Shs

DMa

Rb

a

ARAA

KNILPVS

1

hm[em

PdT

1h

Journal of Chromatography B, 940 (2013) 47– 52

Contents lists available at ScienceDirect

Journal of Chromatography B

j ourna l h om epage: www.elsev ier .com/ locate /chromb

tereoselective analysis of nebivolol isomers in human plasma byigh-performance liquid chromatography–tandem masspectrometry: Application in pharmacokinetics

aniel Valente Nevesa, Carolina Pinto Vieiraa, Eduardo Barbosa Coelhob,aria Paula Marquesa, Vera Lucia Lanchotea,∗

Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo,ibeirão Preto, SP, BrazilDepartamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil

r t i c l e i n f o

rticle history:eceived 17 June 2013ccepted 22 September 2013vailable online 30 September 2013

eywords:ebivolol

somersC–MS/MSharmacokineticsalidationystemic arterial hypertension

a b s t r a c t

Nebivolol is available for clinical use as a racemic mixture. Isomer d-nebivolol (SRRR) is a �1 adrenergicreceptor blocker and its antipode, l-nebivolol (RSSS) is responsible for endothelium-dependent NO liber-ation. This report describes the development and validation of a method of analysis of nebivolol isomersin human plasma by high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS).Nebivolol isomers were extracted from 2 mL aliquots of plasma spiked with tramadol as internal stan-dard, alkalinized and added of sodium chloride and diisopropyl ether:dichloromethane (70:30, v/v).Nebivolol isomers were resolved on a Chirobiotic® V column using methanol:acetic acid:diethylamine(100:0.15:0.05, v/v/v) as mobile phase. Protonated ion and respective ion product were monitored intransitions 406 > 151 for nebivolol and 264 > 58 for internal standard tramadol. There was no racem-ization of nebivolol isomers during the procedures of sample preparation and chromatographic analysisand matrix effect was absent. Analysis of nebivolol isomers showed linearity for plasma concentrations of25–2500 pg/mL of each isomer. The quantification limit was 25 pg of each isomer/mL of plasma. Variationcoefficients and inaccuracy calculated in precision and accuracy determinations were lower than 15%.Nebivolol was stable in human plasma after three successive cycles of freezing and thawing, during 4 hat room temperature and after processing during 12 h in the auto sampler at 5 ◦C showing deviation val-

ues lower than 15%. The method was applied in a study of the kinetic disposition of nebivolol in plasmasamples collected until 48 h after administration of an oral single dose of 10 mg of racemic nebivololhydrochloride to a patient with systemic arterial hypertension. The clinical study demonstrated that thenebivolol pharmacokinetics is stereoselective. Isomer l-nebivolol showed higher AUC0–∞ (9.4 ng/h/mL vs.4.7 ng/h/mL) and smaller apparent clearance (Cl/f) (531.8 L/h vs. 1304.4 L/h) when compared to antipoded-nebivolol.

. Introduction

Nebivolol, a third generation beta blocker with anti-ypertensive activity is available in the clinic as a racemicixture of isomers d-nebivolol [(SRRR)-nebivolol] and l-nebivolol

(RSSS)-nebivolol] [1]. Isomer d- is a potent, selective �1 adren-rgic blocking agent, while isomer l- shows a vasodilator effectediated through l-arginine/endothelial NO [2].

∗ Corresponding author at: Faculdade de Ciências Farmacêuticas de Ribeirãoreto, USP, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Av.o Café, s/n, Campus da USP, Ribeirão Preto 14040-903, SP, Brazil.el.: +55 16 36024699; fax: +55 16 36024725.

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

570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jchromb.2013.09.031

© 2013 Elsevier B.V. All rights reserved.

The enantioselective pharmacokinetics of nebivolol was firstdescribed by Van Peer et al. [3] who employed radioimmunoassayusing enantioselective antibodies [4]. The authors reported iso-meric AUC l/d ratios of about 2 in hypertensive patients orallytreated with the racemic drug. However, enantioselective anti-bodies do not discriminate nebivolol isomers from their aromaticand alicyclic hydroxylated metabolites. In vitro studies show thataffinity of d-isomers of hydroxylated metabolites for the �1-adrenergic receptor is comparable to the one of d-nebivolol [3].Stereospecific radioimmunoassay was also employed by Himmel-mann et al. [5] to determine plasma concentrations of nebivolol

from hypertensive patients treated during 4 weeks with dailydosages p.o. of 5 mg racemic drug. They observed that steady stateplasma concentrations for both isomers (nebivolol plus hydroxy-lated metabolites) were approximately double the ones observed

48 D.V. Neves et al. / J. Chromatogr. B 940 (2013) 47– 52

molec

imbtd

tqicpnOiin

napcwcdh

2

2

f

Fig. 1. Full-scan mass spectra of the protonated

n the administration of the first dose. The stereospecific radioim-unoassay preceded by an extraction procedure was also utilized

y Cheymol et al. [6] to describe the influence of obesity onhe pharmacokinetics of nebivolol in patients treated iv with therug.

High-performance liquid chromatography–tandem mass spec-rometry (LC–MS/MS) was employed by Lindamood et al. [7] touantify nebivolol isomers in the range of 20–1500 pg of each

somer/mL of plasma, but without details of the extraction andhromatographic procedures. Their report only shows a figure withlasma concentrations of the individual isomers while pharmacoki-etic parameters are described as isomeric mixtures of the drug.ther previous studies had already described the use of LC–MS/MS

n the analysis of nebivolol as isomeric mixtures in plasma, employ-ng non chiral columns, in quantification ranges of 10–50 pg ofebivolol/mL of plasma utilizing 200–500 �L of plasma [8–10].

The present study describes the stereoselective analysis ofebivolol in human plasma utilizing a Chirobiotic® V column and

LC–MS/MS detector in quantification limits compatible withharmacokinetic analysis of single dose administration (quantifi-ation limit of 25 pg of each isomer/mL). The validated methodas applied in the determination of nebivolol isomers plasma

oncentrations up to 48 h after the oral administration of a singleose of 10 mg racemic drug to a patient with systemic arterialypertension.

. Materials and methods

.1. Standard solutions and reagents

Racemic nebivolol (nebivolol hydrochloride, 98%) was acquiredrom Toronto Research Chemical (Ontario, Canada). Racemic

ular ion (A) and the ion product (B) of nebivolol.

tramadol hydrochloride was kindly provided by Janssen-Cilag Far-maceutica (São José dos Campos, São Paulo, Brasil). Extractionsolvents, dichloromethane (Philipsburg, NJ, USA) and diisopropylether (Tedia Away, Fairfield, OH, USA) and the mobile phase compo-nents were obtained from Merck (Darmstadt, Germany) and wereof chromatographic grade. Acetic acid (J.T. Baker, Xatoloc, Mexico),diethylamine (J.T. Baker, Phillipsburg, NJ, USA) and sodium hydrox-ide (Synth, Diadema, SP, Brasil) were of P.A. grade. Water utilized toprepare reagents was from Milli-Q® Plus (Millipore Corp. Bedford,MA, USA).

The stock solution of racemic nebivolol was prepared in theconcentration of 100 �g as freebase/mL of methanol. Working solu-tions were prepared by dilution of stock to the final concentrationsof 2, 4, 8, 20, 40, 80 and 200 ng of each nebivolol isomer/mLof methanol. Internal standard (IS) tramadol was prepared as a50 �g/mL solution in methanol. The working solution, 250 ng/mLin methanol, was prepared by appropriate dilution of stock.

2.2. Chromatographic analysis

The high-performance liquid chromatography (HPLC) was con-ducted in a Shimadzu instrument (Kyoto, Japan) equipped witha LC-10 pump and CTO-10 AS oven. Isomers were separatedin a Chirobiotic® V (Sigma–Aldrich) column (250 mm × 4.6 mm,5 �m particles) with a mobile phase of methanol: aceticacid:diethylamine (100%:0.15%:0.05%, v/v/v) in an 1 mL/min fluxat 24 ◦C. The mass spectrometry detection system was a Quattro

Micro LC triple quadrupole (Micromass, Manchester, UK) equippedwith an electrospray interface (ESI). Analysis was conducted in thepositive ionization mode, the capillary voltage in the ESI was 3 kVand the source and desolvation temperatures were 120 and 200 ◦C,

D.V. Neves et al. / J. Chromatogr. B 940 (2013) 47– 52 49

olecul

ro2a

iapwPtfc(

2

wuptattt(ta(

Fig. 2. Full-scan mass spectra of the protonated m

espectively. Nitrogen was the nebulization gas at a release speedf 400 L/h and argon the collision gas at an approximate pressure of.58 × 10−3 mbar. The cone voltage was kept at 40 V for nebivololnd the IS. Collision energy was 30 eV for nebivolol and IS.

Optimization conditions in MS/MS were obtained by directnfusion of standard solutions of nebivolol (20 �g/mL methanol)nd tramadol (50 �g/mL of methanol) incorporated in the mobilehase, through a diffusion pump releasing 10 �L/min. Analysesere conducted in the MRM mode (Multiple Reaction Monitoring).

rotonated ions [M+H]+ and respective ion products were moni-ored in transitions 406 > 151 for nebivolol (see Fig. 1) and 264 > 58or IS tramadol (see Fig. 2). Data acquisition and sample quantifi-ation were made by utilizing the MassLynx program, version 3.5Micromass, Manchester, UK).

.3. Sample preparation

Blank plasma of healthy volunteers (not treated with nebivolol)as provided by the Hemocenter at the University Hospital, Fac-lty of Medicine, University of Sao Paulo, Brazil. Samples wererepared from plasma aliquots (2 mL) containing 25 �L of IS solu-ion, made alkaline by addition of 25 �L of 1 M sodium hydroxide,nd added of 50 mg sodium chloride and 4 mL of the extrac-or solvent diisopropyl ether: dichloromethane (70:30, v/v). Theubes were shaken until vortex formation and kept in a horizon-al shaker (220 ± 10 cycles/min) for 40 min. After centrifugation

10 min, 2500 rpm, 15 ◦C), 3.6 mL of supernatant were transferredo conical tubes and the material concentrated under a flux of airt room temperature. 100 �L aliquots of the resuspended residuesin 130 �L of mobile phase) were submitted to chromatographic

ar ion (A) and the ion product (B) of tramadol (IS).

analysis. Calibration curves were constructed with data from anal-ysis of 2 mL aliquots of blank plasma containing 25 �L of each of theworking nebivolol solutions. Plasma concentrations correspondedto 25, 50, 100, 250, 500, 1000 and 2500 pg of each nebivolol iso-mer/mL. Concentrations in the calibration curves (x) were relatedto ratios of peak areas (y) and equations of linear regression andcorrelation coefficients determined.

2.4. Elution order of nebivolol isomers

Elution order was determined by the individual analysis of thepure d-nebivolol isomer (SRRR) (TRC, Ontario, Canada) using thesame column, mobile phase and flux conditions as described forchromatographic analysis (Section 2.3).

2.5. Determination of the matrix effect

The matrix effect was evaluated by direct comparison of thepeak height of nebivolol isomers and IS, injected directly into themobile phase, with the peak height obtained from standard solu-tions (50 and 2500 pg of each nebivolol isomer/mL of plasma)added to blank plasma extracts from six different volunteers (4normal, 1 hemolyzed and 1 lipemic plasmas). The matrix effect wasevaluated by calculating the internal standard normalized matrixfactor (ISTD) for each nebivolol isomer, as follow: Peak heights of

standard/internal standard added to blank plasma extracts/Peakheights of standard/internal standard injected directly into themobile phase. The variation coefficient obtained for all ISTD valuesobtained should be lower than 15%.

50 D.V. Neves et al. / J. Chromatogr. B 940 (2013) 47– 52

F uman

i ted 1 h(

2

maaeH

2

wflysn2p1t

padla

ig. 3. Chromatographic analysis of nebivolol isomers in plasma. Profiles of (A) hsomer/mL) and IS (3125 pg/mL). (C) Plasma from the hypertensive patient collectramadol), 2 – l-nebivolol and 3 – d-nebivolol.

.6. Racemization tests

Aliquots of blank plasma were enriched with each of the iso-ers d- and l-nebivolol previously collected in a HPLC procedure

s described in Section 2.3 and also with the pure d-nebivololcquired from TRC, North York, Canada. The enriched samples werextracted as described previously and the residues analyzed in thePLC system.

.7. Validation of the method

The analysis of plasma samples containing nebivolol isomersas validated according to recommendations of the USFDA guide

or validation of analytical methods in industry [11]. To determineinearity, calibration curves were constructed with data from anal-sis of 2 mL plasma aliquots containing 25 �L of each of the workingtandard solutions of nebivolol. As such, the concentration of eachebivolol isomer was respectively 25, 50, 100, 250, 500, 1000 and500 pg/mL. The method was considered linear up to the highestlasma concentration showing a deviation equal or smaller than5% in relation to the nominal concentration and a linear correla-ion coefficient equal or higher than 0.98.

The quantification limit was defined as the smallest nebivolollasma concentration analyzed with a 20% precision and an

ccuracy of 80–120%. Samples of plasma containing nebivolol inecreasing concentrations as the ones used to construct the ana-

ytical curve, as low as 25 pg of each isomer/mL of plasma werenalyzed in 10 replicates.

plasma blank, (B) human plasma spiked with racemic nebivolol (2500 pg of each after the administration of a single oral dose of 10 mg racemic nebivolol. 1 – IS

Recovery was evaluated in samples of blank plasma (n = 6),enriched with standard solutions of nebivolol (40, 1000 and2000 pg/mL of each isomer/mL of plasma) submitted to extractionand analysis, by a direct comparison to the peak areas of the drugand the IS added to extracts of blank plasma. Recovery was calcu-lated by comparison of results obtained from extracted samples tothe ones from standard solutions added to extracts of blank plasma,which correspond to 100% recovery.

Precision and accuracy of the analytical method were evaluatedthough intra and inter assay in three concentrations: low qual-ity control (LQC, 40 pg of each nebivolol isomer/mL of plasma);medium quality control (MQC, 1000 pg of each nebivolol isomer/mLof plasma) and high quality control (HQC, 2000 pg of each nebivololisomer/mL plasma). All solutions were divided in aliquots andstored at −70 ◦C until analyzed. Intra-assay precision and accuracywas evaluated by analysis of ten aliquots of these solutions throughonly one analytical curve. For evaluation of inter assay precisionand accuracy, 5 aliquots were analyzed for each nebivolol solutionduring four consecutive assays. Intra and inter assays precision wasevaluated by calculation of the variation coefficient and accuracyexpressed as a ratio between the median concentration experi-mentally determined and the theoretical concentration using thefollowing equation: % inaccuracy = ((median experimental concen-tration − nominal concentration)/nominal concentration) × 100.

The stability of nebivolol in blank plasma samples was evalu-ated by freezing and thawing successive cycles after processing andover short periods. Plasma samples containing nebivolol in low andhigh concentrations (40 and 2000 pg of each isomer/mL of plasma)

D.V. Neves et al. / J. Chromatogr. B 940 (2013) 47– 52 51

Table 1Retention time of l-nebivolol, d-nebivolol and tramadol (IS) determined byLC–MS/MS.

Drug Retention time (min)

Tramadol (IS) 10.20

waweewtot

2

2

mP6taoBsa4t(

3

pCcmn

4

amptp

b(Lrb

raaeim

Table 2Matrix effect of each nebivolol isomer.

Matrix effect l-nebivolol d-nebivolol

CV % 14.08 14.12

CV = coefficient of variation [(standard deviation/mean) × 100] of peak heights of

show as the limit of detection 1 ng of each isomer/mL of plasma[5] or 0.1 ng/mL of plasma [6]. Methods describing analysis ofnebivolol in plasma as isomeric mixture by LC–MS/MS report limits

Table 3Validation parameters of the method of analysis of nebivolol isomers in plasma.

l-Nebivolol d-Nebivolol

Absolute recovery (%)40 pg/mL 94.58 99.221000 pg/mL 97.26 96.682000 pg/mL 94.78 93.99

Linearity (pg/mL)Correlation coefficient (r) 0.997 0.998Linear equation y = 0.0002x + 0.001278 y = 0.0001x + 0.001149Range (pg/mL) 25–2500 25–2500

Limit of quantificationConcentration (pg/mL) 25 25Precision (CV %, n = 10) 14.50 14.92Accuracy (inaccuracy %) 2.41 1.46

Inter-assay precision (CV %)40 pg/mL (n = 5) 8.73 12.061000 pg/mL (n = 5) 7.94 8.812000 pg/mL (n = 5) 7.63 4.62

Intra-assay precision (CV %)40 pg/mL (n = 10) 10.91 5.061000 pg/mL (n = 10) 12.16 8.742000 pg/mL (n = 10) 4.03 5.71

Inter-assay accuracy (% inaccuracy)40 pg/mL (n = 5) 5.83 0.261000 pg/mL (n = 5) −2.54 −1.542000 pg/mL (n = 5) −11.23 −8.17

Intra-assay accuracy (% inaccuracy)40 pg/mL (n = 10) −2.20 −5.08

l-Nebivolol 13.79d-Nebivolol 14.90

ere submitted to three cycles of freezing and thawing (−70 ◦Cnd room temperature) for at least 24 h in each phase and after-ards extracted and analyzed. To evaluate after processing stability

xtracted samples were kept for 12 h in the auto sampler of thequipment before being chromatographed. Short period stabilityas determined by keeping the enriched plasma samples at room

emperature for 4 h before analysis. Results were compared to thenes obtained after analysis of samples immediately after prepara-ion and the differences expressed as deviation percentages.

.8. Clinical protocol

.8.1. Case reportThe clinical protocol was approved by the Research Ethical Com-

ittee of the University Hospital, Faculty of Medicine of Ribeiraoreto, University of Sao Paulo. The patient, 31 years old, weighing7.7 kg, 5 ft 6 in. tall had a diagnostic of systemic arterial hyper-ension and after being briefed about details of the study, durationnd possible risks signed the Term of Informed Consent. After a fastf 12 h, the patient received 10 mg of racemic nebivolol (Nebilet®,iolab, Brazil) in a single oral dose with 200 mL of water. Hospitaltandard food was served 3 h later. Blood samples (10 mL) in hep-rinized syringes were collected at times: 0, 0.5, 1, 1.5, 2, 2.5, 3,, 6, 8, 10, 12, 16, 20, 24, 30, 36, 42 and 48 h after drug adminis-ration. Plasma obtained after centrifugation of the blood samples2500 rpm for 15 min) was stored at −70 ◦C until analyzed.

. Pharmacokinetics of nebivolol

Pharmacokinetics analysis was conducted with the WinNonlinrogram, version 4.0 (Pharsight Corp, Mountain View, CA, USA).alculation of pharmacokinetic parameters was based on plasmaoncentrations determined experimentally. The bi-compartmentalodel was used to analyze data relating plasma concentrations of

ebivolol isomers vs. time.

. Results and discussion

This report describes a method developed and validated fornalysis of nebivolol isomers in human plasma by LC–MS/MS. Theethod, sensitive to drug concentrations as low as 25 pg/mL of

lasma was applied in pharmacokinetic studies after administra-ion of a single oral dose of the racemic drug to a hypertensiveatient.

Nebivolol isomers were separated in a Chirobiotic® V columny a mobile phase consisting of methanol:acetic acid:diethylamine100:0.15:0.05, v/v/v). Isomers l- and d- were eluted from theC–MS/MS system with retention times of 13.79 and 14.90 min,espectively (see Fig. 3 and Table 1). Elution order was determinedy the use of the pure d-isomer.

The first reported separation of nebivolol isomers using chi-al phase columns was reported by Aboul-Enein and Ali [12]. Theuthors employed amylose carbamate columns (Chiralpak® AD

nd Chiralpak® AD-RH) and mobile phases consisting of methanol,thanol, 1-propanol, 2-propanol and 1-butanol. Elution of isomersn the sequence d- and l- were observed with mobile phases

ethanol, ethanol and 1-propanol in Chiralpak®AD columns or

standard/internal standard added to blank plasma extracts/peak heights of stan-dard/internal standard injected directly into the mobile phase for all ISTD values.

with all mobile phases in Chiralpak® AD-RH columns. However,the elution order was inverted by employing Chiralpak®AD columnwith mobile phases 2-propanol and 1-butanol.

In this study nebivolol isomers were analyzed in the LC–MS/MSsystem monitoring transitions 406 > 151 for nebivolol and 264 > 58for tramadol the internal standard. The data confirm resultsreported by Ramakrishna et al. [8] and Selvan et al. [9] employ-ing the same method to analyze racemic mixtures of nebivolol inhuman plasma.

Matrix effect was absent considering that the CV value obtainedfor all ISTD evaluated for each nebivolol isomer was lower than 15%(Table 2). The results also indicate that there is no racemization ofnebivolol isomers during the procedures of sample preparation andchromatographic analysis.

Analysis of nebivolol isomers by the method proposed showedlinearity for plasma concentrations of 25–2500 pg/mL of eachisomer (correlation coefficients above 0.99) (Table 3). The quantifi-cation lower limit (QL) for both isomers was 25 pg/mL of plasmawhen 2 mL plasma aliquots were extracted (Table 3). The QLallowed the analysis of nebivolol isomers up to 48 h after admin-istration of a single dose of 10 mg of racemic drug in all patientsinvestigated. Methods employing radioimmunoassay that do notdifferentiate the parent drug from the hydroxylated metabolites

1000 pg/mL (n = 10) −3.46 0.082000 pg/mL (n = 10) −11.08 −3.80

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

52 D.V. Neves et al. / J. Chromato

Table 4Stability of nebivolol isomers in human plasma.

Deviation (%)a l-Nebivolol d-Nebivolol

Short term (4 h)40 pg/mL 2.96 −10.132000 pg/mL 5.76 −5.34

Cycles freeze/thaw (−70 ◦C)40 pg/mL −14.17 −6.402000 pg/mL −14.81 −13.82

Post processing (12 h)40 pg/mL 4.28 0.982000 pg/mL −13.25 −14.44

a % deviation in relation to recently prepared samples.

Table 5Kinetic disposition of nebivolol isomers in plasma of a hypertensive patient treatedwith a single 10 mg oral dose of the racemic drug.

l-Nebivolol d-nebivolol

Cmax (ng/mL) 2.5 1.2tmax (h) 0.2 0.4AUC0–∞ (ng h/mL) 9.4 4.7t1/2 (h) 14.5 13.8˛ (h−1) 0.7 0.8ˇ (h−1) 0.05 0.05Vd/f (L) 5463.3 10,755.9Vd/f (L/kg) 80.7 158.9Cl/f (L/h) 531.8 1304.4AUC0–∞ l/d 2.0

Fp

o1

drhi1t

[

ig. 4. Plasma nebivolol isomers concentrations vs. time curves in a hypertensiveatient treated with a single oral dose of 10 mg racemic nebivolol.

f detection of 10 pg/mL of plasma and a quantification limit of0 pg/mL of plasma [9].

Variation coefficients calculated in precision and accuracyeterminations (Table 3) were lower than 15%, an assurance ofeproducibility and repeatability of results. Nebivolol was stable in

uman plasma after three successive cycles of freezing and thaw-

ng, during 4 h at room temperature and after processing during2 h in the auto sampler at 5 ◦C showing deviation values lowerhan 15% (Table 4).

[

[

gr. B 940 (2013) 47– 52

Recovery of nebivolol isomers from human plasma by alkalineextraction with diisopropyl ether:dichloromethane (70:30, v/v)was above 90% for both isomers. Results reported by Ramakrishnaet al. [8] in the same conditions are lower, 73.4%, while Selvan et al.[9] obtained a recovery close to 85% by utilizing protein precipita-tion with acetonitrile.

The kinetic disposition of nebivolol isomers determined in thehypertensive patient treated with a single oral dose of 10 mg ofracemic drug was shown to be stereoselective whit a plasma accu-mulation of the l-isomer (Table 5 and Fig. 4). The values of Cmax

were 2.48 ng/mL vs. 1.16 ng/mL and of AUC0→∞ 9.4 ng h/mL vs.4.65 ng h/mL respectively for l- and d-isomers and the AUC ratio l-nebivolol/d-nebivolol was 2.02. Lindamood et al. [7] only reportedvalues of Cmax, approximately 1.3 ng/mL for the l-isomer and 0.7for the d-isomer in healthy volunteers utilizing 10 mg of racemicnebivolol by the oral route.

5. Conclusion

The method of analysis of nebivolol isomers in human plasmaby the LC–MS/MS system employing a chiral Chirobiotic® V columnis sensitive (quantification limit, 25 pg/mL), precise, accurate (vari-ation coefficients and relative errors <15%) and permits nebivololisomers quantification until 48 h after administration of a singleoral dose of 10 mg of the racemic drug to a patient with systemicarterial hypertension.

Nebivolol pharmacokinetics evaluated in the hypertensivepatient was shown to be stereoselective and a plasma accumulationof isomer l-nebivolol when compared to the d-isomer.

Acknowledgments

The authors are grateful to Coordenac ão de Aperfeic oamentode Pessoal de Nivel Superior (CAPES) for financial support and thegranting of a research fellowship.

References

[1] A. Van de Water, R. Xhonneux, R.S. Reneman, P.A. Janssen, Eur. J. Pharmacol.156 (1988) 95.

[2] S. Evangelista, U. Garbin, A.F. Pasini, C. Stranieri, V. Boccioletti, L. Cominacini,Pharmacol. Res. 55 (2007) 303.

[3] A. Van Peer, E. Snoeck, R. Woestenborghs, V. Van de Velde, G. Mannens, W.Meuldermans, J. Heykants, Drug Invest. 3 (1991) 25.

[4] R. Woestenborghs, L. Embrechts, J. Heykants, in: Reid, et al. (Eds.), Method-ological Surveys in Biochemistry and Analysis, Plenum Press, New York, 1994,p. 215.

[5] A. Himmelmann, T. Hedner, E. Snoeck, B. Lundgren, J. Hedner, Eur. J. Clin. Phar-macol. 51 (1996) 259.

[6] G. Cheymol, R. Woestenborghs, E. Snoeck, R. Ianucci, J.P. Le Moing, L. Naditch,J.C. Levron, J.M. Poirier, Eur. J. Clin. Pharmacol. 51 (1997) 493.

[7] C. Lindamood, S. Ortiz, A. Shaw, R. Rackley, J.C. Gorski, J. Clin. Pharmacol. 51(2011) 575.

[8] N.V.S. Ramakrishna, K.N. Vishwottam, M. Koteshwara, S. Manoj, M. Santosh,D.P. Varma, J. Pharm. Biomed. Anal. 39 (2005) 1006.

[9] P.S. Selvan, K.V. Gowda, U. Mandal, W.D.S. Solomon, T.K. Pal, J. Chromatogr. BAnalyt. Technol. Biomed. Life Sci. 858 (2007) 143.

10] J. Nandania, S.J. Rajput, P. Contractor, P. Vasava, B. Solanki, M. Vohra, J. Chro-matogr. B: Analyt. Technol. Biomed. Life Sci. 923/924 (2013) 110.

11] Guidance for Industry Bioanalytical Method Validation, US Department ofHealth and Human Services, Food and Drug Administration, Center for DrugEvaluation and Research, Rockville, 2001 http://www.fda.gov/cder/guidance(accessed 15.12.12).

12] H.Y. Aboul-Enein, I. Ali, J. Biochem. Biophys. Methods 48 (2001) 175.