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Journal of Chromatography B, 971 (2014) 14–19

Contents lists available at ScienceDirect

Journal of Chromatography B

jou rn al hom epage: www.elsev ier .com/ locate /chromb

nalysis of cyclophosphamide and carboxyethylphosphoramideustard enantiomers in human plasma and application to

linical pharmacokinetics

rancine Attié de Castroa, Gabriel dos Santos Scatenab, Quézia Bezerra Cassb,elinda Pinto Simõesc, Vera Lucia Lanchotea,∗

Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo,venida do Café s.n., Campus da USP, 14040-903 Ribeirão Preto, SP, BrazilDepartamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, BrazilDepartamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil

r t i c l e i n f o

rticle history:eceived 6 May 2014ccepted 4 September 2014vailable online 16 September 2014

eywords:yclophosphamidearboxyethylphosphoramide mustardnantiomersC-MS/MSlasma

a b s t r a c t

This study describes for the first time a method for the sequential analysis of the enantiomers ofcyclophosphamide (CY) and its metabolite carboxyethylphosphoramide mustard (CEPM) in humanplasma. The CY and CEPM enantiomers were extracted from plasma using only ethyl acetate and sepa-rated on a Chiralpak® AD-RH column using a mixture of water:acetonitrile:ethanol (45:30:25, v/v/v) plus0.1% trifluoroacetic acid as the mobile phase at a flow rate of 0.5 mL/min. No matrix effect was observedin the analysis of the enantiomers of both analytes and the analytical method was linear in the range of0.05–25.0 �g and 250–1000 ng of each enantiomer/mL plasma. The coefficients of variation and relativeerrors obtained for the assessment of intra- and interassay precision and accuracy were less than 15%.CY and CEPM were found to be stable in human plasma after three successive freeze/thaw cycles, dur-ing storage for 4 h at room temperature, and after 24 h inside the autosampler at 4 ◦C, with deviations

harmacokinetics less than 15%. The method was applied to the study of the pharmacokinetics of CY and its metaboliteCEPM in patients with multiple sclerosis (n = 10) who received a CY pretransplant conditioning regimenfor hematopoietic stem cell transplantation. The pharmacokinetic parameters showed plasma accumu-lation of the (S)-(−)-CY enantiomer (S/R ratio = 1.3) and lack of enantioselective exposure to the CEPMmetabolite (S/R ratio = 1.0).

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Cyclophosphamide (CY) is an alkylating agent commonly usedor the treatment of multiple types of cancer and autoimmune dis-ases in adults and children [1]. It is a prodrug that is metabolized toctive and inactive products. About 80% of the CY dose is activatedo the 4-hydroxycyclophosphamide metabolite, which readily dif-uses into cells and decomposes into phosphoramide mustard, thective cytotoxic metabolite. The main metabolic routes of CY inac-ivation include the formation of 4-ketocyclophosphamide and

arboxyethylphosphoramide mustard (CEPM). The formation of theatter depends on the activity of cytosolic aldehyde dehydrogenaseALDH1) [2,3].

∗ Corresponding author.E-mail address: lanchote@fcfrp.usp.br (V.L. Lanchote).

ttp://dx.doi.org/10.1016/j.jchromb.2014.09.008570-0232/© 2014 Elsevier B.V. All rights reserved.

Several studies have demonstrated that exposure to the CEPMmetabolite can be defined as a biomarker of CY toxicity [4–7].In a study involving 147 patients treated with CY (120 mg/kg),McDonald et al. [4] showed that exposure to the inactive CEPMmetabolite, expressed as AUC, was correlated with liver toxicityand survival, with a 5.9-fold higher risk of mortality among patientswith higher CEPM exposure. Furthermore, in that study, the AUCof the CEPM metabolite showed high variability (up to 16 times)among the patients investigated. As a consequence, researchersstarted to adjust CY doses to achieve a target exposure to CEPMof 325 �mol/L h in order to prevent liver toxicity in the patients [7].

Cyclophosphamide contains a chiral phosphorus (Fig. 1A) andis available in clinical practice as a racemic mixture of the (S)-(−)-

and (R)-(+)-CY enantiomers [8]. Preclinical data show differencesin efficacy and toxicity between CY enantiomers, with (S)-(−)-CY exhibiting a greater antitumor effect and higher therapeuticindex than the (R)-(+) enantiomer [9,10]. Although CY is commonly

F.A.d. Castro et al. / J. Chromato

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ig. 1. (A) Chemical structure of CY. (B) Chemical structure of CEPM (*denotes thehiral center).

sed at different doses for the treatment of different diseases,ew studies have evaluated stereoselectivity in the kinetic dis-osition of unchanged CY [8,11,12]. Clinical data related to thenantioselectivity in CY metabolism show that urinary excretion of-ketocyclophosphamide is greater after intravenous administra-ion of (R)-(+)-CY than that from CY as a racemic mixture, whereashe urinary excretion of CEPM metabolite do not differ among intra-enous administration of (R)-(+)-CY, (S)-(−)-CY or CY as racemicixture [13].Few chromatographic techniques for the analysis of CY enan-

iomers have been described in the literature [8,14–18] and itsEPM metabolite (Fig. 1B) has so far only been evaluated as annantiomeric mixture [19–22]. In view of the lack of analyticalethods of CEPM enantiomers and the importance of knowledge

f CEPM metabolite exposure as a biomarker of liver toxicity, theresent study describes for the first time a simple method forhe simultaneous analysis of CY and CEPM enantiomers in humanlasma using LC-MS/MS and its application to pharmacokinetictudies.

. Materials and methods

.1. Standard solutions and reagents

The stock solution of racemic CY (ISOPAC, purity > 99.5%, mono-ydrated; Sigma, St. Louis, MO, USA) was prepared in ethanol at

concentration of 1 mg/mL. Next, the stock solution was dilutedo obtain the following concentrations: 50, 40, 30, 10, 5, 1, 0.5,nd 0.1 �g/mL ethanol. The standard solutions were stored at20 ◦C. The stock solution of the racemic CEPM metabolite (Torontoesearch Chemicals, Inc., Toronto, Canada) was prepared in watert a concentration of 1 mg/mL and diluted to obtain the follow-ng concentrations: 5000, 3000, 2000, 1000, 500, 200, 100, and0 ng/mL water. The standard solutions were stored at −70 ◦C.ntipyrine (Sigma, St. Louis, MO, USA) was used as the internaltandard (IS). The solution was prepared in methanol at a con-entration of 10 ng/mL and stored at −20 ◦C. All solvents usedere of HPLC grade and were purchased from Merck (Darmstadt,ermany). Trifluoroacetic acid (purity > 99%) was purchased fromigma. The water used was distilled and deionized in a SynergyV® Purification System (Millipore, Molsheim, France). The blanklasma provided by the Blood Center of the School of Medicine ofibeirão Preto, University of São Paulo, was obtained from healthyolunteers.

.2. Chromatographic analysis

The LC-MS/MS system consisted of a binary gradient pumpmodel 1525�, 52777), TCM/CHM column heater, and Xevo TQ-Sriple quadrupole mass spectrometer (all from Waters, Milford, MA,

gr. B 971 (2014) 14–19 15

USA). The CY and CEPM enantiomers and the IS (antipyrine) wereseparated on a Chiralpak® AD-RH column (150 mm × 4.6 mm, 5-�m particle size; Chiral Technologies, Exton, PA, USA) coupled toa LiChrospher® 100 RP 18 pre-column (4 × 4 mm, 5-�m particlesize; Merck, Darmstadt, Germany). The mobile phase consisted ofa mixture of water:acetonitrile:ethanol (45:30:25, v/v/v) plus 0.1%trifluoroacetic acid and was used at a flow rate of 0.5 mL/min. Thecolumn was maintained at a temperature of 25 ± 1 ◦C. The mobilephase originating from the HPLC system was directed to the massspectrometer at a flow rate of 200 �L/min. The desolvation tem-perature was maintained at 200 ◦C, the ionization source at 150 ◦C,and the capillary voltage was 3.4 kV. Nitrogen was used as the neb-ulizer gas at a flow rate of 800 L/h. Argon was used as the collisiongas at a pressure of approximately 7.0 × 10−3 mbar. The cone volt-age was maintained at 6 V for CY and CEPM and at 4 V for the IS.The collision energy was 16 eV for CY and CEPM and 30 eV for theIS. The MS/MS conditions were optimized by direct infusion of thestandard solutions prepared in the mobile phase with an infusionpump. The analyses were carried out in the multiple reaction mon-itoring (MRM) mode. The protonated molecules [MH]+ and theirrespective product ions were monitored at transitions of 261 > 140for CY, 293 > 221 for the CEPM metabolite, and 189 > 104 for theIS. Data acquisition and quantification were performed using theMassLynx 4.1 program (Micromass, Manchester, UK).

2.3. Sample preparation

Plasma aliquots (25 �L) were enriched with 25 �L of the IS(antipyrine) and 1 mL ethyl acetate. The tubes were mixed for 20 sand then centrifuged (Beckman centrifuge, model TJ-6) at 4000 × gfor 5 min at 5 ◦C. The organic phases were transferred to conictubes and evaporated to dryness in a vacuum evaporation sys-tem (RCT90 in the RC10.22 mode, Jouan AS, St. Herblain, France).The residues were resuspended in 300 �L water, mixed for 5 s, andkept in the autosampler at 10 ◦C until injection (20 �L) into thechromatographic column.

2.4. Determination of the elution order of the CY and CEPMenantiomers

The elution order of the CY enantiomers was determinedbased on the individual peaks obtained with the Chiralpak® AD-RH chiral-phase column, followed by injection into a Chiralcel®

OD-RH column as described by de Miranda Silva et al. [14].Aliquots (25 �L) of the racemic CY solution (15 �g of eachenantiomer/mL ethanol) were injected into the Chiralpak® AD-RH column under the previously established conditions usinga mixture of water:acetonitrile:ethanol (45:30:25, v/v/v) plus0.1% trifluoroacetic acid as the mobile phase at a flow rate of0.5 mL/min.

The elution order of the CEPM enantiomers was evaluatedexperimentally by administering the pure CY enantiomers asdescribed in another study from our group [23]. Briefly, Wistarrats (n = 3) fasted for 24 h received a single intravenous dose of20 mg/kg of each CY enantiomer diluted in saline. Blood sampleswere collected by decapitation 30 min after CY administration.Aliquots (5 mL) of the blood samples were transferred to tubescontaining EDTA and the samples were analyzed as described initem 2.3.

2.5. Analysis of racemization

Standard solutions of CY and CEPM at concentrations of30 �g/mL and 1000 ng/mL, respectively, were injected into aChiralpak® AD-RH column. A mixture of water:acetonitrile:ethanol(45:30:25, v/v/v) plus 0.1% trifluoroacetic acid was used as the

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obile phase at a flow rate of 0.5 mL/min. The individual enan-iomers were collected at the column outlet. The individualollected fractions were spiked with 25 �L blank plasma aliquots,nd submitted to the extraction process. The chromatograms wereompared to those obtained for the standard solutions at the sameoncentration.

.6. Validation of the analytical method of CY and CEPMnantiomers in plasma

The method used for the analysis of plasma samples containingY and CEPM enantiomers was validated according to recommen-ations of the US-FDA guide for the validation of analytical methods

n industry [24] and of the European Medicines Agency-Guidelinen bioanalytical method validation [25]. The matrix effect was eval-ated by direct comparison of the peak areas obtained for the CYnd CEPM enantiomers and for the IS (antipyrine) injected directlynto the mobile phase with those obtained for the standard solu-ions (0.05 and 5 �g of each CY enantiomer/mL plasma, 250 ng and

�g of each CEPM enantiomer/mL plasma) added to extracts oflank plasma derived from eight different volunteers (4 normallasma samples, 2 lipemic, and 2 hemolyzed). The coefficient ofariation of the IS normalized matrix factor of all samples shoulde less than 15%. The calibration and linearity curves were con-tructed in triplicate using 25-�L aliquots of blank plasma enrichedith 25 �L of each CY and CEPM standard solution and of the IS and

ubmitted to the extraction process as described above. Thus, theoncentrations were 0.05, 0.25, 0.5, 2.5, 5, 15, 20 and 25 �g of eachY enantiomer/mL plasma and 25, 50, 100, 150, 250, 500, 1000,250, 1500 and 2500 ng of each CEPM enantiomer/mL plasma.he linear regression equations and correlation coefficients werebtained from the ratios of the standard/IS peak areas plotted as

function of the respective plasma concentrations. The limit ofuantification was estimated by the analysis, in eight replicates,f plasma samples enriched with CY (0.05 �g of each CY enan-iomer/mL plasma) and CEPM (25 ng of each CEPM enantiomer/mLlasma). The limit of quantification was defined as the lowestlasma concentration that can be measured with a coefficient ofariation less than 20% and percent inaccuracy less than 15%.

The precision and accuracy of the method were evaluated byntra- and interassay assessments. For this purpose, CY solutions

ere prepared at concentrations of 0.05, 0.15, 10, 21 and 100 �g ofach enantiomer/mL plasma and CEPM solutions at concentrationsf 25, 75, 750, 2000 and 3000 ng of each enantiomer/mL plasma.hese solutions were divided into aliquots and stored at −70 ◦Cntil the time of analysis. For the evaluation of intra-assay pre-ision and accuracy, five aliquots of each of these solutions werenalyzed in the same analytical run. For assessment of interassayrecision and accuracy, five aliquots of each CY and CEPM solutionere analyzed in three different runs on different days.

The freeze/thaw cycle stability, post-processing stability andhort-term stability of CY and CEPM were evaluated. The stability ofY was evaluated by enriching samples with concentrations of 0.15nd 21 �g of each CY enantiomer/mL plasma, whereas for the evalu-tion of CEPM stability samples were enriched with concentrationsf 75 and 2000 ng of each enantiomer/mL plasma. For assessmentf freeze/thaw cycle stability, the enriched samples were frozen at70 ◦C for at least 24 h, then thawed and again frozen for 24 h. Thisrocess was repeated until the third freezing cycle when the sam-les were extracted and analyzed. Post-processing stability wasvaluated by maintaining the extracted samples in the autosampleror 24 h at 4 ◦C before injection into the chromatographic system.

or evaluation of short-term stability, the enriched plasma samplesere kept on the laboratory bench for 3 h at room temperature. The

esults of the stability tests were compared to those obtained forreshly prepared samples.

gr. B 971 (2014) 14–19

2.7. Application of the method

The clinical protocol was approved by the Research EthicsCommittee of the University Hospital, School of Medicine ofRibeirão Preto, University of São Paulo. Ten patients with mul-tiple sclerosis, ranging in age from 27 to 50 years and with aBMI of 18.1–28.4 kg/m2, were studied after they had signed thefree informed consent form. The patients treated for hematopoi-etic stem cell transplantation received 50 mg racemic CY/kg/day(Cycram®, Meizler, Brazil) for 4 days. During infusion of the last CYdose, serial blood samples (2 mL) were collected at times 15, 30,45, 60, 75 and 90 min, and at 2, 3, 5, 8, 12, 14, 16, 20 and 24 h afterinfusion of the drug. Blood samples were collected into tubes con-taining EDTA, centrifuged at 4000 × g for 5 min at 4 ◦C, and plasmawas stored at −70 ◦C until the time of analysis.

2.8. Pharmacokinetics

The pharmacokinetic parameters were calculated based on theplasma CY and CEPM enantiomer concentration versus time curvesusing the WinNonlin 5.2 program (Pharsight Corp., Mountain View,CA, USA). The kinetic disposition of CY and CEPM was calculatedusing first-order kinetics and non-compartmental and monocom-partmental models, respectively.

3. Results and discussion

The present study reports for the first time the developmentand validation of a method for the sequential analysis of theenantiomers of CY and of its metabolite CEPM in plasma usingLC-/MS/MS and its application to clinical pharmacokinetic stud-ies. Few studies describing analytical methods of CY enantiomershave been published [8,14,16–18] and its metabolite CEPM hasso far only been evaluated as the enantiomeric mixture [19–22].Since patients treated with CY generally use different drugs incombination, the analysis of CY and its metabolites by LC-MS/MSis highly recommended mainly because of the selectivity of thistechnique.

The CY and CEPM enantiomers were eluted on a Chiralpak®

AD-RH column using a mixture of water:acetonitrile:ethanol(45:30:25, v/v/v) plus 0.1% trifluoroacetic acid as the mobile phaseat a flow rate of 0.5 mL/min. The chromatographic conditionsfor separation of the CEPM metabolite [resolution of 0.81 basedon the equation R = 1.177 (tR2 − tR1)/(W0.5h1 + W0.5h2)] were exten-sively investigated before optimization, showing no robustness interms of minimal changes in the mobile phase or even flow rate.Fig. 2 shows the chromatograms obtained for blank plasma sam-ple enriched with CY and CEPM at LOQ (50 and 25 ng of eachenantiomer/mL, respectively); (B) blank plasma sample enrichedwith CY and CEPM at concentrations of 1000 and 200 ng of eachenantiomer/mL, respectively and (C) plasma sample obtained froma patient 16 h after the beginning of CY infusion. The retentiontimes were 5.5 min for the IS, 6.8 and 11.0 min for the (S)-(−)-(CY) and (R)-(+)-(CY) enantiomers, respectively, and 4.5 and4.9 min for the (R)-CEPM and (S)-CEPM enantiomers, respectively.The elution order of (R)-CEPM and (S)-CEPM was determinedin an experimental study of rats treated with the pure CYenantiomer.

The CY and CEPM enantiomers and the IS were extracted froma plasma sample (25 �L) in a single extraction step with 1 mL ethylacetate, in contrast to previously published methods in which two

liquid–liquid extraction steps involving mixtures of solvents (ethylacetate and chloroform, followed by a purification step with hex-ane) [14], derivatization techniques [18], or even more complexsolid-phase extraction techniques [16].

F.A.d. Castro et al. / J. Chromatogr. B 971 (2014) 14–19 17

Fig. 2. Chromatograms obtained for (A) blank plasma sample enriched with CY and CEPM at LOQ (50 and 25 ng of each enantiomer/mL, respectively); (B) blank plasmas antiomC S)-CEPf

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accumulation of the (S)-(−)-CY enantiomer, with a median S/Rratio of 1.3 (Table 5 and Fig. 3). This predominance of the (S)-(−)-CY enantiomer in plasma of the patients with multiple sclerosisstudied agrees with the clinical studies of de Miranda Silva et al.

Table 2Validation parameters for the analysis of CY enantiomers in plasma.

(S)-(−)-CY (R)-(+)-CY

Linearity (�g/mL) 0.05–25 0.05–25Equation of the line y = 3.05208x + 0.05733 y = 3.15162x + 0.04873r 0.997 0.997Limit of quantification (ng/mL) 50 50Precision (CV %, n = 6) 12.4 5.2Accuracy (% inaccuracy) 5.2 3.2

Intra-assay precision (CV %)50 ng/mL (n = 5) 8.3 5.6150 ng/mL (n = 5) 12.4 12.4

ample enriched with CY and CEPM at concentrations of 1000 and 200 ng of each enY infusion. The retention times were 4.5 min for the (R)-CEPM (1), 4.9 min for the (

or the IS (5).

The matrix effect was absent considering that the coefficient ofariations of the IS normalized matrix factor obtained for each CYnd CEPM enantiomer were less than 15% (Table 1).

The method used for the analysis of CY and CEPM enan-iomers was linear in the range of 0.05–25 �g/mL and5–2500 ng/mL, respectively (correlation coefficients higherhan 0.99) (Tables 2 and 3).

The coefficient of variations calculated in the precision andccuracy assessments (Tables 2 and 3) were less than 15%, demon-trating the reproducibility and repeatability of the results. CY andts metabolite CEPM were stable in human plasma after three suc-essive freeze/thaw cycles, when kept for 3 h at room temperature,nd after 24 h inside the autosampler at 4 ◦C, showing deviationsess than 15% (Table 4). Carryover effects were analyzed by trip-icate injections of blank plasma samples, one before and twommediately after the injection of a sample in the upper limit of

uantification, and the results observed showed no residual effectdata not shown).

Analysis of the kinetic disposition of the CY enantiomersn patients with multiple sclerosis (n = 10) showed plasma

able 1atrix factor evaluated for each CY and CEPM enantiomer.

Concentration IS normalized matrix factor CV (%)

(S)-(−)-CY0.05 �g/mL 14.95.00 �g/mL

(R)-(+)-CY0.05 �g/mL 12.55.00 �g/mL

(R)-CEPM250 ng/mL 9.71000 ng/mL

(S)-CEPM250 ng/mL 8.41000 ng/mL

S: internal standard.

er/mL, respectively; (C) plasma obtained from a patient 16 h after the beginning ofM (2), 6.8 min for the (S)-(−)-(CY) (3), 11.0 min for the (R)-(+)-(CY) (4) and 5.5 min

10 �g/mL (n = 5) 7.8 9.021 �g/mL (n = 5) 12.6 6.9100 �g/mL (1:5) (n = 5) 3.8 3.0

Interassay precision (CV %)50 ng/mL (n = 15) 5.8 4.1150 ng/mL (n = 15) 13.6 12.810 �g/mL (n = 15) 8.0 7.221 �g/mL (n = 15) 7.2 7.5100 �g/mL (1:5) (n = 15) 6.9 6.5

Intra-assay accuracy (% inaccuracy)50 ng/mL (n = 5) 0.5 10.3150 ng/mL (n = 5) 10.0 10.010 �g/mL (n = 5) −8.2 −5.821 �g/mL (n = 5) −1.8 −4.1100 �g/mL (1:5) (n = 5) 6.3 12.0

Interassay accuracy (% inaccuracy)50 ng/mL (n = 15) 6.2 6.4150 ng/mL (n = 15) 5.7 5.010 �g/mL (n = 15) −2.9 −5.221 �g/mL (n = 15) −4.3 −9.4100 �g/mL (1:5) (n = 15) 7.8 −10.2

CV: coefficient of variation [(standard deviation/mean) × 100]; % inaccu-racy = [(Cobs − Cadded)/Cadded] × 100.

18 F.A.d. Castro et al. / J. Chromatogr. B 971 (2014) 14–19

Table 3Confidence limits of the analytical method of CEPM enantiomers in plasma.

(R)-CEPM (S)-CEPM

Linearity (ng/mL) 25–2500 25–2500Equation of the line y = 0.00013x + 0.00073 y = 0.00057x − 0.00184r 0.999 0.999Limit of quantification (ng/mL) 25 25Precision (CV %, n = 6) 3.0 8.9Accuracy (% inaccuracy) −2.4 −2.6

Intra-assay precision (CV %)25 ng/mL (n = 5) 4.8 3.675 ng/mL (n = 5) 4.4 3.5750 ng/mL (n = 5) 5.7 6.32000 ng/mL (n = 5) 7.8 6.83000 ng/mL (1:5) (n = 5) 4.6 5.7

Interassay precision (CV %)25 ng/mL (n = 15) 5.6 4.175 ng/mL (n = 15) 12.4 14.3750 ng/mL (n = 15) 8.0 9.12000 ng/mL (n = 15) 6.5 11.13000 ng/mL (1:5) (n = 15) 5.3 3.5

Intra-assay accuracy (% inaccuracy)25 ng/mL (n = 5) 0.5 −1.475 ng/mL (n = 5) −7.2 −11.0750 ng/mL (n = 5) −9.4 −13.02000 ng/mL (n = 5) −3.1 0.43000 ng/mL (1:5) (n = 5) −6.4 −12.2

Interassay accuracy (% inaccuracy)25 ng/mL (n = 15) −7.2 −6.875 ng/mL (n = 15) −10.2 −10.8750 ng/mL (n = 15) −6.1 −10.92000 ng/mL (n = 15) −4.5 −7.63000 ng/mL (1:5) (n = 15) −7.8 −10.3

CV: coefficient of variation [(standard deviation/mean) × 100]; % inaccu-racy = [(Cobs − Cadded)/Cadded] × 100.

Table 4Stability testing of CY and CEPM enantiomers in human plasma.

Concentration Short-term stability (4 h) Freeze/tha

Precision (CV %) Accuracy (% inacc.) Precision (CV

CY (150 ng/mL)(S)-(−)-CY 11.3 11.2 7.2

(R)-(+)-CY 10.4 10.4 5.3

CY (21 �g/mL)(S)-(−)-CY 5.3 6.2 6.5

(R)-(+)-CY 2.8 3.5 5.4

CEPM (75 ng/mL)(R)-CEPM 6.5 3.7 7.6

(S)-CEPM 7.3 −14.6 4.2

CEPM (2000 ng/mL)(R)-CEPM 6.6 4.3 10.3

(S)-CEPM 7.8 5.8 8.9

CV: coefficient of variation; % inacc.: percent inaccuracy.

Table 5Kinetic disposition of CY enantiomers in patients with multiple sclerosis (n = 10) receivin

Parameter (S)-(−)-CY

Cmax (�g/mL) 46.0 (37.6–57.0)

tmax (h) 1.0 (1.0–1.3)

AUC0–∞ (�g h/mL) 215.0 (189.2–280.4)

AUCS/AUCR

Cl (mL/h kg) 200.7 (168.7–227.8)

MRT (h) 5.9 (5.7–6.8)

Vss (mL/kg) 1337.1 (965.2–1676.1)

Data are expressed as the median (25–75th percentile).Cmax: maximum plasma concentration; tmax: time to reach Cmax; AUC0–∞: area under the pVss: volume of distribuiton (Vss = Cl.MRT).*Wilcoxon test, p < 0.05 [S-(−)-CY vs R-(+)-CEPM].

Fig. 3. Plasma CY and CEPM enantiomer concentration versus time curves after theinfusion of racemic CY (50 mg/kg/day) to patients with multiple sclerosis (n = 10).Data are expressed as the median.

[12] and Fernandes et al. [11] involving patients with lupusnephritis and breast cancer, respectively. No enantioselectivity wasobserved in the kinetic disposition of the CEPM metabolite (Table 6and Fig. 3). The median Cmax values were 1.11 vs 1.11 �g/mLand the median AUC0–∞ values were 13.8 vs 13.9 �g h/mL for (R)-CEPM and (S)-CEPM, respectively. The median ratios of the AUCof CEPM/CY were 0.19 for the (S)-enantiomer and 0.24 for the

(R)-enantiomer. No data on CEPM enantiomers are available inthe literature; however, the AUC of CEPM calculated as the sumof the two enantiomers (27.7 �g.h/mL) agrees with the valuesreported by McCune et al. [7] (39.8–129.5 �g.h/mL), considering the

w cycle stability (3 cycles) Post-processing stability (24 h)

%) Accuracy (% inacc.) Precision (CV %) Accuracy (% inacc.)

−12.8 13.3 13.31.1 8.3 8.3

−12.3 4.2 −2.52.5 8.4 13.0

3.5 4.4 −4.55.3 0.5 −11.0

10.6 6.4 7.09.3 2.7 13.5

g 50 mg/kg/day of intravenous racemic CY.

(R)-(+)-CY

44.8 (36.9–56.9)1.1 (1.0–1.2)

186.2* (141.7–227.6)1.3 (1.2–1.3)

265.7* (204.3–335.1)4.4* (4.2–5.1)

1284.7 (932.4–1978.4)

lasma concentration versus time curve; Cl—clearance; MRT: mean residence time;

F.A.d. Castro et al. / J. Chromatogr. B 971 (2014) 14–19 19

Table 6Kinetic disposition of CEPM enantiomers in patients with multiple sclerosis (n = 10) receiving 50 mg/kg/day of intravenous racemic CY.

Parameter (R)-CEPM (S)-CEPM

Cmax (ng/mL) 1108.14 (635.79–2343.59) 1137.58 (631.3–2200.07)tmax (h) 3.23 (2.26–3.58) 3.25 (2.56–3.52)Kf (h−1) 0.61 (0.53–1.11) 0.60 (0.52–0.87)t1/2f (h) 1.14 (0.62–1.31) 1.15 (0.79–1.32)AUC0–∞ (ng h/mL) 13810.8 (8580.14–24491.96) 13865.23 (8144.89–22770.23)AUCR/AUCS 1.0 (0.9–1.0)Kel (h−1) 0.13 (0.11–0.14) 0.14 (0.11–0.15)t1/2 (h) 5.43 (4.79–5.99) 5.07 (4.70–6.47)

Data are expressed as the median (25–75th percentile).C constt*

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max: maximum plasma concentration; tmax: time to reach Cmax; Kf: formation rateime curve; Kel: elimination rate constant; t1/2: elimination half-life.Wilcoxon test, p < 0.05 [R-CEPM vs S-CEPM].

ifferences in CY doses (50 vs 60 mg/kg) administered to patientss pretransplant conditioning regimen for hematopoietic stem cellransplantation.

. Conclusion

The present study describes for the first time the developmentnd validation of an analytical method of the enantiomers of CYnd its metabolite CEPM. The method was developed to analyze CYnd CEPM enantiomers in human plasma by LC-MS/MS employing

Chiralcel AD-RH column. Using only 25 �L plasma, the methodhowed sufficient sensitivity to quantify CY and CEPM enantiomersfter 24 h of CY infusion (50 mg/kg) in patients with multipleclerosis submitted to pretransplant conditioning for hematopoi-tic stem cell transplantation. The pharmacokinetic parametershowed plasma accumulation of the (S)-(−)-CY enantiomer andack of enantioselective exposure to the CEPM metabolite.

cknowledgments

The authors thank Fundac ão de Amparo a Pesquisa do Estado deão Paulo (FAPESP) for financial support and for granting a researchellowship.

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