Ionic liquid based dispersive liquid–liquid microextraction followed by RP-HPLC determination of balofloxacin in rat serum

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<ul><li><p>AnalyticalMethods</p><p>PAPER</p><p>Publ</p><p>ishe</p><p>d on</p><p> 06 </p><p>Febr</p><p>uary</p><p> 201</p><p>4. D</p><p>ownl</p><p>oade</p><p>d by</p><p> Kan</p><p>sas </p><p>Stat</p><p>e U</p><p>nive</p><p>rsity</p><p> on </p><p>16/0</p><p>7/20</p><p>14 2</p><p>0:38</p><p>:19.</p><p>View Article OnlineView Journal | View IssueaAnalytical Chemistry Division, Discovery</p><p>Chemical Technology, Tarnaka, Hyderabad</p><p>com; rnrao@iict.res.in; Fax: +91 40 271733bSri Siddhartha Pharmacy College, Nuzvid,cPharmacology Division, Indian Institut</p><p>Hyderabad, 500 607, India</p><p>Cite this: Anal. Methods, 2014, 6, 1674</p><p>Received 17th October 2013Accepted 20th December 2013</p><p>DOI: 10.1039/c3ay41826j</p><p>www.rsc.org/methods</p><p>1674 | Anal. Methods, 2014, 6, 167416Ionic liquid based dispersive liquidliquidmicroextraction followed by RP-HPLCdetermination of balofloxacin in rat serum</p><p>R. Nageswara Rao,*a Ch. Gangu Naidu,a Ch. V. Suresh,b N. Srinathb and Raju Padiyac</p><p>An efficient and environmentally friendly ionic liquid based dispersive liquidliquid microextraction</p><p>procedure for the determination of balofloxacin in rat serum by reverse phase high-performance liquid</p><p>chromatography was developed and validated. The effects of ionic liquids, dispersive solvents,</p><p>extractant/disperser ratios and salt concentrations on sample recovery and enrichment were studied.</p><p>Among the ionic liquids investigated, 1-butyl-3-methylimidazolium hexafluorophosphate was found to</p><p>be the most effective and the recovery was 99.5% at an extractant/disperser ratio of 0.43 by addition of</p><p>5.0% NaCl. A threefold increase in detection (0.01 mg mL1) and quantification (0.035 mg mL1) limits was</p><p>achieved when compared to protein precipitation. A linear relationship in the range of 0.0410.0 mg</p><p>mL1 with a correlation coefficient of (r2) 0.9998 was observed. The method was validated and applied</p><p>to study the pharmacokinetics of balofloxacin in rat serum.Introduction</p><p>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 liquidliquid extraction(LLE).35 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</p><p>500 607, India. E-mail: rnrao55@yahoo.</p><p>87; Tel: +91 40 27193193</p><p>A.P, India</p><p>e of Chemical Technology, Tarnaka,</p><p>83tedious 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 liquidliquid 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.1219</p><p>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 solventThis journal is The Royal Society of Chemistry 2014</p><p>http://dx.doi.org/10.1039/c3ay41826jhttp://pubs.rsc.org/en/journals/journal/AYhttp://pubs.rsc.org/en/journals/journal/AY?issueid=AY006006</p></li><li><p>Paper Analytical Methods</p><p>Publ</p><p>ishe</p><p>d on</p><p> 06 </p><p>Febr</p><p>uary</p><p> 201</p><p>4. D</p><p>ownl</p><p>oade</p><p>d by</p><p> Kan</p><p>sas </p><p>Stat</p><p>e U</p><p>nive</p><p>rsity</p><p> on </p><p>16/0</p><p>7/20</p><p>14 2</p><p>0:38</p><p>:19.</p><p> View Article Onlinebut 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,2426 to the best of the authors'knowledge, their application to blood serum and plasma hasnot been reported so far in the literature.</p><p>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.2733 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</p><p>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) &gt;96% of purity were used.Fig. 1 Chemical structures of a) balofloxacin and b) gemifloxacin (IS).Animals</p><p>Male Wistar rats (200220 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 EthicsThis journal is The Royal Society of Chemistry 2014Committee) were used. Aer a single dose by oral administrationof 100 mg kg1 of balooxacin to healthy Wistar rats (n 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.</p><p>Instrumentation</p><p>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.</p><p>Preparation of stock solutions, calibration standards, andquality control samples</p><p>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 mL1 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 0.0410 mg mL1) 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 mL1) 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.</p><p>Extraction of balooxacin from rat serum</p><p>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 ZICHILIC column for analysis by HPLC. Theblock diagram describing the IL-DLLME of balooxacin fromAnal. Methods, 2014, 6, 16741683 | 1675</p><p>http://dx.doi.org/10.1039/c3ay41826j</p></li><li><p>Fig. 2 Block diagram showing the IL-DLLME process of balofloxacin from rat serum.</p><p>Analytical Methods Paper</p><p>Publ</p><p>ishe</p><p>d on</p><p> 06 </p><p>Febr</p><p>uary</p><p> 201</p><p>4. D</p><p>ownl</p><p>oade</p><p>d by</p><p> Kan</p><p>sas </p><p>Stat</p><p>e U</p><p>nive</p><p>rsity</p><p> on </p><p>16/0</p><p>7/20</p><p>14 2</p><p>0:38</p><p>:19.</p><p> View Article Onlinerat 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 vivoFig. 3 Probable mechanism of phase separation of IL and rat serum co</p><p>1676 | Anal. Methods, 2014, 6, 16741683samples, 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</p><p>Aer several trials, chromatographic separation was achievedon ZICHILIC-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-linentaining balofloxacin.</p><p>This journal is The Royal Society of Chemistry 2014</p><p>http://dx.doi.org/10.1039/c3ay41826j</p></li><li><p>Fig. 4 Schematic representation of IL-based DLLME of balofloxacinfrom rat serum.</p><p>Paper Analytical Methods</p><p>Publ</p><p>ishe</p><p>d on</p><p> 06 </p><p>Febr</p><p>uary</p><p> 201</p><p>4. D</p><p>ownl</p><p>oade</p><p>d by</p><p> Kan</p><p>sas </p><p>Stat</p><p>e U</p><p>nive</p><p>rsity</p><p> on </p><p>16/0</p><p>7/20</p><p>14 2</p><p>0:38</p><p>:19.</p><p> View Article Onlinedegasser. Separation was performed at room temperature usinga 0.8 mL min1 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</p><p>Two important parameters, i.e. ER and EF, were determinedfor evaluation of the proposed method. ER was dened as theTable 1 Density and miscibility of ILs with rat serum used in DLLME</p><p>Name of IL Code Cation</p><p>1-Butyl-3-methylimidazolium hexaurophosphate BmimPF6</p><p>1-Butyl-3-methylimidazolium tetrauoroborate BMITB</p><p>1-Hexyl-3-methylimidazolium hexaurophosphate C6MIMPF6</p><p>1-Butyl-3-methylimidazolium bromide BMIB</p><p>1-Hexyl-3-methylimidazolium chloride HMIC</p><p>1-Ethyl-3-methylimidazolium methylsulfate EMIMS</p><p>This journal is The Royal Society of Chemistry 2014percentage of balooxacin extracted into IL calculated byeqn (1).</p><p>ER (Ci Vi 100)/(C0 V0) (1)</p><p>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).</p><p>EF Ci/C0 (2)Results and discussionOptimization of the extraction conditions</p><p>Generally the application of ILs to DLLME of blood serum islimited practically by two factors: (i) the recovery of IL from ratserum and (ii...</p></li></ul>