Sensitive, selective and rapid determination of bupropion and its major active metabolite,...

Research article

Received 5 March 2011, Revised 28 April 2011, Accepted 2 May 2010 Published online in Wiley Online Library: 8 June 2011

(wileyonlinelibrary.com) DOI 10.1002/bmc.1660

Sensitive, selective and rapid determination ofbupropion and its major active metabolite,hydroxybupropion, in human plasma byLC‐MS/MS: application to a bioequivalencestudy in healthy Indian subjectsJignesh M. Parekha, Dipen K. Sutariyaa, Rajendrasinh N. Vaghelaa,Mallika Sanyalb, Manish Yadavc and Pranav S. Shrivastava,d*

ABSTRACT: A sensitive, selective and rapid liquid chromatography tandem mass spectrometry (LC‐MS/MS) method wasdeveloped for the simultaneous determination of bupropion (BUP) and its major active metabolite hydroxybupropion(HBUP) in human plasma. Separation of both the analytes and venlafaxine as internal standard (IS) from 50 μL human plasmawas carried out by solid‐phase extraction. The chromatographic separation of the analytes was achieved on a Zorbax EclipseXDB C18 (150× 4.6mm, 5µm) analytical column using isocratic mobile phase consisting of 20mM ammonium acetate–methanol (10:90, v/v), with a resolution factor of 3.5. The method was validated over a wide dynamic concentration range of0.1–350ng/mL for BUP and 0.1–600 ng/mL for HBUP. The matrix effect was assessed by post‐column infusion and the meanprocess efficiency was 96.08 and 94.40% for BUP and HBUP, respectively. The method was successfully applied to abioequivalence study of 150mg BUP (test and reference) extended release tablet formulation in 12 healthy Indian malesubjects under fed conditions. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: bupropion; hydroxybupropion; chromatographic separation; LC‐MS/MS; bioequivalence study

* Correspondence to: Pranav S. Shrivastav, Chemistry Department, Schoolof Sciences, Navrangpura, Gujarat University, Ahmedabad‐380009, India.E‐mail: pranav_shrivastav@yahoo.com

a Chemistry Department, Kadi Sarva Vishwavidyalaya, Sarva VidyalayaCampus, Sector 15/23, Gandhinagar‐382015, India

b Chemistry Department, St Xavier’s College, Navrangpura, Ahmedabad‐380009, India

c Bioequivalence and Bioanalytical Department, Cadila Pharmaceuticals Ltd,Ahmedabad‐387810, India

d Department of Chemistry, School of Sciences, Navrangpura, GujaratUniversity, Ahmedabad‐380009, India

Abbreviations used: BUP, bupropion; HBUP, hydroxybupropion; PP,protein precipitation.

IntroductionBupropion (BUP, 2‐tert‐butylamino‐3′‐chloropropiophenone) is asecond‐generation antidepressant used in the management ofsmoking cessation and for the treatment of bipolar disorder,attention‐deficit hyperactivity disorder and weight loss (Colesand Kharasch, 2007; Johnston et al., 2002). It is a monocyclicaminoketone and is a weak inhibitor of dopamine reuptake. Ithas little or no effect on serotonin reuptake or on monoamineoxidase activity (Ferris et al., 1983; Suckow et al., 1997). Owing toits different side effect profile from the conventional tricyclicantidepressants, it serves as an appropriate alternative forpatients who are intolerant to tricyclic side effects (Roose et al.,1991; Suckow et al., 1997; Wenger and Stern, 1983). Bupropion isavailable in three oral formulations: immediate release (giventhree times a day), sustained release (given twice a day) andextended release (given once a day). The extended releaseformulation was approved in 2003 by the US Food and DrugAdministration for the treatment of major depressive disorder.Bupropion (t1/2≈ 21 h) is rapidly absorbed in the gastrointestinaltract after oral administration of the immediate release formu-lation, with a mean Tmax value of 1.5 h. The absorption of BUPis almost complete (~100%); however, the systemic bioavail-ability is less than 100% owing to first‐pass metabolism. Theplasma protein binding of BUP is in the range of 82–88% (Findlayet al., 1981; Jefferson et al., 2005). Bupropion is extensively

metabolized in the liver to give three active metabolites, namelyhydroxybupropion (HBUP), threo‐hydroxybupropion (threo‐HBUP)and erythro‐hydroxybupropion (erythro‐HBUP), with less than0.5% reported to be recovered intact in urine (Coles andKharasch, 2007; Lai and Schroeder, 1983). Hydroxybupropion(t1/2≈ 20 h) is themajor activemetabolite found in human plasma(Johnston et al., 2002; Xu et al., 2007), and is formed viahydroxylation of tert‐butyl group and the amino alcohol isomersand subsequent formation of the morpholinol ring. Threo‐HBUP

Wiley & Sons, Ltd.

Determination of bupropion and hydroxybupropion in human plasma

and erythro‐HBUP are formed by reduction of the carbonyl groupand are analogs of the sympathomimetic amines pseudoephed-rine and ephedrine, respectively (Johnston et al., 2002; Lobozet al., 2005). The activity of HBUP is approximately 50% of theparent drug while the combined activity of the other twometabolites is ~20% of BUP according to in vivo studies using themouse anti‐tetrabenazine model of depression (Coles andKharasch, 2007; Jefferson et al., 2005).

BUP is a chiral compound having one chiral center and is usedclinically as a racemic mixture. Its enantiomers have been shownto undergo rapid racemization in solution (Fang et al., 2000). Thepotency of BUP enantiomers in vitro does not differ from thatof the racemate, although racemization under physiologicalconditions may explain the lack of stereoselectivity (Coles andKharasch, 2007). The major active metabolite, HBUP, has twochiral centers but only two stereoisomers are found in humanplasma out of the possible four diastereoisomers. The (R,R)isomer represents ~96% of HBUP in plasma at steady state afterBUP administration (Loboz et al., 2005; Suckow et al., 1997).Several methods have been developed for the enantiomericdetermination of BUP and/or its metabolites (Coles and Kharasch,2007; Munro and Walker, 2001; Munro et al., 2001; Puyana et al.,2008; Suckow et al., 1997; Xu et al., 2007). Separation of BUPenantiomers was demonstrated using an ovomucoid (Munro andWalker, 2001) and α1‐acid glycoprotein (AGP) chiral column(Munro et al., 2001) within 10min. In both these methods thestationary phase parameters that may influence the separationwere optimized but no application was presented. A coupledachiral–chiral stationary‐phase liquid chromatographic tech-nique was described to separate and quantitate the enantiomersof the phenylmorpholinol metabolite (Suckow et al., 1997). Asensitive and stereoselective assay was developed for thequantitation of the enantiomers of HBUP in human plasma andapplied to study drug interactions with rifampicin (Xu et al., 2007).Separation was achieved on a Cyclobond I 2000 column within20min and the assay was linear over the concentration range of12.5–500 ng/mL for (2R,3R) and (2S,3S)‐HBUP. Coles and Kharasch(2007) demonstrated a stereoselective analysis for BUP and HBUPin human plasma and urine by LC‐MS/MS. The extraction wascarried out on a Waters Oasis MCX solid‐phase 96‐well plate andseparation was done on an AGP chiral column. The limits ofquantitation were 0.5 and 2.5 ng/mL for BUP and HBUPenantiomers, respectively. Electrokinetic chromatography usingcyclodextrins as chiral selectors has also been described todetermine BUP enantiomers in pharmaceutical preparations(Puyana et al., 2008).

Owing to rapid racemization of BUP in vivo and thepredominance of one diastereoisomer of HBUP after BUPadministration, several studies have reported achiral methodsto determine BUP and its active metabolites in differentbiological matrices. Bupropion as a single analyte was deter-mined in human plasma (Al‐khamis, 1989; Jennison et al., 1995),dog plasma (Zhang et al., 2003) and rat everted gut sacs(Arellano et al., 2005). Simultaneous determination of BUP andits metabolites is a subject of several reports (Borges et al. 2004;Cooper et al., 1984; Denooz et al., 2010; Loboz et al., 2005;Suckow et al., 1997; Yeniceli and Dogrukol‐Ak, 2009). Bupropionand its three metabolites were determined in plasma by liquidchromatography with dual wavelength ultraviolet detection(Cooper et al., 1984). Liquid–liquid extraction was done fromalkaline plasma with 1.5% iso‐amyl alcohol in n‐heptane, whilethe separation was achieved on a reversed‐phase column using

phosphate buffer–acetonitrile (80:20, v/v) as the mobile phasecontaining an ion‐pairing reagent and triethyl amine. Thedetection limits were 5 ng/mL for BUP and 100 ng/mL for themajor metabolites. Suckow et al. (1997) studied the pharmaco-kinetics of BUP and its metabolites in plasma and brain of rats,mice and guinea pigs using the reported procedure (Cooperet al., 1984). An HPLC assay for the simultaneous determination ofBUP and its three metabolites in human plasma was describedand used for assessing CYP2B6 activity in vivo following a singledose of BUP (Loboz et al., 2005). The extraction recovery wasgreater than 55% for all the analytes and the method showed alinear response for BUP (2.5–250 ng/mL), threo‐HBUP (5–250 ng/mL), erythro‐HBUP (10–250 ng/mL) and HBUP (10–1000 ng/mL).Yeniceli and Dogrukol‐Ak (2009) developed a simple LC methodfor BUP and HBUP estimation in human plasma, employing one‐step protein precipitation with trichloroacetic acid, followedby separation on an Agilent XDB‐C8 column. The methodwas comparatively less sensitive (BUP, 60 ng/mL; and HBUP,150 ng/mL) with a long chromatographic run time of 15min andthus may not be suitable for high‐throughput analysis. A high‐throughput LC‐MSmethodwas developed and partially validatedto determine BUP and its metabolites in human, mouse and ratplasma using a monolithic column (Borges et al., 2004). Themethodwas linear over a concentration range of 0.25–200 ng/mL(BUP and threo‐HBUP) and 1.25–1000 ng/mL (HBUP). This is themost sensitive method so far for the simultaneous determinationof BUP and its metabolites. Denooz et al. (2010) presented aUPLC‐MS/MS method to quantify BUP and its three activemetabolites in human whole blood. A combination of proteinprecipitation (PP) and solid‐phase extraction (SPE) on Oasis HLBcartridges was employed for sample clean‐up. Separation ofall four analytes was achieved in a run time of 4.1min andthe linearity was established for 5–1000 ng/mL for BUP and10–2000 ng/mL for the metabolites. Very recently, Yeniceli et al.(2011) developed a simple and sensitive LC‐ESI‐MS (ion trap)method to determine BUP and HBUP in rat plasma and brainmicrodialysates. The lower limit of quantitation for both theanalytes was 1.5 ng/mL in these matrices.The aim of the present study was to develop a sensitive and

rapid LC‐MS/MS method for the simultaneous determination ofBUP and HBUP in human plasma for a clinical study. The methodproposed is highly selective for quantification of BUP and HBUPin the presence of seven commonly prescribed antidepressantmedications. The method was successfully used in a clinicalsetting for a bioequivalence study in 12 healthy Indian males fora 150mg BUP extended release tablet formulation under fedconditions.

Experimental

Chemicals and materials

Reference standards of BUP (99.1%), hydroxyboproprion (99.2%) andvenlafaxine (IS, 99.4%) were obtained from Varda Biotech (P) Ltd(Mumbai, India), respectively. HPLC‐grade methanol was procured fromMallinckrodt Baker, S.A.de C.V. (Estado de Mexico, Mexico). Ammoniumacetate and ammonium formate were purchased from Merck SpecialtiesPvt. Ltd (Mumbai, India). Water used in the entire analysis was preparedusing a Milli‐Q water purification system from Millipore (Bangalore,India). Oasis HLB (1 cm3, 30mg) extraction cartridges were from WatersCorporation (Milford, MA, USA). Blank human plasma (K2EDTA asanticoagulant) was obtained from Prathama Blood Centre (Ahmedabad,India) and was stored at −70°C until use.

Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc

J. M. Parekh et al.

Liquid chromatographic conditions

A Shimadzu LC‐VP HPLC system (Kyoto, Japan) consisting of an LC‐10ADVP pump, a SIL‐HTc autosampler, a CTO 10 ASvp column oven anda DGU‐14A degasser, was used for setting the reverse‐phase liquidchromatographic conditions. The separation of BUP, HBUP and IS wasachieved on an Zorbax Eclipse XDB‐C18 (150mm length× 4.6mm innerdiameter and 5.0 µm particle size) analytical column from AgilentTechnologies, Inc. (CA, USA) and maintained at 40°C in a column oven.For isocratic separation, the mobile phase consisted of 20mM

ammonium acetate–methanol (10:90, v/v). The flow‐rate of the mobilephase was kept at 1.0mL/min and the total chromatographic run timewas 2.8min. The autosampler temperature was maintained at 10°C. Thetotal eluant from the column was split in a 50:50 ratio; flow directed tothe ISP interface was equivalent to 500 μL/min.

Mass spectrometric conditions

Ionization and detection of BUP, HBUP and IS was carried out on a triplequadrupole mass spectrometer, MDS SCIEX API‐4000 (Toronto, Canada),equipped with a turbo ion spray interface and operating in positive‐ionmode. Quantitation was performed using multiple reaction monitoring(MRM) mode to monitor precursor→product ion transitions for BUP

Figure 1. Product ion mass spectra of (a) bupropion (m/z 240.3→ 184.1, srange 100–500 amu) and (c) internal standard, venlafaxine (m/z 278.4→ 260

(m/z 240.3→ 184.1), HBUP (m/z 256.1→ 238.1) and IS (m/z 278.4→ 260.3)(Fig. 1). The source‐dependent parameters maintained for BUP, HBUP andIS were: gas 1 and gas 2, 50 psig; ion spray voltage, 3500V; turbo heatertemperature, 500°C; collision activation dissociation, 5 psig; and curtaingas, nitrogen, 35 psig. The optimum values for compound dependentparameters like declustering potential, collision energy, entrancepotential and cell exit potential set were 50, 20, 10 and 5 V for BUP, 51,22, 10 and 6 V for HBUP and 47, 21, 10 and 7 V for IS, respectively.Quadrupoles 1 and 3 were maintained at unit mass resolution and thedwell time was set at 200ms. Analyst software version 1.4.2 was used tocontrol all parameters of LC and MS.

Standard stock, calibration standards and quality controlsample preparation

The standard stock solutions of BUP and HBUP, 1000µg/mL each, wereprepared by dissolving the requisite amount in methanol. Their workingsolutions (100 µg/mL), used for spiking, were prepared in methanol–water (50:50, v/v). Calibration standards and quality control (QC) sampleswere prepared by spiking blank plasma with working solution (5%of total plasma volume). Calibration curve standards of BUP/HBUPwere made at 0.10/0.10, 0.20/0.20, 10.0/30.0, 55.0/95.0, 180/300, 245/420,320/500 and 350/600ng/mL concentrations, respectively, while quality

can range 60–350 amu), (b) hydroxybupropion (m/z 256.1→ 238.1, scan.3, scan range 60–350 amu) in positive ionization mode.

Biomed. Chromatogr. 2012; 26: 314–326Wiley & Sons, Ltd.

control samples were prepared at five levels, 350/600 ng/mL (ULOQQC, upper limit of quantitation quality control), 270/450 ng/mL (HQC,high quality control), 180/310 ng/mL (MQC,mediumquality control), 0.30/0.30 ng/mL (LQC, low quality control) and 0.10/0.10 ng/mL (LLOQ QC,lower limit of quantification quality control). Stock solution (1000 µg/mL)of the internal standard was prepared by dissolving 25.0mg ofvenlafaxine in 25.0mL of methanol. Its working solution (2.0 µg/mL) wasprepared by appropriate dilution of the stock solution in methanol–water(50:50, v/v). Standard stock and working solutions used for spiking werestored at 5°C, while calibration curve and quality control samples inplasma were kept at −70°C until use.

Sample extraction protocol

Prior to analysis, all frozen subject samples, calibration standards andquality control samples were thawed and allowed to equilibrate at roomtemperature. To an aliquot of 50 μL of spiked plasma sample, 25 μL ofinternal standard (2.0 µg/mL) was added and vortexed for 10 s. Further,250 μL of Milli‐Q water was added and vortexed for another 10 s. Thesamples were loaded on Waters Oasis HLB (1 cm3, 30mg) extractioncartridges which were preconditioned with 1.0mL of methanol followedby 1.0mL of water. The cartridges were washed with 1.0mL of MilliQwater followed by 1.0mL, 5% methanol. Drying of cartridges was donefor 2min by applying 20 psi pressure. Elution of analytes and IS from thecartridges was carried out with 500 μL of reconstitution solution (mobilephase) into pre‐labeled tubes and 5 μL was used for injection in thechromatographic system.

Procedures for method validation

The bioanalytical method was thoroughly validated following theUS Food and Drug Administration (FDA) guidelines (US FDA, 2001).System suitability experiment was performed by injecting six consec-utive injections, using the extracted standard mixture of BUP/HBUP(350/600ng/mL) and IS (2.0 µg/mL) at the start of each batch duringmethod validation. System performance was studied by injecting oneextracted LLOQ sample with IS at the beginning of each analytical batchand before re‐injecting any sample during method validation. Theverification of carryover of analytes was also experimentally determined.The design of the experiment comprised the following sequence ofinjections: mobile phase solution [20mM ammonium acetate‐methanol(10:90, v/v)]→ LLOQ sample→ extracted blank plasma→ULOQ sam-ple→ extracted blank plasma→ULOQ sample→ extracted blank plasma.

The selectivity of the method towards endogenous plasma matrixcomponents was assessed in 14 different batches (eight normal K2 EDTA,three hemolysed and 3 lipemic) of blank plasma. As antidepressants arecommonly prescribed in combination with drugs used to treat somaticdisorders (e.g. antipsychotics, anxiolytics and mood stabilizers) Spinaet al., (2008), the check for interference with commonly used antidepres-sant medications (desipramine, imipramine, fluoxetine, trazodone,citalopram, paroxetine and sertraline) was studied for ionization (ionsuppression/enhancement), analytical recovery (precision and accuracy)and chromatographic interference (interference with MRM of metaboliteand IS). Their stock solutions (1000 µg/mL) were prepared by dissolvingrequisite amount in methanol. Further, working solutions (200 ng/mL) ofeach drug were prepared in the methanol–water (50:50, v/v), spiked inplasma and analyzed under the same conditions at LQC and HQC levels.The MRM transitions in the positive ionization mode for desipramine(267.1/72.0), imipramine (281.2/86.1), fluoxetine (310.2/44.4), trazodone(372.2/176.3), citalopram (325.1/109.0), paroxetine (330.0/190.1) andsertraline (306.2/159.2) were studied.

The linearity of the method was determined by analysis of fivecalibration curves containing eight nonzero concentrations. The arearatio response for analyte/IS obtained from multiple reaction monitoringwas used for regression analysis. Each calibration curve was analyzedindividually using least‐square‐weighted (1/x2) linear regression whichwas finalized during pre‐method validation. A correlation coefficient (r2)

value > 0.99 was desirable for all the calibration curves. The loweststandard on the calibration curve was accepted as the LLOQ, if theanalyte response was at least 10 times more than that of drug free(blank) extracted plasma. In addition, the analyte peak of LLOQ sampleshould be identifiable, discrete and reproducible with a precision (%CV)not greater than 20% and accuracy within 80–120%. The deviation ofstandards other than LLOQ from the nominal concentration should notbe more than ±15%.

For determining the intra‐batch accuracy and precision, replicateanalysis of plasma samples of analytes was performed on the same day.The run consisted of a calibration curve and six replicates of LLOQ QC,LQC, MQC, HQC and ULOQ QC samples. The inter‐batch accuracy andprecision were assessed by analyzing five precision and accuracybatches on three consecutive validation days. The deviation at eachconcentration level from the nominal concentration was expected to bewithin ±15% except for LLOQ, for which it should be within ±20%.Similarly, the mean accuracy should not deviate by ±15% except for theLLOQ, where it can be ±20% of the nominal concentration.

Ion suppression/enhancement effects on the MRM LC‐MS/MSsensitivity were evaluated by the post‐column analyte infusionexperiment (King et al. 2000). A standard solution containing BUP andHBUP (at ULOQ QC level) and IS was infused post‐column via a ‘T’connector into the mobile phase at 10 μL/min employing an infusionpump. Aliquots of 5 μL of extracted control plasma were then injectedinto the column by the autosampler and MRM LC‐MS/MS chromato-grams were acquired for analytes and IS. Any dip in the baseline uponinjection of extracted blank plasma (without IS and analyte) indicates ionsuppression, while a peak at the retention time of analytes or IS indicatesion enhancement.

The relative recovery, matrix effect and process efficiency wereassessed as recommended by Matuszewski et al. (2003). All threeparameters were evaluated at HQC, MQC and LQC levels in six replicates.Relative recovery (RE) was calculated by comparing the mean arearesponse of pre‐spiked samples (spiked before extraction) with that ofextracts with post‐spiked samples (spiked after extraction) at each QClevel. The recovery of IS was similarly estimated. Absolute matrix effect(ME) was assessed by comparing the mean area response of unextractedsamples (spiked after extraction) with mean area of neat standardsolutions (in mobile phase). The overall ‘process efficiency’ (%PE) wascalculated as (ME× RE)/100. Further, the effect of plasma matrix (relativematrix effect) on analyte quantification was also checked in six differentlots of plasma. From each batch, six samples at LLOQ level were prepared(spiked after extraction) and checked for the percentage accuracy andprecision (%CV). The deviation of the standards should not be more than±15% and at least 90% of the lots at each QC level should be within theaforementioned criteria.

All stability results were evaluated by measuring the area ratioresponse (analyte–IS) of stability samples against freshly preparedcomparison standards at LQC and HQC levels. Stock solutions of analytesand IS were checked for short‐term stability at room temperature andlong‐term stability at 5°C. The solutions were considered stable if thedeviation from nominal value was within ±15.0%. The autosampler (wetextract), bench‐top (at room temperature) and freeze–thaw stabilitieswere determined at LQC and HQC using six replicates at each level.Freeze–thaw stability was evaluated by successive cycles of freezing (at−20 and −70°C) and thawing (without warming) at room temperature.Long‐term stability of spiked plasma samples stored at −20 and −70°Cwas also studied at both these levels. The samples were consideredstable if the deviation from the mean calculated concentration of freshlythawed quality control samples was within ±15.0%.

To authenticate the ruggedness of the proposed method, it wasperformed on two precision and accuracy batches. The first batch wasanalyzed by different analysts while the second batch was studied on twodifferent columns. Dilution integrity experiment was evaluated by dilutingthe stock solution prepared as spiked standard at 700/1200ng/mL(2×ULOQ) BUP/HBUP concentration in the screenedplasma. Theprecisionand accuracy for dilution integrity standards at 1/5th (140/240ng/mL)

J. M. Parekh et al.

and 1/10th (70/120ng/mL) dilution were determined by analyzing thesamples against freshly prepared calibration curve standards.

Bioequivalence study design and statistical analysis

The design of the study comprised of an open label, balanced, random-ized, two‐treatment, two‐period, two‐sequence, single dose, crossoverbioequivalence study of a test formulation of bupropion hydrochloride(150mg extended release tablets of an Indian Company) and areference formulation (Wellbutrin XL® tablets containing 150mg BUP,manufactured by Biovail Corporation, Canada and manufactured forGlaxoSmithKline, USA) in 12 healthy adult Indian male subjects in the agegroup of 20–40 years (height 160.2–181.5 cm, weight 52.5–81.4 kg, bodymass index 18.9–24.5 kg/m2, all nonsmokers) under fed conditions. Eachsubject was judged to be in good health throughmedical history, physicalexamination and routine laboratory tests. Written consent was taken fromall the subjects after informing them about the objectives and possiblerisks involved in the study. An independent ethics committee constitutedas per Indian Council of Medical Research approved the study protocol.The study was conducted strictly in accordance with guidelines laid downby International Conference on Harmonization and the US FDA (US FDA,1996). The subjects were orally administered a single dose of test andreference formulations after the recommendedwashout period of 2weekswith 200mL of water. Blood samples were collected at 0.0 (pre‐dose), 0.33,0.66, 1.0, 1.33, 1.66, 2.0, 2.33, 2.66, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0,12.0, 16.0, 20.0, 24.0, 48.0, 72.0, 96.0, 120 and 144h after oral administrationof the dose for test and reference formulation in labeled K2EDTA‐vacuettes.The maximum volume of blood withdrawn during the entire study wasapproximately 325mL, which included (other than formeasurement) up to10mL for screening, about 10mL for post‐study safety assessment(hematology and biochemical tests), while 0.5mL of heparinised bloodwas discarded prior to each sampling through venous cannula. Plasmawasseparated by centrifugation and kept frozen at −70°C until analysis.During study, subjects had a standard diet while water intake was free.The pharmacokinetic parameters of BUP and HBUP were estimated usinga noncompartmental model using WinNonlin software version 5.2.1(Scientific Consulting Inc., Apex, NC, USA). The Cmax values and the time toreachmaximum plasma concentration (Tmax) were estimated directly fromthe observed plasma concentration vs time data. The area under theplasma concentration–time curve from time 0 to 144 h (AUC0–144) wascalculated using the linear trapezoidal rule. The AUC0–inf was calculated as:AUC0–inf = AUC0–144 +Ct/Kel, where Ct is the last plasma concentrationmeasured and Kel is the elimination rate constant; Kel was determinedusing linear regression analysis of the logarithm linear part of theplasma concentration–time curve. The t1/2 of BUP and HBUP wascalculated as: t1/2 = ln2/Kel. To determine whether the test and referenceformulations were pharmacokinetically equivalent, Cmax, AUC0–144 andAUC0–inf and their ratios (test/reference) using log‐transformed datawere assessed; their means and 90% CIs were analyzed by using SAS®software version 9.1.3 (SAS Institute Inc., Cary, NC, USA). The drugs wereconsidered pharmacokinetically equivalent if the difference betweenthe compared parameters was statistically nonsignificant (p≥ 0.05) andthe 90% confidence intervals (CI) for these parameters fell within 0.8to 1.25.

An incurred sample re‐analysis was also conducted by computerizedrandom selection of 20 subject samples. The selection criteria includedsamples that were near the Cmax and the elimination phase in thepharmacokinetic profile of the drug. The results obtained werecompared with the data obtained earlier for the same sample usingthe same procedure. The percentage change in the value should not bemore than ±20% (Rocci et al., 2007).

Results and discussionThe present study was intended to chromatographically resolvethe analytes and IS and further to develop a sensitive, selectiveand a high‐throughput method to determine BUP and its major

active metabolite, HBUP for a clinical study involving healthysubjects. To optimize the method, several parameters likecolumn type, organic modifier, mobile phase flow‐rate, pH andconcentration of buffer solution were studied.

Method development

During method development, the electrospray ionization of theanalytes and IS was conducted in positive ionization mode asthey are basic in nature. Bupropion and HBUP have a secondaryamine group while venlafaxine has a tertiary amine groupand can be easily protonated in solution under mild acidicexperimental conditions. Q1 MS full‐scan spectra for BUP, HBUPand IS predominantly contained protonated precursor [M+H]+

ions at m/z 240.3, 256.1 and 278.4, respectively. The mostabundant and consistent product ions in Q3 MS spectra for BUPwere observed at m/z 184.1 owing to the loss of tert‐butylgroup, while for HBUP and IS the stable and consistent productions were found at m/z 238.1 and 260.3 respectively, which canbe attributed to the loss of water molecule. Attempts weremade to have substructures of drug molecule as product ionsfor HBUP and IS, but the product ion fragments observed wereinconsistent, nonreproducible or lacked adequate sensitivitycompared with selected ions. The source‐dependent andcompound‐dependent parameters were suitably optimized toobtain a consistent and adequate response for the analyte.

Reported procedures have employed PP (Yeniceli et al., 2011),liquid–liquid extraction (LLE; Borges et al., 2004), SPE (Coles andKharasch, 2007) or PP in combination with SPE (Denooz et al.,2010) for sample preparation of BUP and HBUP from plasmamatrix. Thus, in the present study all three extractionmethodologies were tested to have the best procedure forquantitative and efficient extraction. PP was studied initiallywith methanol and acetonitrile; however, owing to considerableion suppression (greater than 15% CV), this procedure was notconsidered further. LLE was then initiated with diethyl ether,dichloromethane, methyl tert‐butyl ether, n‐hexane and ethylacetate. Despite a number of trials, it was difficult to obtainclear samples and, moreover, the recovery was not quantitative(60–70%), especially at the LLOQ level. Borges et al. (2004) havestated that LLE with ethyl acetate resulted in ion suppressioneffects owing to matrix components present in the reconsti-tuted extracts. Thus, SPE was conducted on a Waters Oasis HLB(1 cm3, 30mg) extraction cartridges to obtain quantitative andconsistent recovery for BUP and HBUP. The extracts obtainedwere clear, with quantitative and precise recoveries for BUP,HBUP and IS at all QC levels through a simple and straightforwardprocedure employing 50μL human plasma.

As reported previously (Yeniceli et al., 2011; Coles andKharasch, 2007; Borges et al., 2004), the type of column,composition of the mobile phase (organic and aqueous part)and pH play a major role on the selective separation of theseanalytes. Thus, the chromatographic separation of BUP, HBUPand IS was initiated by varying these parameters to achieve ashort run time, symmetric peak shapes, minimum matrixinterference and solvent consumption. Except for the work ofBorges et al. (2004), wherein a monolithic column (ChromolithSpeedROD RP‐18, 50 × 4.6mm) was used for fast chromatograph-ic separation within 1min achieved at a flow‐rate of 5mL/min, allother methods required run times ≥4min. In the present work,chromatographic separation was tried on a variety of columns,like Phenomenex Gemini C18 (100/150 × 4.6mm, 5 µm), Waters

Atlantis T3 C18 (100/150× 4.6mm, 5 µm), ACE C18 (150 × 4.6mm,5 µm), Kromasil C18 (150 × 4.6mm, 3.5 µm), Thermo Hypurity C18(100 × 4.6mm, 5 µm) and Zorbax Eclipse XDB‐C18 (150 × 4.6mm,5 µm) analytical columns. To find the best eluting solvent system,various combinations ofmethanol–acetonitrile with additives likeammonium acetate–ammonium formate in different concentra-tions (2, 5, 10 and 20mM) and volume ratios (70:30, 80:20 and90:10, v/v) were tested. In addition, the effect of flow‐ratewas alsostudied from 0.6 to 1.2mL/min, which was also responsible foracceptable chromatographic separation. On some of thesecolumns the run time was either too long (7–12min) or tooshort (0.8–1.2), almost near the dead time of the columns. Further,on ACE C18, Waters Atlantis and Thermo Hypurity, the resolutionof analytes was between 0.5 and 1.0. Use of ammonium formatein the concentration range from 2 to 20mM resulted in aconsiderable shift in retention time between injections. On theother hand, at lower concentrations of ammonium acetate (2, 5and 10mM) in themobile phase (corresponding pH 6.10, 6.25 and6.35 respectively) adequate separation was obtained; however,the peak shape of IS was affected. Similarly at flow‐rates aboveand below 1mL/min, the resolution of peaks was inadequate.Best results were obtained in terms of higher sensitivity, superiorretention, separation, run time and better peak shapes on ZorbaxEclipse XDB‐C18 column using 20mM ammonium acetate–methanol (10:90, v/v), pH 6.5 as the mobile phase at a flow‐rateof 1mL/min. The total run time of 2.8min ensured separation ofBUP, HBUP and IS at 2.30, 1.60 and 1.90min, respectively. Thecapacity factors, which describe the rate at which the analytesmigrate through the column, were 1.10 and 0.45, respectively, forboth analytes based on the dead time of 1.1min. The selectivityfactor (α) of the column for the chromatographic separation ofBUP and HBUP was 2.4. The numbers of theoretical platesobtained for BUP, HBUP and IS were 2116, 1024 and 1444,respectively, with a resolution factor of 3.5 between the analytes.Also, the reproducibility of retention times for the analytes,expressed as %CV was≤ 1% for 100 injections on the samecolumn. Representative chromatograms in Fig. 2(A and B) ofextracted blank plasma (without IS and analyte), blank plasmafortified with IS, BUP and HBUP at LLOQ and an actual subjectsample at Cmax demonstrates the selectivity of the method. Noneof the antidepressant medications studied showed interferingsignals at the retention time of BUP, HBUP or the IS. Results ofpost‐column infusion experiment in Fig. 3 indicate no ionsuppression or enhancement at the retention time of analytesand IS. The averagematrix factor value calculated as the responseof post‐spiked sample/response of neat solutions inmobile phaseat the LLOQ levels was 0.99 and 0.98 for BUP and HBUP, whichindicates a minor suppression of 1 and 2%, respectively.

A general internal standard was used to minimize any possibleanalytical variation owing to solvent evaporation, extractionefficiency and ionization efficiency of BUP and HBUP owingto nonavailability of deuterated standards. Several drug com-pounds were tested, namely valsartan, irbesartan, telmisartanand venlafaxine for suitable selection of IS. Venlafaxine used as aninternal standard in the present study had similar chromato-graphic behavior, similar protein binding andwas easily extractedby SPE compared with the other drugs. There was no effect of ISon analyte recovery, sensitivity or ion suppression.

Assay performance and validation

During the entire method validation, the precision (%CV) of thesystem suitability test was observed in the range of 0.06–0.20%

for the retention time and 1.2–1.9% for the area response ofboth the analytes and IS. The signal‐to‐noise ratio for systemperformance was ≥50 for both the analytes and IS. Carry‐overevaluation was performed in each analytical run so as to ensurethat it does not affect the accuracy and the precision of theproposed method. There was negligible carry‐over (≤0.09%)observed during auto‐sampler carryover experiment. No en-hancement in the response was observed in extracted blankplasma (without IS and analytes) after subsequent injection ofhighest calibration standard at the retention time of bothanalytes and IS.All five calibration curves were linear over the concentration

range of 0.1–350 ng/mL for BUP and 0.1–600 ng/mL for HBUP. Astraight‐line fit was made through the data points by leastsquare regression analysis to give the mean linear equationy= 0.0028x− 0.0061 and y= 0.0032x – 0.0128 for BUP and HBUP,respectively, where y is the peak area ratio of the analyte/IS andx the concentration of the analyte. The mean standard deviationvalues for slope, intercept and correlation coefficient (r2)observed were 0.00002/0.00003, 0.00005/0.00004 and 0.0003/0.0004 for BUP/HBUP, respectively. The accuracy and precision(%CV) observed for the calibration curve standards ranged from97.2 to 104.0% and from 0.35 to 2.45%, respectively, for both theanalytes. The lowest concentration (LLOQ) in the standard curvethat can be measured with acceptable accuracy and precisionwas found to be 0.1 ng/mL in plasma at a signal‐to‐noise ratio(S/N) of ≥50.The intra‐batch and inter‐batch precision and accuracy were

established from validation runs performed at ULOQ QC, HQC,MQC, LQC and LLOQ QC levels (Table 1). The intra‐batchprecision (%CV) ranged from 1.97 to 6.26 and the accuracy waswithin 96.8–102.7% for both the analytes. Similarly, for the inter‐batch experiments, the precision varied from 2.85 to 4.66 andthe accuracy was within 99.1–102.8%.The relative recovery, absolute matrix effect and process

efficiency data for BUP, HBUP and IS at LQC, MQC and HQClevels are presented in Table 2. The process efficiency/absoluterecovery obtained for both the analytes and IS was greater than91% at all QC levels. Further, the relative matrix effect, whichcompares the precision (%CV) values between different lots(sources) of plasma (spiked after extraction) samples, variedfrom 2.53 to 4.71 for both the analytes at LLOQ level. Theaccuracy results were between 95.22 and 105.65% at the LLOQlevel.The stability of analytes and IS in human plasma and stock

solutions was examined under different storage conditions.Samples for short‐term stability remained unchanged up to52 h, while the long‐term stabilities of the stock solutions forBUP, HBUP and IS were stable for minimum of 126 days atrefrigerated temperature at 5°C. Bupropion and HBUP in controlhuman plasma (bench‐top) at room temperature were stable forat least 12 h at 25°C and for a minimum of four freeze–thawcycles at −20 and −70°C. Spiked plasma samples stored at −20and −70°C, for the long‐term stability experiment were foundto be stable for a minimum period of 124 days. Autosamplerstability (wet extract) of the spiked quality control samplesmaintained at 5°C was determined up to 18 h without significantloss of the analytes. The percentage change for different stabilityexperiments in plasma at two QC levels varied from −8.73 to2.84% for both the analytes, as shown in Table 3.The dilution integrity experiment was performed with the aim

of validating the dilution test to be carried out at higher analyte

Figure 2. (A) MRM ion‐chromatograms of (a) extracted blank plasma (without IS and bupropion), (b) blank plasma with venlafaxine (IS,278.4→ 260.3), (c) bupropion at lower limit of quantitation (LLOQ; m/z 240.3→ 184.1) and IS and (d) real subject sample at Cmax after administration of150mg dose of bupropion. (B) MRM ion‐chromatograms of (a) extracted blank plasma (without IS and hydroxybupropion), (b) blank plasma withvenlafaxine (IS,m/z 278.4→ 260.3), (c) hydroxybupropion at LLOQ (m/z 256.1→ 238.1) and IS and (d) real subject sample at Cmax after administration of150mg dose of bupropion.

J. M. Parekh et al.

Method ruggedness was evaluated using re‐injection ofanalyzed samples on two different columns of the same makeand also with different analysts. The precision (%CV) andaccuracy values for two different columns ranged from 1.2 to3.0% and from 97.8 to 102.5%, respectively, at all five qualitycontrol levels. For the experiment with different analysts, the

results for precision and accuracy were within 2.2–4.1% and96.6–100.4%, respectively at these levels.

Application to a pharmacokinetic/bioequivalence study

The validated method was successfully used to quantify BUPand HBUP concentration in human plasma samples after theadministration of a single 150mg oral dose of BUP. Figure 4shows the plasma concentration of BUP and HBUP vs timeprofile in human subjects under fed conditions. The method wassensitive enough to monitor the BUP and HBUP plasmaconcentration up to 144 h. In all approximately 1800 samplesincluding the calibration, QC and volunteer samples were runand analyzed during a period of 9 days and the precision and

Figure 3. Post‐column analyte infusion experiment for (a) bupropion, (b) hydroxybupropion and (c) venlafaxine.

accuracy were well within the acceptable limits. The meanpharmacokinetic parameters obtained for the test and referenceformulation are presented in Table 4. The equivalence statisticsof bioavailability for the pharmacokinetic parameters of thetwo formulations are summarized in Table 5. No statisticallysignificant differences were found between the two formulationsin any parameter. The mean log‐transformed ratios of theparameters and their 90% CIs were all within the definedbioequivalence range. The mean values of Cmax and t1/2 obtainedfor BUP and HBUP were to some extent different compared withthe work reported by Jefferson et al. (2005) for extended releaseformulation. They reported a 4‐ to 7‐fold HBUP/BUP ratio for Cmax

values; however, in the present study this ratio was 2.3. Similarly,the mean t1/2 values obtained were slightly higher, ~26 ± 6.10 hfor BUP and ~25± 4.13 h for HBUP, compared with reportedvalues of ~21 and ~20 h, respectively. This may be due geneticdifference, race, age, gender (body size and muscle mass) or typeof food, which may result in pharmacokinetic differences

(Jefferson et al., 2005). However, the mean Tmax value wascomparable with the present work. The percentage change in therandomly selected subject samples for incurred samples (assayreproducibility) analysis was within ± 7.5% (Table 6). Thisauthenticates the reproducibility and ruggedness of the pro-posed method. Further, there was no adverse event during thecourse of the study.

Comparison with reported methods

The method presented employs a very small plasma volume (50μL) for processing and has the highest sensitivity compared withall other procedures for the determination of BUP and itsmetabolite, HBUP (Borges et al., 2004; Coles and Kharasch, 2007;Denooz et al., 2010; Yeniceli et al., 2011). Moreover, the totalanalysis time (extraction and chromatography) is the shortestfor BUP and HBUP compared with existing methods. Also, theon‐column loading at LLOQ, only 0.1 pg per sample injection

Table 1. Intra‐batch and inter‐batch precision and accuracy for bupropion and hydroxybupropion

Nominalconcentration

(ng/mL)

Intra‐batch Inter‐batch

Mean concentrationobserved (ng/mL)a

%CV Accuracy(%)

Mean concentrationobserved (ng/mL)b

%CV Accuracy(%)

Bupropionn n

LLOQ QC 0.10 6 0.10 3.49 98.2 30 0.10 3.89 100.1LQC 0.30 6 0.31 3.51 102.7 30 0.31 3.81 102.8MQC 180 6 174 4.26 96.8 30 179 3.34 99.7HQC 270 6 277 3.52 102.6 30 276 3.69 102.3ULOQ QC 350 6 345 2.78 98.6 30 357 2.98 101.9

Hydroxybupropionn n

LLOQ QC 0.10 6 0.10 1.98 99.5 30 0.10 4.12 101LQC 0.30 6 0.31 6.26 102.2 30 0.30 4.66 100.8MQC 310 6 315 1.97 101.5 30 316 2.85 101.9HQC 450 6 461 3.34 102.5 30 446 4.27 99.1ULOQ QC 600 6 611 3.03 101.8 30 608 3.09 101.3

CV, Coefficient of variance; n, total number of observations. aMean of six replicates at each concentration; bmean of six replicatesfor five precision and accuracy batches. LLOQ QC, lower limit of quantitation quality control; LQC, low quality control; MQC,medium quality control; HQC, high quality control; ULOQ QC, upper limit of quantitation quality control.

Table 2. Absolute matrix effect, relative recovery and process efficiency for bupropion and hydroxybupropion

Aa (%CV)b Bc (%CV)b Cd (%CV)b Absolute matrix effecte Relative recoveryf Process efficiencyg

LQCBupropion

0.0051 0.0050 0.0049 98.04 98.83 95.87(1.94) (3.78) (2.12) (94.7)h (95.7)h (92.8)h

Hydroxybupropion0.0037 0.0036 0.0034 97.32 94.49 91.96(2.8) (4.1) (4.8) (94.3)h (93.6)h (92.8)h

MQCBupropion

3.2052 3.1174 3.0612 97.26 98.20 95.51(1.76) (3.06) (2.58) (95.4)h (94.3)h (93.2)h

HQCBupropion

4.5258 4.4713 4.3745 98.80 97.83 96.66(2.69) (3.63) (4.14) (94.3)h (93.8)h (92.4)h

aMean area ratio (analyte/internal standard) response of six replicate samples prepared in mobile phase (neat samples);bcoefficient of variation; cmean area ratio (analyte/internal standard) response of six replicate samples prepared by spiking inextracted blank plasma; dmean area ratio (analyte/internal standard) response of six replicate samples prepared by spiking beforeextraction; eB/A × 100; fC/B × 100; gC/A × 100= (ME× RE)/100; hvalues for internal standard, venlafaxine.

J. M. Parekh et al.

volume, is significantly lower, which helps to maintain thecolumn efficiency for a greater number of injections. A detailedcomparison of LC‐MS based‐procedures for the simultaneousdetermination of BUP and HBUP in biological matrices is givenin Table 7.

Conclusions

The proposed validated method for the simultaneous estimationof BUP and HBUP in human plasma is highly sensitive and rapidcompared with published reports. The method offers significant

Table 3. Stability of bupropion and hydroxybupropion under different conditions (n=6)

Storage condition

Calculated concentration (ng/mL)

Nominal concentration(ng/mL)

Mean, stabilitySamples ± SD

Percentagechange

BUP HBUP BUP HBUP BUP HBUP

Bench‐top stability, 12 hHQC 270 450 268± 10 438± 12 7.75 2.84LQC 0.3 0.3 0.29 ± 0.02 0.30 ± 0.02 −6.45 −4.68

Wet extract stability, 18 hHQC 270 450 265± 7 442± 8 −5.07 −4.92LQC 0.3 0.3 0.28 ± 0.02 0.29 ± 0.01 −6.66 −5.79

Freeze–thaw stability;four cycles, −20°C

HQC 270 450 261± 8 446± 15 −5.33 −5.78LQC 0.3 0.3 0.30 ± 0.01 0.30 ± 0.01 −2.67 −6.12

Freeze–thaw stability;four cycles, −70°C

HQC 270 450 266± 12 436± 22 3.86 7.48LQC 0.3 0.3 0.30 ± 0.03 0.29 ± 0.02 −2.32 −3.49

Long‐term matrix stability,124 days, −20°C

HQC 270 450 272± 10 440± 14 6.24 5.48LQC 0.3 0.3 0.28 ± 0.02 0.31 ± 0.01 −3.51 6.82

Long‐term matrix stability,124 days, −70°C

HQC 270 450 262± 9 432 ± 21 −8.48 −8.73LQC 0.3 0.3 0.29 ± 0.02 0.29 ± 0.02 −4.63 5.87

BUP, Bupropion; HBUP, hydroxybupropion; SD, standard deviation.

Percentage change ¼ mean stability samples−mean comparison samplesmean comparison samples

� 100

Figure 4. Mean plasma concentration–time profiles of bupropion and hydroxybupropion after oral administration of test (150mg extended releasebupropion hydrochloride tablets of an Indian Company) and a reference (Wellbutrin XL® tablets containing 150mg bupropion) formulation to12 healthy volunteers under fed conditions.

advantages over those previously reported, in terms of lowersample requirements (50 μL), simplicity of extraction procedureand overall analysis time. The efficiency of SPE and a chroma-tographic run time of 2.8min per sample make it an attractiveprocedure in high‐throughput bioanalysis of BUP and HBUP. The

linear dynamic range established was adequate to measure theplasma concentration of BUP andHBUP in a clinical study involvingIndian subjects. In addition, the carry‐over test, post‐columninfusion study and the effect of commonly used antidepressantmedications by subjects was also studied in the present work.

Table 4. Mean pharmacokinetic parameters following oral administration of 150mg tablet formulation (test and reference) ofbupropion in 12 healthy human subjects

Parameter Mean± SD

Test Reference

BUP HBUP BUP HBUP

Cmax (ng/mL) 183.88 ± 4.31 423.34 ± 5.23 184.24 ± 6.38 425.74 ± 4.71Tmax (h) 4.17 ± 0.33 6.08 ± 0.36 4.25 ± 0.50 6.17 ± 0.25t½ (h) 26.73 ± 6.10 25.11 ± 4.13 26.55 ± 4.47 24.19 ± 2.98Kel (1/h) 0.027 ± 0.007 0.028 ± 0.004 0.027 ± 0.005 0.029 ± 0.003AUC0–144 (ng h/mL) 2327.92 ± 497.71 12592.16 ± 864.57 2591.32 ± 795.85 13444.12 ± 544.20AUC0–inf (ng h/mL) 2382.83 ± 513.15 12762.90 ± 862.84 2653.21 ± 822.30 13645.85 ± 559.61

BUP, Bupropion; HBUP, hydroxybupropion; Cmax, maximum plasma concentration; Tmax, time point of maximum plasmaconcentration; t1/2, half life of drug elimination during the terminal phase; AUC0–t, area under the plasma concentration–timecurve from 0 to 144 h; AUC0–inf, area under the plasma concentration–time curve from 0h to infinity; SD, standard deviation.

Table 5. Comparison of treatment ratios and 90% CIs of natural log(ln)‐transformed parameters for test and referenceformulations of bupropion in 12 healthy Indian subjects

Parameter Ratio (test/reference) (%) 90% CI

BUP HBUP BUP HBUP

ln Cmax 99.8 99.4 96.3–104.1 97.5–101.6ln AUC0–144 89.8 93.7 87.1–92.7 90.7–96.9ln AUC0–inf 89.8 93.5 87.4–93.1 91.1–95.9

BUP, Bupropion; HBUP, hydroxybupropion; CI, confidence interval.

Table 6. Incurred sample reproducibility data for bupropion and hydroxybupropion

Sample no. Initial Value (ng/mL) Repeat Value (ng/mL) % Change

BUP HBUP BUP HBUP BUP HBUP

1 181.48 412.55 178.76 423.97 −1.51 2.732 1.29 4.21 1.22 4.14 −5.58 −1.683 178.65 430.12 183.66 409.55 2.77 −4.94 1.07 4.34 1.03 4.52 −3.81 4.065 190.66 418.61 181.98 433.65 −4.66 3.536 2.13 3.84 2.23 3.78 4.59 −1.577 188.64 428.31 180.63 418.93 −4.34 −2.218 1.41 6.82 1.49 6.87 5.52 0.739 180.66 421.65 170.87 443.84 −5.57 5.1310 0.78 5.02 0.81 4.83 3.77 −3.8611 187.47 420.55 192.98 402.47 2.9 −4.3912 2.12 3.87 1.97 3.64 −7.33 −6.1313 192.19 425.89 199.03 450.76 3.5 5.6714 2.54 4.91 2.45 4.72 −3.61 −3.9515 185.47 430.77 198.45 457.89 6.76 6.116 0.99 5.72 1.02 5.54 2.99 −3.217 176.32 418.99 182.53 446.71 3.46 6.418 1.02 5.44 1.09 5.29 6.64 −2.819 186.79 422.89 178.42 401.55 −4.58 −5.1820 1.88 7.91 1.94 7.52 3.14 −5.06

BUP, Bupropion; HBUP, hydroxybupropion.

Percentage change ¼ repeat value−initial valueð Þmean of initial and repeat values

� 100

J. M. Parekh et al.

parison

‐MSmetho

dsforthesimultane

inationof

buprop

droxyb

upropion

inbiolog

matrices

Sampleno

.Extractio

nproced

ure(biologicalsam

volume);m

eanrecovery

n;elution

Injectionvo

lume;maxim

‐colum

atLLOQ;a

nalytical

runtim

ference

1Semi‐a

96‐w

ellliq

uid–

liquid

extractio

lacetate(150

dratplasma);9

8–10

2%forBU

dthreo‐HBU

SpeedRO

(50×4.6mm);isocratic;8

acetate–

aceton

itrile

(55:45

,v/v);5.0mL/min

dthreo‐HBU

P;1.0min;0

.25–

/mLforBU

dthreo‐HBU

P,1.25

–100

/mLforHBU

Borges

etal.(20

2WatersOasisMCXsolid

‐pha

‐well

plate;

plasma,

urine);5

8–80

%forBU

mersin

plasmaan

–84%

mersin

ChiralAGP(100

×2.0mm,5

ient;2

ffer,p

H5.7an

dmetha

nol;0.22

mL/min

pgforBU

plasmaan

durine

forHBU

plasmaan

durine,

respectiv

15.0min;0

.5–2

and2.5–

LforBU

mersin

plasma,

respectiv

5–20

–10,00

/mLforBU

mersin

urine,

respectiv

arasch

3Proteinprecipita

tionwith

combine

‐pha

seextractio

OasisHLB

lebloo

BUP,63

%;erytho‐

ndthreo‐HBU

WatersAcq

UPLCBE

column(100

×2.1mm,1

);grad

ient,2

buffer,pH4.0an

daceton

itrile,0.4mL/

formetab

s;4.0min;5

.0–1

/mLforBU

–200

/mLformetab

al.(20

4Proteinprecipita

tionwith

trichloroa

μLratplasma,

dialysate);BU

.1;HBU

inplasma;

.9–3

.5–2

inmicrodialysate

Zorbax

×2.1mm,

3.5µm

);grad

ient,1

pH4.0an

daceton

itrile,

0.4mL/min

P;9.0min;

–400

LforBU

thematrices

Yeniceliet

al.(20

lid‐pha

seextractio

WatersOasis

cartrid

ge(50μ

plasma);

96.3%;H

Zorbax

BC18(150

×4.6mm,

5.0µm

);isocratic;20

acetate–metha

(90:10

,v/v);1.0mL/min

2.8min;0

.1–3

/mLforBU

d0.1–

/mLforHBU

PPresen

tmetho

Buprop

droxyb

upropion

J. M. Parekh et al.

AcknowledgmentsWe thank Dr Dhaval K. Shah, Senior Scientist at GrotonLaboratories, Pfizer Inc., USA, for his help in the pharmacokineticstudy. The authors are thankful to Cadila Pharmaceuticals Ltd,Ahmedabad, India for providing infrastructure facility to carryout this work.

ReferencesAl‐khamis KI. Rapid determination of bupropion in human plasma by

high performance liquid chromatography. Journal of Liquid Chroma-tography and Related Technologies 1989; 12: 645–655.

Arellano C, Philibert C, Vachoux C, Woodley J and Houin G. Validation ofa liquid chromatography–mass spectrometry method to assess themetabolism of buproprion in rat everted gut sacs. Journal ofChromatography B 2005; 829: 50–55.

Borges V, Yang E, Dunn J and Henion J. High‐throughput liquidchromatography–tandem mass spectrometry determination ofbupropion and its metabolites in human, mouse and rat plasma usingamonolithic column. Journal of Chromatography B 2004; 804: 277–287.

Coles R and Kharasch ED. Stereoselective analysis of bupropion andhydroxybupropion in human plasma and urine by LC‐MS/MS. Journalof Chromatography B 2007; 857: 67–75.

Cooper TB, Suckow RF and Glassman A. Determination of bupropion andits major basic metabolites in plasma by liquid chromatography withdual‐wavelength ultraviolet detection. Journal of PharmaceuticalScience 1984; 73:1104–1107.

Denooz R, Mercerolle M, LachÃ¢tre G and Charlier C. Ultra‐performanceliquid chromatography–tandem mass spectrometry method for thedetermination of bupropion and its main metabolites in humanwhole blood. Journal of Analytical Toxicology 2010; 34: 280–286.

Fang QK, Han Z, Grover P, Kessler D, Senanayake CH and Wald SA. Rapidaccess to enantiopure bupropion and its major metabolite bystereospecific nucleophilic substitution on an α‐ketotriflate. Tetrahe-dron 2000; 11: 3659–3663.

Ferris RM, Cooper BR and Maxwell RA. Studies of bupropion’smechanism of antidepressant activity. Journal of Clinical Psychiatry1983; 44: 74–78.

Findlay JW, Van Wyck Fleet J, Smith PG, Butz RF, Hinton ML, Blum MRand Schroeder DH. Pharmacokinetics of bupropion, a novel antide-pressant agent, following oral administration to healthy subjects.European Journal of Clinical Pharmacology 1981; 21: 127–135.

Jefferson JW, Pradko JF and Muir KT. Bupropion for major depressivedisorder: Pharmacokinetic and formulation considerations. ClinicalTherapeutics 2005; 27: 1685–1695.

Jennison TA, Brown P, Crossett J and Urry FM. A high performance liquidchromatographic method for quantitating bupropion in humanplasma or serum. Journal of Analytical Toxicology 1995; 19: 69–72.

Johnston JA, Ascher J, Leadbetter R, Schmith VD, Patel DK, Durcan M andBentley B. Pharmacokinetic optimisation of sustained releasebupropion for smoking cessation. Drugs 2002; 62: S11–S24.

King R, Bonfiglio R, Fernandez‐Metzler C, Miller‐Stein C and Olah T.Mechanistic investigation of ionization suppression in electrosprayionization. Journal of American Society for Mass Spectrometry 2000;11: 942–950.

Lai AA and Schroeder DH. Clinical pharmacokinetics of bupropion: areview. Journal of Clinical Psychiatry 1983; 44: 82–84.

Loboz KK, Gross AS, Ray J and McLachlan AJ. HPLC assay for buproprionand its major metabolites in human plasma. Journal of Chromatog-raphy B 2005; 823: 115–121.

Matuszewski BK, Constanzer ML and Chavez‐Eng CM. Strategies forthe assessment of matrix effect in quantitative bioanalyticalmethods based on HPLC‐MS/MS. Analytical Chemistry 2003; 75:3019–3030.

Munro JS and Walker TA. Bupropion hydrochloride: the development ofa chiral separation using an ovomucoid column. Journal ofChromatography A 2001; 913: 275–282.

Munro JS, Gormley JP and Walker TA. Bupropion hydrochloride: Thedevelopment of a chiral separation using a chiral AGP column.Journal of Liquid Chromatography and Related Technologies 2001; 24:327–339.

Puyana MC, Garcia MA and Marina ML. Enantiomeric separation ofbupropion enantiomers by electrokinetic chromatography: quanti-tative analysis in pharmaceutical formulations. Journal of Chroma-tography B 2008; 875: 260–265.

Rocci Jr ML, Devanarayan V, Haughey DB and Jardieu P. ConfirmatoryReanalysis of Incurred Bioanalytical Samples. The American Associa-tion of Pharmaceutical Scientists 2007; 9: E336–E343.

Roose SP, Dalack GW, Glassman AH, Woodring S, Walsh BT andGiardina EGV. Cardiovascular effects of bupropion in depressedpatients with heart disease. American Journal of Psychiatry 1991;148: 512–516.

Spina E, Santoro V and D’Arrigo C. Clinically relevant pharmacokineticdrug interactions with second‐generation antidepressants: anupdate. Clinical Therapeutics 2008; 30: 1260–1227.

Suckow RF, Zhang MF and Cooper TB. Enantiomeric determination ofthe phenylmorpholinol metabolite of bupropion in human plasmausing coupled achiral–chiral liquid chromatography. BiomedicalChromatography 1997; 11: 174–179.

US FDA. Guidance for Industry: Bionanlytical Method Validation. USDepartment of Health and Human Services, Food and DrugAdministration Centre for Drug Evaluation and Research and Centrefor Veterinary Medicine, May 2001.

US FDA. Guidance for Industry: ICH E6 Good Clinical Practice. USDepartment of Health and Human Services, Food and DrugAdministration, Centre for Drug Evaluation and Research and Centrefor Biologics Evaluation and Research, April 1996.

Wenger TL and Stern WC. The cardiovascular profile of bupropion.Journal of Clinical Psychiatry 1983; 44: 176–182.

Xu H, Loboz KK, Gross AS and McLachlan AJ. Stereoselective analysis ofhydroxybupropion and application to drug interaction studies.Chirality 2007; 19: 163–170.

Yeniceli D and Dogrukol‐Ak D. An LC method for the determination ofbupropion and its main metabolite, hydroxybupropion in humanplasma. Chromatographia 2009; 70: 1703–1708.

Yeniceli D, Sener E, Korkmaz OT, Dogrukol‐Ak D and Tuncel N. A simpleand sensitive LC–ESI‐MS (ion trap) method for the determination ofbupropion and its major metabolite, hydroxybupropion in rat plasmaand brain microdialysates. Talanta 2011; 84: 19–26.

Zhang D, Yuan B, Qiao M and Li F. HPLC determination andpharmacokinetics of sustained release bupropion tablets in dogs.Journal of Pharmaceutical and Biomedical Analysis 2003; 33: 287–293.

Sensitive, selective and rapid determination of bupropion and its major active metabolite,...

Documents

Detection of Outliers in Bioequivalence Studies Data Analysis with Williams Design

Ms. power poin

Ultrasensitive Proteomics Using High-Efficiency On-Line Micro-SPE-NanoLC-NanoESI MS and MS/MS

MS-9600LS/MS-9600LSE MS-9600UDLS/MS-9600UDLSE

TENtec Private Portal IReport User Manual MS Editors and MS Validators

MS MAGAGULA

Cover Ms Project 2007.indd 1 !Cover Ms

A Comparison of nLC-ESI-MS/MS and nLC-MALDI-MS/MS for GeLC-Based Protein Identification and iTRAQ-Based Shotgun Quantitative Proteomics

Discovering Known and Unanticipated Protein Modifications Using MS/MS Database Searching

HPLC-UV and LC-MS/MS analysis to study formulation and

MS -220 MS -230 MS -240

Clinical Pharmacokinetic and Bioequivalence Studies of Two

Kogod School of Business 2010 MS Finance and MS Real Estate

MS project Handouts

MS-DRG Crosswalk

Date - Ms Abadie

A Method for LC-MS/MS Profiling of Coumarins in ... - MDPI

LC-MS/MS-Bestimmung von Kokzidiostatika in Futtermittel

ASEAN GUIDELINES FOR THE CONDUCT OF BIOAVAILABILITY AND BIOEQUIVALENCE STUDIES

Manual MS PROYECT