Journal of Chromatography B, 877 (2009) 31943200

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Journal of Chromatography B

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Rapid a ndmethod hu

Yan ZhanShanghai Instit 03, PR

a r t i c l

Article history:Received 12 AAccepted 12 AAvailable onlin

Keywords:Liquid chromaspectrometryLeuprolidePharmacokine

thatanalyeenl-indrolidrupolf acein theeuprond acble anethod

after a single subcutaneous injection of 1mg leuprolide acetate in healthy male Chinese volunteers. 2009 Elsevier B.V. All rights reserved.

1. Introdu

Peptidesthe pharmapeutic agenare often sypharmacolodevelopme

LeuproliH5), a synthhormone (and afnityical castratof sex hormcancer, endlow bioavaiinjection [3or intramus

Single daistered evercancer, end[5]. The rec

CorresponE-mail add

1570-0232/$ doi:10.1016/j.ction

and proteins are becoming increasingly important inceutical industry, especially as a novel class of thera-ts for cancer therapy [1]. Many peptide therapeuticsnthetic analogs of endogenous peptides. The studies ofgically active peptides are therefore signicant for thent of new therapeutic methods for disease therapy.de (pGlu-His-Trp-Ser-Tyr-d-Leu-Leu-Arg-Pro-NHC2etic nonapeptide, is a potent gonadotropin releasing

GnRH) analog that has increased duration of effectfor the pituitary receptor, and that can induce med-

ion [2]. Leuprolide has been used in the treatmentone-related disorders, including advanced prostateometriosis, and precocious puberty [2]. Due to thelability after oral administration or intraduodenal (ID)], leuprolide is presently administered by subcutaneouscular injection [4].ily injections of leuprolide or its retard formula, admin-y 4 or more weeks, have been introduced for prostateometriosis, and other sex hormone-related disordersommended dose for Lupron (leuprolide acetate injec-

ding author. Tel.: +86 21 50800738; fax: +86 21 50800738.ress: dfzhong@mail.shcnc.ac.cn (D. Zhong).

tion) is 1mg administered as a single daily subcutaneous injection[6]. In patients with advanced prostate cancer, leuprolide acetate3 month depot (11.25mg leuprolide acetate) is effective in sup-pressing the testosterone levels for at least 13 weeks [7]. At week13, therapeutic effective levels of the mean values of leuprolide inserum were reported as 17477pg/mL in 19 patients [7].

To date, the main method for studying the pharmacokineticsof leuprolide is by immunoassay [2,811]. More recently, So-anos et al. [12] established a method using HPLC, coupled to ahybridquadrupole linear ion trap, todetermine leuprolide inmouseplasma. They obtained a lower limit of quantication (LLOQ) of0.100ng/mL using 100Lmouse plasma, but separation required arelatively long chromatographic run time (12min). Therefore, ana-lytical methods of more sensitivity are still required for studyingleuprolide and its pharmacokinetic proles in human plasma orserum.

To our knowledge, determination of leuprolide in human serumor plasma by a LCMS/MSmethod has not yet been reported. In thepresent study, we have optimized the chromatographic conditionsand serumsamplepreparation to initiate amore rapid and sensitiveLCMS/MS method for determining leuprolide in human serum.The LLOQ of this method has been decreased to 0.018ng/mL andthe chromatographic run time was less than 5min. The validatedmethod was applied successfully to a pharmacokinetic study ofleuprolide after a single subcutaneous injection of 1mg leuprolideacetate.

see front matter 2009 Elsevier B.V. All rights reserved.jchromb.2009.08.023nd sensitive liquid chromatographytafor the determination of leuprolide in

, Xiaoyan Chen, Xiaohua Zhao, Dafang Zhong

ute of Materia Medica, Chinese Academy of Sciences, 646 Songtao Road, Shanghai 2012

e i n f o

pril 2009ugust 2009e 21 August 2009

tographytandem mass

tics

a b s t r a c t

Leuprolide is a synthetic nonapeptideture. By selection of an appropriatean improved analytical method has btions in human serum. After methanocartridge solid-phase extraction, leupcolumn interfaced with a triple quadization. The mobile phase consisted ointernal standard were both detectedlinear range of 0.01845.2ng/mL for lrelative standard deviation (R.S.D.), aof quantication (LLOQ) was identiaaccuracy. The validated LCMS/MS m/ locate /chromb

em mass spectrometryman serum

China

has two basic amino acids, arginine and histidine, in its struc-tical column and optimizing the mobile phase composition,developed and validated to determine leuprolide concentra-uced protein precipitation of serum samples and Oasis HLBe and an internal standard (alarelin) were analyzed on a C18e tandem mass spectrometer with positive electrospray ion-tonitrilewaterpropionic acid (20:80:0.05). The analyte andselective reactionmonitoringmode. Themethod exhibited alide. The intra- and inter-assay precisions were 11.5% or lesscuracy was within 2.8% relative error (RE). The lower limitd reproducible at 0.018ng/mL, with acceptable precision andwas tested to a clinical pharmacokinetic study of leuprolide

Y. Zhan et al. / J. Chromatogr. B 877 (2009) 31943200 3195

2. Experimental

2.1. Materials

Leuprolide acetate (chemical purity 99.3%, peptide content90.2%) was kindly provided by H&J Beijing Clinical ResearchLtd. (Beijintent 86.7%)Ltd. (Hefeiacid, and pMO, USA).(Faireld, O(Molsheim,HLB cartrid(Milford, Mthe FourthChina).

2.2. Instrum

The Agiequipped wdegasser, aautosampleric detectioUltra tripleChemstatioto control thprocessing.

2.3. Chrom

ChromatASB-C18 coDE, USA)(4mm3.0ture of aused as tThe columperature (2direct HPLC2min of thsource.

2.4. Mass sp

The masusing an eleters werecontainingof 20L/mcolumn Tauxiliary gaulization anthe capillarinduced dispressure ofreaction m(221.0 +249for alarelinoptimized cIS. The mation (peakQ3.

2.5. Preparation of calibration standards and quality control (QC)samples

Astock solution of leuprolidewith a concentration of 376g/mL(calculatedrately weig

dard226tockweren ints we.6ngce cl stasolusoluraturopyling ted on

mple

zen sd to0LhanoL oin anixedhasewithg thenolwwithwasn, anonitrt was

ethod

ensuthe msele

serumix dingwLOQl star thele ints durlinefor lblanlide,es. Thquarepeakfor eion fr(LLOhat cas dg, China). Alarelin (internal standard, peptide con-was obtained from Anhui Xinli Pharmaceutical Co.,

, China). HPLC-grade methanol, acetonitrile, formicropionic acid were purchased from Sigma (St. Louis,HPLC-grade acetic acid was purchased from TediaH, USA). Millipore Milli-Q gradient puried waterFrance) was used throughout the study. An Oasis

ge (30mg, 1mL) was purchased from Waters Corp.A, USA). Drug-free human serum was supplied byMilitary Medical University (Xijing Hospital, Xian,

ent

lent 1100 liquid chromatography system used wasith a G1311A quaternary pump, a G1322A vacuumG1316A thermostated column oven and a G1367Ar (Agilent, Waldbronn, Germany). Mass spectromet-n was performed on a Thermo Finnigan TSQ Quantumquadrupole instrument (San Jose, CA, USA). The Agilentn and Finnigan Xcalibur software packages were usede LCMS/MS system, aswell as for data acquisition and

atographic conditions

ographic separation was achieved on a Venusillumn (150mm4.6mm i.d., 5m, Agela, Newark,protected by a SecurityGuard C18 guard columnmm i.d.; Phenomenex, Torrance, CA, USA). A mix-cetonitrilewaterpropionic acid (20:80:0.05) washe mobile phase at a ow-rate of 0.50mL/min.n temperature was maintained at ambient tem-5 C). A post-column diverter valve was used tocolumn eluate to a waste container for the rst

e chromatographic run and then to the ionization

ectrometric conditions

s spectrometer was operated in the positive ion modeectrospray ionization (ESI) source. The tuning param-optimized for leuprolide and IS by infusing a solution226ng/mL of both the analyte and IS, at a ow-ratein into the mobile phase (0.50mL/min), using a post-connection. Nitrogen was used as sheath gas (35Arb),s (5Arb), and ion sweep gas (2Arb), to assist with neb-d desolvation. The spray voltage was set at 4200V andy temperature was maintained at 320 C. For collision-sociation (CID), argon was used as the collision gas at a1.2mTorr. Quanticationwas performed using selectedonitoring (SRM) of the transitions m/z 605.5m/z.0) for leuprolide and m/z 584.5m/z (221.0 +249.0)(IS), with a dwell time of 200ms per transition. Theollision energy of 35eV was used for the analyte andss spectrometer was operated at unit mass resolu-width at half-height set at 0.7Da) for both Q1 and

of stan0.090of the scurvessolutiosampleand 40refereninternaa stockof thetempepolypr

Durobserv

2.6. Sa

Frothaweing, a 4of metto 200for 1mwas msolid-ptiallyloadinmethaelutedeluatenitrogeof acetaliquo

2.7. M

Totivity,

Theblankfrom scoelutiof the Linternasate fovariabproces

Thecurvestion, aleuproferencleast sof theteriondeviatcationcurve tsion, was peptide content)was prepared bydissolving an accu-hed sample of leuprolide acetate in methanol. A seriesworking solutions with concentrations in the range of

ng/mL for leuprolide were obtained by further dilutionsolutionwithmethanolwater (50:50, v/v). Calibrationprepared by spiking 40L of the appropriate standardo 200L of blank human serum. Quality control (QC)re similarly prepared at concentrations of 0.045, 1.80,/mL in human serum, by a separate weighing of theompound. A 50.0ng/mL (calculated as peptide content)ndard (IS) working solution was prepared by dilutingtion of alarelin with methanolwater (50:50, v/v). Alltions were stored at 4 C and were brought to roome before use. QC samples were aliquoted into 1.5mLene vials and stored at 20 C until analysis.he study, no adsorption of leuprolide or alarelin wasthe different tube materials.

preparation

erum samples from the human study subjects wereroom temperature prior to preparation. After vortex-aliquot of the IS solution (alarelin, 50.0ng/mL), 40Llwater (50:50, v/v), and 200L methanol were addedf serum sample. The mixture was vigorously vortexedd centrifuged at 11,300 g for 5min. The supernatantwith 300L water and then transferred to Oasis HLBextraction tubes that had been pre-treated sequen-2 1mL of methanol and 2 1mL of water. Afterserum sample, the cartridge was washed with 1mLater solution (10:90, v/v), then leuprolide and IS were2 1mL of methanol containing 1% formic acid. The

evaporated to dryness at 40 C under a gentle stream ofd the residue was reconstituted by addition of 100Lilewaterpropionic acid (15:85:0.15, v/v/v). A 20Linjected onto the LCMS/MS system for analysis.

validation

re the selectivity, accuracy, reproducibility, and sensi-ethod was validated described as follows.ctivity of the method was evaluated by analyzing sixsamples and six spiked serum samples at LLOQ level

fferent sources. Peak areas of endogenous compoundsith the analytes should be less than 20% of the peak areastandard according to international guidelines [13]. Thendardwaspresent in the bioanalytical assay to compen-variability of extraction in LC-MS/MS analysis. A highlyernal standard can be an indication of an uncontrolleding sample analysis [14].arity was evaluated by analyzing duplicate calibrationeuprolide in human serum on 3 separate days. In addi-k (no leuprolide or IS) and a zero serum sample (nobutwith IS)were run to eliminate the presence of inter-e standard curves were calculated by aweighted (1/x2)s linear regression method through the measurement-area ratio of the analyte to IS. The acceptance cri-ach back-calculated standard concentration was 15%om the nominal value [13]. The lower limit of quanti-Q), taken as the lowest concentration on the calibrationould be measured with acceptable accuracy and preci-etermined in six replicates on 3 consecutive validation

3196 Y. Zhan et al. / J. Chromatogr. B 877 (2009) 31943200

days. The precision should be equal or less than 20% and accuracybetween 80% and 120% of nominal concentrations for both withinand between-assay [13].

Precision and accuracy of the method were assessed by thedeterminat1.80 and 40precision wand calcula[15]. The ai.e. (observconcentratirequired to

To evalusample dilu4-fold befopharmacok

Themattor, as denmatrix fromof serum mtive to the ndeterminedinter-subjecshould be le

The recotion levels (ratios of thelyte prior toanalyte wasets of samwas determconcentrati

The stabanalyzing rtions of 0.0conditionsdeterminedperature forextraction,temperaturstorage of thThe freeze/freeze/thaware consideconcentrati

2.8. Applica

The LCmine thepharmacokteers werethe Medicaconsent waand risks oflide acetateinto each subefore andand 24h afthen centriffractions. Thanalysis.

The phabynon-comgram WinNmaximum

rence (Tmax) were both obtained directly from the measured data.The area under the serumconcentrationtime curve from time zeroto the time of the last measurable concentration (AUC0t) was cal-culated by the linear trapezoidal method. The terminal elimination

nstant (ke) was estimated by log-linear regression of con-tions observed during the terminal phase of elimination. Theonding elimination half-life (t1/2) was then calculated as

ke.

ults and discussion

timization of the mass spectrometric condition

prolide is an oligopeptide containing a histidine (His) andinine (Arg) in its structure. The presence of these two basicacids resulted in favorable sensitivity for leuprolide in thee ESI ionizationmode. Alarelin is the analogue of leuprolide,y difference in their structure is that a leucine of leupro-oup is substituted by an alanine group. In the positive ESIce, both leuprolide and IS formedpredominantlydoublepro-d molecules [M+2H]2+ at m/z 605.5 and m/z 584.5 in Q1n mass spectra, respectively (see Fig. 1), while the [M+H]+

m/z 1209.9 and m/z 1168.6 were less than 5% relative abun-of [M+2H]2+. The correspondingproduct ionmass spectra areed in Fig. 2, where [M+2H]2+ of each compoundwas selectedprecursor ion. Leuprolide and IS both have fragment ions at1.0 and m/z 249.0. In order to improve the sensitivity, theajor diagnostic fragment ionswere both acquired in the SRMprolide and IS. The optimal collision energy for leuprolidewere both set at 35 eV.

Fig.ion of QC samples at three concentration levels (0.045,.6 ng/mL), in six replicates, on 3 validation days. Theas expressed by relative standard deviation (R.S.D.)ted using the one-way analysis of variance (ANOVA)ssay accuracy was expressed as relative error (RE),ed concentrationnominal concentration)/(nominalon)100%. The intra- and inter-day precisions werebe below 15%, and the accuracy to be within 15% [13].ate the precision and accuracy of the method in thetion process, QC samples at 90.2ng/mL were dilutedre being analyzed in six replicates on the rst day ofinetic study.rix effect was investigated bymeasuring thematrix fac-ed by the ratio (analyte peak area in presence of serumsix different sources)/(analyte peak area in absenceatrix), and was expressed as percent response rela-eat solution. In our experiment, the matrix effect wasat twoconcentration levels (0.045and40.6ng/mL). Thet variability ofmatrix effect at each concentration levelss than 15% [16].very of leuprolide was estimated at three concentra-0.045, 1.80 and40.6ng/mL) by comparing thepeak-areaanalyte to IS. Samples that were spiked with the ana-extraction were compared with samples to which the

s added post-extraction. The IS was added to the twoples post-extraction. The extraction recovery of the ISined in a similar way, using the QC samples at mediumon as a reference.ility of leuprolide in human serum was assessed byeplicates (n=3) of serum samples at the concentra-45 and 40.6ng/mL, which were exposed to different(time and temperature). The short-term stability wasafter the exposure of the spiked samples to room tem-2h, orbyexposureof the ready-to-inject samples (afterin the mobile phase) to the autosampler rack (roome) for 24h. The long-term stability was evaluated aftere standard spiked serum samples at20 C for 21 days.thaw cycle stability was assessed after three completecycles (20 to 25 C) on consecutive days. The analytesred to be stable in serum when 85115% of the initialons were found.

tion to a pharmacokinetic study

MS/MS method described above was applied to deter-serum concentrations of leuprolide from a clinicalinetic study in which 10 healthy male Chinese volun-enrolled. The pharmacokinetic study was approved byl Ethics Committee of Xian Xijing Hospital. Informeds obtained from all subjects after explaining the aimsthe study. A single subcutaneous dose of 1mg leupro-, equivalent to leuprolide base at 0.95mg, was injectedbject. Venous blood samples were collected into tubes0.17, 0.33, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10, 14ter dosing. Samples were kept at 4 C for 30min anduged at 2000 g (4 C) for 10min to separate the serume collected serum samples were stored at 20 C until

rmacokinetic parameters of leuprolide were calculatedpartmental assessment of datausing the computer pro-onlin (V5.0.1, Pharsight, Mountain View, CA, USA). Theserum concentrations (Cmax) and their time of occur-

rate cocentracorresp0.693/

3. Res

3.1. Op

Leuan argaminopositivthe tinlide grinterfatonatefull-scaions atdancedepictas them/z 22twomfor leuand IS1. Full-scan mass spectra of leuprolide (A) and alarelin (B).

Y. Zhan et al. / J. Chromatogr. B 877 (2009) 31943200 3197

3.2. Optimichromatogr

Currentlextractionextractionextraction (used by Soplasma samtested a PPbut we encsubstancesisocratic HPsion mightas describedshort analy

The useenrichmentmination. Sfrom biologment,we inStrata-ph caOasis HLBdifferent lotridge provformic acidery of leupmethanol a1% formic aFig. 2. Product ion mass spectra of [M+2H]2+ ion of leupro

zation of the sample preparation andaphic condition

y, the most commonly employed techniques for theof peptides from biological matrices are solid-phase(SPE), protein precipitation (PPT) and liquidliquidLLE) [17]. PPT, a simple pre-treatment technique, wasanos et al. [12] to separate leuprolide from mouseples. During our initial method development, we alsoT method to separate leuprolide from serum samples,ountered strong ion suppression from the endogenousin serum samples and a broad peak occurred underLC conditions. Although improvements in ion suppres-be improved by gradient chromatographic conditions,in the literature, thiswould havemeant sacricing our

tical run time.of solid-phase extraction (SPE) cartridges also allowsof analytes and therefore better sensitivity for deter-

PEhasbeenwidely chosen for the separationofpeptidesical matrixes [1820]. During our method develop-vestigatedavarietyof SPEcartridges (Strata-Ecartridge,rtridge, Orochem C18 cartridge, Strata-X cartridge andcartridge) for serum pre-treatment and optimized theading, washing and elution steps. The Oasis HLB car-ided the highest and most stable recovery. When 1%in methanol was used as elution solvent, the recov-rolide could be improved from the 50% seen withlone toabout80%. Throughoptimization,weestablishedcid in methanol (2 1mL) to be the elution solvent that

achieved thand IS.

A watersuccessfullyHowever, aneeded toLeuprolidethe study,high-polariSynergi 4(Shiseido, JASB-C18 (ADiamonsildouble peaon Phenomalso attemp(HILIC) col10min. Aftconsideredon the coluand MS resmobile pha

Acetonitlower backorganic phaphase wasThe MS resier, was aacid, but thlide (A) and alarelin (B).

e highest recovery (more than 80%) for both leuprolide

s symmetry C4 column using gradient elution has beenused to separate leuprolide from mouse plasma [12].relatively long analytical run time (about 12min) wasequilibrate the column and to avoid ion suppression.is a strongly hydrophilic and basic compound. Inall the tested columns were suitable for analysis ofty compounds, including Diamonsil C18 (Dikma, China),Hydro-RP (Phenomenex, USA), Capcell PAK AQ C18

apan), Atlantis HILIC Silica (Waters, USA) and Venusilgela, USA). Strong ion suppression was observed onC18 column. As shown in Fig. 3, there was an obviousk on the Capcell PAK C18 AQ column and the peak shapeenex Synergi 4Hydro-RP 80 A columnwas tailed. Weted a hydrophilic-interaction liquid chromatography

umn, but the retention time of leuprolide was overer a series of trials, the Venusil ASB-C18 column wasto be optimal and leuprolide and IS were well retainedmn (Fig. 3). However, the retention time, peak shape,ponse were easily affected by the composition of these.rile led to a higher mass spectrometric response and aground noise than did methanol, and was chosen as these. A small amount of acidic modiers in the mobilefound to improve MS response of leuprolide and IS.ponse of leuprolide using acetic acid as acidic mod-bout 2 times higher than that obtained with formice leuprolide peak was split with mobile phases con-

3198 Y. Zhan et al. / J. Chromatogr. B 877 (2009) 31943200

Fig. 3. Typicalcolumns to evPeak A: acetoC18 column (4B: acetonitrilecolumn (4.6mC: acetonitrileergi 4 HydrotR,leuprolide = 6.2

taining 0.05could be remobile phaacid in placbut a reasoand no peakand the aciion-pairingof leuprolidincrease wphase systemay have ptributed toof this peak

As showlide was imreconstitutiable retentacetonitrilereconstitutiless than 5m

3.3. Method

3.3.1. AssayAs for th

with incurrtypical chrosample spikfrom a volulide. No sigobserved attion times f

The absoof human s141% and 1and 8.6%, re(10.0ng/ml

Typicaon a

.05, vtutionacetonacetonacetoe =4.6

as at conrolid

Lineacatilineere prolin a v

2 y reps theed inchromatograms of leuprolide in human serum acquired at differentaluate the chromatographic performance of leuprolide and alarelin.nitrilewaterpropionic acid (20:80:0.05, v/v/v) on a Venusil ASB-.6mm150mm i.d., 5m, Agela, USA), tR,leuprolide = 3.71min; Peakwaterpropionic acid (20:80:0.05, v/v/v) on a Capcell PAK C18 AQm150mm i.d., 5m, Shiseido, Japan), tR,leuprolide = 4.86min; Peakwaterpropionic acid (30:70:0.05, v/v/v) on a Phenomenex Syn--RP column 80 A (4.6mm150mm i.d., 4m, Phenomenex, USA),7min.

% acetic acid. The peak-splitting issue for leuprolidesolved by introducing 5mM ammonium acetate to these, but then the IS peak became broadened. Propionice of acetic acid resulted in a slightly lowerMS response,nable peak shape and retention time was achieved,-splitting was observed. We supposed that leuprolidedic modier might have a transition process betweenand dynamic ion-exchange mechanism. The retentione in the ion-pair chromatography system tended toith longer carbon chain acidic modiers. The mobilem of acetonitrilewaterpropionic acid (20:80:0.05)rovided a condition where only one mechanism con-chromatographic separations. However, other aspects-splitting phenomenon are the subject of further study.

Fig. 4.obtained(20:80:0reconstiPeak A:Peak B:Peak C:tR,leuprolid

therewpresenof leup

3.3.2.quanti

Thetionswfor leucurve o

y = 1.0whereresentobtainn in Fig. 4, the MS response and peak shape of leupro-proved by increasing propionic acid content of theon solvent. A sharp chromatographic peak with suit-ion time (4.6min) was obtained when a solution ofwaterpropionic acid (15:85:0.15) was used as theon solvent. The analytical run time of each sample wasin.

validation

selectivity and matrix effecte impact of internal standard variability, the variabilityed samples of leuprolide was insignicant. Fig. 5 showsmatograms of a blank human serum, human serumed with leuprolide at LLOQ and IS, and a serum samplenteer 4.0h after subcutaneous injection with leupro-nicant interference from endogenous substances wasthe retention times of leuprolide and IS. Typical reten-or leuprolide and IS were 4.5 and 3.0min, respectively.lute matrix effects of leuprolide in six different lotserum at concentrations of 0.045 and 40.6ng/mL were46%, respectively. The relative matrix effect was 6.5%spectively. The absolute and relativematrix effect for ISin serum)were 126% and 10.5%, respectively. Although

The lowefor leuproliLLOQ are shthe serum c24h after aacetate tohto investigastudies.

3.3.3. PreciThe met

marizes the

Table 1Precision andserum (in 3 co

Concentratio

Added

0.0180.0451.80

40.690.2l chromatograms of leuprolide and alarelin in human serumVenusil ASB-C18 column when acetonitrilewaterpropionic acid

/v/v) was used as the mobile phase to evaluate the effect of thesolvent on the chromatographic peak shape and MS response;itrilewaterpropionic acid (20:80:0.05, v/v/v), tR,leuprolide = 3.73min;itrilewaterpropionic acid (15:85:0.15, v/v/v), tR,leuprolide = 4.55min.nitrilewaterpropionic acidacetic acid (15:85:0.04:0.11, v/v/v/v),4min.

n extent to thematrix enhancement for leuprolide in thedition, it did not inuence the accurate determinatione in human serum.

rity of calibration curve and lower limit ofonar regression of the peak-area ratios versus concentra-tted over the concentration range of 0.01845.2ng/mL

de in human serum. A typical equation of the calibrationalidation run was as follows:

103 + 0.402x (r2 = 0.9923)resents the peak-area ratio of analyte to IS and x rep-serum concentration of leuprolide. Good linearity wasthis concentration range.r limit of quanticationwas established as 0.018ng/mL

de. The precision and accuracy values corresponding toown in Table 1. Under the present LLOQ of 0.018ng/mL,oncentration of leuprolide could be determined up tosingle subcutaneous administration of 1mg leuprolideealthymaleChinese volunteers. This is sensitive enoughte thepharmacokinetic behavior of leuprolide in clinical

sion and accuracyhod showed good precision and accuracy. Table 1 sum-intra- and inter-day precision and accuracy values for

accuracy of the LCMS/MSmethod to determine leuprolide in humannsecutive days, six replicates for each day).

n (ng/mL) R.S.D. (%) RE (%)

Found Intra-day Inter-day

0.0177 0.0020 11.5 6.6 1.80.0444 0.0036 7.9 10.4 1.6

1.77 0.14 8.5 5.8 1.940.4 1.85 4.3 6.1 0.592.9 9.02 9.8 2.8

Y. Zhan et al. / J. Chromatogr. B 877 (2009) 31943200 3199

Fig. 5. Represhuman serumleuprolide atsample of 14.9leuprolide ace

leuprolide fcisions werwith relativthe acceptaably accura

3.3.4. ExtraThe rec

81.63.2%,1.80, and 40was 81.0

3.3.5. StabiThe stab

ipated cond

Table 2Summery of stability of leuprolide under various storage conditions (n=3).

Storage conditions Concetrations (ng/mL) R.S.D. (%) RE (%)

Added Found

freezethaw 0.045 0.0444 0.0040 8.9 1.640.6 40.1 1.9 4.8 1.2

ng for 21 daysC)

0.045 0.0451 0.0048 10.6 0.240.6 39.0 3.5 8.9 3.9

mpler for5 C)

0.045 0.0408 0.0015 3.7 9.640.6 41.0 3.4 8.4 0.9

term (2h at 0.045 0.0464 0.0062 13.4 2.940.6 39.9 1.4 3.6 1.8Threecycles

Freezi(20

Autosa24h (2

Short-25 C)entative SRM chromatograms for leuprolide (I) and alarelin (IS, II) in: (A) a blank serum sample; (B) a blank serum sample spiked with0.018ng/mL and alarelin (IS) at 10.0ng/mL; (C) a volunteer serumng/mL obtained at 4.0h after a single subcutaneous injection of 1mgtate (equivalent to 0.95mg leuprolide base).

rom QCs. In this assay, the intra- and inter-assay pre-e measured to be below 11.5% and 10.4%, respectively,e errors from 1.9% to 2.8%. These values were withinble range, and the method was thus judged to be suit-te and precise.

ction recoveryoveries of leuprolide extracted from serum were86.87.2% and 83.61.9% at concentrations of 0.045,.6 ng/mL, respectively (n=6). Mean recovery for the IS

2.5% (n=6).

lityility tests of the leuprolideweredesigned to cover antic-itions of handling of typical clinical samples. As seen in

Fig. 6. Mean stion of 1mg lemale Chinese

Table 2, theprocessing

3.4. Applicahealthy volu

The valia pharmacsubjects. Upost-injectisubjects, aftlide acetatequantify thserum samanalyzed. Ficentrationfor leuprolicokinetic re

TaMsin(eneerum concentrations of leuprolide after a single subcutaneous injec-

uprolide acetate (equivalent to 0.95mg leuprolide base) to 10 healthyvolunteers (mean S.D.).

analyte was stable under the typical serum storage andconditions used throughout the current study.

tion of the method to a pharmacokinetic study innteers

dated LCMS/MS method was successfully applied tookinetic study of leuprolide in healthy male Chinesesing this analytical method, we were able to measureon serumconcentrationof leuprolide forup to24h inaller a single subcutaneous administrationof 1mg leupro-(equivalent to 0.95mg leuprolide base). In order to

e higher leuprolide concentrations observed in humanples, the samples were diluted 2-fold before beingg. 6 shows theproleof themean leuprolide serumcon-versus time and the main pharmacokinetic parametersde are presented in Table 3. Compared with pharma-sults reported previously [8], the values of AUC024 and

ble 3ean pharmacokinetics parameters for leuprolide after agle subcutaneous injection of 1mg leuprolide acetatequivalent to 0.95mg leuprolide base) to healthymale Chi-se volunteers (mean S.D., n=10).

Parameters Mean S.D.Cmax (ng/mL) 64.9 8.36Tmax (h) 0.407 0.178AUC024 (ngh/mL) 164 21.3AUC0 (ngh/mL) 164 21.6t1/2 (h) 3.47 0.732

3200 Y. Zhan et al. / J. Chromatogr. B 877 (2009) 31943200

Cmax from the present study were about 2 times higher, while thet1/2 did not differ signicantly. The reportedmethod [8] was deter-minedbyamodied radioimmunoassayprocedure. Thedifferencesof pharmacokinetic parameters might result from the differentassay methods or inter-ethnic differences.

According to the literature [7], the therapeutic effective levels ofleuprolide acetate depot in serum were 17477pg/mL. Thereforehigh detection sensitivity will be necessary for any pharmacoki-netic study of leuprolide depot.

4. Conclusion

By optimizing the chromatographic conditions, a sensitive andrapid LCMS/MS method for the quantication of leuprolide inhuman serumwas developed and validated. This method was sen-sitive enough to monitor low-dosage pharmacokinetic or depotformulation studies of leuprolide in human serum. Compared withpreviously reported analytical methods, this method showed highthroughput (4.8min each sample) and greater sensitivity, with anLLOQ of 0.018ng/mL. It was successfully applied to characterizethe pharmacokinetics of leuprolide in healthymale Chinese volun-teers.

Acknowledgments

This study was supported by the Grant No. 30472053 of theNational Natural Sciences Foundation of China. We thank Prof.Aidong Wen at Xian Xijing Hospital in the conduct of the clinicalstudy.

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Rapid and sensitive liquid chromatography-tandem mass spectrometry method for the determination of leuprolide in human serumIntroductionExperimentalMaterialsInstrumentChromatographic conditionsMass spectrometric conditionsPreparation of calibration standards and quality control (QC) samplesSample preparationMethod validationApplication to a pharmacokinetic study

Results and discussionOptimization of the mass spectrometric conditionOptimization of the sample preparation and chromatographic conditionMethod validationAssay selectivity and matrix effectLinearity of calibration curve and lower limit of quantificationPrecision and accuracyExtraction recoveryStability

Application of the method to a pharmacokinetic study in healthy volunteers

ConclusionAcknowledgmentsReferences