AnalyticalMethods
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aAnalytical Chemistry Division, Discovery
Chemical Technology, Tarnaka, Hyderabad
com; [email protected]; Fax: +91 40 271733bSri Siddhartha Pharmacy College, Nuzvid,cPharmacology Division, Indian Institut
Hyderabad, 500 607, India
Cite this: Anal. Methods, 2014, 6, 1674
Received 17th October 2013Accepted 20th December 2013
DOI: 10.1039/c3ay41826j
www.rsc.org/methods
1674 | Anal. Methods, 2014, 6, 1674–16
Ionic liquid based dispersive liquid–liquidmicroextraction followed by RP-HPLCdetermination of balofloxacin in rat serum
R. Nageswara Rao,*a Ch. Gangu Naidu,a Ch. V. Suresh,b N. Srinathb and Raju Padiyac
An efficient and environmentally friendly ionic liquid based dispersive liquid–liquid microextraction
procedure for the determination of balofloxacin in rat serum by reverse phase high-performance liquid
chromatography was developed and validated. The effects of ionic liquids, dispersive solvents,
extractant/disperser ratios and salt concentrations on sample recovery and enrichment were studied.
Among the ionic liquids investigated, 1-butyl-3-methylimidazolium hexafluorophosphate was found to
be the most effective and the recovery was 99.5% at an extractant/disperser ratio of 0.43 by addition of
5.0% NaCl. A threefold increase in detection (0.01 mg mL�1) and quantification (0.035 mg mL�1) limits was
achieved when compared to protein precipitation. A linear relationship in the range of 0.04–10.0 mg
mL�1 with a correlation coefficient of (r2) 0.9998 was observed. The method was validated and applied
to study the pharmacokinetics of balofloxacin in rat serum.
Introduction
Animal blood uids are the most complex biological matricesdue to the presence of endogenous substances.1 Their analysisposes several problems and challenges to chemists involved inbioanalytical method development. Of late, the potent drugdiscovery protocols made it possible to administer smallamounts of drug dosages to animals resulting in low drugconcentrations in biological matrices. Thus, determination ofdrugs at trace levels in such complex biological uids requiresefficient sample preparation procedures. Further, the analysisof highly hydrophilic molecules in blood serum and plasma is achallenging task due to low extraction recovery (ER) from suchmaterials.2 The commonly used techniques to extract drugsfrom biological matrices include protein precipitation (PP),solid phase extraction (SPE) and liquid–liquid extraction(LLE).3–5 Among these approaches, PP is routinely used in earlydrug discovery due to its simplicity and rapidity. However, itsuffers not only from specicity but also from selectivity due toco-precipitation of drugs along with the proteins.6 SPE offersdifferent retention mechanisms to retain the desired analytesand extract them out of biomatrices. Oen this involves time-consuming method development and requires skilledpersonnel.7 LLE is also considered to be time consuming,
Laboratory D215, Indian Institute of
500 607, India. E-mail: rnrao55@yahoo.
87; Tel: +91 40 27193193
A.P, India
e of Chemical Technology, Tarnaka,
83
tedious as it employs large amounts of toxic organic solventsnot only harmful to workers but also resulting in environmen-tally hazardous waste.8 LLE using solvents, such as methylt-butyl ether, 1-chlorobutane, and ethyl acetate, cannot beapplied due to their low log P index. Thus, either developmentof new or improvement of the existing extraction procedures toaddress the bioanalytical requirements of new drug discoveryand development processes is of great interest in today'sbiomedical and pharmaceutical industry. Dispersive liquid–liquid microextraction (DLLME), introduced recently by Rezaeeet al., is an alternative sample preparation method for extrac-tion of organic molecules from environmental as well as bio-logical samples.9 It offers several advantages such as simplicityin operation, rapid extraction, high enrichment factors (EF) andlow consumption of organic solvents.10,11 DLLME offers thepossibility of enhancing the sensitivity and simplication of theextraction procedure. Because of its advantages, DLLME hasbeen successfully used for the enrichment and sensitive deter-mination of triazine herbicides, organophosphorus pesticides,chlorobenzenes, chlorophenols, PAHs and metal ions.12–19
However, its main limitation was the use of high-densityorganic solvents such as chlorobenzene,13 chloroform,20 andcarbon tetrachloride,21 which are highly toxic. Of late, room-temperature ionic liquids (RTILs) are used as extractionsolvents due to their unique physicochemical properties andeco-friendly nature. Recently, a new extraction techniquetermed as temperature-controlled ionic liquid dispersive liquid-phase microextraction using [C6MIM][PF6] was also tried foranalysis of some of the organophosphorus pesticides in envi-ronmental samples. Its mechanism was very much like DLLME,but the dispersion realized was not by injection of the solvent
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but by dissolution with increase in temperature. Anderson et al.have comprehensively reviewed the recent applications of ILs inanalytical chemistry.22 Accordingly, the number of investiga-tions using ILs for extraction, separation, and enrichment oftraces of analytes from complex matrices has been increasingyear by year.23 Although the ILs were used for extraction of drugsand their metabolites from urine,24–26 to the best of the authors'knowledge, their application to blood serum and plasma hasnot been reported so far in the literature.
The present manuscript describes the development of ionicliquid based DLLME of balooxacin from rat serum. Balo-oxacin is used as an antibiotic for treatment of uncomplicatedurinary tract infections. Several methods for analysis of balo-oxacin in biological matrices were reported.27–33 However, athorough literature search revealed that there were no reportson the extraction of balooxacin from rat serum using anIL-based DLLME. This is the rst report on extraction of balo-oxacin from rat serum by IL-DLLME followed by RP-HPLC. Todevelop the method, ve different ILs and a series of dispersivesolvents in different ratios and salt concentrations were tried.The IL-DLLME was optimized and validated via systematicexperimentation and successfully applied to pharmacokineticstudies in rats.
ExperimentalChemicals and reagents
All the reagents were of analytical grade unless stated otherwise.High-purity water prepared using a Millipore Milli-Q waterpurication system (Millipore synergy, Billerica, MA, USA), HPLCgrade acetonitrile and methanol (MeOH) (Merck Fine chemicals,Mumbai, India), and AR grade acetic acid (SD Fine Chemicals,Mumbai, India) were used. Membrane lter papers (0.45 mm)(Whatmann, Sanford, FL, USA) were used. Ionic liquids viz.1-butyl-3-methylimidazolium tetrauoroborate (BMITB), 1-butyl-3-methylimidazolium hexauorophosphate (BmimPF6), 1-butyl-3-methylimidazolium bromide (BMIB), (Fluka, Steinheim,Switzerland), 1-hexyl-3-methylimidazolium chloride (HMIC) and1-ethyl-3-methylimidazolium methyl sulfate (EMIMS) (Aldrich,Deisenhofen, Germany) >96% of purity were used.
Fig. 1 Chemical structures of a) balofloxacin and b) gemifloxacin (IS).
Animals
Male Wistar rats (200–220 g) (Pharmacology division, IndianInstitute of Chemical Technology, Tarnaka, Hyderabad, India)housed under standard conditions and had ad libitum access towater and standard laboratory diet throughout the experiments(rats obtained from National Centre for Laboratory AnimalSciences (NCLAS), National Institute of Nutrition (NIN), Hyder-abad-500 007, India, (Reg. no. 154, dt. 22-10-1999)) and animalswere kept and experiments were performed in compliance withthe relevant laws and institutional guidelines (“Bio-Safe”, Phar-macology Division, Indian Institute of Chemical Technology(IICT), Hyderabad 500 007, Reg. no. 97/1999/CPCSEA (Committeefor the Purpose of Control and Supervision on Experiments onAnimals), dated 28.04.1999) and also approved by the IICTbasic research committee, IAEC (Institutional Animal Ethics
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Committee) were used. Aer a single dose by oral administrationof 100 mg kg�1 of balooxacin to healthy Wistar rats (n ¼ 6),blood samples (0.2 mL) were collected into the processed testtubes at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 24 h post-dose.The blood samples were centrifuged at 4500 � g rpm for 5 minand the serum samples were stored at �20 �C until analysis.
Instrumentation
The HPLC system consisting of two LC-20AT pumps, an SPD-M20A diode array detector, a DGU-20A3 degasser, and a CBM-20Asystem controller (all from Shimadzu, Kyoto, Japan) was used.The chromatographic data were recorded using an HP-Vectra(Hewlett Packard, Waldron, Germany) computer system with LCsolution data acquiring soware (Shimadzu). A vortex shaker,sample tubes, a repeater (Tarsons, Chennai, India), and acentrifuge (model2-16P) (Sigma, Zurich, Switzerland) were used.
Preparation of stock solutions, calibration standards, andquality control samples
Accurately weighed 10.0 mg of each balooxacin and gemi-oxacin (IS) (Fig. 1) were separately dissolved in 10.0 mL ofMeOH in two volumetric asks to give 1.0 mg mL�1 individualstock solutions. Working standards of gemioxacin wereprepared by appropriate dilutions of stock solution with MeOH.The working standards were used for the preparation of wholeserum calibration standards and also quality control samples.All the stock solutions, calibration standards, and qualitycontrol samples were stored at 4 �C. Calibration standards(range 0.04–10 mg mL�1) were prepared by spiking workingstandard solutions into serum. These calibration standardswere used to test the linearity of the method. The quality controlsamples (0.05, 0.1, 2.0 and 7.5 mg mL�1) were prepared byspiking 100 mL of appropriate working standard into 200 mL ofserum. The QC samples were used to estimate the extractionrecovery (ER) and enrichment factor (EF)s of IL-based DLLMEextraction of balooxacin and gemioxacin from rat serum.
Extraction of balooxacin from rat serum
The extraction was performed in Eppendorf tubes. The tubeswere lled with quality control samples followed by addition ofIS, IL (BMITB/BmimPF6/C6MIMPF6/BMIB/HMIC/EMIMS), ACN,and NaCl solutions. The abovemixture was vortexed for 60 s andcentrifuged at 4000 rpm for 5.0 min. The bottom layer con-sisting of IL was collected using a micro-syringe, and injecteddirectly onto the ZIC®HILIC column for analysis by HPLC. Theblock diagram describing the IL-DLLME of balooxacin from
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Fig. 2 Block diagram showing the IL-DLLME process of balofloxacin from rat serum.
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rat serum is shown in Fig. 2. In case of in vitro samples, 200 mLof the serum spiked with 100 mL of balooxacin working stan-dard was taken in a 1.0 mL centrifuge tube. To this, 60 mL ACN,20 mL of IS, 50 mL of 5% NaCl solution, and 40 mL of IL wereadded, then vortexed for 60 s and centrifuged at 4000 rpm for5.0 min. The IL was found to be as a ne drop at the bottom ofthe tube well separated from the serum. The IL was initiallycolorless, but aer extraction it appeared as yellow in color dueto transfer of balooxacin whose color was yellow. The IL wasseparated carefully from the tube using the micro-syringe andinjected directly onto an RP-HPLC column. A probable mecha-nism of phase separation of IL and rat serum containing balo-oxacin is shown in Fig. 3. The chromatogram obtained showsthat the proteins present in the serum do not interact with theIL selected in the present investigation. In the case of in vivo
Fig. 3 Probable mechanism of phase separation of IL and rat serum co
1676 | Anal. Methods, 2014, 6, 1674–1683
samples, 200 mL of the serum was mixed directly with 40 mL ofIL, 60 mL of ACN, 20 mL of IS, and 5%NaCl solution. Themixturewas vortexed for 60 s, followed by centrifugation at 4000 rpm for5.0 min. The remaining procedure was the same as in the caseof in vitro samples. A schematic representation of IL-basedDLLME of balooxacin from rat serum is shown in Fig. 4.
Chromatographic conditions
Aer several trials, chromatographic separation was achievedon ZIC®HILIC-C18 (250 � 4.6 mm; 5 mm) (Merck, Darmstadt,Germany) in isocratic mode of elution. The mobile phase was amixture of 10 mM ammonium acetate : acetonitrile (20 : 80,v/v). It was freshly prepared, ltered through a Millipore lter(pore size 0.45 mm) and degassed continuously using an on-line
ntaining balofloxacin.
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Fig. 4 Schematic representation of IL-based DLLME of balofloxacinfrom rat serum.
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degasser. Separation was performed at room temperature usinga 0.8 mL min�1
ow-rate and 10 min run time. The injectionvolume was 10 mL and the detection wavelengths were set at287 nm. The chromatographic and integrated data were recor-ded using an HP-Vectra (Hewlett Packard, Waldron, Germany)computer system using LC-Solution data acquiring soware(Shimadzu, Kyoto, Japan).
Analytical parameters
Two important parameters, i.e. ER and EF, were determinedfor evaluation of the proposed method. ER was dened as the
Table 1 Density and miscibility of ILs with rat serum used in DLLME
Name of IL Code Cation
1-Butyl-3-methylimidazolium hexaurophosphate BmimPF6
1-Butyl-3-methylimidazolium tetrauoroborate BMITB
1-Hexyl-3-methylimidazolium hexaurophosphate C6MIMPF6
1-Butyl-3-methylimidazolium bromide BMIB
1-Hexyl-3-methylimidazolium chloride HMIC
1-Ethyl-3-methylimidazolium methylsulfate EMIMS
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percentage of balooxacin extracted into IL calculated byeqn (1).
ER ¼ (Ci � Vi � 100)/(C0 � V0) (1)
where Ci and C0 are the concentration of balooxacin in the ILand the initial concentration of the rat serum, respectively. Viand V0 are the volumes of the phases separated. The EF wasdened as the ratio of the concentration of balooxacin in IL tothe initial concentration of the sample. The EF was calculatedby eqn (2).
EF ¼ Ci/C0 (2)
Results and discussionOptimization of the extraction conditions
Generally the application of ILs to DLLME of blood serum islimited practically by two factors: (i) the recovery of IL from ratserum and (ii) the IL/dispersive solvent ratio. The miscibility ofIL with serum plays an important role in its recovery.
Selection of IL
Dispersive liquid–liquid microextraction (DLLME) has theadvantages of simplicity, rapidity, low sample volume, low cost,high recovery, and high enrichment factors. In DLLME, it isconsiderably important to select an extraction solvent withhigher density than water, high extraction capability of
AnionMiscibility withrat serum
Density (g mL�1)at 20 �C
Immiscible 1.38
Partial 1.21
Partial 1.41
Br� Miscible 1.30
Cl� Miscible 1.05
Partial 1.28
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compounds of interest and good chromatographic behavior.Toxic solvents such as chlorobenzene, carbon tetrachloride,tetrachloroethylene and carbon disulde have been oen usedas extraction solvents. However, environmental friendlysolvents are preferably used in place of chlorinated organicsolvents in order to protect the environment from toxic effects.34
Room temperature ionic liquids (RTILs), known as a new andnovel generation of solvents, have been widely applied inseparation and many other elds. They have many propertiesincluding low volatility, chemical and thermal stability, andgood solubility for heterocyclic insecticides.35 Moreover, by ne-tuning the structure, these properties can be designed to matchthe specic application requirements. The main reason thatmade them useful in analytical chemistry is the negligible vaporpressure of most RTILs.
The ILs viz. BMITB/BmimPF6/C6MIMPF6/BMIB/HMIC/EMIMS were initially selected to check their suitability asextraction solvents in DLLME of rat serum. Their miscibility andvolume added and recovered were studied. The density andmiscibility of ILs are given in Table 1. The ILs, BMIB and HMIC,have shown complete miscibility indicating their strong inter-actions with serum. This property is against the LLE principle,hence these ILs were not selected. Another three ILs, BMITB,EMIMS and C6MIMPF6, have shown partial miscibility withserum. In addition, the color of the recovered EMIMS alsochanged showing its partial interaction with serum. Based onthe above observations, EMIMS has been omitted, even though itcould partially recover balooxacin. Similarly, BMITB andC6MIMPF6 (1-hexyl-3-methylimidazolium hexauorophosphate)were also eliminated due to lower recoveries and partial misci-bility compared to BmimPF6. The IL BmimPF6 was immisciblewith rat serum and yielded a recovery of 97%. The recovered IL(BmimPF6) was directly injected into a restricted access mediumcolumn. The resulted chromatogram did not show any peakcorresponding to protein. It conrms that BmimPF6 was notinteracting with proteins present in the serum. So, Finally, theBmimPF6 IL was found to be a suitable extraction solvent for thepresent work.
Fig. 5 Optimization of IL-DLLME conditions.
Selection of dispersive solvent and optimization of extractant/disperser ratio
A ow chart of the optimization of an extraction procedure isshown in Fig. 5. The choice of a dispersive solvent with appro-priate miscibility in both the IL and serum phase plays animportant role because of the fact that the IL could completelydisperse into the aqueous phase of serum with the help of anappropriate solvent used as a disperser. In the present investi-gation, solvents, such as ACN, ethanol, and acetone, were triedfor experimentation as they fullled both the considerations.Since the partition coefficients and recovery (%) of balooxacinwere high with ACN (Fig. 6a), it was selected as a dispersivesolvent (Table 1). Mixtures containing ACN as a dispersivesolvent and BmimPF6 as an extractant in different volumeratios were selected. The extractant/disperser volume ratio wasoptimized by evaluating not only the ER and EFs but also therepeatability of the IL volume recovered. Different ratios of ACN
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and BmimPF6 were added to 200 mL of serum spiked with 100mL of balooxacin. EF and ERs were calculated using eqn (1) and(2) as described in the above section. Fig. 6b compares ER and
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Fig. 6 Effects of (a) dispersive solvents, (b) BmimPF6 : ACN ratios, and (c) %NaCl on extraction of balofloxacin.
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EF at different ratios BmimPF6/ACN used as extractant anddispersive solvents. The partition coefficient and recoveryvalues are recorded in Table 2. It could be seen from the results(Table 2 and Fig. 6b) that the highest partition ratio andrecoveries were obtained at a dispersive ratio of 0.43.
Effect of extraction and centrifugal time
In a DLLME procedure, the surface area between the extrac-tion solvent and the sample is innitely large aer theformation of a cloudy solution. Thus, the transfer of analytesfrom an aqueous phase to an extraction phase is quick andthe extraction process reaches equilibrium very fast. Theeffect of the extraction time was investigated between 0.0 and
Table 2 Partition ratio and recovery (%) of balofloxacina into ionic liquid
Dispersive solvent
Balooxacinconcentration (mM) Peak area
IL Serum IL
Without solvent 0.0162 0.0469 53 219Acetone 0.0188 0.0440 641 632Ethanol 0.0429 0.0199 1 459 038Acetonitrile 0.0621 0.0017 2 085 497
a Spiked concentration ¼ 0.0635 mM.
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10.0 min and observed that there was no considerable effectaer 60 s of the extraction time on the efficiency. The masstransfer of analytes could slow down due to the high viscosityof ILs, but IL-DLLME is a kind of fast equilibrium extractionand the extraction time was very short as reported in theliterature.34,35 Therefore, aer injection of IL and ACN intoserum, the mixture was vortexed for 60 s. Centrifugation is acritical step in the DLLME technique because it facilitates theseparation of the IL extraction phase from the sample phase,and it also affects the size of the settled phase and theconcentration of analyte in the extraction phase. A series ofextractions were performed by varying the centrifugationtime from 2.0 to 14.0 min at an interval of 3.0 min. Theresults showed that the volume of the IL phase increased
s
Partition ratio (p) Recovery (%)Serum
2 092 124 0.3454 25.51 506 012 0.4272 29.6688 926 2.1557 67.559 968 36.176 97.7
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Table 3 Partition ratio and recovery (%) of balofloxacina at different IL : ACN ratios and NaCl concentrations
Conditions
Concentration ofbalooxacin (mM) Peak area
Partition ratio (p) Recovery (%)IL Serum IL Serum
IL : ACN (v/v)0.11 0.0335 0.0292 1 141 265 1 006 199 1.147 52.70.25 0.0419 0.0219 1 425 917 723 549 1.913 65.90.43 0.0628 0.0010 2 117 120 30 542 62.80 98.80.67 0.0512 0.0129 1 702 775 445 802 3.968 80.61.00 0.0478 0.0161 1 591 352 557 268 2.968 75.2
NaCl (%)1.0 0.0629 0.0009 2 127 963 20 546 69.8 99.02.0 0.0630 0.0007 2 132 467 16 942 90.0 99.25.0 0.0632 0.0004 2 138 682 10 345 158 99.510.0 0.0632 0.0004 2 138 682 10 345 158 99.5
a Spiked concentration ¼ 0.0635 mM.
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from 28.4 to 29.5 mL by increasing the centrifugal time from2.0 to 5.0 min. The highest extraction efficiencies of balo-oxacin were obtained at a centrifugation time of 5.0 min,and a further increase of centrifugation time did not result inany better extraction efficiencies. Accordingly, 5.0 min wasselected as an optimum centrifugal time.
Effect of salt
The effect of salt on IL-based DLLME efficiency (Fig. 6c) wasevaluated by adding 1–10% (w/v) of NaCl to the rat serumunder the optimum experimental conditions. There was asignicant improvement in the partition ratio as shown inTable 3. It could be seen from Table 3 that the maximumrecovery (99.5%) of balooxacin was found at 5% (w/v) NaCl.Within the tested range, further addition of NaCl could notenhance the extraction efficiency of balooxacin. So, NaCl aslow as 5% was selected as an optimized condition. Thepartition coefficients and ERs could not be further improvedby increasing the NaCl concentration beyond 5.0%. Once theextraction conditions were optimized, its repeatability wasevaluated. Seven consecutive extractions were performedusing the optimum conditions and the overall recovery wasmeasured. It was found that the balooxacin recovery fromrat serum was repeatable with a relative standard deviation(RSD%) was <5%.
Table 4 Extraction recovery (ER) and enrichment factor (EF) ofbalofloxacin
Balooxacinconc. (mg mL�1)
Rat serum
ER � SD (%) EF � SD (%)
0.05 99.3 � 1.1 10.4 � 1.50.1 98.5 � 0.8 10.1 � 1.42.0 99.5 � 0.9 10.7 � 2.37.5 99.1 � 1.0 10.5 � 1.8
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Analytical performance of the method
The proposed IL-DLLME method was characterized by extrac-tion parameters (ER and EF) as well as the analytical gures ofmerit. Table 4 summarizes the ER and EFs of balooxacinconcentrations (0.05, 0.1, 2.0 and 7.5 mg mL�1) calculated underthe optimal experimental conditions. Both the parameters wereevaluated at four levels of concentration. The EFs variedbetween 10.1 and 10.7, while the ERs varied between 98.5 and99.5%. The results are given in Table 4. The calibration curvewas generated by plotting the ratio of the balooxacin and ISpeak areas against the concentration of balooxacin. Workingstandards containing balooxacin at ve concentration levelswere in the range 0.04–10 mg mL�1 (linear regression equationy ¼ 33.294x + 53.864) and the correlation coefficient was0.9998. The collected extracts of different concentrations ofbalooxacin with gemioxacin (IS) were subsequently analyzed
Fig. 7 Typical chromatograms of IL-DLLME extracts of rat serumspiked with balofloxacin at different concentrations: (a) blank, (b) 0.1,(c) 1.0, (d) 5.0, and (e) 10.0 ppm.
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Table 6 Intra and inter-day assay variation
Analyte
Intra-day
Inter-day0 1 2
BalooxacinConcentrationa 0.05 0.04 0.07 0.06RSD 1.98 2.05 2.46 2.01Concentrationa 0.10 0.09 0.11 0.11RSD 2.98 3.26 4.12 2.95Concentrationa 2.01 1.98 2.14 2.12RSD 1.51 3.05 4.21 2.35Concentrationa 7.52 7.60 7.48 7.54RSD 2.82 3.65 4.10 2.50
Gemioxacin (IS)Concentrationa 0.06 0.05 0.04 0.07RSD 2.14 1.86 1.65 2.38Concentrationa 0.10 0.12 0.09 0.08RSD 2.57 3.96 4.08 2.87Concentrationa 2.01 1.98 2.14 2.10RSD 4.65 3.27 2.55 1.50Concentrationa 7.52 7.60 7.48 7.53RSD 3.25 1.98 4.17 2.99
a Mean of concentration (mg mL�1; n ¼ 6); RSD, relative standarddeviation.
Table 7 Stability data of balofloxacin in rat seruma
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by RP-HPLC. Gemioxacin was used as an internal standard (IS)in order to minimize or reduce the matrix interference. Therepresentative chromatograms are shown in Fig. 7. The chro-matograms of the blank and spiked serum indicate that therewas no interference from blank (IL) peaks. The detection levelswere also enhanced when compared with the PP method asshown in Fig. 8. The results obtained by the proposed methodwere comparable with the PP method of extracting balooxacinfrom rat serum. Good linearity between the corresponding peakareas and the concentration was obtained (R > 0.999). The LODcalculated according to the S/N ¼ 3 ratio is 0.01 mg mL�1. Thelimit of quantication (LOQ) also calculated according to theS/N ¼ 10 ratio is 0.035 mg mL�1 and the data are given in Table5. The precision of the extraction method expressed as RSD% ofbalooxacin was <5%. The intra-day and inter-day assay varia-tion data are given in Table 6. Recovery studies were carried outby analyzing serum samples spiked with balooxacin at fourdifferent concentration levels. The relative recoveries werecalculated and found to be in the range of 98.5–99.5%.
The stability of balooxacin was investigated in the stock,working solutions, serum and extracts during storage and pro-cessing. The results are presented in Table 7. In rat serum thestability of balooxacin was examined during storage and aerfour freeze–thaw cycles at �20 �C with a minimal interval of24 h. The quality control samples that had been frozen andthawed three times were compared with the freshly prepared
Fig. 8 Typical chromatograms of PP and IL-DLLLME extracts ofbalofloxacin from rat serum.
Table 5 Validation parametersa
Parameter Obtained value
Linear range (mg mL�1) 0.04–10LOD (mg mL�1) 0.01LOQ (mg mL�1) 0.035RSD (%) <5RR (%) 99.5
a LOD, limit of detection; RSD, relative standard deviation; RR, relativerecovery.
Storage conditionsAdded conc.(mg mL�1)
Calculated conc.(mg mL�1) (mean � S.D) RE (%)
Ambient (6 h) 0.05 0.07 � 1.92 2.40.1 0.12 � 2.34 2.92.0 1.95 � 3.02 2.57.5 7.52 � 3.83 3.5
3 freeze (�20 �C)thaw cycles
0.05 0.04 � 3.07 2.10.1 0.09 � 4.41 2.62.0 2.05 � 3.80 3.17.5 7.48 � 4.29 4.2
2–10 �C, 3 days 0.05 0.06 � 3.54 2.90.1 0.13 � 4.07 3.82.0 1.96 � 3.45 2.47.5 7.42 � 4.21 3.7
Autosamplerstability (24 h)
0.05 0.08 � 2.90 3.20.1 0.15 � 3.42 2.92.0 2.10 � 2.61 2.27.5 7.44 � 4.22 3.8
a SD: standard deviation; RE: relative error (n ¼ 6).
This journal is © The Royal Society of Chemistry 2014
quality control samples and found to be stable. Balooxacin wasstable in rat serum during processing for 6 h at room temper-ature and 3 days at 2–10 �C. The reinjection reproducibility aerstorage of the samples in glass vials in the autosampler for 24 hwas also determined. Stability experiments were performed atfour concentrations (0.05, 0.1, 2.0 and 7.5 mg mL�1) in triplicate.Balooxacin was considered to be stable in stock and workingsolutions having the mean recoveries of 98.5–99.5% of theoriginal concentration. Whereas in biological matrices, therecoveries were 88–99.5% of the initial concentration.
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Fig. 9 Concentration vs. time profiles over 24 h of balofloxacin inserum of rat (n ¼ 6) receiving a single 100 mg kg�1 dose ofbalofloxacin.
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Application to pharmacokinetic studies
The developed IL-DLLME followed by the HPLC method wassuccessfully used for quantication of the balooxacinconcentration in rat serum aer oral administration. Balo-oxacin was extracted from serum using the proposedILDLLME procedure and analyzed by HPLC. The concentration–time prole of balooxacin is shown in Fig. 9. It shows that thebalooxacin concentration was <0.5 mg mL�1 at initial hours ofdosage and the rate of absorption was poor with serum. But, at 1h the concentration was maximum and then graduallydecreased up to 24 h. The maximum absorbance of balooxacinin rat serum was found to be 0.64 mg mL�1.
Conclusions
The present investigation explores for the rst time a novelcombination of ionic liquids with DLLME as a high-performanceand powerful preconcentration microextraction technique forextraction of balooxacin from rat serum. In DLLME, theextraction time was very short because of large surface areacontact between the analyte and extraction solvent. Additionally,ILs are not only green solvents that replace environmentallyhazardous organic solvents but also interact with a variety ofanalytes with different polarities due to their unique physico-chemical properties. Some of the advantages of this techniqueinclude: (i) compatibility with HPLC, (ii) no time-consumingprocedures aer extraction for cleanup and reducing the volumeof extracted solution and (iii) environmentally friendly. Theperformance of the proposed method in extraction of balo-oxacin from rat serum was found to be excellent. It can be usedas a simple, effective, reproducible and rapid method. Finally,when compared with other extractionmethods, it provides lowerLODs in shorter time. This study demonstrates that IL-DLLME isan accurate and reliable method for preconcentration of balo-oxacin from rat serum for analysis by RP-HPLC.
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Abbreviations
BmimPF6
1-Butyl-3-methylimidazolium hexauorophosphate DLLME Dispersive liquid–liquid microextraction EF Enrichment factor ER Extraction recovery IL Ionic liquid RTILs Room-temperature ionic liquids IS Internal standard LLE Liquid–liquid extraction MeOH Methanol ACN Acetonitrile PP Protein precipitationAcknowledgements
The authors thank the Director, IICT for constant encourage-ment and permission to communicate the manuscript forpublication. Mr Ch. Gangu Naidu, thanks the Council ofScientic and Industrial Research (CSIR), New Delhi, India for aresearch fellowship.
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