002 29 98 25GN-Impurity C 0005 0002 30 98 31GN-Impurity D 0003 0001 29 97 25DN-Impurity A 0014 0004 28 101 11DN-Impurity B 0021 0006 27 104 34DN-Impurity C 0023 0007 28 99 49DN-N-oxide 0016 0005 31 98 22DN-NFM Impurity 0016 0005 27 101 15DN-NFO Impurity 0041 0012 29 106 42GN 0006 0002 31 98 21DN 0015 0005 28 96 28

replicate injections is notmore than 50 resolution betweenimpurities 20 the tailing factor for GN and DN is not morethan 15 and the theoretical plates are not less than 5000

422 Specificity All forced degradation samples were ana-lyzed with the aforementioned HPLC conditions using aPDA detector to monitor the homogeneity and purity of theGN DN and their related impurities Individual impuritiesplacebo GN and DN were verified and proved to be nonin-terfering with each other thus proving the specificity of themethod

Figure 3 shows that there is no interference at the RT(retention time) of GN DN and all known impurities fromthe other excipients Degradation was not observed in pho-tolytic stress humidity acid hydrolysis base hydrolysis waterhydrolysis and thermal stress studies Significant degradationwas observed in oxidative conditions The typical oxidativestressed chromatogram was shown in Figure 4 It was inter-esting to note that all the peaks due to degradation were well

resolved from the peaks of GN DN and their impuritiesFurther the peak purity of GN DN and their impuritieswas found to be homogeneous based on the evaluationparameters such as purity angle and purity threshold usingWaters Empower Networking Software The verification ofpeak purity indicates that there is no interference fromdegradants facilitating error-free quantification of GN andDN impurities Also themass balance of stressed samples wasfound to be more than 98 Thus the method is consideredto be ldquostability-indicatingrdquo The specificity results were shownin Tables 4(a) and 4(b)

423 Precision The RSD for the individual of allimpurities in impurities method precision study was within34 The results obtained in the intermediated precisionstudy for the RSD of the individual of all impurities werewell within 41 conforming high precision of the methodThe results are shown in Tables 5 and 6

10 Chromatography Research International

Table 8 Accuracy of the method

Compound Recovery at each levela

LOQ 01 02 04 08 10GN-Impurity A 985 995 1012 967 999 1035GN-Impurity B 1056 1027 997 1051 985 975GN-Impurity C 1026 1056 1019 1061 1035 1021GN-Impurity D 956 972 965 958 965 985DN-Impurity A 935 965 952 942 985 967DN-Impurity B 985 967 999 989 975 965DN-Impurity C 1011 1023 1025 1037 1025 1018DN-N-oxide 1056 1065 1051 1045 1029 1037DN-NFM Impurity 1015 985 1025 1012 995 986DN-NFO Impurity 1071 1056 1069 1058 1065 1058GN 985 965 972 968 974 987DN 992 985 971 995 1005 1012aAverage of six determinations at LOQ level amp 10 level and three determinations at remaining levels

Table 9 Regression statistics

Substance Linearity range (120583gmLminus1) Correlation coefficient (119877) 119884-intercept bias in GN-Impurity A 093 to 23961 0999 09GN-Impurity B 201 to 24278 0999 10GN-Impurity C 126 to 24192 1000 07GN-Impurity D 079 to 24255 0999 08DN-Impurity A 017 to 1410 1000 04DN-Impurity B 025 to 1010 1000 01DN-Impurity C 027 to 1223 1000 08DN-N-oxide 019 to 1249 1000 06DN-NFM Impurity 019 to 1219 0998 15DN-NFO Impurity 048 to 1222 1000 09GN 153 to 24478 0999 16DN 018 to 1215 1000 01

Table 10 Results of robustness study

S no Impurity name

RRTrsquos of impurities

As suchmethod

At06mLminflow rate

At10mLminflow rate

At 50∘Ccolumn

temperature

At 60∘Ccolumn

temperature

At pH 28(buffer pH)

At pH 32(buffer pH)

1 GN-Impurity A 074 076 073 072 071 074 073

2 GN-Impurity B 112 111 113 111 110 112 113

3 GN-Impurity C 204 206 205 205 202 204 205

4 GN-Impurity D 214 212 215 213 211 213 214

5 DN-Impurity B 083 082 081 082 082 083 082

6 DN-Impurity C 098 097 098 098 097 097 098

7 DN-N-oxide 101 102 103 102 103 102 104

8 DN-Impurity A 103 104 105 104 105 104 106

9 DN-NFM Impurity 128 131 130 131 129 128 119

10 DN-NFO Impurity 138 141 142 139 139 139 141Note GN known impurities RRTs were calculated against GN main peak and DN known impurities RRTs were calculated against DN main peak

Chromatography Research International 11

424 Limit of Detection (LOD) and Limit of Quantification(LOQ) The determined limit of detection limit of quantifi-cation and precision at LOQ values for GN DN and theirimpurities were reported inTable 7TheRSD for peak areas ofGNDN and their related impurities at limit of quantificationlevel was within 100

425 Accuracy The recovery of all the impurities fromfinished pharmaceutical dosage form ranged from 850 to1150 The summary of recovery for individual impuritywas mentioned in Table 8

426 Linearity Linear calibration plot for the related sub-stance method was obtained over the calibration rangestested that is LOQ to 10 of the target test concentrationThe correlation coefficient obtained was greater than 0997for all the componentsThe slope and y-intercept values werealso provided in Table 9 which confirmed good linearitybetween peak areas and concentration The linearity graphswere shown in Figure 5(a) to Figure 5(l)

427 Robustness No significant effect was observed onsystem suitability parameters such as resolution RSD tailingfactor RRTs of impurities or the theoretical plates of GNand DN when small but deliberate changes were made tochromatographic conditions The results were presented inTables 3 and 10 along with the system suitability parametersof normal conditions Thus the method was found to berobust with respect to variability in applied conditions

428 Stability in Solution and in the Mobile Phase Nosignificant changeswere observed in the content of impuritiesduring solution stability and mobile phase stability exper-iments when performed using the impurities method Thesolution stability and mobile phase stability experiment dataconfirms that the sample solution and mobile phases usedduring the impurity determination were stable for at least48 h

5 Conclusions

The gradient HPLC method developed for the simultaneousdetermination of GN and DN impurities in pharmaceuticaldosage form was precise accurate and specific The methodis validated as per ICH guidelines and found to be specificprecise linear accurate rugged and robust The developedmethod can be used for the stability analysis of GN and DNeither individually or in their combination dosage forms

Acknowledgments

The authors wish to thank the management of Dr Reddyrsquosgroup for supporting this workThe authors wish to acknowl-edge the formulation development group for providing thesamples for their research They would also like to thankcolleagues in bulkmanufacturers for providing chemicals andimpurity standards for their research work

References

[1] httpwwwrxlistcomorganidin-nr-drughtm[2] W Zhang TWang L Qin et al ldquoNeuroprotective effect of dex-

tromethorphan in the MPTP Parkinsonrsquos disease model role ofNADPHoxidaserdquoTheFASEB Journal vol 18 no 3 pp 589ndash5912004

[3] Dextromethorphan NHTSA[4] ldquoChild deaths lead to FDA hearing on cough cold medsrdquo 2007

httpwwwcnncom[5] B KuKanich and M G Papich ldquoPlasma profile and pharmac-

okinetics of dextromethorphan after intravenous and oraladministration in healthy dogsrdquo Journal of Veterinary Pharma-cology andTherapeutics vol 27 no 5 pp 337ndash341 2004

[6] ldquoKidsrsquo cough medicine no better than placebordquo San FranciscoChronicle 2004

[7] I M Paul K E Yoder K R Crowell et al ldquoEffect of dextro-methorphan diphenhydramine and placebo on nocturnalcough and sleep quality for coughing children and their par-entsrdquo Pediatrics vol 114 no 1 pp e85ndashe90 2004

[8] S Gorog M Babjak G Balogh et al ldquoDrug impurity profilingstrategiesrdquo Talanta vol 44 no 9 pp 1517ndash1526 1997

[9] B B Sanjay R K Bharati S J Yogini and A S Atul ldquoImpurityprofile significance in active pharmaceutical ingredientrdquo Eura-sian Journal of Analytical Chemistry vol 2 pp 32ndash53 2007

[10] ICH ldquoImpurities in new drug substances Q3A (R2)rdquo 2006[11] ICH ldquoImpurities in new drug products Q3B (R2)rdquo 2006[12] G Grosa E D Grosso R Russo and G Allegrone ldquoSimulta-

neous stability indicating HPLC-DAD determination ofguaifenesin and methyl and propyl-parabens in cough syruprdquoJournal of Pharmaceutical and Biomedical Analysis vol 41 no3 pp 798ndash803 2006

[13] M Senthilraja and P Giriraj ldquoReverse phase hplc method forthe simultaneous estimation of terbutanile sulphate bromhex-ine HCl and guaifenesin in cough syruprdquo Asian Journal ofPharmaceutical and Clinical Research vol 4 no 2 pp 13ndash152011

[14] M L Wilcox and J T Stewart ldquoHPLC determination of guaife-nesin with selected medications on underivatized silica with anaqueous-organic mobile phaserdquo Journal of Pharmaceutical andBiomedical Analysis vol 23 no 5 pp 909ndash916 2000

[15] A Ozdemir H Aksoy E Dinc D Baleanu and S Dermis ldquoDe-termination of guaifenesin and dextromethorphan in a coughsyrup by HPLC with fluorometric detectionrdquo Revue Roumainede Chimie vol 51 no 2 pp 117ndash122 2006

[16] I Demian ldquoHigh-performance liquid chromatography (HPLC)chiral separations of guaifenesin methocarbamol and race-morphanrdquo Chirality vol 5 no 4 pp 238ndash240 1993

[17] S Suzen C Akay and S Cevheroglu ldquoSimultaneous determi-nation of guaiphenesin and codeine phosphate in tablets byhigh-performance liquid chromatographyrdquo Farmaco vol 54no 10 pp 705ndash709 1999

[18] S M Amer S S Abbas M A Shehata and NM Ali ldquoSimulta-neous determination of phenylephrine hydrochloride guaifen-esin and chlorpheniraminemaleate in cough syrup by gradientliquid chromatographyrdquo Journal of AOAC International vol 91no 2 pp 276ndash284 2008

[19] V Galli andC Barbas ldquoHigh-performance liquid chromatogra-phic analysis of dextromethorphan guaifenesin and benzoate ina cough syrup for stability testingrdquo Journal of ChromatographyA vol 1048 no 2 pp 207ndash211 2004

12 Chromatography Research International

[20] X Chen J Huang Z Kong and D Zhong ldquoSensitive liquidchromatography-tandem mass spectrometry method for thesimultaneous determination of paracetamol and guaifenesin inhuman plasmardquo Journal of Chromatography B vol 817 no 2 pp263ndash269 2005

[21] R M Gudipati J E Wallace and S A Stavchansky ldquoHigh per-formance liquid chromatography determination of guaifenesinin dog plasmardquo Analytical Letters vol 24 pp 265ndash274 1991

[22] Q-H Ge Z Zhou X-J Zhi L-L Ma and C-G Ding ldquoSim-ultaneous determination of guaifenesin bromhexine andambroxol in human plasma by LC-MSMS and its pharmacoki-netic studiesrdquo Chinese Pharmaceutical Journal vol 44 no 13pp 1025ndash1028 2009

[23] R Rajagopalan ldquoReview of regulatory guidance on impuritiesrdquoSeparation Science and Technology vol 5 pp 27ndash37 2004

[24] ICH Harmonized Tripartite Guideline Validation of AnalyticalProcedures Text and Methodology Q2(R1) 2005

[25] ICH ldquoStability testing of new drug substances and productsQ1A (R2)rdquo 2003

[26] ICH ldquoPhotostability testing of new drug substances and prod-ucts Q1Brdquo 1996

[27] The United States Pharmacopeia USP35-NF30 3829ndash3837[28] European Pharmacopeia 70 1821-1822 amp 2128-2129[29] P Sunil Reddy K Sudhakar Babu N Kumar and Y V V Sasi

Sekhar ldquoDevelopment and validation of stability indicating theRP-HPLC method for the estimation of related compounds ofguaifenesin in pharmaceutical dosage formsrdquo PharmaceuticalMethods vol 2 pp 229ndash234 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

8 Chromatography Research International

Table 3 System suitability results

Parameter RSDlowast of standard Theoretical plateslowast Tailing factorlowast Resolution 1 Resolution 2GN DN GN DN GN DN

As such method 05 07 45320 498659 10 10 27 23At 06mLmin flow rate 12 06 41091 495497 10 10 29 25At 10mLmin flow rate 09 04 45828 515551 10 10 25 22At 50∘C column temperature 15 08 45321 508966 10 10 26 23At 60∘C column temperature 21 23 40929 515807 10 10 29 26At pH 28 (buffer pH) 11 05 43970 463987 10 10 24 23At pH 32 (buffer pH) 07 15 44619 532891 10 10 26 27lowastAverage of six replicate standard injections

Table 4 (a) Forced degradation data for Guaifenesin (b) Forced degradation data for Dextromethorphan

(a)

Degradation conditions Guaifenesin degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 million lux hours followed by 200Watt hours) 006 3585 4142 1002Exposed to humidity at 25∘C and 90 RH for about 7 days 007 3881 4241 998Refluxed with purified water for about 12 hours at 60∘C 005 3285 3845 986Refluxed with 05 N HCL solution for about 2 hours at 60∘C 010 5293 7073 992Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 006 3067 3489 989Refluxed with 10 H2O2 solution for about 30min at 60∘C 013 2755 3607 983Exposed to dry heat for about 15 hours at 105∘C 007 3346 4331 994

(b)

Degradation conditions Dextromethorphan degraded Purity angle Purity threshold Mass balance ()

Photo stress (12 Million lux hours followed by 200Watt hours) 001 0115 0242 998Exposed to humidity at 25∘C and 90 RH for about 7 days 001 0124 0261 995Refluxed with purified water for about 12 hours at 60∘C 001 0101 0261 989Refluxed with 05 N HCL solution for about 2 hours at 60∘C 002 0154 0262 981Refluxed with 05 N NaOH solution for about 2 hours at 60∘C 001 0103 0256 992Refluxed with 10 H2O2 solution for about 30min at 60∘C 105 0102 0250 997Exposed to dry heat for about 15 hours at 105∘C 001 0099 0261 987

Table 5 Results of precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0198 0201 0189 0213 0205 0209 0203 42GN-Impurity B 0209 0195 0192 0201 0206 0194 0200 35GN-Impurity C 0189 0195 0186 0192 0198 0184 0191 28GN-Impurity D 0208 0190 0192 0193 0188 0186 0193 41DN-Impurity A 0215 0214 0215 0209 0212 0205 0212 19DN-Impurity B 0204 0200 0202 0203 0205 0191 0201 25DN-Impurity C 0195 0186 0198 0199 0184 0194 0193 32DN-N-oxide 0216 0214 0218 0219 0211 0210 0215 17DN-NFM Impurity 0216 0218 0215 0215 0216 0212 0215 09DN-NFO Impurity 0198 0195 0213 0192 0214 0215 0205 52GN 0203 0212 0204 0206 0205 0210 0207 17DN 0182 0189 0194 0188 0187 0201 0190 34

Chromatography Research International 9

Table 6 Results of intermediate precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0191 0204 0201 0199 0210 0212 0203 38GN-Impurity B 0212 0204 0210 0209 0211 0204 0208 17GN-Impurity C 0192 0198 0191 0194 0202 0209 0198 35GN-Impurity D 0185 0192 0191 0194 0193 0192 0192 15DN-Impurity A 0202 0202 0203 0202 0202 0204 0203 04DN-Impurity B 0191 0188 0204 0209 0189 0202 0197 45DN-Impurity C 0183 0189 0191 0186 0205 0206 0193 51DN-N-oxide 0197 0210 0213 0202 0198 0212 0205 35DN-NFM Impurity 0183 0187 0179 0184 0185 0188 0184 17DN-NFO Impurity 0206 0197 0209 0212 0194 0208 0204 35GN 0199 0205 0201 0204 0207 0208 0204 17DN 0187 0185 0192 0186 0194 0191 0189 19

Table 7 LOD LOQ and precision data

Compound LOD () LOQ () SN ratio RSD at LOQ Level precisionLOD LOQ

GN-Impurity A 0004 0001 27 95 42GN-Impurity B 0008 0002 29 98 25GN-Impurity C 0005 0002 30 98 31GN-Impurity D 0003 0001 29 97 25DN-Impurity A 0014 0004 28 101 11DN-Impurity B 0021 0006 27 104 34DN-Impurity C 0023 0007 28 99 49DN-N-oxide 0016 0005 31 98 22DN-NFM Impurity 0016 0005 27 101 15DN-NFO Impurity 0041 0012 29 106 42GN 0006 0002 31 98 21DN 0015 0005 28 96 28

replicate injections is notmore than 50 resolution betweenimpurities 20 the tailing factor for GN and DN is not morethan 15 and the theoretical plates are not less than 5000

422 Specificity All forced degradation samples were ana-lyzed with the aforementioned HPLC conditions using aPDA detector to monitor the homogeneity and purity of theGN DN and their related impurities Individual impuritiesplacebo GN and DN were verified and proved to be nonin-terfering with each other thus proving the specificity of themethod

Figure 3 shows that there is no interference at the RT(retention time) of GN DN and all known impurities fromthe other excipients Degradation was not observed in pho-tolytic stress humidity acid hydrolysis base hydrolysis waterhydrolysis and thermal stress studies Significant degradationwas observed in oxidative conditions The typical oxidativestressed chromatogram was shown in Figure 4 It was inter-esting to note that all the peaks due to degradation were well

resolved from the peaks of GN DN and their impuritiesFurther the peak purity of GN DN and their impuritieswas found to be homogeneous based on the evaluationparameters such as purity angle and purity threshold usingWaters Empower Networking Software The verification ofpeak purity indicates that there is no interference fromdegradants facilitating error-free quantification of GN andDN impurities Also themass balance of stressed samples wasfound to be more than 98 Thus the method is consideredto be ldquostability-indicatingrdquo The specificity results were shownin Tables 4(a) and 4(b)

423 Precision The RSD for the individual of allimpurities in impurities method precision study was within34 The results obtained in the intermediated precisionstudy for the RSD of the individual of all impurities werewell within 41 conforming high precision of the methodThe results are shown in Tables 5 and 6

10 Chromatography Research International

Table 8 Accuracy of the method

Compound Recovery at each levela

LOQ 01 02 04 08 10GN-Impurity A 985 995 1012 967 999 1035GN-Impurity B 1056 1027 997 1051 985 975GN-Impurity C 1026 1056 1019 1061 1035 1021GN-Impurity D 956 972 965 958 965 985DN-Impurity A 935 965 952 942 985 967DN-Impurity B 985 967 999 989 975 965DN-Impurity C 1011 1023 1025 1037 1025 1018DN-N-oxide 1056 1065 1051 1045 1029 1037DN-NFM Impurity 1015 985 1025 1012 995 986DN-NFO Impurity 1071 1056 1069 1058 1065 1058GN 985 965 972 968 974 987DN 992 985 971 995 1005 1012aAverage of six determinations at LOQ level amp 10 level and three determinations at remaining levels

Table 9 Regression statistics

Substance Linearity range (120583gmLminus1) Correlation coefficient (119877) 119884-intercept bias in GN-Impurity A 093 to 23961 0999 09GN-Impurity B 201 to 24278 0999 10GN-Impurity C 126 to 24192 1000 07GN-Impurity D 079 to 24255 0999 08DN-Impurity A 017 to 1410 1000 04DN-Impurity B 025 to 1010 1000 01DN-Impurity C 027 to 1223 1000 08DN-N-oxide 019 to 1249 1000 06DN-NFM Impurity 019 to 1219 0998 15DN-NFO Impurity 048 to 1222 1000 09GN 153 to 24478 0999 16DN 018 to 1215 1000 01

Table 10 Results of robustness study

S no Impurity name

RRTrsquos of impurities

As suchmethod

At06mLminflow rate

At10mLminflow rate

At 50∘Ccolumn

temperature

At 60∘Ccolumn

temperature

At pH 28(buffer pH)

At pH 32(buffer pH)

1 GN-Impurity A 074 076 073 072 071 074 073

2 GN-Impurity B 112 111 113 111 110 112 113

3 GN-Impurity C 204 206 205 205 202 204 205

4 GN-Impurity D 214 212 215 213 211 213 214

5 DN-Impurity B 083 082 081 082 082 083 082

6 DN-Impurity C 098 097 098 098 097 097 098

7 DN-N-oxide 101 102 103 102 103 102 104

8 DN-Impurity A 103 104 105 104 105 104 106

9 DN-NFM Impurity 128 131 130 131 129 128 119

10 DN-NFO Impurity 138 141 142 139 139 139 141Note GN known impurities RRTs were calculated against GN main peak and DN known impurities RRTs were calculated against DN main peak

Chromatography Research International 11

424 Limit of Detection (LOD) and Limit of Quantification(LOQ) The determined limit of detection limit of quantifi-cation and precision at LOQ values for GN DN and theirimpurities were reported inTable 7TheRSD for peak areas ofGNDN and their related impurities at limit of quantificationlevel was within 100

425 Accuracy The recovery of all the impurities fromfinished pharmaceutical dosage form ranged from 850 to1150 The summary of recovery for individual impuritywas mentioned in Table 8

426 Linearity Linear calibration plot for the related sub-stance method was obtained over the calibration rangestested that is LOQ to 10 of the target test concentrationThe correlation coefficient obtained was greater than 0997for all the componentsThe slope and y-intercept values werealso provided in Table 9 which confirmed good linearitybetween peak areas and concentration The linearity graphswere shown in Figure 5(a) to Figure 5(l)

427 Robustness No significant effect was observed onsystem suitability parameters such as resolution RSD tailingfactor RRTs of impurities or the theoretical plates of GNand DN when small but deliberate changes were made tochromatographic conditions The results were presented inTables 3 and 10 along with the system suitability parametersof normal conditions Thus the method was found to berobust with respect to variability in applied conditions

428 Stability in Solution and in the Mobile Phase Nosignificant changeswere observed in the content of impuritiesduring solution stability and mobile phase stability exper-iments when performed using the impurities method Thesolution stability and mobile phase stability experiment dataconfirms that the sample solution and mobile phases usedduring the impurity determination were stable for at least48 h

5 Conclusions

The gradient HPLC method developed for the simultaneousdetermination of GN and DN impurities in pharmaceuticaldosage form was precise accurate and specific The methodis validated as per ICH guidelines and found to be specificprecise linear accurate rugged and robust The developedmethod can be used for the stability analysis of GN and DNeither individually or in their combination dosage forms

Acknowledgments

The authors wish to thank the management of Dr Reddyrsquosgroup for supporting this workThe authors wish to acknowl-edge the formulation development group for providing thesamples for their research They would also like to thankcolleagues in bulkmanufacturers for providing chemicals andimpurity standards for their research work

References

[1] httpwwwrxlistcomorganidin-nr-drughtm[2] W Zhang TWang L Qin et al ldquoNeuroprotective effect of dex-

tromethorphan in the MPTP Parkinsonrsquos disease model role ofNADPHoxidaserdquoTheFASEB Journal vol 18 no 3 pp 589ndash5912004

[3] Dextromethorphan NHTSA[4] ldquoChild deaths lead to FDA hearing on cough cold medsrdquo 2007

httpwwwcnncom[5] B KuKanich and M G Papich ldquoPlasma profile and pharmac-

okinetics of dextromethorphan after intravenous and oraladministration in healthy dogsrdquo Journal of Veterinary Pharma-cology andTherapeutics vol 27 no 5 pp 337ndash341 2004

[6] ldquoKidsrsquo cough medicine no better than placebordquo San FranciscoChronicle 2004

[7] I M Paul K E Yoder K R Crowell et al ldquoEffect of dextro-methorphan diphenhydramine and placebo on nocturnalcough and sleep quality for coughing children and their par-entsrdquo Pediatrics vol 114 no 1 pp e85ndashe90 2004

[8] S Gorog M Babjak G Balogh et al ldquoDrug impurity profilingstrategiesrdquo Talanta vol 44 no 9 pp 1517ndash1526 1997

[9] B B Sanjay R K Bharati S J Yogini and A S Atul ldquoImpurityprofile significance in active pharmaceutical ingredientrdquo Eura-sian Journal of Analytical Chemistry vol 2 pp 32ndash53 2007

[10] ICH ldquoImpurities in new drug substances Q3A (R2)rdquo 2006[11] ICH ldquoImpurities in new drug products Q3B (R2)rdquo 2006[12] G Grosa E D Grosso R Russo and G Allegrone ldquoSimulta-

neous stability indicating HPLC-DAD determination ofguaifenesin and methyl and propyl-parabens in cough syruprdquoJournal of Pharmaceutical and Biomedical Analysis vol 41 no3 pp 798ndash803 2006

[13] M Senthilraja and P Giriraj ldquoReverse phase hplc method forthe simultaneous estimation of terbutanile sulphate bromhex-ine HCl and guaifenesin in cough syruprdquo Asian Journal ofPharmaceutical and Clinical Research vol 4 no 2 pp 13ndash152011

[14] M L Wilcox and J T Stewart ldquoHPLC determination of guaife-nesin with selected medications on underivatized silica with anaqueous-organic mobile phaserdquo Journal of Pharmaceutical andBiomedical Analysis vol 23 no 5 pp 909ndash916 2000

[15] A Ozdemir H Aksoy E Dinc D Baleanu and S Dermis ldquoDe-termination of guaifenesin and dextromethorphan in a coughsyrup by HPLC with fluorometric detectionrdquo Revue Roumainede Chimie vol 51 no 2 pp 117ndash122 2006

[16] I Demian ldquoHigh-performance liquid chromatography (HPLC)chiral separations of guaifenesin methocarbamol and race-morphanrdquo Chirality vol 5 no 4 pp 238ndash240 1993

[17] S Suzen C Akay and S Cevheroglu ldquoSimultaneous determi-nation of guaiphenesin and codeine phosphate in tablets byhigh-performance liquid chromatographyrdquo Farmaco vol 54no 10 pp 705ndash709 1999

[18] S M Amer S S Abbas M A Shehata and NM Ali ldquoSimulta-neous determination of phenylephrine hydrochloride guaifen-esin and chlorpheniraminemaleate in cough syrup by gradientliquid chromatographyrdquo Journal of AOAC International vol 91no 2 pp 276ndash284 2008

[19] V Galli andC Barbas ldquoHigh-performance liquid chromatogra-phic analysis of dextromethorphan guaifenesin and benzoate ina cough syrup for stability testingrdquo Journal of ChromatographyA vol 1048 no 2 pp 207ndash211 2004

12 Chromatography Research International

[20] X Chen J Huang Z Kong and D Zhong ldquoSensitive liquidchromatography-tandem mass spectrometry method for thesimultaneous determination of paracetamol and guaifenesin inhuman plasmardquo Journal of Chromatography B vol 817 no 2 pp263ndash269 2005

[21] R M Gudipati J E Wallace and S A Stavchansky ldquoHigh per-formance liquid chromatography determination of guaifenesinin dog plasmardquo Analytical Letters vol 24 pp 265ndash274 1991

[22] Q-H Ge Z Zhou X-J Zhi L-L Ma and C-G Ding ldquoSim-ultaneous determination of guaifenesin bromhexine andambroxol in human plasma by LC-MSMS and its pharmacoki-netic studiesrdquo Chinese Pharmaceutical Journal vol 44 no 13pp 1025ndash1028 2009

[23] R Rajagopalan ldquoReview of regulatory guidance on impuritiesrdquoSeparation Science and Technology vol 5 pp 27ndash37 2004

[24] ICH Harmonized Tripartite Guideline Validation of AnalyticalProcedures Text and Methodology Q2(R1) 2005

[25] ICH ldquoStability testing of new drug substances and productsQ1A (R2)rdquo 2003

[26] ICH ldquoPhotostability testing of new drug substances and prod-ucts Q1Brdquo 1996

[27] The United States Pharmacopeia USP35-NF30 3829ndash3837[28] European Pharmacopeia 70 1821-1822 amp 2128-2129[29] P Sunil Reddy K Sudhakar Babu N Kumar and Y V V Sasi

Sekhar ldquoDevelopment and validation of stability indicating theRP-HPLC method for the estimation of related compounds ofguaifenesin in pharmaceutical dosage formsrdquo PharmaceuticalMethods vol 2 pp 229ndash234 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

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Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Chromatography Research International 9

Table 6 Results of intermediate precision

Compound Prep-1 Prep-1 Prep-3 Prep-4 Prep-5 Prep-6 Avg RSDGN-Impurity A 0191 0204 0201 0199 0210 0212 0203 38GN-Impurity B 0212 0204 0210 0209 0211 0204 0208 17GN-Impurity C 0192 0198 0191 0194 0202 0209 0198 35GN-Impurity D 0185 0192 0191 0194 0193 0192 0192 15DN-Impurity A 0202 0202 0203 0202 0202 0204 0203 04DN-Impurity B 0191 0188 0204 0209 0189 0202 0197 45DN-Impurity C 0183 0189 0191 0186 0205 0206 0193 51DN-N-oxide 0197 0210 0213 0202 0198 0212 0205 35DN-NFM Impurity 0183 0187 0179 0184 0185 0188 0184 17DN-NFO Impurity 0206 0197 0209 0212 0194 0208 0204 35GN 0199 0205 0201 0204 0207 0208 0204 17DN 0187 0185 0192 0186 0194 0191 0189 19

Table 7 LOD LOQ and precision data

Compound LOD () LOQ () SN ratio RSD at LOQ Level precisionLOD LOQ

GN-Impurity A 0004 0001 27 95 42GN-Impurity B 0008 0002 29 98 25GN-Impurity C 0005 0002 30 98 31GN-Impurity D 0003 0001 29 97 25DN-Impurity A 0014 0004 28 101 11DN-Impurity B 0021 0006 27 104 34DN-Impurity C 0023 0007 28 99 49DN-N-oxide 0016 0005 31 98 22DN-NFM Impurity 0016 0005 27 101 15DN-NFO Impurity 0041 0012 29 106 42GN 0006 0002 31 98 21DN 0015 0005 28 96 28

replicate injections is notmore than 50 resolution betweenimpurities 20 the tailing factor for GN and DN is not morethan 15 and the theoretical plates are not less than 5000

422 Specificity All forced degradation samples were ana-lyzed with the aforementioned HPLC conditions using aPDA detector to monitor the homogeneity and purity of theGN DN and their related impurities Individual impuritiesplacebo GN and DN were verified and proved to be nonin-terfering with each other thus proving the specificity of themethod

Figure 3 shows that there is no interference at the RT(retention time) of GN DN and all known impurities fromthe other excipients Degradation was not observed in pho-tolytic stress humidity acid hydrolysis base hydrolysis waterhydrolysis and thermal stress studies Significant degradationwas observed in oxidative conditions The typical oxidativestressed chromatogram was shown in Figure 4 It was inter-esting to note that all the peaks due to degradation were well

resolved from the peaks of GN DN and their impuritiesFurther the peak purity of GN DN and their impuritieswas found to be homogeneous based on the evaluationparameters such as purity angle and purity threshold usingWaters Empower Networking Software The verification ofpeak purity indicates that there is no interference fromdegradants facilitating error-free quantification of GN andDN impurities Also themass balance of stressed samples wasfound to be more than 98 Thus the method is consideredto be ldquostability-indicatingrdquo The specificity results were shownin Tables 4(a) and 4(b)

423 Precision The RSD for the individual of allimpurities in impurities method precision study was within34 The results obtained in the intermediated precisionstudy for the RSD of the individual of all impurities werewell within 41 conforming high precision of the methodThe results are shown in Tables 5 and 6

10 Chromatography Research International

Table 8 Accuracy of the method

Compound Recovery at each levela

LOQ 01 02 04 08 10GN-Impurity A 985 995 1012 967 999 1035GN-Impurity B 1056 1027 997 1051 985 975GN-Impurity C 1026 1056 1019 1061 1035 1021GN-Impurity D 956 972 965 958 965 985DN-Impurity A 935 965 952 942 985 967DN-Impurity B 985 967 999 989 975 965DN-Impurity C 1011 1023 1025 1037 1025 1018DN-N-oxide 1056 1065 1051 1045 1029 1037DN-NFM Impurity 1015 985 1025 1012 995 986DN-NFO Impurity 1071 1056 1069 1058 1065 1058GN 985 965 972 968 974 987DN 992 985 971 995 1005 1012aAverage of six determinations at LOQ level amp 10 level and three determinations at remaining levels

Table 9 Regression statistics

Substance Linearity range (120583gmLminus1) Correlation coefficient (119877) 119884-intercept bias in GN-Impurity A 093 to 23961 0999 09GN-Impurity B 201 to 24278 0999 10GN-Impurity C 126 to 24192 1000 07GN-Impurity D 079 to 24255 0999 08DN-Impurity A 017 to 1410 1000 04DN-Impurity B 025 to 1010 1000 01DN-Impurity C 027 to 1223 1000 08DN-N-oxide 019 to 1249 1000 06DN-NFM Impurity 019 to 1219 0998 15DN-NFO Impurity 048 to 1222 1000 09GN 153 to 24478 0999 16DN 018 to 1215 1000 01

Table 10 Results of robustness study

S no Impurity name

RRTrsquos of impurities

As suchmethod

At06mLminflow rate

At10mLminflow rate

At 50∘Ccolumn

temperature

At 60∘Ccolumn

temperature

At pH 28(buffer pH)

At pH 32(buffer pH)

1 GN-Impurity A 074 076 073 072 071 074 073

2 GN-Impurity B 112 111 113 111 110 112 113

3 GN-Impurity C 204 206 205 205 202 204 205

4 GN-Impurity D 214 212 215 213 211 213 214

5 DN-Impurity B 083 082 081 082 082 083 082

6 DN-Impurity C 098 097 098 098 097 097 098

7 DN-N-oxide 101 102 103 102 103 102 104

8 DN-Impurity A 103 104 105 104 105 104 106

9 DN-NFM Impurity 128 131 130 131 129 128 119

10 DN-NFO Impurity 138 141 142 139 139 139 141Note GN known impurities RRTs were calculated against GN main peak and DN known impurities RRTs were calculated against DN main peak

Chromatography Research International 11

424 Limit of Detection (LOD) and Limit of Quantification(LOQ) The determined limit of detection limit of quantifi-cation and precision at LOQ values for GN DN and theirimpurities were reported inTable 7TheRSD for peak areas ofGNDN and their related impurities at limit of quantificationlevel was within 100

425 Accuracy The recovery of all the impurities fromfinished pharmaceutical dosage form ranged from 850 to1150 The summary of recovery for individual impuritywas mentioned in Table 8

426 Linearity Linear calibration plot for the related sub-stance method was obtained over the calibration rangestested that is LOQ to 10 of the target test concentrationThe correlation coefficient obtained was greater than 0997for all the componentsThe slope and y-intercept values werealso provided in Table 9 which confirmed good linearitybetween peak areas and concentration The linearity graphswere shown in Figure 5(a) to Figure 5(l)

427 Robustness No significant effect was observed onsystem suitability parameters such as resolution RSD tailingfactor RRTs of impurities or the theoretical plates of GNand DN when small but deliberate changes were made tochromatographic conditions The results were presented inTables 3 and 10 along with the system suitability parametersof normal conditions Thus the method was found to berobust with respect to variability in applied conditions

428 Stability in Solution and in the Mobile Phase Nosignificant changeswere observed in the content of impuritiesduring solution stability and mobile phase stability exper-iments when performed using the impurities method Thesolution stability and mobile phase stability experiment dataconfirms that the sample solution and mobile phases usedduring the impurity determination were stable for at least48 h

5 Conclusions

The gradient HPLC method developed for the simultaneousdetermination of GN and DN impurities in pharmaceuticaldosage form was precise accurate and specific The methodis validated as per ICH guidelines and found to be specificprecise linear accurate rugged and robust The developedmethod can be used for the stability analysis of GN and DNeither individually or in their combination dosage forms

Acknowledgments

The authors wish to thank the management of Dr Reddyrsquosgroup for supporting this workThe authors wish to acknowl-edge the formulation development group for providing thesamples for their research They would also like to thankcolleagues in bulkmanufacturers for providing chemicals andimpurity standards for their research work

References

[1] httpwwwrxlistcomorganidin-nr-drughtm[2] W Zhang TWang L Qin et al ldquoNeuroprotective effect of dex-

tromethorphan in the MPTP Parkinsonrsquos disease model role ofNADPHoxidaserdquoTheFASEB Journal vol 18 no 3 pp 589ndash5912004

[3] Dextromethorphan NHTSA[4] ldquoChild deaths lead to FDA hearing on cough cold medsrdquo 2007

httpwwwcnncom[5] B KuKanich and M G Papich ldquoPlasma profile and pharmac-

okinetics of dextromethorphan after intravenous and oraladministration in healthy dogsrdquo Journal of Veterinary Pharma-cology andTherapeutics vol 27 no 5 pp 337ndash341 2004

[6] ldquoKidsrsquo cough medicine no better than placebordquo San FranciscoChronicle 2004

[7] I M Paul K E Yoder K R Crowell et al ldquoEffect of dextro-methorphan diphenhydramine and placebo on nocturnalcough and sleep quality for coughing children and their par-entsrdquo Pediatrics vol 114 no 1 pp e85ndashe90 2004

[8] S Gorog M Babjak G Balogh et al ldquoDrug impurity profilingstrategiesrdquo Talanta vol 44 no 9 pp 1517ndash1526 1997

[9] B B Sanjay R K Bharati S J Yogini and A S Atul ldquoImpurityprofile significance in active pharmaceutical ingredientrdquo Eura-sian Journal of Analytical Chemistry vol 2 pp 32ndash53 2007

[10] ICH ldquoImpurities in new drug substances Q3A (R2)rdquo 2006[11] ICH ldquoImpurities in new drug products Q3B (R2)rdquo 2006[12] G Grosa E D Grosso R Russo and G Allegrone ldquoSimulta-

neous stability indicating HPLC-DAD determination ofguaifenesin and methyl and propyl-parabens in cough syruprdquoJournal of Pharmaceutical and Biomedical Analysis vol 41 no3 pp 798ndash803 2006

[13] M Senthilraja and P Giriraj ldquoReverse phase hplc method forthe simultaneous estimation of terbutanile sulphate bromhex-ine HCl and guaifenesin in cough syruprdquo Asian Journal ofPharmaceutical and Clinical Research vol 4 no 2 pp 13ndash152011

[14] M L Wilcox and J T Stewart ldquoHPLC determination of guaife-nesin with selected medications on underivatized silica with anaqueous-organic mobile phaserdquo Journal of Pharmaceutical andBiomedical Analysis vol 23 no 5 pp 909ndash916 2000

[15] A Ozdemir H Aksoy E Dinc D Baleanu and S Dermis ldquoDe-termination of guaifenesin and dextromethorphan in a coughsyrup by HPLC with fluorometric detectionrdquo Revue Roumainede Chimie vol 51 no 2 pp 117ndash122 2006

[16] I Demian ldquoHigh-performance liquid chromatography (HPLC)chiral separations of guaifenesin methocarbamol and race-morphanrdquo Chirality vol 5 no 4 pp 238ndash240 1993

[17] S Suzen C Akay and S Cevheroglu ldquoSimultaneous determi-nation of guaiphenesin and codeine phosphate in tablets byhigh-performance liquid chromatographyrdquo Farmaco vol 54no 10 pp 705ndash709 1999

[18] S M Amer S S Abbas M A Shehata and NM Ali ldquoSimulta-neous determination of phenylephrine hydrochloride guaifen-esin and chlorpheniraminemaleate in cough syrup by gradientliquid chromatographyrdquo Journal of AOAC International vol 91no 2 pp 276ndash284 2008

[19] V Galli andC Barbas ldquoHigh-performance liquid chromatogra-phic analysis of dextromethorphan guaifenesin and benzoate ina cough syrup for stability testingrdquo Journal of ChromatographyA vol 1048 no 2 pp 207ndash211 2004

12 Chromatography Research International

[20] X Chen J Huang Z Kong and D Zhong ldquoSensitive liquidchromatography-tandem mass spectrometry method for thesimultaneous determination of paracetamol and guaifenesin inhuman plasmardquo Journal of Chromatography B vol 817 no 2 pp263ndash269 2005

[21] R M Gudipati J E Wallace and S A Stavchansky ldquoHigh per-formance liquid chromatography determination of guaifenesinin dog plasmardquo Analytical Letters vol 24 pp 265ndash274 1991

[22] Q-H Ge Z Zhou X-J Zhi L-L Ma and C-G Ding ldquoSim-ultaneous determination of guaifenesin bromhexine andambroxol in human plasma by LC-MSMS and its pharmacoki-netic studiesrdquo Chinese Pharmaceutical Journal vol 44 no 13pp 1025ndash1028 2009

[23] R Rajagopalan ldquoReview of regulatory guidance on impuritiesrdquoSeparation Science and Technology vol 5 pp 27ndash37 2004

[24] ICH Harmonized Tripartite Guideline Validation of AnalyticalProcedures Text and Methodology Q2(R1) 2005

[25] ICH ldquoStability testing of new drug substances and productsQ1A (R2)rdquo 2003

[26] ICH ldquoPhotostability testing of new drug substances and prod-ucts Q1Brdquo 1996

[27] The United States Pharmacopeia USP35-NF30 3829ndash3837[28] European Pharmacopeia 70 1821-1822 amp 2128-2129[29] P Sunil Reddy K Sudhakar Babu N Kumar and Y V V Sasi

Sekhar ldquoDevelopment and validation of stability indicating theRP-HPLC method for the estimation of related compounds ofguaifenesin in pharmaceutical dosage formsrdquo PharmaceuticalMethods vol 2 pp 229ndash234 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

10 Chromatography Research International

Table 8 Accuracy of the method

Compound Recovery at each levela

LOQ 01 02 04 08 10GN-Impurity A 985 995 1012 967 999 1035GN-Impurity B 1056 1027 997 1051 985 975GN-Impurity C 1026 1056 1019 1061 1035 1021GN-Impurity D 956 972 965 958 965 985DN-Impurity A 935 965 952 942 985 967DN-Impurity B 985 967 999 989 975 965DN-Impurity C 1011 1023 1025 1037 1025 1018DN-N-oxide 1056 1065 1051 1045 1029 1037DN-NFM Impurity 1015 985 1025 1012 995 986DN-NFO Impurity 1071 1056 1069 1058 1065 1058GN 985 965 972 968 974 987DN 992 985 971 995 1005 1012aAverage of six determinations at LOQ level amp 10 level and three determinations at remaining levels

Table 9 Regression statistics

Substance Linearity range (120583gmLminus1) Correlation coefficient (119877) 119884-intercept bias in GN-Impurity A 093 to 23961 0999 09GN-Impurity B 201 to 24278 0999 10GN-Impurity C 126 to 24192 1000 07GN-Impurity D 079 to 24255 0999 08DN-Impurity A 017 to 1410 1000 04DN-Impurity B 025 to 1010 1000 01DN-Impurity C 027 to 1223 1000 08DN-N-oxide 019 to 1249 1000 06DN-NFM Impurity 019 to 1219 0998 15DN-NFO Impurity 048 to 1222 1000 09GN 153 to 24478 0999 16DN 018 to 1215 1000 01

Table 10 Results of robustness study

S no Impurity name

RRTrsquos of impurities

As suchmethod

At06mLminflow rate

At10mLminflow rate

At 50∘Ccolumn

temperature

At 60∘Ccolumn

temperature

At pH 28(buffer pH)

At pH 32(buffer pH)

1 GN-Impurity A 074 076 073 072 071 074 073

2 GN-Impurity B 112 111 113 111 110 112 113

3 GN-Impurity C 204 206 205 205 202 204 205

4 GN-Impurity D 214 212 215 213 211 213 214

5 DN-Impurity B 083 082 081 082 082 083 082

6 DN-Impurity C 098 097 098 098 097 097 098

7 DN-N-oxide 101 102 103 102 103 102 104

8 DN-Impurity A 103 104 105 104 105 104 106

9 DN-NFM Impurity 128 131 130 131 129 128 119

10 DN-NFO Impurity 138 141 142 139 139 139 141Note GN known impurities RRTs were calculated against GN main peak and DN known impurities RRTs were calculated against DN main peak

Chromatography Research International 11

424 Limit of Detection (LOD) and Limit of Quantification(LOQ) The determined limit of detection limit of quantifi-cation and precision at LOQ values for GN DN and theirimpurities were reported inTable 7TheRSD for peak areas ofGNDN and their related impurities at limit of quantificationlevel was within 100

425 Accuracy The recovery of all the impurities fromfinished pharmaceutical dosage form ranged from 850 to1150 The summary of recovery for individual impuritywas mentioned in Table 8

426 Linearity Linear calibration plot for the related sub-stance method was obtained over the calibration rangestested that is LOQ to 10 of the target test concentrationThe correlation coefficient obtained was greater than 0997for all the componentsThe slope and y-intercept values werealso provided in Table 9 which confirmed good linearitybetween peak areas and concentration The linearity graphswere shown in Figure 5(a) to Figure 5(l)

427 Robustness No significant effect was observed onsystem suitability parameters such as resolution RSD tailingfactor RRTs of impurities or the theoretical plates of GNand DN when small but deliberate changes were made tochromatographic conditions The results were presented inTables 3 and 10 along with the system suitability parametersof normal conditions Thus the method was found to berobust with respect to variability in applied conditions

428 Stability in Solution and in the Mobile Phase Nosignificant changeswere observed in the content of impuritiesduring solution stability and mobile phase stability exper-iments when performed using the impurities method Thesolution stability and mobile phase stability experiment dataconfirms that the sample solution and mobile phases usedduring the impurity determination were stable for at least48 h

5 Conclusions

The gradient HPLC method developed for the simultaneousdetermination of GN and DN impurities in pharmaceuticaldosage form was precise accurate and specific The methodis validated as per ICH guidelines and found to be specificprecise linear accurate rugged and robust The developedmethod can be used for the stability analysis of GN and DNeither individually or in their combination dosage forms

Acknowledgments

The authors wish to thank the management of Dr Reddyrsquosgroup for supporting this workThe authors wish to acknowl-edge the formulation development group for providing thesamples for their research They would also like to thankcolleagues in bulkmanufacturers for providing chemicals andimpurity standards for their research work

References

[1] httpwwwrxlistcomorganidin-nr-drughtm[2] W Zhang TWang L Qin et al ldquoNeuroprotective effect of dex-

tromethorphan in the MPTP Parkinsonrsquos disease model role ofNADPHoxidaserdquoTheFASEB Journal vol 18 no 3 pp 589ndash5912004

[3] Dextromethorphan NHTSA[4] ldquoChild deaths lead to FDA hearing on cough cold medsrdquo 2007

httpwwwcnncom[5] B KuKanich and M G Papich ldquoPlasma profile and pharmac-

okinetics of dextromethorphan after intravenous and oraladministration in healthy dogsrdquo Journal of Veterinary Pharma-cology andTherapeutics vol 27 no 5 pp 337ndash341 2004

[6] ldquoKidsrsquo cough medicine no better than placebordquo San FranciscoChronicle 2004

[7] I M Paul K E Yoder K R Crowell et al ldquoEffect of dextro-methorphan diphenhydramine and placebo on nocturnalcough and sleep quality for coughing children and their par-entsrdquo Pediatrics vol 114 no 1 pp e85ndashe90 2004

[8] S Gorog M Babjak G Balogh et al ldquoDrug impurity profilingstrategiesrdquo Talanta vol 44 no 9 pp 1517ndash1526 1997

[9] B B Sanjay R K Bharati S J Yogini and A S Atul ldquoImpurityprofile significance in active pharmaceutical ingredientrdquo Eura-sian Journal of Analytical Chemistry vol 2 pp 32ndash53 2007

[10] ICH ldquoImpurities in new drug substances Q3A (R2)rdquo 2006[11] ICH ldquoImpurities in new drug products Q3B (R2)rdquo 2006[12] G Grosa E D Grosso R Russo and G Allegrone ldquoSimulta-

neous stability indicating HPLC-DAD determination ofguaifenesin and methyl and propyl-parabens in cough syruprdquoJournal of Pharmaceutical and Biomedical Analysis vol 41 no3 pp 798ndash803 2006

[13] M Senthilraja and P Giriraj ldquoReverse phase hplc method forthe simultaneous estimation of terbutanile sulphate bromhex-ine HCl and guaifenesin in cough syruprdquo Asian Journal ofPharmaceutical and Clinical Research vol 4 no 2 pp 13ndash152011

[14] M L Wilcox and J T Stewart ldquoHPLC determination of guaife-nesin with selected medications on underivatized silica with anaqueous-organic mobile phaserdquo Journal of Pharmaceutical andBiomedical Analysis vol 23 no 5 pp 909ndash916 2000

[15] A Ozdemir H Aksoy E Dinc D Baleanu and S Dermis ldquoDe-termination of guaifenesin and dextromethorphan in a coughsyrup by HPLC with fluorometric detectionrdquo Revue Roumainede Chimie vol 51 no 2 pp 117ndash122 2006

[16] I Demian ldquoHigh-performance liquid chromatography (HPLC)chiral separations of guaifenesin methocarbamol and race-morphanrdquo Chirality vol 5 no 4 pp 238ndash240 1993

[17] S Suzen C Akay and S Cevheroglu ldquoSimultaneous determi-nation of guaiphenesin and codeine phosphate in tablets byhigh-performance liquid chromatographyrdquo Farmaco vol 54no 10 pp 705ndash709 1999

[18] S M Amer S S Abbas M A Shehata and NM Ali ldquoSimulta-neous determination of phenylephrine hydrochloride guaifen-esin and chlorpheniraminemaleate in cough syrup by gradientliquid chromatographyrdquo Journal of AOAC International vol 91no 2 pp 276ndash284 2008

[19] V Galli andC Barbas ldquoHigh-performance liquid chromatogra-phic analysis of dextromethorphan guaifenesin and benzoate ina cough syrup for stability testingrdquo Journal of ChromatographyA vol 1048 no 2 pp 207ndash211 2004

12 Chromatography Research International

[20] X Chen J Huang Z Kong and D Zhong ldquoSensitive liquidchromatography-tandem mass spectrometry method for thesimultaneous determination of paracetamol and guaifenesin inhuman plasmardquo Journal of Chromatography B vol 817 no 2 pp263ndash269 2005

[21] R M Gudipati J E Wallace and S A Stavchansky ldquoHigh per-formance liquid chromatography determination of guaifenesinin dog plasmardquo Analytical Letters vol 24 pp 265ndash274 1991

[22] Q-H Ge Z Zhou X-J Zhi L-L Ma and C-G Ding ldquoSim-ultaneous determination of guaifenesin bromhexine andambroxol in human plasma by LC-MSMS and its pharmacoki-netic studiesrdquo Chinese Pharmaceutical Journal vol 44 no 13pp 1025ndash1028 2009

[23] R Rajagopalan ldquoReview of regulatory guidance on impuritiesrdquoSeparation Science and Technology vol 5 pp 27ndash37 2004

[24] ICH Harmonized Tripartite Guideline Validation of AnalyticalProcedures Text and Methodology Q2(R1) 2005

[25] ICH ldquoStability testing of new drug substances and productsQ1A (R2)rdquo 2003

[26] ICH ldquoPhotostability testing of new drug substances and prod-ucts Q1Brdquo 1996

[27] The United States Pharmacopeia USP35-NF30 3829ndash3837[28] European Pharmacopeia 70 1821-1822 amp 2128-2129[29] P Sunil Reddy K Sudhakar Babu N Kumar and Y V V Sasi

Sekhar ldquoDevelopment and validation of stability indicating theRP-HPLC method for the estimation of related compounds ofguaifenesin in pharmaceutical dosage formsrdquo PharmaceuticalMethods vol 2 pp 229ndash234 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Chromatography Research International 11

424 Limit of Detection (LOD) and Limit of Quantification(LOQ) The determined limit of detection limit of quantifi-cation and precision at LOQ values for GN DN and theirimpurities were reported inTable 7TheRSD for peak areas ofGNDN and their related impurities at limit of quantificationlevel was within 100

425 Accuracy The recovery of all the impurities fromfinished pharmaceutical dosage form ranged from 850 to1150 The summary of recovery for individual impuritywas mentioned in Table 8

426 Linearity Linear calibration plot for the related sub-stance method was obtained over the calibration rangestested that is LOQ to 10 of the target test concentrationThe correlation coefficient obtained was greater than 0997for all the componentsThe slope and y-intercept values werealso provided in Table 9 which confirmed good linearitybetween peak areas and concentration The linearity graphswere shown in Figure 5(a) to Figure 5(l)

427 Robustness No significant effect was observed onsystem suitability parameters such as resolution RSD tailingfactor RRTs of impurities or the theoretical plates of GNand DN when small but deliberate changes were made tochromatographic conditions The results were presented inTables 3 and 10 along with the system suitability parametersof normal conditions Thus the method was found to berobust with respect to variability in applied conditions

428 Stability in Solution and in the Mobile Phase Nosignificant changeswere observed in the content of impuritiesduring solution stability and mobile phase stability exper-iments when performed using the impurities method Thesolution stability and mobile phase stability experiment dataconfirms that the sample solution and mobile phases usedduring the impurity determination were stable for at least48 h

5 Conclusions

The gradient HPLC method developed for the simultaneousdetermination of GN and DN impurities in pharmaceuticaldosage form was precise accurate and specific The methodis validated as per ICH guidelines and found to be specificprecise linear accurate rugged and robust The developedmethod can be used for the stability analysis of GN and DNeither individually or in their combination dosage forms

Acknowledgments

The authors wish to thank the management of Dr Reddyrsquosgroup for supporting this workThe authors wish to acknowl-edge the formulation development group for providing thesamples for their research They would also like to thankcolleagues in bulkmanufacturers for providing chemicals andimpurity standards for their research work

References

[1] httpwwwrxlistcomorganidin-nr-drughtm[2] W Zhang TWang L Qin et al ldquoNeuroprotective effect of dex-

tromethorphan in the MPTP Parkinsonrsquos disease model role ofNADPHoxidaserdquoTheFASEB Journal vol 18 no 3 pp 589ndash5912004

[3] Dextromethorphan NHTSA[4] ldquoChild deaths lead to FDA hearing on cough cold medsrdquo 2007

httpwwwcnncom[5] B KuKanich and M G Papich ldquoPlasma profile and pharmac-

okinetics of dextromethorphan after intravenous and oraladministration in healthy dogsrdquo Journal of Veterinary Pharma-cology andTherapeutics vol 27 no 5 pp 337ndash341 2004

[6] ldquoKidsrsquo cough medicine no better than placebordquo San FranciscoChronicle 2004

[7] I M Paul K E Yoder K R Crowell et al ldquoEffect of dextro-methorphan diphenhydramine and placebo on nocturnalcough and sleep quality for coughing children and their par-entsrdquo Pediatrics vol 114 no 1 pp e85ndashe90 2004

[8] S Gorog M Babjak G Balogh et al ldquoDrug impurity profilingstrategiesrdquo Talanta vol 44 no 9 pp 1517ndash1526 1997

[9] B B Sanjay R K Bharati S J Yogini and A S Atul ldquoImpurityprofile significance in active pharmaceutical ingredientrdquo Eura-sian Journal of Analytical Chemistry vol 2 pp 32ndash53 2007

[10] ICH ldquoImpurities in new drug substances Q3A (R2)rdquo 2006[11] ICH ldquoImpurities in new drug products Q3B (R2)rdquo 2006[12] G Grosa E D Grosso R Russo and G Allegrone ldquoSimulta-

neous stability indicating HPLC-DAD determination ofguaifenesin and methyl and propyl-parabens in cough syruprdquoJournal of Pharmaceutical and Biomedical Analysis vol 41 no3 pp 798ndash803 2006

[13] M Senthilraja and P Giriraj ldquoReverse phase hplc method forthe simultaneous estimation of terbutanile sulphate bromhex-ine HCl and guaifenesin in cough syruprdquo Asian Journal ofPharmaceutical and Clinical Research vol 4 no 2 pp 13ndash152011

[14] M L Wilcox and J T Stewart ldquoHPLC determination of guaife-nesin with selected medications on underivatized silica with anaqueous-organic mobile phaserdquo Journal of Pharmaceutical andBiomedical Analysis vol 23 no 5 pp 909ndash916 2000

[15] A Ozdemir H Aksoy E Dinc D Baleanu and S Dermis ldquoDe-termination of guaifenesin and dextromethorphan in a coughsyrup by HPLC with fluorometric detectionrdquo Revue Roumainede Chimie vol 51 no 2 pp 117ndash122 2006

[16] I Demian ldquoHigh-performance liquid chromatography (HPLC)chiral separations of guaifenesin methocarbamol and race-morphanrdquo Chirality vol 5 no 4 pp 238ndash240 1993

[17] S Suzen C Akay and S Cevheroglu ldquoSimultaneous determi-nation of guaiphenesin and codeine phosphate in tablets byhigh-performance liquid chromatographyrdquo Farmaco vol 54no 10 pp 705ndash709 1999

[18] S M Amer S S Abbas M A Shehata and NM Ali ldquoSimulta-neous determination of phenylephrine hydrochloride guaifen-esin and chlorpheniraminemaleate in cough syrup by gradientliquid chromatographyrdquo Journal of AOAC International vol 91no 2 pp 276ndash284 2008

[19] V Galli andC Barbas ldquoHigh-performance liquid chromatogra-phic analysis of dextromethorphan guaifenesin and benzoate ina cough syrup for stability testingrdquo Journal of ChromatographyA vol 1048 no 2 pp 207ndash211 2004

12 Chromatography Research International

[20] X Chen J Huang Z Kong and D Zhong ldquoSensitive liquidchromatography-tandem mass spectrometry method for thesimultaneous determination of paracetamol and guaifenesin inhuman plasmardquo Journal of Chromatography B vol 817 no 2 pp263ndash269 2005

[21] R M Gudipati J E Wallace and S A Stavchansky ldquoHigh per-formance liquid chromatography determination of guaifenesinin dog plasmardquo Analytical Letters vol 24 pp 265ndash274 1991

[22] Q-H Ge Z Zhou X-J Zhi L-L Ma and C-G Ding ldquoSim-ultaneous determination of guaifenesin bromhexine andambroxol in human plasma by LC-MSMS and its pharmacoki-netic studiesrdquo Chinese Pharmaceutical Journal vol 44 no 13pp 1025ndash1028 2009

[23] R Rajagopalan ldquoReview of regulatory guidance on impuritiesrdquoSeparation Science and Technology vol 5 pp 27ndash37 2004

[24] ICH Harmonized Tripartite Guideline Validation of AnalyticalProcedures Text and Methodology Q2(R1) 2005

[25] ICH ldquoStability testing of new drug substances and productsQ1A (R2)rdquo 2003

[26] ICH ldquoPhotostability testing of new drug substances and prod-ucts Q1Brdquo 1996

[27] The United States Pharmacopeia USP35-NF30 3829ndash3837[28] European Pharmacopeia 70 1821-1822 amp 2128-2129[29] P Sunil Reddy K Sudhakar Babu N Kumar and Y V V Sasi

Sekhar ldquoDevelopment and validation of stability indicating theRP-HPLC method for the estimation of related compounds ofguaifenesin in pharmaceutical dosage formsrdquo PharmaceuticalMethods vol 2 pp 229ndash234 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

12 Chromatography Research International

[20] X Chen J Huang Z Kong and D Zhong ldquoSensitive liquidchromatography-tandem mass spectrometry method for thesimultaneous determination of paracetamol and guaifenesin inhuman plasmardquo Journal of Chromatography B vol 817 no 2 pp263ndash269 2005

[21] R M Gudipati J E Wallace and S A Stavchansky ldquoHigh per-formance liquid chromatography determination of guaifenesinin dog plasmardquo Analytical Letters vol 24 pp 265ndash274 1991

[22] Q-H Ge Z Zhou X-J Zhi L-L Ma and C-G Ding ldquoSim-ultaneous determination of guaifenesin bromhexine andambroxol in human plasma by LC-MSMS and its pharmacoki-netic studiesrdquo Chinese Pharmaceutical Journal vol 44 no 13pp 1025ndash1028 2009

[23] R Rajagopalan ldquoReview of regulatory guidance on impuritiesrdquoSeparation Science and Technology vol 5 pp 27ndash37 2004

[24] ICH Harmonized Tripartite Guideline Validation of AnalyticalProcedures Text and Methodology Q2(R1) 2005

[25] ICH ldquoStability testing of new drug substances and productsQ1A (R2)rdquo 2003

[26] ICH ldquoPhotostability testing of new drug substances and prod-ucts Q1Brdquo 1996

[27] The United States Pharmacopeia USP35-NF30 3829ndash3837[28] European Pharmacopeia 70 1821-1822 amp 2128-2129[29] P Sunil Reddy K Sudhakar Babu N Kumar and Y V V Sasi

Sekhar ldquoDevelopment and validation of stability indicating theRP-HPLC method for the estimation of related compounds ofguaifenesin in pharmaceutical dosage formsrdquo PharmaceuticalMethods vol 2 pp 229ndash234 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of