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Chiral analysis of carvedilol and its metabolites hydroxyphenyl carvedilol and O-desmethyl carvedilol in human plasma by liquid chromatography-tandem mass spectrometry: Application to a clinical pharmacokinetic study
Journal of Chromatography B, 1015 (2016) 173–180
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Chiral analysis of carvedilol and its metabolites hydroxyphenyl carvedilol and O-desmethyl carvedilol in human plasma by liquid chromatography-tandem mass spectrometry: Application to a clinical pharmacokinetic study Glauco Henrique Balthazar Nardotto a , Eduardo Barbosa Coelho b , Maria Paula Marques a , 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 Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil
a r t i c l e
i n f o
Article history: Received 26 October 2015 Received in revised form 15 February 2016 Accepted 17 February 2016 Available online 23 February 2016 Keywords: Carvedilol Enantiomers Pharmacokinetics Metabolism Plasma Diabetes
a b s t r a c t Carvedilol is an antihypertensive drug, which is available in clinical practice as a racemic mixture. (S)-(−)carvedilol is a - and ␣1-adrenergic antagonist, while (R)-(+)-carvedilol only acts as an ␣1-adrenergic antagonist. Carvedilol is metabolized mainly by glucuronidation and, to a lesser extent, by CYP2D6 to hydroxyphenyl carvedilol (OHC) and by CYP2C9 to O-desmethyl carvedilol (DMC). This study describes the development and validation of a method for the sequential analysis of the enantiomers of carvedilol, OHC and DMC in plasma using a Chirobiotic® V chiral-phase column coupled to an LC–MS/MS system. The method was linear in the range of 0.05–100, 0.05–10 and 0.02–10 ng/mL for the carvedilol, OHC and DMC enantiomers, respectively. Application of the method to the investigation of a patient with type 2 diabetes mellitus treated with a single oral dose of 25 mg racemic carvedilol showed plasma accumulation of the (R)-(+)-carvedilol, (R)-(+)-DMC and (R)-(+)-OHC enantiomers. These results suggest that plasma accumulation of (R)-(+)-carvedilol cannot be explained by its oxidative metabolism. © 2016 Published by Elsevier B.V.
1. Introduction Carvedilol, a third-generation -adrenergic antagonist, is used for the treatment of hypertension, angina pectoris, cardiac arrhythmias, and congestive heart failure [1,2]. Carvedilol is available in clinical practice as racemic mixture of the (S)-(−) and (R)-(+)carvedilol enantiomers, which have distinct pharmacokinetic and pharmacodynamic properties. (S)-(−)-carvedilol is an ∝1- and  -adrenergic antagonist, while (R)-(+)-carvedilol only exhibits ∝1 antagonism [3]. The clearance of (S)-(−)-carvedilol (662 mL/min) is greater than that of (R)-(+)-carvedilol (605 mL/min). The oral bioavailability of (R)-(+)-carvedilol is about twice that of its antipode (31.1% for (R)-(+) and 15.1% for (S)-(−)-carvedilol) [4] and the plasma concentrations of (R)-(+)-carvedilol are approximately three times higher than those of (S)-(−)-carvedilol [5].
∗ Corresponding author. E-mail address: [email protected] (V.L. Lanchote). http://dx.doi.org/10.1016/j.jchromb.2016.02.028 1570-0232/© 2016 Published by Elsevier B.V.
Carvedilol is mainly eliminated by conjugation with glucuronic acid [6,7] and by oxidation reactions to hydroxyphenyl carvedilol (OHC), (4 -hydroxyphenyl carvedilol and 5 -hydroxyphenyl carvedilol), and to O-desmethyl carvedilol (DMC) [5,8,9]. Studies using human liver microsomes have shown that (S)-(−)-carvedilol is metabolized faster than the (R)-(+)carvedilol through oxidative reactions, although the same CYP enzymes are involved in the metabolism of both enantiomers. CYP2D6 is the main enzyme responsible for the formation of 4-OHC and 5-OHC, although CYP2E1, CYP2C9 and CYP3A4 also contribute in the production of these metabolites. CYP2C9 is responsible for the formation of DMC with additional contribution from CYP2D6, CYP1A2 and CYP2E1 [8]. In human liver microsomes, glucuronidation of (S)-(−)-carvedilol is also faster than (R)-(+)-carvedilol in the racemate, but true activities of both glucuronidations are approximately the same for the pure enantiomers. UGT1A1 and UGT2B7 are main responsible for the glucuronidation of (S)-(−) and (R)-(+)carvedilol in human intestinal microsomes [10]. The sequential analysis of carvedilol and its metabolites 4-OHC and DMC is only described for the enantiomeric mixture in rat
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plasma using UPLC–MS/MS, with quantification limits of 0.5 ng/mL for carvedilol and of 0.05 ng/mL for 4-OHC and DMC [11]. The enantioselective analysis of carvedilol and its metabolite DMC in human plasma has only been reported by Eisenberg et al. [12] with quantification limits of 0.625 ng of each enantiomer/mL, whereas the enantioselective analysis of carvedilol and its metabolite 4-OHC has only been reported by Furlong et al. [13] with quantification limits of 0.2 and 0.02 ng of each enantiomer/mL plasma, respectively. Therefore, the present study reports for the first time the development, validation and clinical application of a method for sequential analysis of the enantiomers of carvedilol, OHC (4OHC + 5-OHC) and DMC in human plasma using a chiral-phase column coupled to an LC–MS/MS system. The method was applied to investigate the enantioselective carvedilol metabolism in one patient with type 2 diabetes mellitus who was treated with a single oral dose of racemic carvedilol. 2. Methods 2.1. Standard solutions and quality control samples
Table 1 Quality controls concentrations of carvedilol enantiomers and their metabolites. Sample
carvedilol
OHC
DMC
0.05 0.08 0.4 8 40
0.02 0.04 0.4 8 40
concentration (ng/mL) LLOQC LQC MQC HQC DQCa
0.05 0.08 4 80 400
LLOQC, LQC, MQC, HQC e DQC are the quality controls of Lower limit of quantification, low, medium and high concentrations and dilution. a DQC: the samples were diluted with blank plasma in the proportion 1/5 immediately before the sample preparation. OHC: hydroxyphenylcarvedilol. DMC: O-desmethylcarvedilol. The samples were prepared as described in Section 2.3 sample preparation and 70 L were injected.
collision gas (argon) was maintained at 0.19 mL/min. Protonated carvedilol, OHC, DMC and metoprolol and their respective product ions were monitored at transitions of m/z 407 > 100, 423 > 222, 393 > 210 and 268 > 116, respectively.
The stock solutions were prepared using carvedilol standard purchased from Sigma (purity: 99.1%; St. Louis, MO, USA), and metoprolol, DMC and 4-OHC standards purchased from Toronto Research Chemicals (purity: 98%; TRC, North York, Canada). The carvedilol stock solution was prepared at a concentration of 100 g of each enantiomer/mL methanol (J.T. Backer, Mexico City, Mexico) and the stock solutions of OHC and DMC at a concentration of 10 g of each enantiomer/mL methanol. Using these stock solutions, standard solutions diluted in methanol were prepared at the following concentrations: 4000, 2000, 400, 200, 40, 20, 8, 4 and 2 ng of each carvedilol enantiomer/mL; 400, 200, 40, 20, 4, 2, 0.8, 0.4 and 0.2 ng of each OHC and DMC enantiomer/mL. The standard solution of racemic metoprolol used as internal standard (IS) was prepared at a concentration of 10 g/mL in methanol.
2.3. Sample preparation
2.2. Chromatographic analysis
2.4. Determination of elution order
The ACQUITY UPLC® H-Class chromatographic system coupled to a Xevo TQ-S® triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA) equipped with Zspray® electrospray ionization (ESI) was used for analysis. The devices were controlled with the MassLynx 4.1 program (Waters Corp.). The carvedilol, OHC and DMC enantiomers were separated on an Astec Chirobiotic® V column (0.46 × 25 cm, 5-m particle size; Supelco, Bellefonte, PA, USA) maintained at a temperature of 24 ◦ C. The mobile phase consisted of 99% of mixture A and 1% of mixture B and was eluted at a flow rate of 0.8 mL/min. Mixture A consisted of 60% ethanol (purity: 99.9%; Panreac, Barcelona, Spain) and 40% methanol (purity: 99.95%; J.T. Backer, Mexico City, Mexico) plus 1.8 mL/L glacial acetic acid (purity: 99.7%; Synth, Diadema, Brazil) and 0.2 mL/L diethylamine (purity: 100%; J.T. Baker, Phillipsburg, NJ, USA). Mixture B consisted of purified water plus 1.8 mL/L acetic acid and 0.2 mL/L diethylamine. The water was purified using the Synergy® UV system (Millipore, Molsheim, France). The operating conditions of the Xevo TQ-S® system were optimized in the multiple reaction-monitoring mode by direct infusion of the standard solutions of carvedilol, OHC, DMC and metoprolol (IS) into the mobile phase. The capillary voltage was set at 3.30 kV and the source and desolvation temperatures at 150 ◦ C and 550 ◦ C, respectively. The collision energy was 15, 20, 22 and 25 V for metoprolol, DMC, OHC and carvedilol, respectively. The cone voltages were 50 and 30 V for metoprolol and DMC, respectively, and 35 V for OHC and carvedilol. The desolvation and cone gas (nitrogen) flow rates were 1000 and 150 L/h, respectively, and the flow rate of the
The standard solutions in methanol at concentrations of 2, 4 and 3 g/mL, respectively for (R)-(+)-carvedilol, (R)-(+)-DMC and (R)(+)-OHC were prepared with standards purchased from Toronto Research Chemicals (purity: 98%; TRC, North York, Canada). Aliquots (25 L) of the standard solutions were evaporated to dryness and submitted to chromatographic analysis. The retention times were compared to those obtained by analyzing the racemic standards in methanol.
Plasma aliquots (1 mL) were added to 25 L of the IS solution and 5 mL di-isopropyl ether (purity: 98.5%; Sigma-Aldrich, St. Louis, MO, USA). The samples were shaken for 50 min in a mechanical horizontal shaker at 220 ± 10 cycles/min (MA 139/CFT, Marconi, Piracicaba, Brazil) and centrifuged at 1800 g for 10 min at 5 ◦ C in a refrigerated centrifuge (Himac CF 8DL, Hitachi, Tokyo, Japan). Volumes of 4 mL of the organic phase were separated and evaporated to dryness in a vacuum evaporator (Christ RVC 2–25 CD and Christ CT 04–50 SR, Osterode am Harz, Germany). The residues were resuspended in 100 L of mixture A and 70 L was injected into a Chirobiotic® V column. The processed samples were kept in the automatic injector at 12 ◦ C.
2.5. Validation of the method The analytical method was validated according to the recommendations of the US-FDA guidance for industry [14] for the validation of analytical methods and of the guidelines of the European Medicines Agency for the validation of bioanalytical methods [15]. The calibration curves were prepared in triplicate using 1 mL aliquots of blank plasma spiked with 25 L of each standard solution of carvedilol and its metabolites, including a blank sample and a zero sample. The calibration curves were constructed in the range of 0.05–100 ng of each carvedilol enantiomer, 0.05–10 ng of each OHC enantiomer, and 0.02–10 ng of each DMC enantiomer per mL plasma. Quality control samples were prepared from the stock solutions in blank plasma, corresponding to the lower limit of quantification (LLOQC), low (LQC), medium (MQC) and high (HQC) concentrations, and dilution quality control (DQC). The concentrations are shown
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in Table 1. The quality control samples in blank plasma were stored at −70 ◦ C. Selectivity was evaluated using 1 mL aliquots of blank plasma obtained from six different volunteers, including four normal, two lipemic and two hemolyzed samples. The chromatograms obtained were compared to LLOQC samples. Carry-over was analyzed by injecting blank samples before and after a sample at the upper limit of quantification (ULOQ). The chromatogram of the blank sample following the ULOQ standard was compared to that of the LLOQC. The matrix effect was evaluated using eight 1 mL aliquots of blank plasma obtained from different volunteers, including two lipemic, two hemolyzed and four normal samples. The blank plasma extracts were spiked with standard solutions at the concentrations corresponding to the HQC and LQC and with the IS solution. Additionally, the same standard solutions in methanol spiked with the IS solution were analyzed. The IS-normalized matrix factor (MF) was calculated for each sample according to the equation below. The matrix effect was determined as the coefficient of variation (CV) of all MFs obtained. MF=
area of matrix /area of IS in matrix area of analyte in solution /area of IS in solution
The efficiency of the extraction process was evaluated in eight 1 mL aliquots of blank plasma (2 lipemic, 2 hemolyzed and 4 normal) spiked with the standard solutions containing carvedilol and its metabolites at concentrations corresponding to the HQC and LQC. The results were compared to those obtained by analyzing the same standard solutions in methanol spiked with the IS. The percentage of recovery was determined using the following equation:
Recovery=
area of extracted analyte / area of extracted IS area of analyte in methanol / area of IS in methanol
× 100
Precision and accuracy were evaluated using six replicates of LLOQC, LQC, MQC, HQC and DQC analyzed in a single analytical run (intra-assay) and in three different analytical runs (interassay). The precision and accuracy results are reported as CV and relative standard error (RSE).
RSE =
mean experimental concentration − nominal concentration nominal concentration
× 100
Stability testing in the biological matrix was performed using four replicates of the LQC and HQC. For the evaluation of freeze/thaw stability, the HQC and LQC replicates were frozen at −70 ◦ C for 24 h, thawed at room temperature and again frozen at −70 ◦ C for 24 h. This cycle was repeated two more times and the samples were analyzed at the end of the three cycles. Short-term stability was evaluated by maintaining the LQC and HQC samples at room temperature for 1 h before preparation and analysis. For the evaluation of post-processing stability, the processed LQC and HQC samples were stored in the automatic injector for 24 h at 12 ◦ C before analysis. The results are expressed as CV and RSE. 2.6. Application of the method The clinical trial was approved by the Research Ethics Committee of the School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, and by the University Hospital of the Ribeirão Preto School of Medicine, University of São Paulo. After providing free informed consent, a 48-year-old patient with type 2 diabetes mellitus and hepatic and renal function within the normal range was investigated. The patient received one tablet of 25 mg racemic carvedilol (Carvedilat® , EMS, Hortolândia, Brazil) with 200 mL water after a 12-h fast. A breakfast was served 3 h after administration of the drug. Serial blood samples (approximately 5 mL) were collected at times zero, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5,
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4, 5, 6, 8, 10, 12, 15, 18 and 24 h after administration of the drug. After centrifugation for 10 min at 2500 rpm (Himac CF 8DL, Hitachi, Tokyo, Japan), the plasma samples were separated and stored at −70 ◦ C until the time of analysis. The pharmacokinetic parameters of the carvedilol, OHC and DMC enantiomers were calculated using the WinNonLin 4.0 program (Pharsight Corp, Mountain View, CA, USA). 3. Results Figs. 1 and 2 show the protonated ions and their respective products obtained by the analysis of carvedilol (406.8 > 100), OHC (423.0 > 222), DMC (393.2 > 210), and metoprolol (IS, 268 > 116). The product ions were selected as a function of signal intensity and of the separation of other carvedilol metabolites of the same mass [13,16]. Fig. 3 illustrates the chromatograms of the carvedilol, OHC, DMC and metoprolol (IS) enantiomers in plasma. As can be seen, there are no interfering peaks in the blank samples eluting together with the enantiomers of the IS or of carvedilol and its metabolites. The first eluted peak of metoprolol enantiomers, indicated in Fig. 3 as number 7, was used as internal standard. The elution order of carvedilol, DMC and OHC enantiomers is shown in Fig. 4 as the retention times of the pure (R)-(+) enantiomers of carvedilol and its metabolites. The results permit to infer that the carvedilol, DMC and OHC enantiomers are eluted in the sequence (S)-(−) and (R)-(+). Supplement 1 shows the matrix effect, recovery, linearity, precision, accuracy and stability obtained by the analysis of the carvedilol, OHC and DMC enantiomers. The method was applied to the study of pharmacokinetics and metabolism in a patient with type 2 diabetes mellitus treated with a single oral dose of 25 mg racemic carvedilol. The plasma concentration versus time curves for each enantiomer are shown in Fig. 5 and the pharmacokinetic parameters are given in Table 2. 4. Discussion The present study reports the sequential analysis of the carvedilol enantiomers and their metabolites OHC and DMC, as well as application of the method to the clinical study of administration of a single oral dose of racemic carvedilol. Since the different chiral columns experimented did not separate 4-OHC from 5-OHC, which only differ in the position of the hydroxyl group in the phenol ring and are therefore not separated in the MS/MS detector [13], the method was validated using 4-OHC as standard. However, the concentrations in the plasma sample of the investigated patient correspond to the sum of 4-OHC and 5-OHC and are simply referred to as OHC. The time of the chromatographic run using a Chirobiotic V® chiral column was 26 min (Fig. 3) and resolution of the enantiomers was 1.07 for carvedilol and OHC and 0.8 for DMC. Although the separation is not complete, it is noticed that peaks with a resolution of at least 0.8 can be distinguished when the concentration ratio between them is up to 4:1 [17,18], so the carvedilol, OHC and DMC enantiomers were identified considering that the (R)-(+)/(S)-(−) AUC ratios were lower than 3:1 (Table 2). Analysis of different blank plasma samples (Fig. 3) did not show interference of endogenous components with the carvedilol enantiomers and their metabolites, indicating adequate selectivity. The coefficients of variation obtained for all MFs were less than 15% (Supplement 1), demonstrating the absence of a matrix effect. The blank plasma samples analyzed immediately after the ULOQ sample did not contain interfering peaks, indicating the absence of carry-over of previous injections.
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Fig. 1. Mass spectra of carvedilol, OHC and DMC (A1, B1, C1) and their fragments (A2, B2, C2) [11,13,16]. Carvedilol (406.8 > 100.1), OHC (423.0 > 222.0), DMC (393.2 > 210.1).
Since the plasma concentrations of the OHC and DMC enantiomers are approximately 10 times lower than those of the carvedilol enantiomers [5,9,12,13], low LLOQC values are funda-
mental for application of the method to the clinical study of the pharmacokinetics of a single dose of the racemic drug. The method developed and validated in the present study exhibits lower limits
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Fig. 2. Mass spectra of metoprolol (IS, A1) and its fragment (A2) [21]. (268 > 116).
Table 2 Pharmacokinetic parameters of carvedilol, OHC and DMC enantiomers in a type II diabetes mellitus patient after administration of a single oral dose of racemic carvedilol 25 mg. Parameters
(R)-(+)- carvedilol
(S)-(−)- carvedilol
(R)-(+)-OHC
(S)-(−)-OHC
(R)-(+)-DMC
(S)-(−)-DMC
Cmax (ng/mL) tmax (h) AUC0− ∞ (ng h/mL) CL/F (L/h) V1/F (L) V2/F (L) t1/2 (h) AUC (R)/(S)
29.08 0.94 132.69 94.21 311.16 417.03 6.75 2.87
14.51 0.89 46.18 270.69 399.69 1164.67 5.18
3.91 0.65 12.57 – – – 6.52 1.77
1.13 1.08 7.09 – – – 11.92
3.19 1.14 26.96 – – – 6.75 2.93
1.36 1.09 9.21 – – – 4.05
OHC: hydroxyphenylcarvedilol, DMC: O-desmethylcarvedilol, AUC: area under the plasma concentration versus time curve, CL/F: apparent clearance, V1/F: apparent central volume of distribution, V2/F: apparent peripheral volume of distribution, t1/2: half-life, Cmax : maximum plasma concentration, tmax : time to achieve Cmax .
of quantification of 0.05 ng/mL for the carvedilol and OHC enantiomers and of 0.02 ng/mL for the DMC enantiomers (Supplement 1). Hence, the present method is more sensitive than those reported by Eisenberg et al. [12] for the analysis of carvedilol and DMC enantiomers using HPLC with fluorescence detection and by Furlong et al. [13] for the analysis of carvedilol and 4-OHC enantiomers using LC–MS/MS. The method described here permitted to quantify the carvedilol enantiomers and their metabolites OHC and DMC up to 24 h after administration of a single oral dose of 25 mg racemic carvedilol (Fig. 5). Precision and accuracy studies revealed coefficients of variation and standard errors of the mean of less than 15%, indicating that the method is precise and accurate. The stability tests provided coefficients of variation of less than 15%, guaranteeing stability after three freeze/thaw cycles, after 1 h on the bench at room tempera-
ture, and 24 h after processing (samples kept at 12 ◦ C) (Supplement 1). The method developed and validated here was applied to the investigation of enantioselectivity in the pharmacokinetics of carvedilol and its metabolites in a patient with type 2 diabetes mellitus. The data shown in Table 2 permit to infer higher plasma concentrations of the (R)-(+)-carvedilol enantiomer, with the observation of approximately 2-fold higher Cmax and 3-fold higher AUC values when compared to (S)-(−)-carvedilol. Apparent clearance and the apparent volume of distribution of (S)-(−)carvedilol were also approximately 3 times higher when compared to (R)-(+)-carvedilol. The pharmacokinetic parameters obtained for the enantiomers of unchanged carvedilol in the patient with type 2 diabetes mellitus agree with studies in the literature involving healthy volunteers or hypertensive patients [4,5,9,13,19,20].
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Fig. 3. Chromatograms of plasma samples obtained at 1.5 h after administration of a single 25 mg oral racemic carvedilol dose (A1–A4), blank plasma added of 50, 5 and 5 ng/mL of each enantiomer of carvedilol, DMC and OHC (B1–B4) and blank samples (C1–C4). Peaks: 1: (S)-(−)-carvedilol, 2: (R)-(+)-carvedilol, 3: (S)-(−)-DMC, 4 (R)-(+)-DMC, 5: (S)-(−)-OHC, 6: (R)-(+)-OHC, 7: metoprolol (IS).
The results also show higher plasma concentrations of (R)-(+)OHC (Fig. 5 and Table 2), with the AUC value of (R)-(+)-OHC being almost the double of that observed for (S)-(−)-OHC. Higher plasma concentrations of (R)-(+)-OHC has been described by Furlong et al.
[13] in the investigation of a healthy volunteer. Furthermore, the AUC value of (R)-(+)-DMC was approximately three times higher than that obtained for (S)-(−)-DMC, similar to the lower plasma concentrations of (S)-(−)-DMC reported by Eisenberg et al. [12].
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Fig. 4. Elution order of carvedilol enantiomers and their metabolites. Standard solutions of (R)-(+)-carvedilol (A1), (R)-(+)-DMC (B1) and (R)-(+)-OHC (C1). Racemic standard solutions of carvedilol (A2), DMC (B2) and OHC (C2).
explained by enantioselectivity in oxidative metabolism but by other metabolism pathways such as glucuronidation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb.2016. 02.028. References
Fig. 5. Plasma concentrations (ng/mL) versus time (h) curves of carvedilol, OHC and DMC obtained from a type II diabetes mellitus patient following administration of a single oral dose of racemic carvedilol 25 mg.
5. Conclusion The method developed and validated here for the sequential analysis of carvedilol, OHC and DMC enantiomers in plasma using a chiral column and LC–MS/MS exhibits sensitivity, linearity, selectivity, precision and accuracy compatible with the application in clinical studies on the enantioselective pharmacokinetics and oxidative metabolism of a single oral dose of racemic drug. Despite the limitation of the method, which does not separate 4-OHC from 5-OHC, the pharmacokinetic parameters permit to infer higher plasma concentrations of the (R)-(+)-carvedilol and its metabolites (R)-(+)-OHC and (R)-(+)-DMC in a patient with type 2 diabetes mellitus treated with a single oral dose of racemic carvedilol. Hence, the higher plasma concentrations of (R)-(+)-carvedilol cannot be
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Chiral analysis of carvedilol and its metabolites hydroxyphenyl carvedilol and O-desmethyl carvedilol in human plasma by liquid chromatography-tandem mass spectrometry: Application to a clinical pharmacokinetic study Glauco Henrique Balthazar Nardotto a , Eduardo Barbosa Coelho b , Maria Paula Marques a , 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 Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil
a r t i c l e
i n f o
Article history: Received 26 October 2015 Received in revised form 15 February 2016 Accepted 17 February 2016 Available online 23 February 2016 Keywords: Carvedilol Enantiomers Pharmacokinetics Metabolism Plasma Diabetes
a b s t r a c t Carvedilol is an antihypertensive drug, which is available in clinical practice as a racemic mixture. (S)-(−)carvedilol is a - and ␣1-adrenergic antagonist, while (R)-(+)-carvedilol only acts as an ␣1-adrenergic antagonist. Carvedilol is metabolized mainly by glucuronidation and, to a lesser extent, by CYP2D6 to hydroxyphenyl carvedilol (OHC) and by CYP2C9 to O-desmethyl carvedilol (DMC). This study describes the development and validation of a method for the sequential analysis of the enantiomers of carvedilol, OHC and DMC in plasma using a Chirobiotic® V chiral-phase column coupled to an LC–MS/MS system. The method was linear in the range of 0.05–100, 0.05–10 and 0.02–10 ng/mL for the carvedilol, OHC and DMC enantiomers, respectively. Application of the method to the investigation of a patient with type 2 diabetes mellitus treated with a single oral dose of 25 mg racemic carvedilol showed plasma accumulation of the (R)-(+)-carvedilol, (R)-(+)-DMC and (R)-(+)-OHC enantiomers. These results suggest that plasma accumulation of (R)-(+)-carvedilol cannot be explained by its oxidative metabolism. © 2016 Published by Elsevier B.V.
1. Introduction Carvedilol, a third-generation -adrenergic antagonist, is used for the treatment of hypertension, angina pectoris, cardiac arrhythmias, and congestive heart failure [1,2]. Carvedilol is available in clinical practice as racemic mixture of the (S)-(−) and (R)-(+)carvedilol enantiomers, which have distinct pharmacokinetic and pharmacodynamic properties. (S)-(−)-carvedilol is an ∝1- and  -adrenergic antagonist, while (R)-(+)-carvedilol only exhibits ∝1 antagonism [3]. The clearance of (S)-(−)-carvedilol (662 mL/min) is greater than that of (R)-(+)-carvedilol (605 mL/min). The oral bioavailability of (R)-(+)-carvedilol is about twice that of its antipode (31.1% for (R)-(+) and 15.1% for (S)-(−)-carvedilol) [4] and the plasma concentrations of (R)-(+)-carvedilol are approximately three times higher than those of (S)-(−)-carvedilol [5].
∗ Corresponding author. E-mail address: [email protected] (V.L. Lanchote). http://dx.doi.org/10.1016/j.jchromb.2016.02.028 1570-0232/© 2016 Published by Elsevier B.V.
Carvedilol is mainly eliminated by conjugation with glucuronic acid [6,7] and by oxidation reactions to hydroxyphenyl carvedilol (OHC), (4 -hydroxyphenyl carvedilol and 5 -hydroxyphenyl carvedilol), and to O-desmethyl carvedilol (DMC) [5,8,9]. Studies using human liver microsomes have shown that (S)-(−)-carvedilol is metabolized faster than the (R)-(+)carvedilol through oxidative reactions, although the same CYP enzymes are involved in the metabolism of both enantiomers. CYP2D6 is the main enzyme responsible for the formation of 4-OHC and 5-OHC, although CYP2E1, CYP2C9 and CYP3A4 also contribute in the production of these metabolites. CYP2C9 is responsible for the formation of DMC with additional contribution from CYP2D6, CYP1A2 and CYP2E1 [8]. In human liver microsomes, glucuronidation of (S)-(−)-carvedilol is also faster than (R)-(+)-carvedilol in the racemate, but true activities of both glucuronidations are approximately the same for the pure enantiomers. UGT1A1 and UGT2B7 are main responsible for the glucuronidation of (S)-(−) and (R)-(+)carvedilol in human intestinal microsomes [10]. The sequential analysis of carvedilol and its metabolites 4-OHC and DMC is only described for the enantiomeric mixture in rat
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plasma using UPLC–MS/MS, with quantification limits of 0.5 ng/mL for carvedilol and of 0.05 ng/mL for 4-OHC and DMC [11]. The enantioselective analysis of carvedilol and its metabolite DMC in human plasma has only been reported by Eisenberg et al. [12] with quantification limits of 0.625 ng of each enantiomer/mL, whereas the enantioselective analysis of carvedilol and its metabolite 4-OHC has only been reported by Furlong et al. [13] with quantification limits of 0.2 and 0.02 ng of each enantiomer/mL plasma, respectively. Therefore, the present study reports for the first time the development, validation and clinical application of a method for sequential analysis of the enantiomers of carvedilol, OHC (4OHC + 5-OHC) and DMC in human plasma using a chiral-phase column coupled to an LC–MS/MS system. The method was applied to investigate the enantioselective carvedilol metabolism in one patient with type 2 diabetes mellitus who was treated with a single oral dose of racemic carvedilol. 2. Methods 2.1. Standard solutions and quality control samples
Table 1 Quality controls concentrations of carvedilol enantiomers and their metabolites. Sample
carvedilol
OHC
DMC
0.05 0.08 0.4 8 40
0.02 0.04 0.4 8 40
concentration (ng/mL) LLOQC LQC MQC HQC DQCa
0.05 0.08 4 80 400
LLOQC, LQC, MQC, HQC e DQC are the quality controls of Lower limit of quantification, low, medium and high concentrations and dilution. a DQC: the samples were diluted with blank plasma in the proportion 1/5 immediately before the sample preparation. OHC: hydroxyphenylcarvedilol. DMC: O-desmethylcarvedilol. The samples were prepared as described in Section 2.3 sample preparation and 70 L were injected.
collision gas (argon) was maintained at 0.19 mL/min. Protonated carvedilol, OHC, DMC and metoprolol and their respective product ions were monitored at transitions of m/z 407 > 100, 423 > 222, 393 > 210 and 268 > 116, respectively.
The stock solutions were prepared using carvedilol standard purchased from Sigma (purity: 99.1%; St. Louis, MO, USA), and metoprolol, DMC and 4-OHC standards purchased from Toronto Research Chemicals (purity: 98%; TRC, North York, Canada). The carvedilol stock solution was prepared at a concentration of 100 g of each enantiomer/mL methanol (J.T. Backer, Mexico City, Mexico) and the stock solutions of OHC and DMC at a concentration of 10 g of each enantiomer/mL methanol. Using these stock solutions, standard solutions diluted in methanol were prepared at the following concentrations: 4000, 2000, 400, 200, 40, 20, 8, 4 and 2 ng of each carvedilol enantiomer/mL; 400, 200, 40, 20, 4, 2, 0.8, 0.4 and 0.2 ng of each OHC and DMC enantiomer/mL. The standard solution of racemic metoprolol used as internal standard (IS) was prepared at a concentration of 10 g/mL in methanol.
2.3. Sample preparation
2.2. Chromatographic analysis
2.4. Determination of elution order
The ACQUITY UPLC® H-Class chromatographic system coupled to a Xevo TQ-S® triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA) equipped with Zspray® electrospray ionization (ESI) was used for analysis. The devices were controlled with the MassLynx 4.1 program (Waters Corp.). The carvedilol, OHC and DMC enantiomers were separated on an Astec Chirobiotic® V column (0.46 × 25 cm, 5-m particle size; Supelco, Bellefonte, PA, USA) maintained at a temperature of 24 ◦ C. The mobile phase consisted of 99% of mixture A and 1% of mixture B and was eluted at a flow rate of 0.8 mL/min. Mixture A consisted of 60% ethanol (purity: 99.9%; Panreac, Barcelona, Spain) and 40% methanol (purity: 99.95%; J.T. Backer, Mexico City, Mexico) plus 1.8 mL/L glacial acetic acid (purity: 99.7%; Synth, Diadema, Brazil) and 0.2 mL/L diethylamine (purity: 100%; J.T. Baker, Phillipsburg, NJ, USA). Mixture B consisted of purified water plus 1.8 mL/L acetic acid and 0.2 mL/L diethylamine. The water was purified using the Synergy® UV system (Millipore, Molsheim, France). The operating conditions of the Xevo TQ-S® system were optimized in the multiple reaction-monitoring mode by direct infusion of the standard solutions of carvedilol, OHC, DMC and metoprolol (IS) into the mobile phase. The capillary voltage was set at 3.30 kV and the source and desolvation temperatures at 150 ◦ C and 550 ◦ C, respectively. The collision energy was 15, 20, 22 and 25 V for metoprolol, DMC, OHC and carvedilol, respectively. The cone voltages were 50 and 30 V for metoprolol and DMC, respectively, and 35 V for OHC and carvedilol. The desolvation and cone gas (nitrogen) flow rates were 1000 and 150 L/h, respectively, and the flow rate of the
The standard solutions in methanol at concentrations of 2, 4 and 3 g/mL, respectively for (R)-(+)-carvedilol, (R)-(+)-DMC and (R)(+)-OHC were prepared with standards purchased from Toronto Research Chemicals (purity: 98%; TRC, North York, Canada). Aliquots (25 L) of the standard solutions were evaporated to dryness and submitted to chromatographic analysis. The retention times were compared to those obtained by analyzing the racemic standards in methanol.
Plasma aliquots (1 mL) were added to 25 L of the IS solution and 5 mL di-isopropyl ether (purity: 98.5%; Sigma-Aldrich, St. Louis, MO, USA). The samples were shaken for 50 min in a mechanical horizontal shaker at 220 ± 10 cycles/min (MA 139/CFT, Marconi, Piracicaba, Brazil) and centrifuged at 1800 g for 10 min at 5 ◦ C in a refrigerated centrifuge (Himac CF 8DL, Hitachi, Tokyo, Japan). Volumes of 4 mL of the organic phase were separated and evaporated to dryness in a vacuum evaporator (Christ RVC 2–25 CD and Christ CT 04–50 SR, Osterode am Harz, Germany). The residues were resuspended in 100 L of mixture A and 70 L was injected into a Chirobiotic® V column. The processed samples were kept in the automatic injector at 12 ◦ C.
2.5. Validation of the method The analytical method was validated according to the recommendations of the US-FDA guidance for industry [14] for the validation of analytical methods and of the guidelines of the European Medicines Agency for the validation of bioanalytical methods [15]. The calibration curves were prepared in triplicate using 1 mL aliquots of blank plasma spiked with 25 L of each standard solution of carvedilol and its metabolites, including a blank sample and a zero sample. The calibration curves were constructed in the range of 0.05–100 ng of each carvedilol enantiomer, 0.05–10 ng of each OHC enantiomer, and 0.02–10 ng of each DMC enantiomer per mL plasma. Quality control samples were prepared from the stock solutions in blank plasma, corresponding to the lower limit of quantification (LLOQC), low (LQC), medium (MQC) and high (HQC) concentrations, and dilution quality control (DQC). The concentrations are shown
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in Table 1. The quality control samples in blank plasma were stored at −70 ◦ C. Selectivity was evaluated using 1 mL aliquots of blank plasma obtained from six different volunteers, including four normal, two lipemic and two hemolyzed samples. The chromatograms obtained were compared to LLOQC samples. Carry-over was analyzed by injecting blank samples before and after a sample at the upper limit of quantification (ULOQ). The chromatogram of the blank sample following the ULOQ standard was compared to that of the LLOQC. The matrix effect was evaluated using eight 1 mL aliquots of blank plasma obtained from different volunteers, including two lipemic, two hemolyzed and four normal samples. The blank plasma extracts were spiked with standard solutions at the concentrations corresponding to the HQC and LQC and with the IS solution. Additionally, the same standard solutions in methanol spiked with the IS solution were analyzed. The IS-normalized matrix factor (MF) was calculated for each sample according to the equation below. The matrix effect was determined as the coefficient of variation (CV) of all MFs obtained. MF=
area of matrix /area of IS in matrix area of analyte in solution /area of IS in solution
The efficiency of the extraction process was evaluated in eight 1 mL aliquots of blank plasma (2 lipemic, 2 hemolyzed and 4 normal) spiked with the standard solutions containing carvedilol and its metabolites at concentrations corresponding to the HQC and LQC. The results were compared to those obtained by analyzing the same standard solutions in methanol spiked with the IS. The percentage of recovery was determined using the following equation:
Recovery=
area of extracted analyte / area of extracted IS area of analyte in methanol / area of IS in methanol
× 100
Precision and accuracy were evaluated using six replicates of LLOQC, LQC, MQC, HQC and DQC analyzed in a single analytical run (intra-assay) and in three different analytical runs (interassay). The precision and accuracy results are reported as CV and relative standard error (RSE).
RSE =
mean experimental concentration − nominal concentration nominal concentration
× 100
Stability testing in the biological matrix was performed using four replicates of the LQC and HQC. For the evaluation of freeze/thaw stability, the HQC and LQC replicates were frozen at −70 ◦ C for 24 h, thawed at room temperature and again frozen at −70 ◦ C for 24 h. This cycle was repeated two more times and the samples were analyzed at the end of the three cycles. Short-term stability was evaluated by maintaining the LQC and HQC samples at room temperature for 1 h before preparation and analysis. For the evaluation of post-processing stability, the processed LQC and HQC samples were stored in the automatic injector for 24 h at 12 ◦ C before analysis. The results are expressed as CV and RSE. 2.6. Application of the method The clinical trial was approved by the Research Ethics Committee of the School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, and by the University Hospital of the Ribeirão Preto School of Medicine, University of São Paulo. After providing free informed consent, a 48-year-old patient with type 2 diabetes mellitus and hepatic and renal function within the normal range was investigated. The patient received one tablet of 25 mg racemic carvedilol (Carvedilat® , EMS, Hortolândia, Brazil) with 200 mL water after a 12-h fast. A breakfast was served 3 h after administration of the drug. Serial blood samples (approximately 5 mL) were collected at times zero, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5,
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4, 5, 6, 8, 10, 12, 15, 18 and 24 h after administration of the drug. After centrifugation for 10 min at 2500 rpm (Himac CF 8DL, Hitachi, Tokyo, Japan), the plasma samples were separated and stored at −70 ◦ C until the time of analysis. The pharmacokinetic parameters of the carvedilol, OHC and DMC enantiomers were calculated using the WinNonLin 4.0 program (Pharsight Corp, Mountain View, CA, USA). 3. Results Figs. 1 and 2 show the protonated ions and their respective products obtained by the analysis of carvedilol (406.8 > 100), OHC (423.0 > 222), DMC (393.2 > 210), and metoprolol (IS, 268 > 116). The product ions were selected as a function of signal intensity and of the separation of other carvedilol metabolites of the same mass [13,16]. Fig. 3 illustrates the chromatograms of the carvedilol, OHC, DMC and metoprolol (IS) enantiomers in plasma. As can be seen, there are no interfering peaks in the blank samples eluting together with the enantiomers of the IS or of carvedilol and its metabolites. The first eluted peak of metoprolol enantiomers, indicated in Fig. 3 as number 7, was used as internal standard. The elution order of carvedilol, DMC and OHC enantiomers is shown in Fig. 4 as the retention times of the pure (R)-(+) enantiomers of carvedilol and its metabolites. The results permit to infer that the carvedilol, DMC and OHC enantiomers are eluted in the sequence (S)-(−) and (R)-(+). Supplement 1 shows the matrix effect, recovery, linearity, precision, accuracy and stability obtained by the analysis of the carvedilol, OHC and DMC enantiomers. The method was applied to the study of pharmacokinetics and metabolism in a patient with type 2 diabetes mellitus treated with a single oral dose of 25 mg racemic carvedilol. The plasma concentration versus time curves for each enantiomer are shown in Fig. 5 and the pharmacokinetic parameters are given in Table 2. 4. Discussion The present study reports the sequential analysis of the carvedilol enantiomers and their metabolites OHC and DMC, as well as application of the method to the clinical study of administration of a single oral dose of racemic carvedilol. Since the different chiral columns experimented did not separate 4-OHC from 5-OHC, which only differ in the position of the hydroxyl group in the phenol ring and are therefore not separated in the MS/MS detector [13], the method was validated using 4-OHC as standard. However, the concentrations in the plasma sample of the investigated patient correspond to the sum of 4-OHC and 5-OHC and are simply referred to as OHC. The time of the chromatographic run using a Chirobiotic V® chiral column was 26 min (Fig. 3) and resolution of the enantiomers was 1.07 for carvedilol and OHC and 0.8 for DMC. Although the separation is not complete, it is noticed that peaks with a resolution of at least 0.8 can be distinguished when the concentration ratio between them is up to 4:1 [17,18], so the carvedilol, OHC and DMC enantiomers were identified considering that the (R)-(+)/(S)-(−) AUC ratios were lower than 3:1 (Table 2). Analysis of different blank plasma samples (Fig. 3) did not show interference of endogenous components with the carvedilol enantiomers and their metabolites, indicating adequate selectivity. The coefficients of variation obtained for all MFs were less than 15% (Supplement 1), demonstrating the absence of a matrix effect. The blank plasma samples analyzed immediately after the ULOQ sample did not contain interfering peaks, indicating the absence of carry-over of previous injections.
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Fig. 1. Mass spectra of carvedilol, OHC and DMC (A1, B1, C1) and their fragments (A2, B2, C2) [11,13,16]. Carvedilol (406.8 > 100.1), OHC (423.0 > 222.0), DMC (393.2 > 210.1).
Since the plasma concentrations of the OHC and DMC enantiomers are approximately 10 times lower than those of the carvedilol enantiomers [5,9,12,13], low LLOQC values are funda-
mental for application of the method to the clinical study of the pharmacokinetics of a single dose of the racemic drug. The method developed and validated in the present study exhibits lower limits
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Fig. 2. Mass spectra of metoprolol (IS, A1) and its fragment (A2) [21]. (268 > 116).
Table 2 Pharmacokinetic parameters of carvedilol, OHC and DMC enantiomers in a type II diabetes mellitus patient after administration of a single oral dose of racemic carvedilol 25 mg. Parameters
(R)-(+)- carvedilol
(S)-(−)- carvedilol
(R)-(+)-OHC
(S)-(−)-OHC
(R)-(+)-DMC
(S)-(−)-DMC
Cmax (ng/mL) tmax (h) AUC0− ∞ (ng h/mL) CL/F (L/h) V1/F (L) V2/F (L) t1/2 (h) AUC (R)/(S)
29.08 0.94 132.69 94.21 311.16 417.03 6.75 2.87
14.51 0.89 46.18 270.69 399.69 1164.67 5.18
3.91 0.65 12.57 – – – 6.52 1.77
1.13 1.08 7.09 – – – 11.92
3.19 1.14 26.96 – – – 6.75 2.93
1.36 1.09 9.21 – – – 4.05
OHC: hydroxyphenylcarvedilol, DMC: O-desmethylcarvedilol, AUC: area under the plasma concentration versus time curve, CL/F: apparent clearance, V1/F: apparent central volume of distribution, V2/F: apparent peripheral volume of distribution, t1/2: half-life, Cmax : maximum plasma concentration, tmax : time to achieve Cmax .
of quantification of 0.05 ng/mL for the carvedilol and OHC enantiomers and of 0.02 ng/mL for the DMC enantiomers (Supplement 1). Hence, the present method is more sensitive than those reported by Eisenberg et al. [12] for the analysis of carvedilol and DMC enantiomers using HPLC with fluorescence detection and by Furlong et al. [13] for the analysis of carvedilol and 4-OHC enantiomers using LC–MS/MS. The method described here permitted to quantify the carvedilol enantiomers and their metabolites OHC and DMC up to 24 h after administration of a single oral dose of 25 mg racemic carvedilol (Fig. 5). Precision and accuracy studies revealed coefficients of variation and standard errors of the mean of less than 15%, indicating that the method is precise and accurate. The stability tests provided coefficients of variation of less than 15%, guaranteeing stability after three freeze/thaw cycles, after 1 h on the bench at room tempera-
ture, and 24 h after processing (samples kept at 12 ◦ C) (Supplement 1). The method developed and validated here was applied to the investigation of enantioselectivity in the pharmacokinetics of carvedilol and its metabolites in a patient with type 2 diabetes mellitus. The data shown in Table 2 permit to infer higher plasma concentrations of the (R)-(+)-carvedilol enantiomer, with the observation of approximately 2-fold higher Cmax and 3-fold higher AUC values when compared to (S)-(−)-carvedilol. Apparent clearance and the apparent volume of distribution of (S)-(−)carvedilol were also approximately 3 times higher when compared to (R)-(+)-carvedilol. The pharmacokinetic parameters obtained for the enantiomers of unchanged carvedilol in the patient with type 2 diabetes mellitus agree with studies in the literature involving healthy volunteers or hypertensive patients [4,5,9,13,19,20].
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Fig. 3. Chromatograms of plasma samples obtained at 1.5 h after administration of a single 25 mg oral racemic carvedilol dose (A1–A4), blank plasma added of 50, 5 and 5 ng/mL of each enantiomer of carvedilol, DMC and OHC (B1–B4) and blank samples (C1–C4). Peaks: 1: (S)-(−)-carvedilol, 2: (R)-(+)-carvedilol, 3: (S)-(−)-DMC, 4 (R)-(+)-DMC, 5: (S)-(−)-OHC, 6: (R)-(+)-OHC, 7: metoprolol (IS).
The results also show higher plasma concentrations of (R)-(+)OHC (Fig. 5 and Table 2), with the AUC value of (R)-(+)-OHC being almost the double of that observed for (S)-(−)-OHC. Higher plasma concentrations of (R)-(+)-OHC has been described by Furlong et al.
[13] in the investigation of a healthy volunteer. Furthermore, the AUC value of (R)-(+)-DMC was approximately three times higher than that obtained for (S)-(−)-DMC, similar to the lower plasma concentrations of (S)-(−)-DMC reported by Eisenberg et al. [12].
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Fig. 4. Elution order of carvedilol enantiomers and their metabolites. Standard solutions of (R)-(+)-carvedilol (A1), (R)-(+)-DMC (B1) and (R)-(+)-OHC (C1). Racemic standard solutions of carvedilol (A2), DMC (B2) and OHC (C2).
explained by enantioselectivity in oxidative metabolism but by other metabolism pathways such as glucuronidation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb.2016. 02.028. References
Fig. 5. Plasma concentrations (ng/mL) versus time (h) curves of carvedilol, OHC and DMC obtained from a type II diabetes mellitus patient following administration of a single oral dose of racemic carvedilol 25 mg.
5. Conclusion The method developed and validated here for the sequential analysis of carvedilol, OHC and DMC enantiomers in plasma using a chiral column and LC–MS/MS exhibits sensitivity, linearity, selectivity, precision and accuracy compatible with the application in clinical studies on the enantioselective pharmacokinetics and oxidative metabolism of a single oral dose of racemic drug. Despite the limitation of the method, which does not separate 4-OHC from 5-OHC, the pharmacokinetic parameters permit to infer higher plasma concentrations of the (R)-(+)-carvedilol and its metabolites (R)-(+)-OHC and (R)-(+)-DMC in a patient with type 2 diabetes mellitus treated with a single oral dose of racemic carvedilol. Hence, the higher plasma concentrations of (R)-(+)-carvedilol cannot be
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