G Model ARTICLE IN PRESS PBA-11061; No. of Pages 6 Journal of Pharmaceutical and Biomedical Analysis xxx (2017) xxx–xxx Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba An indirect stereoselective analysis of nebivolol glucuronides in plasma by LC–MS/MS: Application to clinical pharmacokinetics Carolina Pinto Vieira a , Daniel Valente Neves a , Evandro José Cesarino a , Adriana Rocha a , Séverine Poirier b , Vera Lucia Lanchote a,∗ a Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil b Spécialité Conception & Développement, École de Biologie Industrielle, Cergy-Pontoise, France a r t i c l e i n f o Article history: Received 7 October 2016 Received in revised form 12 January 2017 Accepted 29 January 2017 Available online xxx Keywords: Nebivolol Isomers Metabolism Pharmacokinetics Patient a b s t r a c t Nebivolol is a racemate of the d-isomer responsible for ␤1 adrenergic receptor antagonism and the l-isomer responsible for the release of nitric oxide from endothelial cells. Nebivolol is mainly metabolized to nebivolol glucuronide, which also contribute to the nebivolol ␤1 adrenoreceptor antagonism. This study reports the development and validation of an indirect stereoselective method of analysis of nebivolol glucuronides in plasma by LC–MS/MS. The method was applied to the investigation of stereoselectivity in the glucuronidation of nebivolol in elderly hypertensive patients (n = 11) CYP2D6 phenotyped as EM and treated with a single oral dose of the racemate. One-milliliter plasma aliquots spiked with internal standard (S)-(−)-metoprolol were incubated with 25 ␮L of ␤-glucuronidase (final concentration 2500 unit/mL) at pH 5.0 for 16 h at 37 ◦ C. Linearity for total nebivolol was 0.2–125 ng of each isomer per mL plasma and permitted analysis of nebivolol glucuronide isomers up to 48 h after administration of a single oral dose of 10 mg racemate. Regarding to the nebivolol glucuronide isomers, higher plasma concentrations of the d-isomer were observed compared to the l-isomer (d/l AUC = 5.4), explaining at least in part the plasma accumulation of unchanged l-nebivolol (l/d AUC = 1.8). This study also showed metabolic glucuronide nebivolol/unchanged nebivolol ratios of approximately 6.5 for the l-isomer (AUC 65.3 vs 10.1 ng h/mL) and approximately 62.1 (335.2 vs 5.4 ng h/mL) for the d-isomer. Considering that d-nebivolol glucuronide also contributes for ␤1 adrenergic receptor antagonism, future studies regarding PK-PD of nebivolol should evaluate not only plasma concentrations of unchanged nebivolol isomers but also glucuronide nebivolol isomers. © 2017 Published by Elsevier B.V. 1. Introduction Nebivolol, a drug with four chiral centers, is available for clinical use as a racemate of the d-nebivolol (SRRR) and l-nebivolol (RSSS) isomers [1,2]. Nebivolol is used to treat systemic arterial hypertension and congestive heart failure. Nebivolol is a third-generation and highly selective ␤1 adrenoreceptor antagonist with vasodilatoy effects mediated by nitric oxide via ␤3 receptor agonism [3]. The d-isomer is a highly selective ␤1 adrenoreceptor antagonist, while l-nebivolol promotes the release of nitric oxide from endothelial cells [4–6]. ∗ Corresponding author at: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto – USP, Avenida do Café s.n., Campus da USP, 14040-903 Ribeirão Preto, SP, Brazil. E-mail address: lanchote@fcfrp.usp.br (V.L. Lanchote). Nebivolol is mainly metabolized by direct glucuronidation and secondarily by aromatic hydroxylation and N-dealkylation. The major metabolites found in plasma are glucuronides in addition to the oxidative N-dealkylated acid. N-dealkylation and hydroxylation depend on the activity of CYP2D6, an enzyme that exhibits genetic polymorphism. Some oxidative hydroxylated conjugates are only found in the plasma of extensive metabolizers (EM). In poor metabolizers (PM), the glucuronidation of unchanged nebivolol is considered a primary metabolic pathway. The hydroxylated and glucuronide metabolites also contribute to the nebivolol ␤1 adrenoreceptor antagonism [3,6–8]. Previous clinical trials assessing the nebivolol metabolism both as monotherapy or in combination with CYP2D6 inhibitors have been conducted evaluating only its hydroxylated metabolite, 4-hydroxynebivolol. The plasma concentrations of 4-hydroxynebivolol evaluated as isomeric mixture are lower than those of unchanged nebivolol, with nebivolol/4-hydroxynebivolol http://dx.doi.org/10.1016/j.jpba.2017.01.054 0731-7085/© 2017 Published by Elsevier B.V. Please cite this article in press as: C.P. Vieira, et al., An indirect stereoselective analysis of nebivolol glucuronides in plasma by LC–MS/MS: Application to clinical pharmacokinetics, J. Pharm. Biomed. Anal. (2017), http://dx.doi.org/10.1016/j.jpba.2017.01.054 G Model ARTICLE IN PRESS PBA-11061; No. of Pages 6 C.P. Vieira et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2017) xxx–xxx 2 metabolic ratios close to 2. Treatment of healthy volunteers with CYP2D6 inhibitors (paroxetine or bupropion) for 7 days results in an approximately six-fold increase in the area under the plasma concentration versus time curve (AUC) for both the unchanged drug and its 4-hydroxynebivolol metabolite [9–11]. A previous study [12] described the development and validation of an analytical method for measuring unchanged nebivolol isomers in human plasma by LC–MS/MS coupled to a chiral ® Chirobiotic V stationary-phase column using a mobile phase of methanol:acetic acid:diethylamine (100:0.15:0.05, v:v:v). In the present study, we describe for the first time an indirect method for the analysis of nebivolol glucuronide isomers in plasma by LC–MS/MS. The nebivolol glucuronide isomers concentrations were calculated as the difference between total nebivolol concentrations and unchanged nebivolol concentrations for each isomer. For the determination of the total nebivolol concentration plasma samples were treated with ␤-glucuronidase before nebivolol quantitation while unchanged nebivolol was directly quantified. The indirect analytical method for measuring nebivolol glucuronide isomers in plasma was applied to the investigation of stereoselectivity in the metabolism of nebivolol in hypertensive elderly patients treated with a single oral dose of the racemic nebivolol. 2. Experimental 2.1. Analysis of total nebivolol isomers 2.1.1. Chemicals Racemic nebivolol (98% nebivolol hydrochloride) was obtained from Toronto Research Chemicals (Ontario, Canada). (S)-(−)metoprolol, used as internal standard (IS), was kindly provided from AstraZeneca (Mölndal, Sweden). HPLC-grade LiChrosolv methanol was purchased from Merck (Darmstadt, Germany). Diethylamine and sodium acetate were obtained from J.T. Baker (Phillipsburg, EUA). ␤-Glucuronidase (type HP-2 from Helix pomatia, glucuronidase activity ≥ 100,000 units per mL and sulfatase activity ≤7500 units per mL) was purchased from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade. Water was obtained from Milli-Q system (Millipore, Bedford, MA, USA). 2.1.2. Calibration and quality control samples The stock solution of racemic nebivolol was prepared at a concentration of 100 ␮g of the free base per mL methanol. This solution was diluted to obtain working solutions of 8, 20, 80, 200, 400, 1000 and 5000 ng of each nebivolol isomer per mL methanol. One milliliter of blank human plasma samples were spiked with 25 ␮L of each working solution to obtain nebivolol plasma concentrations of 0.2, 0.5, 2, 5, 10, 25 and 125 ng of each nebivolol isomer per mL plasma. Quality control (QC) samples were prepared separately, at five different plasma concentrations (0.2, 0.5, 50, 100 and 5000 ng of each nebivolol isomer per mL, respectively as lower limit of quantification (LLOQ), low (LQC), medium (MQC), high (HQC) and dilution (DQC). Calibration curves included a blank sample (a blank plasma sample processed without the IS), a zero sample (a blank plasma sample processed with the IS) and non-zero samples (0.2, 0.5, 2, 5, 10, 25 and 125 ng per mL). The calibration curves were constructed by plotting the peak area ratios (each nebivolol isomer/IS) versus corresponding concentrations by weighted (1/x2 ) least-squares linear regression. 2.1.3. Enzymatic hydrolysis and sample preparation Aliquots of blank human plasma (1 mL) were spiked with 25 ␮L of (S)-(−)-metoprolol solution (internal standard, 300 ng per mL in methanol), 500 ␮L of sodium acetate buffer 0.75 M, pH 5.0, and 25 ␮L of ␤-glucuronidase (final concentration of 2500 units per mL). The tubes were vortexed for 30 s and the samples were kept in a water bath at 37 ◦ C for 16 h. After enzymatic hydrolysis, the samples were spiked with 200 ␮L of a 1 M NaOH solution, 50 mg of NaCl and 4 mL of diisopropyl:dichloromethane (70:30, v:v). The tubes were kept in a horizontal shaker (MA 139/CFT, Marconi, Piracicaba, São Paulo, Brazil) for 40 min. After centrifugation at 2500 rpm for 10 min at 15 ◦ C (Himac CF 8DL, Hitachi, Tokyo, Japan), 3.6 mL of the supernatants were transferred to conical tubes and evaporated to dryness in a vacuum evaporation system (Christ RVC 2-25 CD and Christ CT 04-50 SR, Osterode am Harz, Germany). The residues were resuspended in 200 ␮L of the mobile phase and 40 ␮L were used for chromatographic analysis. 2.1.4. LC–MS/MS instrument and conditions ® The nebivolol isomers were separated on a Chirobiotic V column (particle size of 5 ␮m, 250 mm × 4.6 mm, Astec, Whippany, NJ, USA) maintained at 24 ◦ C. The mobile phase consisted of 95% of a mixture of methanol:acetic acid:diethylamine (100:0.15:0.05, v:v:v) and 5% of a mixture of water:acetic acid:diethylamine (100:0.15:0.05, v:v:v) eluted at a flow rate of 0.8 mL/min. The mass spectrometry detection system was a Quattro Micro LC triple quadrupole spectrometer (Micromass, Manchester, United Kingdom) equipped with an electrospray interface (ESI). The analyses were carried out in the positive ion mode. The capillary voltage in ESI was 3 kV. The source and desolvation temperatures were maintained at 150 and 550 ◦ C, respectively. Nitrogen was used as the nebulization gas at a flow rate of 150 L/h. Argon was used as the collision gas at a pressure of approximately 3 × 10−4 mbar. The cone voltage was maintained at 40 V for nebivolol and at 25 V for the internal standard (S)-(−)-metoprolol. The collision energy was 30 eV for nebivolol and 15 eV for the internal standard (S)-(−)metoprolol. The optimal conditions of tandem mass spectrometry (MS/MS) were determined by direct infusion of the standard solutions of nebivolol (170 ng per mL methanol) and (S)-(−)-metoprolol (internal standard, 250 ng per mL methanol) prepared in the mobile phase and injected with an infusion pump at a flow rate of 10 ␮L/min. The analysis was carried out in the multiple reaction monitoring (MRM) mode. The protonated ions [M+H]+ and their respective product ions were monitored at transitions of 406 > 151 for nebivolol and of 268 > 116 for (S)-(−)-metoprolol. The elution order and racemization test of the nebivolol isomers had been determined in a previous study [12]. 2.2. Analysis of unchanged nebivolol isomers The analysis of unchanged nebivolol isomers in plasma using LC–MS/MS was conducted as previously described [12] with some modifications. Briefly, plasma aliquots of 500 ␮L were spiked with 25 ␮L of (S)-(−)-metoprolol solution (internal standard, 600 pg per mL in methanol), 25 ␮L of an aqueous solution of 1 M NaOH, 50 mg of NaCl and 4 mL of diisopropyl:dichloromethane (70:30, v:v). The plasma samples were extracted according to Section 2.1.3 and the nebivolol isomers were separated on a chiral column as previously described in Section 2.1.4. ® The analyses were carried out on an ACQUITY UPLC System coupled to a Xevo TQ-S Triple Quadrupole mass spectrometer (Waters, Milford, MA, USA) equipped with an electrospray ionization interface (ESI). Analysis was performed in the positive ion mode. The capillary voltage in ESI was 2 kV. The source and desolvation temperatures were maintained at 150 and 550 ◦ C, respectively. Nitrogen was used as the nebulization gas at a flow rate of 150 L/h. Argon was used as the collision gas at a pressure of approximately 7 × 10−4 mbar. The cone voltage and collision Please cite this article in press as: C.P. Vieira, et al., An indirect stereoselective analysis of nebivolol glucuronides in plasma by LC–MS/MS: Application to clinical pharmacokinetics, J. Pharm. Biomed. Anal. (2017), http://dx.doi.org/10.1016/j.jpba.2017.01.054 G Model PBA-11061; No. of Pages 6 ARTICLE IN PRESS C.P. Vieira et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2017) xxx–xxx 3 energy were maintained at 25 V and 15 eV, respectively, for nebivolol and internal standard (S)-(−)-metoprolol. 2.3. Validation of the methods The methods for analysis of total nebivolol isomers and unchanged nebivolol isomers in human plasma were validated according to the USFDA guidelines for validation of bioanalytical methods. The calibration curves were constructed from 0.2 to 125 ng of each isomer per mL plasma for total nebivolol and from 15 to 3000 pg of each isomer per mL plasma for unchanged nebivolol. The methods were validated based on matrix effect, precision, accuracy, sensitivity and linearity. 2.4. Method application 2.4.1. Clinical protocol The clinical protocol was approved by the Research Ethical Committee of the School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo. After signing the free informed consent form, a total of eleven elderly patients with arterial hypertension were investigated. After an 8-h fast, the patients received two ® tablets of 5 mg racemic nebivolol (Nebilet , Biolab) with 200 mL water in the morning. The standard diet of the hospital was served 2 h after drug administration. Blood samples (5 mL) were collected ® with heparinized syringes (5000 IU Liquemine , Roche) at times 0, 0.5, 1, 1,5, 2, 2,5, 3, 4, 6, 8, 10, 12, 24, 30, 36, and 48 h. Plasma was separated by centrifugation (1.800 × g, 20 min) of the blood samples and stored at −80 ◦ C until the time of analysis. After 48 h nebivolol administration, all patients received a single oral dose of 100 mg metoprolol tartrate as a probe of CYP2D6 phenotype. All urine was collected from zero to 8 h after metoprolol administration. The total volume of urine was measured and a sample of 10 mL was kept at −80 ◦ C until the time of analysis. 2.4.2. Pharmacokinetics and statistical analyses The pharmacokinetic parameters of the unchanged nebivolol isomers and their glucuronide metabolites were calculated using a two-compartmental model with lag time (Phoenix WinNonlin 6.4; Pharsight Corp, Mountain View, CA, USA). Statistical analysis ® was performed using the R software for the calculation of medians, means and 95% confidence intervals. The Wilcoxon test for paired data was used to evaluate isomer ratios different from unity for unchanged and glucuronide nebivolol. The level of significance was set at 5%. 3. Results and discussion 3.1. Method development The present study is the first to validate an indirect method for the analysis of nebivolol glucuronide isomers in human plasma by LC–MS/MS. A previous study regarding plasma analysis of total nebivolol did not describe sample preparation and method validation [8]. ® The nebivolol isomers were separated on a Chirobiotic V column using a mobile phase consisting of 95% of a mixture of methanol:acetic acid:diethylamine (100:0.15:0.05, v:v:v) and 5% of a mixture of water:acetic acid:diethylamine (100:0.15:0.05, v:v:v). Fig. 1 illustrates the chromatograms of the nebivolol isomers in plasma after hydrolysis with ␤-glucuronidase. The chromatographic run lasted less less than 12 min. The elution order was determined by analysis of pure d-nebivolol carried out in a previous study [12]. A higher plasma concentration of total d-nebivolol was observed, while analysis of the nebivolol isomers not preceded Fig. 1. Chromatographic analysis of total nebivolol isomers in plasma. Profiles of (A) human blank plasma (B) human blank plasma spiked with racemate nebivolol (25 ng of each isomer/mL); (C) human blank plasma spiked with (S)-(−)-metoprolol; (D) Plasma sample from an elderly hypertensive patient collected 1 h after the administration of a single oral dose of 10 mg racemate; (E) Plasma sample from the elderly hypertensive patient spiked with (S)-(−)-metoprolol). 1 – (S)-(−)-metoprolol (internal standard), 2 – l-nebivolol and 3 – d-nebivolol. by enzymatic hydrolysis resulted in higher plasma concentrations of unchanged l-nebivolol (data not shown). Development of the analytical method for total nebivolol isomers preceded by enzymatic hydrolysis required evaluation of the ␤-glucuronidase activity and of the time of enzymatic hydrolysis using plasma samples collected at Cmax (maximum plasma concentrations) from patients treated with 10 mg racemic nebivolol. The data in Fig. 2 show that an activity of 2500 unit per mL of ␤glucuronidase is sufficient to hydrolyze plasma aliquots of 1 mL. Hydrolysis for 16 h at 37 ◦ C (Fig. 3) resulted in the highest total concentration of both nebivolol isomers. Please cite this article in press as: C.P. Vieira, et al., An indirect stereoselective analysis of nebivolol glucuronides in plasma by LC–MS/MS: Application to clinical pharmacokinetics, J. Pharm. Biomed. Anal. (2017), http://dx.doi.org/10.1016/j.jpba.2017.01.054 G Model ARTICLE IN PRESS PBA-11061; No. of Pages 6 C.P. Vieira et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2017) xxx–xxx 4 Table 1 Validation parameters of the method of analysis of unchanged nebivolol isomers in plasma. 20 Concentration (ng/mL) 18 16 14 12 d-nebivolol 10 l-nebivolol 8 6 4 2 0 0 2000 4000 6000 Units/mL 8000 10000 12000 Fig. 2. Enzymatic hydrolysis procedure for the analysis of total nebivolol in plasma: assessment of activity of ␤-glucuronidase. Selectivity was evaluated using blank plasma samples obtained from eight different volunteers, including 4 normal samples, 2 lipemic samples and 2 hemolyzed samples. The chromatograms compared to the LLOQC standard showed no peak eluted at the same retention times of nebivolol isomers or internal standard (S)(−)-metoprolol. The residual effect was analyzed by injecting blank plasma samples before and after the HQC standard. The chromatograms of the blank plasma sample followed by the HQC standard were compared to the LLOQC standard. No residual effect of previous injections was observed in the present study. The matrix effect was evaluated using 8 plasma samples, including 4 normal samples, 2 lipemic samples and 2 hemolyzed samples. The plasma extracts obtained were spiked with (S)-(−)metoprolol (internal standard) and with the nebivolol isomers at concentrations corresponding to the LQC and HQC. The internal standard-normalized matrix factor (MF = area of matrix/area of IS in matrix/area of the analyte in solution/area of IS in solution) was evaluated for each sample. The results expressed as the coefficient of variation (CV) of all MFs obtained values show the absence of a matrix effect (CV of MF values <15%; Tables 1 and 2). Validation parameters of the analytical methods for nebivolol isomers preceded (total nebivolol) or not (unchanged nebivolol) by enzymatic hydrolysis are presented, respectively, in Tables 1 and 2. The methods were linear over the concentration range of 0.2–125 ng of each isomer per mL plasma for total nebivolol and of 15–3000 pg of each isomer per mL plasma for unchanged nebivolol. The quantification limits were defined as the lowest concentrations analyzed with a 20% precision and an accuracy of 80–120%. In comparison to the previously published method [12], in the present work, smaller aliquots of plasma (500 ␮L) were used and a lower limit of quantification (15 pg of each isomer per mL plasma; Table 1) Concentration (ng/mL) 20 18 16 14 12 10 l-nebivolol 8 d-nebivolol 6 4 l-Nebivolol d-Nebivolol MF (CV%) Linearity (pg/mL) Linear equation Correlation coefficient 11.19 15–3000 y = 0.0067x + 0.00388 r = 0.995 13.37 15–3000 y = 0.0082x + 0.01121 r = 0.998 Limit of quantification (pg/mL) Precision (CV % n = 10) Accuracy (inaccuracy %) 15 10.22 2.84 15 8.24 −6.27 Inter-assay precision (CV %) 15 pg/mL (LLOQC – n = 15) 45 pg/mL (LQC – n = 15) 1500 pg/mL (MQC – n = 15) 2400 pg/mL (HQC – n = 15) 5000 pg/mL (DQC – n = 15) (1:2) 10.00 5.44 1.84 2.87 9.98 4.69 6.51 2.06 3.81 3.62 Intra-assay precision (CV %) 15 pg/mL (LLOQC – n = 5) 45 pg/mL (LQC – n = 5) 1500 pg/mL (MQC – n = 5) 2400 pg/mL (HQC – n = 5) 5000 pg/mL (DQC – n = 5) (1:2) 2.84 8.47 6.99 7.37 6.79 2.53 9.78 8.08 7.13 3.58 Inter-assay accuracy (inaccuracy %) 6.80 15 pg/mL (LLOQC – n = 15) 45 pg/mL (LQC – n = 15) −4.82 1500 pg/mL (MQC – n = 15) −8.38 −0.30 2400 pg/mL (HQC – n = 15) 5000 pg/mL (DQC – n = 15) (1:2) 6.21 −2.53 −5.24 −7.51 0.32 −3.41 Intra-assay accuracy (inaccuracy %) 15 pg/mL (LLOQC – n = 5) 0.80 45 pg/mL (LQC – n = 5) −7.33 1500 pg/mL (MQC – n = 5) −10.76 −0.30 2400 pg/mL (HQC – n = 5) 7.65 5000 pg/mL (DQC – n = 5) (1:2) −2.53 −5.24 −6.70 0.32 −3.68 MF = normalized matrix factor; CV = coefficient of variation. LLOQC, LQC, MQC, HQC and DQC are the quality controls of lower limit of quantification, low, medium, high and dilution concentrations. was achieved. Regarding total nebivolol isomers extracted from plasma samples of 1 mL we observed a LLOQ of 0.2 ng of each isomer per mL plasma. A previous study regarding plasma analysis of total nebivolol isomers reported a limit of quantification of 1 ng of each isomer per mL plasma [8]. The intra-assay precision and accuracy were determined by analyzing 5 aliquots of each QC using a single calibration curve. For the evaluation of inter-assay precision and accuracy, 5 aliquots of each QC were analyzed in 3 different analytical runs. The intraand inter-assay precision and accuracy, expressed as the CV of the results and presented in Tables 1 and 2 show that both methods are precise (CV < 15%) and accurate (relative standard error < 15%). Stability testing was performed in the previous study [12] and revealed CVs less than 15% for the analysis of stability after three freeze-thaw cycles (−70 ◦ C and room temperature), short-term stability (4 h at room temperature), and post-processing stability (12 h in the autoinjector at 4 ◦ C). In the present work, the total nebivolol isomers were extracted from plasma aliquots of 1 mL and we obtained a limit of quantification of 0.2 ng of each isomer per mL plasma. The method is precise, accurate and sensitive, permitting their quantification up to 48 h after the administration of a single oral dose of 10 mg of the racemic nebivolol. 2 3.2. Method application 0 0 5 10 15 20 25 Hydrolysis time (h) Fig. 3. Enzymatic hydrolysis procedure for the analysis of total nebivolol in plasma: assessment of hydrolysis time reaction with ␤-glucuronidase. The method of the analysis of total nebivolol isomers in plasma was applied to the investigation of stereoselectivity in the metabolism of nebivolol in eleven elderly hypertensive patients Please cite this article in press as: C.P. Vieira, et al., An indirect stereoselective analysis of nebivolol glucuronides in plasma by LC–MS/MS: Application to clinical pharmacokinetics, J. Pharm. Biomed. Anal. (2017), http://dx.doi.org/10.1016/j.jpba.2017.01.054 G Model PBA-11061; No. of Pages 6 ARTICLE IN PRESS C.P. Vieira et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2017) xxx–xxx 5 Table 2 Validation parameters of the method of analysis of total nebivolol isomers in plasma. l-Nebivolol d-Nebivolol MF (CV%) Linearity (ng/mL) Linear equation Correlation coefficient 13.03 0.2–125 y = 0.012556x + 0.00207 r = 0.989 12.52 0.2–125 y = 0.014581x + 0.00087 r = 0.992 Inter-assay precision (CV %) 0.2 ng/mL (LLOQC – n = 15) 0.5 ng/mL (LQC – n = 15) 50 ng/mL (MQC – n = 15) 100 ng/mL (HQC – n = 15) 500 ng/mL (DQC – n = 15) (1:10) 9.60 9.58 5.72 6.15 5.89 12.51 9.08 5.03 5.74 6.14 Intra-assay precision (CV %) 0.2 ng/mL (LLOQC – n = 5) 0.5 ng/mL (LQC – n = 5) 50 ng/mL (MQC – n = 5) 100 ng/mL (HQC – n = 5) 500 ng/mL (DQC – n = 5) (1:10) 9.41 7.64 3.70 8.21 5.14 13.72 4.53 3.85 8.72 2.72 Inter-assay accuracy (inaccuracy %) 0.2 ng/mL (LLOQC – n = 15) 0.5 ng/mL (LQC – n = 15) 50 ng/mL (MQC – n = 15) 100 ng/mL (HQC – n = 15) 500 ng/mL (DQC – n = 15) (1:10) 0.00 2.00 5.48 10.38 8.46 0.00 2.00 4.64 4.06 8.96 Intra-assay accuracy (inaccuracy %) 0.2 ng/mL (LLOQC – n = 5) 0.5 ng/mL (LQC – n = 5) 50 ng/mL (MQC – n = 5) 100 ng/mL (HQC – n = 5) 500 ng/mL (DQC – n = 5) (1:10) −5.00 12.00 7.06 9.01 13.52 −5.00 10.00 4.84 −5.76 12.60 MF = normalized matrix factor; CV = coefficient of variation. LLOQC, LQC, MQC, HQC and DQC are the quality controls of lower limit of quantification, low, medium, high and dilution concentrations. treated with a single oral dose of 10 mg racemic nebivolol. Demographic data of the investigated patients are shown in Table 3 as median (minimum-maximum). Patients of both sex (6 men and 5 women), aged from 65 to 74 years and phenotyped as EM for CYP2D6 were enrolled. The pharmacokinetics of unchanged nebivolol is stereoselective with plasma concentrations of l-nebivolol higher than d-nebivolol (l/d AUC = 1.8) in hypertensive patients (n = 11) orally treated with racemic nebivolol (Table 4 and Fig. 4). Higher AUC (10.1 vs 5.4 ng h/mL) and lower apparent clearance (495 vs 922 L/h) values were observed in the present study for the unchanged l-nebivolol isomer compared to d-nebivolol (Table 4). A previous investigation on the pharmacokinetics of unchanged nebivolol isomers in hypertensive patients showed a higher AUC (9.4 vs 4.7 ng h/mL) and lower apparent clearance (531.8 vs 1304.4 L/h) values for l-nebivolol compared to d-nebivolol [12]. Stereoselectivity in nebivolol pharmacokinetics with higher plasma concentrations of the unchanged l-nebivolol compared to d-nebivolol was also observed in healthy volunteers and hypertensive patients (AUC 6.8 vs 4.2 ng h/mL), patients with chronic kidney disease (AUC 9.9 vs 7.3 ng h/mL) and hemodialysis patients (AUC 6.4 vs 4.9 ng h/mL) treated with a single oral dose of 10 mg racemic nebivolol [13]. Regarding the nebivolol glucuronide, the results also indicate stereoselectivity favoring the formation of d-glucuronide isomer (d/l AUC = 5.4). Higher plasma concentrations of the d-nebivolol glucuronide compared to the l-nebivolol glucuronide (335.2 vs 65.3 ng h/mL) were observed, a finding that explains in part the higher plasma concentrations of unchanged l-nebivolol (Fig. 4 and Table 4). The present study highlighted stereoselectivity in Table 3 Demographic data of the enrolled hypertensive patients (n = 11). Data are expressed as median (minimum − maximum). Elderly hypertensive patients (n = 11) Gender Men Women Age CYP2D6 phenotype EM PM Metabolic ratio Weight (kg) 6 5 69 (65–74) 11 0 1.07 (0.47–4.26) 74 (42–86) Metabolic ratio: metoprolol/␣-hidroximetoprolol in urine; EM: extensive metabolizers; PM: poor metabolizers. Fig. 4. Plasma concentrations of unchanged nebivolol isomers and glucuronide nebivolol isomers vs time in eleven elderly hypertensive patients following the administration of a single oral dose of 10 mg racemate. Data are expressed as median and interquartile range. Please cite this article in press as: C.P. Vieira, et al., An indirect stereoselective analysis of nebivolol glucuronides in plasma by LC–MS/MS: Application to clinical pharmacokinetics, J. Pharm. Biomed. Anal. (2017), http://dx.doi.org/10.1016/j.jpba.2017.01.054 G Model ARTICLE IN PRESS PBA-11061; No. of Pages 6 C.P. Vieira et al. / Journal of Pharmaceutical and Biomedical Analysis xxx (2017) xxx–xxx 6 Table 4 Pharmacokinetic parameters for unchanged nebivolol isomers and glucuronide nebivolol isomers following a single oral dose of 10 mg of the racemate to elderly hypertensive patients (n = 11). Data are expressed as median, mean and CI (95%). l-Nebivolol d-Nebivolol l-Glucuronide d-Glucuronide Cmax (ng/mL) 2.1* 2.5 (1.8–3.2) 1.3 1.3 (1.0–1.5) 7.8* 9.1 (7.0–11.1) 54.9 58.6 (43.3–73.8) Tmax (h) 0.7 0.8 (0.6–1.2) 0.8 0.9 (0.6–1.1) 1.6 1.7 (1.3–2.1) 1.7 1.8 (1.4–2.2) AUC0–∞ (ng h/mL) 10.1* 10.3 (8.6–12.0) 5.4 5.4 (4.5–6.4) 65.3* 71.5 (58.1–85) 335.2 393.6 (236.8–550.3) t1/2 (h) 14.6 14.2 (12.0–16.3) 12.7 12.7 (11.6–13.8) 9.8 9.5 (8.3–10.8) 9.2 9.5 (7.5–11.6) Vd/F (L) 5035.8* 5524.5 (4363.6–6685.3) 8403.9 9034.2 (6647.3–11421.2) – – Cl/F (L/h) 495.3* 515.7 (423.2–608.4) 922.1 979.8 (786.0–1173.7) – – Razão AUC (L/d)AUC ratio (l/d) 1.8 1.9 (1.6-2.2) 5.4 5.4 (4.0–6.8) * Wilcoxon test for paired data, p < 10.05. Cmax: maximum plasma concentration; Tmax: time to reach Cmax; AUC0–∞ : area under the concentration–time curve from time zero extrapolated to infinity; t1/2 : terminal elimination half-life. the conjugation of nebivolol with glucuronic acid in hypertensive elderly patients CPY2D6 phenotyped as EM, with preponderance of the d-glucuronide isomer. Regarding glucuronidation, this study shows for the first time the plasma metabolic ratios glucuronide nebivolol to unchanged nebivolol. It is interesting to observe metabolic ratios of approximately 6.5 for the l-isomer (AUC 65.3 vs 10.1 ng h/mL) and approximately 62.1 (335.2 vs 5.4 ng h/mL) for d-isomer; Table 4). Considering that nebivolol glucuronide is pharmacologically active [3,8] and considering that the unchanged d-isomer is responsible for ␤1 adrenergic receptor antagonism [3–6], future studies regarding PK-PD of nebivolol should evaluate not only plasma concentrations of unchanged nebivolol isomers but also glucuronide nebivolol isomers. 4. Conclusion The analytical indirect method developed and validated for measuring nebivolol glucuronide isomers in plasma is precise, accurate and sensitive, permitting their quantification up to 48 h after the administration of a single oral dose of 10 mg of racemic nebivolol. Unchanged nebivolol pharmacokinetics is stereoselective with higher AUC and lower apparent clearance for l-nebivolol compared to the d-isomer in hypertensive elderly patients CYP2D6 phenotyped as EM. 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