A stereospecific high-performance liquid chromatographic assay for the determination of ketoconazole enantiomers in rat plasma

Copyright © 2008 John Wiley & Sons, Ltd. Biomed. Chromatogr. 22: 542–547 (2008)DOI: 10.1002/bmc

542 D. A. Hamdy and D. R. BrocksORIGINAL RESEARCH ORIGINAL RESEARCH

Copyright © 2008 John Wiley & Sons, Ltd.

BIOMEDICAL CHROMATOGRAPHYBiomed. Chromatogr. 22: 542–547 (2008)Published online 18 January 2008 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/bmc.967

A stereospecific high-performance liquid chromatographicassay for the determination of ketoconazole enantiomersin rat plasma

Dalia A. Hamdy and Dion R. Brocks*

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada

Received 9 August 2007; revised 5 October 2007; accepted 18 October 2007

ABSTRACT: A stereospecific high-performance liquid chromatographic assay was developed for the quantitation of ketoconazoleenantiomers (KTZ) in rat plasma. After protein precipitation of 100 μL plasma using acetonitrile, a wash step was performedusing hexane. The supernatant was removed and KTZ enantiomers and amiodarone, the internal standard, were extracted usingliquid–liquid extraction with tert-butyl methyl ether. After transfer and evaporation of the organic layer, the residue was reconsti-tuted in mobile phase and injected into the HPLC through a chiral column. The mobile phase consisted of hexane:ethanol:2-propanol with diethyl amine, pumped at 1.5 mL/min. All components eluted within 18 min. KTZ enantiomers were baselineresolved and peaks were symmetrical in appearance with no interferences. Calibration curves were linear over the range 62.5–5000 ng/mL of enantiomer. The intraday and interday CV% assessments were ≤19 and <13%, respectively, and mean error was<4% for both enantiomers. The lower limit of quantitation was 62.5 ng/mL for each enantiomer based on 100 μL rat plasma.In rats, plasma concentrations of (+)-KTZ were higher than those of antipode after single oral doses. The assay was shown to besensitive and appropriate for use in pharmacokinetics study of KTZ in rat. Copyright © 2008 John Wiley & Sons, Ltd.

KEYWORDS: enantiospecific; HPLC; antifungals; pharmacokinetics

*Correspondence to: D. R. Brocks, Faculty of Pharmacy and Phar-maceutical Sciences, 3118 Dentistry/Pharmacy Centre, University ofAlberta, Edmonton, AB, Canada T6G 2N8.E-mail: [email protected]

Abbreviations used: AM, amiodarone; KTZ, ketoconazole; SFC,supercritical fluid chromatography; TBME, tert-butyl methyl ether.

Contract/grant sponsor: CIHR; Contract/grant number: MOP 67169.

INTRODUCTION

Over the last two decades, there has been a dramaticincrease in the incidence of serious mucosal and systemicfungal infections. This is mainly due to immunosuppres-sion associated with human immunodeficiency virus,organ transplantation and anticancer chemotherapy(Thienpont et al., 1999; Dilmaghanian et al., 2004; Castro-Puyana et al., 2005). Amongst the different antifungalagents, the azoles (imidazole- or triazole-based drugs)constitute an important class due to their broad spectrumof antifungal activity and reasonably good oral bioavai-lability (Como and Dismukes, 1994). Ketoconazole (KTZ)was the first introduced orally administered azole.However, due to the risk of KTZ-induced hepatotoxi-city and serious drug–drug interactions, its use tends tobe restricted to serious infections resistant to othersafer azoles (Velikinac et al., 2004; Korashy et al., 2007).

KTZ, cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3dioxolan-4-yl]methoxy]phenyl]-piperazine, is a chiral drug administered clinically as a

racemic (1:1) mixture of the enantiomers of the cisconfiguration (Fig. 1). The cis configuration indicatesthat the hydrogen and the 2,4-dichlorophenyl group atthe two chiral centers are on the same side of the five-membered (dioxolane) ring. Their absolute configura-tion has been reported via synthesis as (+)-(2R,4S)dextrorotatory enantiomer and (−)-(2S,4R) levorotatoryenantiomer (Rotstein et al., 1992).

Since stereochemistry is an important modulatorof drug biological effects, for new drug candidates theUS Food and Drug administration (FDA) has recom-mended the development of quantitative assays forindividual enantiomers in in vivo samples early in drugdevelopment (Anonymous, 1992). For many enantio-meric pairs of drugs, the pharmacological activity ofone enantiomer may differ from that of its antipode(Jamali et al., 1989; Brocks and Jamali, 1995). In manycases as well, the pharmacokinetic behaviour of oneenantiomer may differ from the other (Brocks, 2006;Lu, 2007). Thus, it is worth examining the potentialof stereoselectivity in KTZ pharmacokinetic and phar-macodynamic properties. To date, however, there hasbeen no published report of the stereoselective phar-macokinetics of KTZ.

The enantiomeric separation of KTZ has beensuccessfully achieved in solutions or pharmaceuticalformulations using supercritical fluid chromatography(SFC) with a ChiralPak AD column (Thienpont


Determination of ketoconazole enantiomers in rat plasma 543ORIGINAL RESEARCH

Figure 1. Ketoconazole enantiomer chemical structures.

et al., 1999; Bernal et al., 2000) and electrokinetic capil-lary electrophoresis (Castro-Puyana et al., 2005). SomeHPLC separations have been reported using chiralcolumns (Thienpont et al., 1999; Bernal et al., 2002).Although some success in separating the enantiomersof KTZ has been reported, to our knowledge a methodfor quantifying the enantiomers in biological specimenshas to date not been published. Therefore, in thisreport we describe a sensitive and specific HPLC methodfor the determination of KTZ enantiomers in rat plasma.

EXPERIMENTAL

Materials and reagents. Ketoconazole and amiodarone HClwere obtained from Sigma (St Louis, MO, USA). Hexane,isopropyl alcohol, absolute ethanol, tert-butyl methyl ether(TBME) (all HPLC grade), and diethylamine were purchasedfrom Fisher Scientific (Fair Lawn, NJ, USA).

Chromatographic conditions. The chromatography systemconsisted of a Waters 710B WISP auto-injector, Waters510 pump (Waters, Milford, MA, USA) and an HP 1050 UVdetector (Hewlett Packard, Palo Alto, CA, USA). The UVwavelength was set at 240 nm. The chromatographic separa-tion of amiodarone (IS) and ketoconazole enantiomers wasaccomplished using a 250 × 4.6 mm ChiralPak AD column(Diacel Chemical Industries Ltd, NJ, USA) attached to apre-column holder for PVDF-Cardridges 4-4 containingLichrospher 100 Diol 5 μm guard column (Merck, KGaA,Darmstadt, Germany). Chromatographic data were collectedand compiled by use of EZChrom software (Scientific Soft-ware, Pleasanton, CA, USA). The mobile phase used was a70:20:10 (v/v) mixture of hexane, absolute ethanol and 2-propanol to which 0.1% diethylamine. The mobile phase waspumped at a flow rate of 1.5 mL/min.

Standard and stock solutions. A 100 mg/L stock solution ofracemic KTZ was prepared by dissolving 10 mg of (±)-KTZin 100 mL methanol. A 100 mg/L stock solution of AM

(amiodarone, internal standard) was prepared by dissolving10.6 mg of AM HCl in 100 mL methanol. To prepare samplesfor the calibration curve and validation assessment, threeworking solutions of 0.1, 1 and 10 mg/L of (±)-KTZ were pre-pared by successive 1:10 dilutions of the stock solution withmethanol. The AM stock solution was stored at −30°C whileKTZ stock and working solutions were stored at 2°C betweenuses.

Extraction procedure. IS (0.02 mL) was added to each 0.1 mLplasma sample in a 2 mL polypropylene microcentrifuge tube.To this, 0.3 mL of acetonitrile was added to precipitate theplasma proteins. Because methanol was present in the stand-ard curve samples, an equivalent amount was added to thetest samples as well (0.125 mL). The tubes were briefly vortexmixed (4 s) at high speed then subsequently centrifuged for3 min at ~2500g. The supernatants were carefully transferredto new glass tubes using Pasteur pipettes. Then 0.05 mLof 0.1 M HCl and 1 mL of hexane were subsequently added toeach tube as a washing step. The tubes were then vortexmixed for 30 s and centrifuged at ~2500g for 3 min. Theorganic hexane supernatant was carefully aspirated and then0.075 mL of 0.1 M NaOH and 5 mL of TBME were added.The tubes were then vortex mixed again for 30 s and centri-fuged at ~2500g for 3 min. The organic layer was transferredto new glass tubes and evaporated to dryness in vacuo. Theresidues were reconstituted in 150 μL mobile phase, of which75–125 μL volumes were injected into the HPLC.

Recovery. The plasma recoveries were determined at KTZenantiomer concentrations of 250, 1000 and 5000 ng/mL, usingfour replicates for each concentration. The extraction efficiencywas determined by comparing the peak areas of analyte tothe peak areas of the same amounts directly injected to theinstrument, without extraction. Recovery of IS was performedusing the concentrations used in the assay (20 μg/mL).

Calibration, accuracy and validation. Calibration curves wereconstructed using samples of 0.1 mL rat plasma containingKTZ and IS. The curve ranged from 62.5 to 5000 ng/mL ofeach KTZ enantiomer. The ratio of KTZ enantiomers to IS


544 D. A. Hamdy and D. R. BrocksORIGINAL RESEARCH

peak height was calculated and plotted vs nominal KTZenantiomer concentration. Owing to the wide range of con-centrations, the calibration curve data were weighed by afactor of 1/KTZ enantiomer concentration.

Intraday accuracy and precision of the assay were deter-mined using five sample replicates of 62.5, 125, 500 and2500 ng/mL of each KTZ enantiomer in rat plasma. Topermit the assessment of interday accuracy and precision, theassay was repeated on three separate days. For each dailyrun, concentrations were determined by comparison with acalibration curve prepared on the day of the analysis. Preci-sion was determined using percentage coefficient of variation(CV %) and bias was assessed using mean intra- or interdaypercentage error of the mean.

Assignment of KTZ enantiomer optical rotation. Theorder of elution of (+) and (−) enantiomers was determinedby collecting eluent fractions from repeated injections. Therewere ~25 injections made into the HPLC, with ~180 μgracemate per injection. After compiling the eluent fractionscorresponding to each enantiomer, they were dried in vacuo,reconstituted in methanol and the optical rotation determinedusing a Perkin Elmer 241 polarimeter (PerkinElmer Life andAnalytical Sciences, Waltham, MA, USA).

Test for racemization. Ketoconazole enantiomer fractionsof 10,000 ng each were collected. Each enantiomer fractionwas divided into three aliquots, and then dried in vacuo. Thecontents of the tubes were extracted according to the pro-cedure described above. The samples were reconstitutedin 150 μL of mobile phase, and 125 μL was injected into theHPLC.

Assessment of chromatographic separation. The capacityfactor (K′) was calculated using the equation K′ = (tr − tm)/tm,where tr and tm are the retention times of the peak of interestand the non-retained peak (solvent front). The separationfactor (α) was calculated as α = K′J/K′I, where component Jis the more strongly retained compound. The resolutionfactor (Rs) was calculated as Rs = (tR,J − tR,I)/0.5(Wt,J − Wt,I),where Wt is the width at the base of the peak. The symmetryindex was determined by first determining the peak width at10% of peak height. The latter part of the width from time ofpeak height onwards was divided by the first part of the widthup to the time of peak height.

Animal study. In order to assess the applicability of thismethod in vivo, two rats (280–300 g) were administered10 mg/kg (±)-KTZ orally. The protocol was approved by theUniversity of Alberta Health Sciences Animal Policy andWelfare Committee. The day before the pharmacokineticstudy, the right jugular vein of each rat was cannulated withMicro-Renathane tubing (Braintree Scientific, Braintree, MA,USA) under isoflurane anesthesia. The cannula was filledwith 100 U/mL heparin in 0.9% saline. After cannula implan-tation, the rats were transferred to regular holding cages andallowed free access to water, but food was withheld overnight.The next morning, animals were transferred to metaboliccages for conduct of the pharmacokinetic experiments.

Serial blood samples were collected at 0.5, 1, 2, 3, 4, 6, 8, 10and 24 h postdose into heparinized 2 mL polypropylene

microcentrifuge tubes. Plasma was separated by centrifuga-tion of the blood at 2500g for 3 min. The samples were keptat −30°C until assayed using the developed HPLC method.The Cmax and tmax were examined from visual examinationof the data, and the area under the plasma concentrations vstime curve from time of dosing to last measured concentra-tion was calculated using log–linear trapezoidal rule.

RESULTS

The IS and KTZ enantiomers eluted chromatographi-cally at approximately 3.8, 11.9 and 15.6 min, respec-tively (Fig. 2). From the polarimetry assessment, thedextro (+) enantiomer was found to be eluting first.KTZ enantiomer peaks showed baseline resolution andwere symmetrical in appearance with no interferencesfrom endogenous substances in plasma. The assayshowed no observable racemization. The total analyti-cal run time was 18 min.

The column capacity factors (K′) for IS, (+)- and (−)-KTZ were calculated to be 0.3, 3.1 and 4.4 respectively.The column separation factor (α) and resolution factorfor the KTZ enantiomers were calculated to be 1.4 and2.3, respectively. The symmetry index were 1.1 and 1.2for the (+) and (−) enantiomers, respectively.

The average extraction recoveries in plasma of (+)-KTZ were 89.0, 81.3 and 82.6% for the 250, 1000 and5000 ng/mL enantiomer concentrations, respectively.For the (−) enantiomer, extraction efficiencies were87.9, 79.8 and 82.0%, respectively, at concentrations of250, 1000 and 5000 ng/mL enantiomer. The recovery ofIS was 81.8%. Highly linear relationships were notedbetween the analyte/IS peak height or area ratios andrat plasma enantiomer concentrations ranging from62.5 to 5000 ng/mL. The mean r2 for the three standardcurves were 0.999 for each enantiomer. The standardcurve in plasma plotting concentration (ng/mL) vs peakheight ratio yielded slopes of 1.17 × 10−5 ± 8.15 × 10−7

and 9.31 × 10−6 ± 7.33 × 10−7 for (+)- and (−)-KTZ,respectively. The corresponding intercepts were 2.87 ×10−4 ± 1.65 × 10−4 and 3.02 × 10−4 ± 1.69 × 10−4, respec-tively for the (+) and (−) enantiomers.

The validation data showed the assay to be sensitive,accurate and precise, with the intraday and interdayassessment CV% less than or equal to 19 and 13%,respectively, for both enantiomers (Table 1). The meaninterday error in rat plasma was less than 4% for bothenantiomers. Since both CV% of interday and intradayassessment and interday mean error yielded valuesless that 20% at the lowest concentration tested, thelower limit of quantitation (LLQ) based on 100 μLof rat plasma was found to be 62.5 ng/mL for eachenantiomer

In the rats dosed with (±)-KTZ orally, the concentra-tions of (+) enantiomer were much higher than those of(−)-KTZ (Fig. 3). The Cmax of the (+) enantiomer was



Figure 2. Chromatograms of (A) blank rat plasma sample, (B) rat plasma spikedwith 2500 ng/mL of (±)-KTZ extracted from rat plasma samples, (C) rat plasma ob-tained after 2 h of 10 mg (±)-KTZ/kg. The inset shows a section of a chromatogramof a plasma sample spiked with 125 ng/mL of (±)-KTZ (the validated LLQ of theassay). IS denotes internal standard (amiodarone).

Figure 3. Plasma KTZ enantiomer concentrations vs time curvesfrom two rats given an oral dose of 10 mg/kg KTZ racemate.

5246 and 1389 ng/mL for the two rats while those of the(−) enantiomer were 2424 and 614 ng/mL, respectively.The tmax for both enantiomers were similar, occurring at~1 h post-dose in both rats. The KTZ enantiomer con-centrations occurring after 4 h post-dosing below theLLQ of 62.5 ng/mL, thus limiting the AUC calculationfrom 0 to 4 h post-dose. The AUC for KTZ enantiomerconcentrations in the two rats studied was 10596 and2346 ng h/mL for the (+)-KTZ and 4404 and 945 ng h/mL for the (−)-KTZ.

DISCUSSION

The described assay was capable of quantifying theenantiomers of KTZ in a biological specimen, ratplasma. The assay was sensitive and specific, affordingbaseline resolution of the enantiomers. For an analyti-cal run of 20 samples, it took approximately 90 minto prepare the samples for injection into the HPLC. Toour knowledge, this is the first stereospecific assay forKTZ in plasma. However, it is not the first methodreported for separation of the KTZ enantiomers. Thefirst HPLC separation utilized ChiralPak AS andChiralCel OD columns with combinations of hexanesand alcohols with octanoic acid. (Thienpont et al., 1999)Although a chromatogram was not presented, the authorsreported a baseline separation of KTZ enantiomers,albeit with a very long analysis time (~50 min). Similarly,Bernal et al. reported that KTZ enantiomers werestrongly retained (over 60 min) on both ChiralPak ADand Chiracel OD columns, using either hexane:ethanol


546 D. A. Hamdy and D. R. BrocksORIGINAL RESEARCH

or hexane:2-propanol as mobile phase (Bernal et al.,2002). Both authors reported better or similar resolu-tion and shorter analytical run (~7 min) upon usingSFC (Thienpont et al., 1999; Bernal et al., 2002). It wasconcluded by one group that SFC was the only tech-nique that can provide appropriate enantiomeric sepa-ration for KTZ (Bernal et al., 2002). However, ourHPLC method provided sensitive and precise means ofmeasuring KTZ enantiomers with sufficient resolutionand sensitivity for application in biological specimens.Although the analytical run time was somewhat longerthan determined by SFC (Bernal et al., 2000, 2002),it was still entirely acceptable for an HPLC method(~18 min; Fig. 2).

The current method provided resolution parametersthat were quite comparable and in some ways superiorto those calculated from or reported in previously forSFC using similar chiral columns. Thienpont et al.reported an α of 1.3 and a resolution factor of 0.83.From the chromatograms presented by Bernal et al., anα of 1.6 and a resolution factor of >2 were calculated(Thienpont et al., 1999; Bernal et al., 2000). In bothcases our values of α and resolution factor were equiva-lent or superior. Both of the SFC methods were separa-tion technique optimizations and were not tested forquantification; neither standard curves nor validationdata were reported.

A capillary electrophoresis technique has been re-ported which was capable of measuring KTZ enantiomersin pharmaceutical formulations, and had superior sepa-ration qualities of 3 min elution time and resolutionfactor >2. However, the stated lower limit of detectionwas 250 ng/mL for each enantiomer and there was nomention of extraction from biological fluids, limiting itsapplication to drug solutions only (Castro-Puyana et al.,2005).

Several non-stereospecific analytical methods for thedetermination of KTZ in biological specimens havebeen reported. Using HPLC the non-stereospecific LLQof KTZ ranged from 62.5 ng/mL using fluorescencedetection, to 100 ng/mL using UV detection, based on100 μL of human plasma (Alton, 1980; Yuen and Peh,1998). In canine plasma, a LLQ of 15 ng/mL was re-ported using HPLC and UV detection based on 100 μLplasma (Vertzoni et al., 2006). In rat plasma the LLQranged from 2 ng/mL using LC/MS to more than200 ng/mL using UV detection based on 100 μL and1 mL plasma, respectively (Riley and James, 1986;Huang et al., 2007). Thus our reported LLQ is withinthe ranges reported using HPLC and UV detection inbiological specimens.

In working up the method, we found that there wasa need to incorporate a hexane wash step to removean interfering peak from plasma which eluted at theinitial part of the (+)-KTZ peak. Hexane was an excel-lent solvent for this purpose, as it caused virtually noT

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extraction of KTZ enantiomers from the acetonitrile–aqueous phase. There was some loss of IS, but it didnot have any adverse effects on linearity or validationparameters. We noticed that there was some noise inthe baseline underlying the amiodarone peak, but dueto the excess of amiodarone added, it similarly did notadversely affect KTZ enantiomer validation param-eters. The optical rotation testing showed that (+)-KTZis eluted first. This is in accordance with a reportedpreparative method for separation of KTZ enantiomersusing the same HPLC column but different mobilephase (Gal et al., 1993; Dilmaghanian et al., 2004).

In vitro studies applying KTZ enantiomers tocytochrome P4503A4 using testosterone and metha-done as substrates suggested stereoselective inhibition,wherein the (−)-KTZ enantiomer showed ~2 fold moreinhibitory potency (Dilmaghanian et al., 2004). In ourreport based on Cmax and AUC, we found that theplasma concentrations of (+)-KTZ were 2.1-2.4 foldhigher than those of the (−)-KTZ, indicating a highlevel of stereoselectivity in KTZ pharmacokineticsin vivo after single oral doses. The tmax values of thetwo enantiomers were similar, indicating similar ratesof absorption (Fig. 3).

(±)-KTZ plasma concentrations are reported to bemuch lower than in tissues (Riley and James, 1986). Aprevious report could not determine any measurableKTZ racemate plasma levels after 6 h of an i.v. admin-istration of 5 mg/kg (±)-KTZ using nonstereospecificHPLC assay with 100 ng/mL LLQ based on 200 μLplasma (Remmel et al., 1987). Our method could quan-tify KTZ enantiomer concentrations for 4 h post-doseusing an oral dose of 10 mg/kg (±)-KTZ (Fig. 3). It isof note that (±)-KTZ is associated with nonlinearclearance (Sjoberg et al., 1988; Matthew et al., 1993),and therefore with repeated dosing it is likely that thetime duration over which our assay could measure thedrug would be longer than 4 h.

In conclusion, our method is the first to reportseparation and determination of KTZ enantiomersin plasma biological specimen. The method was sensi-tive and specific, with a lower limit of quantitationof 62.5 ng/mL, which is sufficient to permit use forpharmacokinetic studies involving rats.

Acknowledgments

Funding for this study came from CIHR MOP 67169.The authors thank Dr Neal Davies for his helpful dis-cussions regarding the stereospecific separation.

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A stereospecific high-performance liquid chromatographic assay for the determination of ketoconazole enantiomers in rat plasma