- Email: [email protected]
MS method for the simultaneous determination of agomelatine and its metabolites, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma: Application to a bioequivalence study
Accepted Manuscript Title: Development and Validation a LC-MS/MS method for the simultaneous determination of agomelatine and its metabolites, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma: application to a bioequivalence study Author: Meizhen Li Fang Tang Feifan Xie Yisha Lv Peng Yu Zhi Liu Zeneng Cheng PII: DOI: Reference:
S1570-0232(15)30193-8 http://dx.doi.org/doi:10.1016/j.jchromb.2015.09.018 CHROMB 19627
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
19-3-2015 11-9-2015 15-9-2015
Please cite this article as: Meizhen Li, Fang Tang, Feifan Xie, Yisha Lv, Peng Yu, Zhi Liu, Zeneng Cheng, Development and Validation a LC-MS/MS method for the simultaneous determination of agomelatine and its metabolites, 7-desmethyl-agomelatine and 3hydroxy-agomelatine in human plasma: application to a bioequivalence study, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2015.09.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development and Validation a LC-MS/MS method for the simultaneous determination
of
agomelatine
and
its
metabolites,
7-desmethyl-
agomelatine and 3-hydroxy-agomelatine in human plasma: application to a bioequivalence study Meizhen Li1, #, Fang Tang1, #, Feifan Xie1,*, Yisha Lv1, Peng Yu1, Zhi Liu2, Zeneng Cheng1,* 1 School of Pharmaceutical Sciences, Central South University, Changsha 410013, PR China. 2. Hunan Tiger-Xiangya R&D Company Ltd., Changsha 410013, China. # These authors contributed equally. *Corresponding author: Zeneng Cheng, School of Pharmaceutical Sciences, Central South University, Tongzipo Road 172, Changsha 410013, PR China. Tel.&Fax: +86 731 8265 0451; E-mail: [email protected] *Corresponding author: Feifan Xie, School of Pharmaceutical Sciences, Central South University, Tongzipo Road 172, Changsha 410013, PR China. Tel.&Fax: +86 137 8702 4726; E-mail: [email protected]
Highlights
Simultaneous determination of agomelatine and its metabolites in human plasma by LC-MS/MS. Simple protein precipitation procedure was employed. Wide linear ranges for the target analytes were obtained. Successfully applied to a bioequivalence study in healthy Chinese volunteers.
Abstract A novel sensitive and selective LC-MS/MS method for the determination of agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma was developed and validated. After simple protein precipitation, the analytes were separated on a Phenomenex ODS3 column (4.6×150 mm，5μm，Phenomenex, USA) with mobile phase consisted of methanol and 5 mM ammonium formate solution (containing 0.2% formic acid) at a ratio of 70:30 (v/v) with a flow rate of 0.8 mL/min. The MS acquisition was performed in multiple reactions monitoring (MRM) mode with a positive electrospray ionization source. The mass transitions monitored were m/z 244.1→185.1, m/z 230.1→171.1, m/z 260.1→201.1 and m/z 180.1→110.1 for agomelatine, 7-desmethyl-agomelatine, 3-hydroxy-agomelatine and internal standard (phenacetin), respectively. The method was validated for specificity, linearity and lower limit of quantification, precision and accuracy, extraction recovery, matrix effect and stability. The calibration curves for agomelatine, 7desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma were linear over concentration ranges of 0.0457~100ng/mL, 0.1372~300ng/mL and
0.4572~1000ng/mL,
respectively.
Intra-
and
inter-day
precisions
and
accuracies data met the acceptance criteria of FDA guideline for bioanalytical method validation. The developed method has been successfully applied to a bioequivalence study in healthy Chinese volunteers. Keywords: LC-MS/MS; Agomelatine; 7-desmethyl-agomelatine; 3-hydroxy agomelatine; Metabolite; Plasma; Bioequivalence
1. Introduction Agomelatine is a specific agonist of MT1 and MT2, and a selective antagonist of 5-HT2c receptors[1, 2]. It is a napthalenic compound chemically designated as N-[2-(7-Methoxy-1-naphthyl)ethyl] acetamide (Fig.1) with a selectivity of large than 100-fold for MT1 and MT2 receptors with no significant affinities to muscarinic, adrenergic, dopaminergic or histaminergic receptors, which has strong effects on improving depression and fewer adverse reactions[3, 4]. Agomelatine has a short half-life of about 2 hours in human beings. It is rapidly absorbed from the gastrointestinal tract and immediately transported to the liver[5], where it is extensively metabolized by the P450 (CYP) isoenzymes namely CYPA1, CYPA2, and CYP2C9. 7-desmethyl-, 3-hydroxy-, and 3-hydroxy-7-desmethyl-agomelatine (Fig.1) were identified as the three metabolites of agomelatine, which have less activity than the parent drug[6-8]. For new generic agomelatine products with clinical trial permission of China Food and Drug Administration (CFDA), bioequivalence (BE) evaluation is one of the pivotal clinical studies required in support of its marketing application[9]. Our preliminary BE experiment indicated that the within-subject variability of AUC and Cmax were 53% and 70% for agomelatine, 21.2% and 37.8% for
3-hydroxy-agomelagtine, 42.6% and 61.4% for 7-desmethyl-
agomelatine. In addition, the AUC in plasma of 3-hydroxy-agomelagtine and 7-desmethyl-agomelatine were found about 15-fold and 3/4 of that of
agomelagtine. Due to the smaller within-subject variability and high in vivo concentrations of 3-hydroxy-agomelagtine and 7-desmethyl-agomelatine, these two metabolites were selected as auxiliary index for BE assessment in the present study, which can provide ample information for the BE study, thus the in vivo data of these two metabolites are needed. To our knowledge, several LC-MS/MS methods for the determination of agomelatine in human plasma have been published[10-12]. Two of the reported bioanalytical methods[10, 11] employed a liquid-liquid extraction procedure and a time-costing evaporation process, and the linear ranges were relatively narrow, which are not adequate for the determination of this high variability drug. In addition, only the parent drug agomelatine was determined in these two reported LC-MS/MS methods. Ogawa T, et al [12]reported a UPLC-MS/MS method for determination of ramelteon, agomelatine, and melatonin in human plasma, and it employed tedious and costly sample preparation technique (solid-phase extraction) and timeconsuming evaporating procedure. Up to date, there were no reports described a method for the simultaneous determination of agomelatine and its major metabolites using LC-MS/MS. In the present study, we developed and fully validated a simple, rapid and sensitive enough LC-MS/MS method for the simultaneous determination of agomelatine, 7-desmethyl-agomelatine and 3hydroxy-agomelatine in human plasma for the first time. This assay employed a simple protein precipitation procedure and obtained high extraction
recoveries and wide linear ranges, which was successfully applied to a bioequivalence study in healthy Chinese volunteers. 2. Experimental 2.1 Chemicals and reagents Reference standards of agomelatine (purity 99.5%), 7-desmethylagomelatine (purity 98.2%) and 3-hydroxy-agomelatine (purity 98.0%) were all provided by Chongqing Fuke pharmaceutical group Co., Ltd (Chongqing, China). Reference standard of phenacetin (internal standard, IS, purity 99.8%) was purchased from National Institutes for Food and Drug Control (Beijing, China). HPLC grade methanol and formic acid were supplied from Merck (Darmstadt, Germany) and Tianjin Kermel Chemical Reagent Co., Ltd (Tianjin, China), respectively. Ammonium formate (analytical grade) and hydrochloric acid (analytical grade) were obtained from Sinopharm Chemcial Reagent Co., Ltd. (Shanghai, China). Sodium metabisulphite (analytical grade) was purchased from Tianjin Fengchuan Chemical Reagent Science And Technology Co., Ltd (Tianjin, China). Blank human plasma was obtained from healthy volunteers with heparin used as anticoagulant. Purified water was made by AHL-2001-P trace and analysis type of ultra-pure water machine (Echo pu, Shanghai, China). 2.2 Chromatographic and mass spectrometric condition An Agilent 6460 triple-quadrupole LC-MS/MS system was employed comprising degasser, binary pump, autosampler, thermostatted column
compartment, electrospray ionization source (ESI) and a triple-quadrupole MS detector.
Data
acquisition
was
performed
with
Agilent
MassHunter
Workstation Software (Agilent, USA). The separation was carried out on a Phenomenex ODS3 column (4.6×150 mm, 5μm, Phenomenex, USA) with a Gemini C18 column (4×3.0 mm, 5 μm, Phenomenex, USA) employed as guard column. The mobile phase was methanol-5 mM ammonium formate solution (containing 0.2% formic acid) in a ratio 70:30 (v/v), which was delivered at a flow rate of 0.8mL/min. The injection volume was 5 μL and the column temperature was maintained at 35°C. The mass spectrometer was equipped with electrospray ionization source and operated in positive ion mode using multiple reactions monitoring (MRM). The mass transition ion pairs were selected as m/z 244.1→185.1, m/z 230.1→171.1, m/z 260.1→201.1 and m/z 180.1→110.1 for agomelatine, 7desmethyl-agomelatine, 3-hydroxy-agomelatine and phenacetin, respectively. The other optimized MS/MS parameters were as follows: fragmentor voltage 90V for agomelatine, 50V for 7-desmethyl-agomelatine and 3-hydroxyagomelatine, and 70V for phenacetin, collision energy (CE) 10eV for agomelatine and 3-hydroxy-agomelatine, 15eV for 7-desmethyl-agomelatine and 20eV for phenacetin, Gas Temp，350°C；Gas Flow 8L·min-1, Nebulizer 45psi, Sheath Temp 350°C, Sheath Gas Flow 11 L·min-1, and Capillary 4000V. 2.3 Preparation of stock reference solutions and standard plasma
samples The stock reference solutions containing agomelatine (1mg·mL-1), 7desmethyl-agomelatine (1mg·mL-1) and 3-hydroxy-agomelatine (1mg·mL-1) were separately prepared in methanol. Stock solution of IS (1 mg·mL-1) was prepared in methanol, and was further diluted with methanol to prepare the IS working solution at the concentration of 100 ng/mL. Eight
standard
agomelatine
and
working
solutions
3-hydroxy-agomelatine
of
agomelatine,
were
serially
7-desmethylprepared
by
appropriately mixing these three analytes stock solutions at the beginning and then appropriately diluting the mixture solution with methanol, which contains agomelatine at the concentrations of 1000, 333.3, 111.1, 37.04,12.35, 4.115, 1.372 and 0.4572 ng/mL, 7-desmethyl-agomelatine at the concentrations of 3000, 1000, 333.3, 111.1, 37.04, 12.35, 4.115 and 1.372 ng/mL, and 3hydroxy-agomelatine at the concentrations of 10000, 3333, 1111, 370.4, 123.5, 41.15, 13.72 and 4.572 ng/mL. A 20μL of working standard solution was added to 180μL of blank plasma for preparing calibration standard samples. The agomelatine,
calibration
curves
300~0.1372ng/mL
ranges were for
100~0.0457ng/mL for
7-desmethyl-agomelatine,
and
1000~0.4572ng/mL for 3-hydroxy-agomelatine, respectively. Quality control (QC) samples were independently prepared in the same way at three concentration levels (0.1097, 3.704 and 80ng/mL for agomelatine, 0.3292, 11.11 and 240ng/mL for 7-desmethyl-agomelatine, 1.097, 37.04 and
800ng/mL for 3-hydroxy-agomelatine). The stock and working solutions were all stored at 4°C, and the IS working solution and the calibration standards were freshly prepared for each analytical run. 2.4 Sample preparation Each 200μL human plasma sample was added with 20μL sodium metabisulphite solution (20g·L-1) and vortex-mixed for 30s. Subsequently, 30μL IS working solution (100ng/mL) was added into the plasma sample and vortex-mixed for 30 s, then the plasma sample was precipitated with 600μL 15% hydrochloric acid methanol solution, vortex-mixed for 5 min. The treated plasma samples were centrifuged at 15700×g for 10 min, then 5μL of supernatant was injected to the LC-MS/MS system.
2.5 Method validation 2.5.1 Specificity The specificity is the ability of an analytical method to differentiate and quantify the analytes in the presence of other components in the sample. Specificity was assessed by comparing the chromatograms of extracted blank plasma from six different healthy volunteers with the corresponding plasma samples added with agomelatine, 7-desmethyl-agomelatine, 3-hydroxyagomelatine and IS. 2.5.2 Linearity and lower limit of quantification (LLOQ) The linearity of the calibration curves for agomelatine and its two
metabolites were assessed by analyzing eight standard samples in which the concentrations ranged over 0.0457~100 ng/mL, 0.1372~300 ng/mL and 0.4572~1000 ng/mL for agomelatine, 7-desmethyl-agomelatine and 3hydroxy-agomelatine,respectively. The calibration curves were established through weighed linear least-squares regression (1/X2 weighing) of the peak area ratios (Y) of the analytes to the IS versus the respective standard concentrations (X), respectively. The acceptance criterion for each backcalculated standard concentration was ±15% deviation from the nominal value, except at LLOQ. The LLOQ was defined as the lowest point on the calibration curve. Six different standard plasma samples at this concentration level should be quantified with less than ±20% bias of the nominal value in accuracy and less than 20% relative standard deviation (RSD) in precision while providing a signal-to-noise ratio ≥10. 2.5.3 Precision and accuracy Precision and accuracy were evaluated by analyzing standard plasma samples at three concentration QC levels (0.1097, 3.704 and 80 ng/mL for agomelatine, 0.3292, 11.11 and 240 ng/mL for 7-desmethyl-agomelatine, 1.097, 37.04 and 800 ng/mL for 3-hydroxy-agomelatine) on five replicates of each level for three batches successively. The concentration of each sample was determined using the calibration curve prepared on the same batch. Precision was expressed using the relative standard deviation, and accuracy
was defined as the relative deviation in the determined concentration of a standard from that of its nominal concentration expressed as a percentage. The precisions were required within 15%, and accuracies were required not to exceed ±15%. 2.5.4 Recovery and matrix effect The extraction recoveries of agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine
were
determined
at
corresponding
three
QC
concentrations respectively by comparing the peak areas that were extracted from plasma samples with peak areas of these three analytes reconstituted in blank plasma extracts at the equivalent concentrations. The recovery of the IS was evaluated at 100 ng/mL. To evaluate the matrix effects, blank human plasma samples were processed according to the sample preparation procedure described above and then spiked with analytes and IS at the final concentration after extraction. The matrix effects of the plasma were expressed as the ratio of the mean peak area of analytes spiked postextraction to that of the neat standard solution with mobile phase at the same three concentration levels. When the ratios were within 85-115%, the matrix effects were considered as negligible. 2.5.5 Stability Stability tests for agomelatine, 7-desmethyl-agomelatine and 3-hydroxyagomelatine were performed using analytes-spiked plasma samples under various conditions: ambient temperature storage (at 22°C for 4 h), through
three freeze/thaw cycles (from -40°C to room temperature), and for long-term storage (frozen at -40°C for 90 days), post-preparative storage (at 4°C for 24 h) by analyzing three replicates at low, medium and high QC concentrations, respectively. 2.6 Pharmacokinetic study This method was successfully applied to determine the plasma levels of agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine from a bioequivalence study involving 44 healthy Chinese volunteers aged 18-26 years and weighed 53-72 kg. The study design was a randomized, openlabel, two-treatment and four-period crossover study, and each subject was fasted and orally administered 25 mg test or reference formulation according to the design. The test formulation was agomelatine tablet supplied from Chongqing
Fuke
pharmaceutical
group
Co.,
Ltd
and
VALDOXAN®
manufactured by pharmaceutical company Servier as the reference formulation. The ethics committee approved the protocol and the volunteers provided with informed written consent. A total of 15 blood samples (5mL each) were collected into heparinized polypropylene tubes at 0 (pre-dose), 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7 and 8 h after administration. Plasma was separated by centrifugation at 3000×g for 10 min and stored at -40°C until analysis. The pharmacokinetics parameters were acquired and analyzed by a non-compartmental model performed on Phoenix WinNonlin 6.1 software.
3. Results and discussion 3.1 Method development As to mass spectrometric detection, both positive and negative MRM modes were tested, and positive MRM mode was chosen over negative MRM mode because of its improved signal to noise ratio (S/N) for analytes in extracted samples. In the full scan Q1 mass spectrum, the most abundant ions were the protonated molecular ions [M+H]+ found at m/z = 244.1, 230.1, 260.1 and 180.1 for agomelatine, 7-desmethyl-agomelatine, 3-hydroxyagomelatine and IS, respectively. The corresponding most prominent product ions were shown at m/z = 185.1, 171.1, 201.1 and 110.1, respectively. All mass spectrometric parameters were optimized for optimal intensity of the protonated molecular ions and productions. Subsequently, the mass transitions were monitored at m/z 244.1→185.1, 230.1→171.1, 260.1→201.1 and 180.1→110.1 for agomelatine, 7-desmethyl-agomelatine, 3-hydroxyagomelatine and IS, respectively. The addition of 0.2% formic acid in the mobile phase was found to give higher MS response as it favors ionization. Compared with liquid-liquid extraction, protein precipitation was found to offer satisfactory recovery and desired extraction efficiency in determining the levels of the analytes. There is a phenolic hydroxyl group in both 7-desmethyl-agomelatine and 3-hydroxyagomelatine, thus 20μL sodium metabisulphite solution (20g·L-1) was added during the protein precipitation process to prevent the oxidation of phenolic
hydroxyl group based on our previous bioanalytical experience. Despite using methanol solution (~600µL) as protein-precipitant, the extraction efficiency couldn't be improved. Therefore, stronger precipitating agent like hydrochloric acid was used for harvesting the plasma. Several amounts of hydrochloric acid dissolved in methanol solution were investigated to effectively extract the analytes from plasma, and eventually 600μL 15% hydrochloric acid methanol solution gave the desired extraction efficiency. 3.2 Specificity The typical chromatograms of blank plasma, blank plasma spiked with analytes and IS are shown in the Fig.2A and Fig.2B. The retention times of agomelatine, 7-desmethyl-agomelatine, 3-hydroxy-agomelatine and IS were approximately 5.5 min, 3.4min, 3.1min and 3.1 min, respectively. No obvious interferences from endogenous substance were observed. Other typical chromatograms are presented in Fig. 2C. 3.3 Linearity and lower limit of quantification (LLOQ) The calibration curves for agomelatine, 7-desmethyl-agomelatine and 3hydroxy-agomelatine
were
linear
over
the
concentration
ranges
of
0.0457~100 ng/mL, 0.1372~300 ng/mL and 0.4572~1000 ng/mL in human plasma respectively, and the mean regression equations of the calibration curves for these analytes were Y=0.1188X-0.0005 (r2=0.9962), Y=0.0734X0.0003 (r2=0.9975), and Y=0.0543X-0.0007 (r2=0.9978). The LLOQ for agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in plasma
were 0.0457 ng/mL, 0.1372 ng/mL and 0.4572 ng/mL respectively on the basis that the accuracies were 102.1%, 92.9% and 86.2%, and the precisions were 7.5%, 8.1% and 3.8%, respectively. 3.4 Precision and accuracy The intra- and inter-day precisions and accuracies for agomelatine, 7desmethyl-agomelatine and 3-hydroxy-agomelatine are presented in Table 1. As shown, all the intra- and inter-day precisions were less than 6.6% and accuracies were at the range of 90.2%~105.1%. The data demonstrated that the precisions and accuracies of this method were within the acceptable range. 3.5 Recovery and matrix effect The mean extraction recoveries for the analytes at corresponding low, medium and high QC concentration levels were 88.5%, 87.7% and 90.7% for agomelatine, 99.5%, 91.4% and 95.3% for 7-desmethyl-agomelatine, and 104.6%, 99.9% and 100.3% for 3-hydroxy-agomelatine, while the mean recovery of IS was 93.6%. These results indicated that the analytes showed satisfactory extraction recoveries and not significant concentration dependent. The average matrix effects (%) for the analytes at corresponding low, medium and high QC concentration levels were found to be 112.1%, 102.7% and 107.5% for agomelatine, 107.0%, 103.3% and 108.0% for 7-desmethylagomelatine, and 104.6%, 110.1% and 111.7% for 3-hydroxy-agomelatine. The average matrix effects of IS was 105.3%. These results indicated that
matrix effects were similar and not significant concentration dependent, thus not an issue in the current method. 3.6 Stability In terms of stability, all the results had accuracy values ranged from 89.4% to 111.8% with RSD% ≤ 12.4% (shown in Table 2), indicating that agomelatine,
7-desmethyl-agomelatine and 3-hydroxy-agomelatine were
stable in human plasma and processed samples under the conditions described above. 3.7 Incurred samples reanalysis(ISR) Sixty clinical study samples for agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine were selected for the incurred samples reanalysis study plan. The acceptance criteria specify that the percent differences, i.e. 100×(repeat value−original value)/the mean of original value and repeat value, of at least 2/3 of all the analyzed ISR samples should be within ±20%. The percent differences ranged from −4.72 to 3.23% with the average of −0.37% for agomelatine, from −5.67 to 9.21% with the average of 2.82% for 7-desmethyl-agomelatine and from −6.17 to 5.34% with the average of 2.43% for 3-hydroxy-agomelatine. The ISR results for all analytes demonstrated this method was reproducible and robust. 3.8 Pharmacokinetic study All the subjects completed the study and were incorporated in the pharmacokinetics analysis. The average concentration-time profiles of
agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma are presented in Fig.3. Peak concentrarion (Cmax) and time to peak concentration (Tmax) were directly obtained from experimental observations. The main pharmacokinetics parameters of agomelatine, 7-desmethylagomelatine and 3-hydroxy-agomelatine obtained from 44 Chinese subjects administered 25mg test or reference agomelatine tablet are summarized in Table 3. The mean Tmax and t1/2 of reference tablet (Valdoxan) obtained in this study were similar to those values (1h for Tmax and 0.9h for t1/2) of CHMP Assessment Report for Valdoxan [13]and reported values (1.1h for Tmax and 1.1h for t1/2) in healthy Chinese volunteers by Wang X, et al[11]. The mean Cmax and AUCinf in this study were 1.9-fold and 1.7-fold higher than those values (3.0ng/mL for Cmax and 4.9ng•h/mL for AUCinf) of CHMP Assessment Report for Valdoxan[13], however, the Cmax and AUCinf were much lower compared with the reported values (15.1ng/mL for Cmax and 16.4ng•h/mL for AUCinf) by Wang X, et al[11]. The mean Cmax for agomelatine, 7-desmethyl-agomelatine and 3hydroxy-agomelatine of test formulation were 1.32, 1.15 and 1.17 fold respectively higher than those of reference formulation. The bioequivalence acceptance criteria for Cmax, AUClast and AUCinf of agomelatine are 48.99%~204.13%, 58.48%~170.99% and 61.38%~162.91% respectively based on reference-scaled average bioequivalence approach[14]. The 90%
confidence intervals of the ratios of means Cmax, AUClast and AUCinf for agomelatine
were
104.42%~139.86%,
101.33%~123.83%
and
97.90%~117.94%, respectively, which all falls within the corresponding acceptance range thus the test formulation was considered bioequivalent to the reference formulation.
4. Conclusion A rapid, simple and sensitive LC-MS/MS method for the simultaneous determination of agomelatine, 7-desmethyl-agomelatine and 3-hydroxyagomelatine in human plasma was developed and fully validated for the first time on the basis of specificity, linearity, sensitivity, precision and accuracy, recovery, matrix effect and stability. The method provided satisfactory sensitive, high extraction recoveries, and wide linear ranges, which is suitable for the analysis of this high variability drug. The method was successfully applied to a bioequivalence study in healthy Chinese volunteers.
Acknowledgements This research was supported by National Science and Technology Major Projects for "Major New Drugs Innovation and Development" (No. 2012ZX09303014-001, Beijing, China), and the National Natural Science Foundation of China (Beijing, China) for Project No. 81273585.
Reference [1] S. Yous, J. Andrieux, H.E. Howell, P.J. Morgan, P. Renard, B. Pfeiffer, D. Lesieur, B. Guardiola-Lemaitre, J Med Chem, 35 (1992) 1484-1486. [2] M.J. Millan, A. Gobert, F. Lejeune, A. Dekeyne, A. Newman-Tancredi, V. Pasteau, J.M. Rivet, D. Cussac, J Pharmacol Exp Ther, 306 (2003) 954-964. [3] D. Eser, T.C. Baghai, H.-J.U.R. M O Ller, Core evidence, 4 (2009) 171. [4] B. Green, Current Medical Research \& Opinion, 27 (2011) 745--749. [5] V. Srinivasan, D.P. Cardinali, S.R. Pandi-Perumal, G.M. Brown, Journal of Experimental and Integrative Medicine, 1 (2011) 149--158. [6] J.J.P. Bogaards, E.M. Hissink, M. Briggs, R. Weaver, R. Jochemsen, P. Jackson, M. Bertrand, P.J. van Bladeren, European Journal of Pharmaceutical Sciences, 12 (2000) 117 - 124. [7] S. Conway, S.J. Canning, H.E. Howell, E.S. Mowat, P. Barrett, J.E. Drew, P. Delagrange, D. Lesieur, P.J. Morgan, European Journal of Pharmacology, 390 (2000) 15 - 24. [8]
M.
Fornaro,
D.
Prestia,
S.
Colicchio,
G.
Perugi,
Current
neuropharmacology, 8 (2010) 287-304. [9] Q. Pei, Y. Wang, Z.Y. Hu, S.K. Liu, H.Y. Tan, C.X. Guo, R.R. Zhang, Y.X. Xiang, J. Huang, L. Huang, H. Yuan, G.P. Yang, PLoS One, 9 (2014) e109300. [10] S.R. Patil, K.K. Nerurkar, A.M. Kalamkar, V. Pukale, K.V. Mangaonkar, S.G. Pingale, J Mass Spectrom, 47 (2012) 23-28.
[11] X. Wang, D. Zhang, M. Liu, H. Zhao, D. A, L. Meng, H. Liu, Biomed Chromatogr, 28 (2014) 218-222. [12] T. Ogawa, M. Iwai, H. Hattori, K. Zaitsu, T. Kumazawa, A. Ishii, O. Suzuki, H. Seno, Forensic Toxicology, 32 (2014) 126-131. [13] Agency EM. CHMP Assessment Report for Valdoxan. Document Reference EMEA/655251/2008. [14] B.M. Davit, M.L. Chen, D.P. Conner, S.H. Haidar, S. Kim, C.H. Lee, R.A. Lionberger, F.T. Makhlouf, P.E. Nwakama, D.T. Patel, D.J. Schuirmann, L.X. Yu, The AAPS journal, 14 (2012) 915-924.
Figure captions Fig.1 Chemical structures of agomelatine (A), 7-desmethyl-agomelatine (B), 3-hydroxy-agomelatine (C), 3-hydroxy-7-desmethyl-agomelatine (D) and phenacetin (IS) (E). Fig.2 Typical chromatograms of agomelatine, 7-desmethyl-agomelatine and 3hydroxy-agomelatine in human plasma: (A) blank human plasma from healthy volunteers; (B) blank human plasma spiked with agomelatine (0.0457μg·L-1), 7-desmethyl-agomelatine
metabolite
(0.1372μg·L-1)
and
3-hydroxy-
agomelatine (0.4572μg·L-1) and IS; (C) Plasma sample from a healthy
volunteer 4 h after orally administrated of 25mg agomelatine tablet. For each chromatogram, the panels from top to bottom represent 3-hydroxyagomelatine, agomelatine, 7-desmethyl-agomelatine and IS, respectively. Fig.3
Mean plasma concentration-time curves of agomelatine (A), 7-
desmethyl-agomelatine (B) and 3-hydroxy-agomelatine(C) for test (T) and reference (R) formulations in healthy Chinese subjects after orally administrated of 25mg agomelatine tablet (n=44). Each data represents the mean+SD.
Fig.1
A
B
O
N H HO
OH
C
E
D
Fig.2
A
B
C
Fig.3
A
B
C
Table 1 Inter- and intra-day precisions and accuracies of agomelatine, 7-desmethylagomelatine and 3-hydroxy-agomelatine in QC samples of human plasma. Intra-day(n=5) Mean oncentrati Accuracy on (%) found(ng/ mL)
Inter-day(n=15) Mean concentra Accur RS tion acy D found(ng/ (%) (%) mL)
Analytes
Nominal concentration(n g/mL)
agomelat ine
0.1097
0.1046
95.3
3.4
0.1066
97.2
6.6
3.704 80
3.391 84.01
91.5 105.1
2.9 2.1
3.360 82.59
90.7 103.3
2.9 3.3
0.3292
0.3000
91.1
5.1
0.3066
93.1
4.7
11.11
10.18
91.6
2.7
10.12
91.1
2.5
240
249.0
103.8
2.0
245.5
102.3
2.3
1.097
1.007
91.8
3.0
0.9897
90.2
3.9
37.04
34.79
93.9
2.5
34.61
93.4
2.4
800
825.2
103.2
1.8
813.2
101.7
2.7
7desmeth ylagomelat ine 3hydroxyagomelat ine
RS D (%)
Table 2 Stability studies on QC samples for agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma. Storage conditions
agomelatine (ng/mL)
7-desmethyl-agomelatine(ng/mL)
0.1097 3.704 80 0.3292 Ambient temperature storage stability (at 22°C for 4 h)
11.11
240
3-hydroxy-agomelatine(ng/mL) 1.097
37.04
800
Mean(ng/mL) 0.1107 3.388 82.06 0.3308 Accuracy (%) 101.0 91.4 102.6 100.5 RSD (%) 4.9 0.8 3.0 4.4 Post-preparative storage stability (at 4°C for 24 h) Mean(ng/mL) 0.1226 4.001 81.37 0.3513 Accuracy (%) 111.8 108.1 101.7 106.8 RSD (%) 1.6 1.5 4.5 5.3 Freeze/thaw stability (from -40°C to room temperature)
9.938 89.4 1.6
241.7 100.7 2.1
1.075 97.9 1.6
34.35 92.7 0.4
795 99.3 2.1
11.57 104.3 2.4
230.7 96 2.5
1.149 104.8 2.4
39.39 106.4 1.7
762.3 95.2 3.5
Mean(ng/mL) 0.1112 3.836 75.57 0.3358 Accuracy (%) 101.4 103.6 94.4 102 RSD (%) 12.4 5.2 0.8 4.2 Long-term storage stability (at -40°C for 90 days)
11.47 103.2 1.5
227.8 94.8 0.5
1.061 96.7 2.9
38.75 104.7 1.3
752.9 94.1 0.2
10.82 97.3 2.1
224.5 93.5 8.6
1.121 102.2 11.1
35.99 97.1 1.5
764.5 95.5 2.1
Mean(ng/mL) Accuracy (%) RSD (%)
0.1109 101.1 1.9
3.766 101.7 0.9
80.77 101.0 7.2
0.3391 103.0 6.3
Table 3 Main pharmacokinetics parameters of agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine obtained from 44 Chinese subjects after orally administered 25mg test or reference agomelatine tablet.
Parameters
Cmax(ng/mL) Tmax(h) AUClast (ng•h/mL) AUCinf (ng•h/mL) t1/2(h)
Reference formulation 73desmethyl agomelat hydroxyine agomelat agomelati ine ne 5.74 ± 43.30 ± 3.77 ± 6.91 22.45 2.44 1.22 ± 1.25 ± 1.23 ± 0.86 0.81 0.80 7.99 ± 84.44 ± 5.34 ± 10.15 32.49 2.38 8.31 ± 86.11 ± 5.76 ± 10.23 33.06 2.51 1.58 ± 1.29 ± 1.47 ± 1.32 0.26 0.92
Test formulation 73desmethyl agomelat hydroxyine agomelat agomelati ine ne 7.55 ± 50.09 ± 4.43 ± 10.01 25.45 3.04 1.14 ± 1.13 ± 1.07 ± 0.75 0.72 0.72 8.59 ± 88.30 ± 5.75 ± 10.17 31.81 2.64 8.72 ± 89.78 ± 6.16 ± 10.16 32.27 2.76 1.24 ± 1.24 ± 1.55 ± 1.40 0.24 1.97
S1570-0232(15)30193-8 http://dx.doi.org/doi:10.1016/j.jchromb.2015.09.018 CHROMB 19627
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
19-3-2015 11-9-2015 15-9-2015
Please cite this article as: Meizhen Li, Fang Tang, Feifan Xie, Yisha Lv, Peng Yu, Zhi Liu, Zeneng Cheng, Development and Validation a LC-MS/MS method for the simultaneous determination of agomelatine and its metabolites, 7-desmethyl-agomelatine and 3hydroxy-agomelatine in human plasma: application to a bioequivalence study, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2015.09.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development and Validation a LC-MS/MS method for the simultaneous determination
of
agomelatine
and
its
metabolites,
7-desmethyl-
agomelatine and 3-hydroxy-agomelatine in human plasma: application to a bioequivalence study Meizhen Li1, #, Fang Tang1, #, Feifan Xie1,*, Yisha Lv1, Peng Yu1, Zhi Liu2, Zeneng Cheng1,* 1 School of Pharmaceutical Sciences, Central South University, Changsha 410013, PR China. 2. Hunan Tiger-Xiangya R&D Company Ltd., Changsha 410013, China. # These authors contributed equally. *Corresponding author: Zeneng Cheng, School of Pharmaceutical Sciences, Central South University, Tongzipo Road 172, Changsha 410013, PR China. Tel.&Fax: +86 731 8265 0451; E-mail: [email protected] *Corresponding author: Feifan Xie, School of Pharmaceutical Sciences, Central South University, Tongzipo Road 172, Changsha 410013, PR China. Tel.&Fax: +86 137 8702 4726; E-mail: [email protected]
Highlights
Simultaneous determination of agomelatine and its metabolites in human plasma by LC-MS/MS. Simple protein precipitation procedure was employed. Wide linear ranges for the target analytes were obtained. Successfully applied to a bioequivalence study in healthy Chinese volunteers.
Abstract A novel sensitive and selective LC-MS/MS method for the determination of agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma was developed and validated. After simple protein precipitation, the analytes were separated on a Phenomenex ODS3 column (4.6×150 mm，5μm，Phenomenex, USA) with mobile phase consisted of methanol and 5 mM ammonium formate solution (containing 0.2% formic acid) at a ratio of 70:30 (v/v) with a flow rate of 0.8 mL/min. The MS acquisition was performed in multiple reactions monitoring (MRM) mode with a positive electrospray ionization source. The mass transitions monitored were m/z 244.1→185.1, m/z 230.1→171.1, m/z 260.1→201.1 and m/z 180.1→110.1 for agomelatine, 7-desmethyl-agomelatine, 3-hydroxy-agomelatine and internal standard (phenacetin), respectively. The method was validated for specificity, linearity and lower limit of quantification, precision and accuracy, extraction recovery, matrix effect and stability. The calibration curves for agomelatine, 7desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma were linear over concentration ranges of 0.0457~100ng/mL, 0.1372~300ng/mL and
0.4572~1000ng/mL,
respectively.
Intra-
and
inter-day
precisions
and
accuracies data met the acceptance criteria of FDA guideline for bioanalytical method validation. The developed method has been successfully applied to a bioequivalence study in healthy Chinese volunteers. Keywords: LC-MS/MS; Agomelatine; 7-desmethyl-agomelatine; 3-hydroxy agomelatine; Metabolite; Plasma; Bioequivalence
1. Introduction Agomelatine is a specific agonist of MT1 and MT2, and a selective antagonist of 5-HT2c receptors[1, 2]. It is a napthalenic compound chemically designated as N-[2-(7-Methoxy-1-naphthyl)ethyl] acetamide (Fig.1) with a selectivity of large than 100-fold for MT1 and MT2 receptors with no significant affinities to muscarinic, adrenergic, dopaminergic or histaminergic receptors, which has strong effects on improving depression and fewer adverse reactions[3, 4]. Agomelatine has a short half-life of about 2 hours in human beings. It is rapidly absorbed from the gastrointestinal tract and immediately transported to the liver[5], where it is extensively metabolized by the P450 (CYP) isoenzymes namely CYPA1, CYPA2, and CYP2C9. 7-desmethyl-, 3-hydroxy-, and 3-hydroxy-7-desmethyl-agomelatine (Fig.1) were identified as the three metabolites of agomelatine, which have less activity than the parent drug[6-8]. For new generic agomelatine products with clinical trial permission of China Food and Drug Administration (CFDA), bioequivalence (BE) evaluation is one of the pivotal clinical studies required in support of its marketing application[9]. Our preliminary BE experiment indicated that the within-subject variability of AUC and Cmax were 53% and 70% for agomelatine, 21.2% and 37.8% for
3-hydroxy-agomelagtine, 42.6% and 61.4% for 7-desmethyl-
agomelatine. In addition, the AUC in plasma of 3-hydroxy-agomelagtine and 7-desmethyl-agomelatine were found about 15-fold and 3/4 of that of
agomelagtine. Due to the smaller within-subject variability and high in vivo concentrations of 3-hydroxy-agomelagtine and 7-desmethyl-agomelatine, these two metabolites were selected as auxiliary index for BE assessment in the present study, which can provide ample information for the BE study, thus the in vivo data of these two metabolites are needed. To our knowledge, several LC-MS/MS methods for the determination of agomelatine in human plasma have been published[10-12]. Two of the reported bioanalytical methods[10, 11] employed a liquid-liquid extraction procedure and a time-costing evaporation process, and the linear ranges were relatively narrow, which are not adequate for the determination of this high variability drug. In addition, only the parent drug agomelatine was determined in these two reported LC-MS/MS methods. Ogawa T, et al [12]reported a UPLC-MS/MS method for determination of ramelteon, agomelatine, and melatonin in human plasma, and it employed tedious and costly sample preparation technique (solid-phase extraction) and timeconsuming evaporating procedure. Up to date, there were no reports described a method for the simultaneous determination of agomelatine and its major metabolites using LC-MS/MS. In the present study, we developed and fully validated a simple, rapid and sensitive enough LC-MS/MS method for the simultaneous determination of agomelatine, 7-desmethyl-agomelatine and 3hydroxy-agomelatine in human plasma for the first time. This assay employed a simple protein precipitation procedure and obtained high extraction
recoveries and wide linear ranges, which was successfully applied to a bioequivalence study in healthy Chinese volunteers. 2. Experimental 2.1 Chemicals and reagents Reference standards of agomelatine (purity 99.5%), 7-desmethylagomelatine (purity 98.2%) and 3-hydroxy-agomelatine (purity 98.0%) were all provided by Chongqing Fuke pharmaceutical group Co., Ltd (Chongqing, China). Reference standard of phenacetin (internal standard, IS, purity 99.8%) was purchased from National Institutes for Food and Drug Control (Beijing, China). HPLC grade methanol and formic acid were supplied from Merck (Darmstadt, Germany) and Tianjin Kermel Chemical Reagent Co., Ltd (Tianjin, China), respectively. Ammonium formate (analytical grade) and hydrochloric acid (analytical grade) were obtained from Sinopharm Chemcial Reagent Co., Ltd. (Shanghai, China). Sodium metabisulphite (analytical grade) was purchased from Tianjin Fengchuan Chemical Reagent Science And Technology Co., Ltd (Tianjin, China). Blank human plasma was obtained from healthy volunteers with heparin used as anticoagulant. Purified water was made by AHL-2001-P trace and analysis type of ultra-pure water machine (Echo pu, Shanghai, China). 2.2 Chromatographic and mass spectrometric condition An Agilent 6460 triple-quadrupole LC-MS/MS system was employed comprising degasser, binary pump, autosampler, thermostatted column
compartment, electrospray ionization source (ESI) and a triple-quadrupole MS detector.
Data
acquisition
was
performed
with
Agilent
MassHunter
Workstation Software (Agilent, USA). The separation was carried out on a Phenomenex ODS3 column (4.6×150 mm, 5μm, Phenomenex, USA) with a Gemini C18 column (4×3.0 mm, 5 μm, Phenomenex, USA) employed as guard column. The mobile phase was methanol-5 mM ammonium formate solution (containing 0.2% formic acid) in a ratio 70:30 (v/v), which was delivered at a flow rate of 0.8mL/min. The injection volume was 5 μL and the column temperature was maintained at 35°C. The mass spectrometer was equipped with electrospray ionization source and operated in positive ion mode using multiple reactions monitoring (MRM). The mass transition ion pairs were selected as m/z 244.1→185.1, m/z 230.1→171.1, m/z 260.1→201.1 and m/z 180.1→110.1 for agomelatine, 7desmethyl-agomelatine, 3-hydroxy-agomelatine and phenacetin, respectively. The other optimized MS/MS parameters were as follows: fragmentor voltage 90V for agomelatine, 50V for 7-desmethyl-agomelatine and 3-hydroxyagomelatine, and 70V for phenacetin, collision energy (CE) 10eV for agomelatine and 3-hydroxy-agomelatine, 15eV for 7-desmethyl-agomelatine and 20eV for phenacetin, Gas Temp，350°C；Gas Flow 8L·min-1, Nebulizer 45psi, Sheath Temp 350°C, Sheath Gas Flow 11 L·min-1, and Capillary 4000V. 2.3 Preparation of stock reference solutions and standard plasma
samples The stock reference solutions containing agomelatine (1mg·mL-1), 7desmethyl-agomelatine (1mg·mL-1) and 3-hydroxy-agomelatine (1mg·mL-1) were separately prepared in methanol. Stock solution of IS (1 mg·mL-1) was prepared in methanol, and was further diluted with methanol to prepare the IS working solution at the concentration of 100 ng/mL. Eight
standard
agomelatine
and
working
solutions
3-hydroxy-agomelatine
of
agomelatine,
were
serially
7-desmethylprepared
by
appropriately mixing these three analytes stock solutions at the beginning and then appropriately diluting the mixture solution with methanol, which contains agomelatine at the concentrations of 1000, 333.3, 111.1, 37.04,12.35, 4.115, 1.372 and 0.4572 ng/mL, 7-desmethyl-agomelatine at the concentrations of 3000, 1000, 333.3, 111.1, 37.04, 12.35, 4.115 and 1.372 ng/mL, and 3hydroxy-agomelatine at the concentrations of 10000, 3333, 1111, 370.4, 123.5, 41.15, 13.72 and 4.572 ng/mL. A 20μL of working standard solution was added to 180μL of blank plasma for preparing calibration standard samples. The agomelatine,
calibration
curves
300~0.1372ng/mL
ranges were for
100~0.0457ng/mL for
7-desmethyl-agomelatine,
and
1000~0.4572ng/mL for 3-hydroxy-agomelatine, respectively. Quality control (QC) samples were independently prepared in the same way at three concentration levels (0.1097, 3.704 and 80ng/mL for agomelatine, 0.3292, 11.11 and 240ng/mL for 7-desmethyl-agomelatine, 1.097, 37.04 and
800ng/mL for 3-hydroxy-agomelatine). The stock and working solutions were all stored at 4°C, and the IS working solution and the calibration standards were freshly prepared for each analytical run. 2.4 Sample preparation Each 200μL human plasma sample was added with 20μL sodium metabisulphite solution (20g·L-1) and vortex-mixed for 30s. Subsequently, 30μL IS working solution (100ng/mL) was added into the plasma sample and vortex-mixed for 30 s, then the plasma sample was precipitated with 600μL 15% hydrochloric acid methanol solution, vortex-mixed for 5 min. The treated plasma samples were centrifuged at 15700×g for 10 min, then 5μL of supernatant was injected to the LC-MS/MS system.
2.5 Method validation 2.5.1 Specificity The specificity is the ability of an analytical method to differentiate and quantify the analytes in the presence of other components in the sample. Specificity was assessed by comparing the chromatograms of extracted blank plasma from six different healthy volunteers with the corresponding plasma samples added with agomelatine, 7-desmethyl-agomelatine, 3-hydroxyagomelatine and IS. 2.5.2 Linearity and lower limit of quantification (LLOQ) The linearity of the calibration curves for agomelatine and its two
metabolites were assessed by analyzing eight standard samples in which the concentrations ranged over 0.0457~100 ng/mL, 0.1372~300 ng/mL and 0.4572~1000 ng/mL for agomelatine, 7-desmethyl-agomelatine and 3hydroxy-agomelatine,respectively. The calibration curves were established through weighed linear least-squares regression (1/X2 weighing) of the peak area ratios (Y) of the analytes to the IS versus the respective standard concentrations (X), respectively. The acceptance criterion for each backcalculated standard concentration was ±15% deviation from the nominal value, except at LLOQ. The LLOQ was defined as the lowest point on the calibration curve. Six different standard plasma samples at this concentration level should be quantified with less than ±20% bias of the nominal value in accuracy and less than 20% relative standard deviation (RSD) in precision while providing a signal-to-noise ratio ≥10. 2.5.3 Precision and accuracy Precision and accuracy were evaluated by analyzing standard plasma samples at three concentration QC levels (0.1097, 3.704 and 80 ng/mL for agomelatine, 0.3292, 11.11 and 240 ng/mL for 7-desmethyl-agomelatine, 1.097, 37.04 and 800 ng/mL for 3-hydroxy-agomelatine) on five replicates of each level for three batches successively. The concentration of each sample was determined using the calibration curve prepared on the same batch. Precision was expressed using the relative standard deviation, and accuracy
was defined as the relative deviation in the determined concentration of a standard from that of its nominal concentration expressed as a percentage. The precisions were required within 15%, and accuracies were required not to exceed ±15%. 2.5.4 Recovery and matrix effect The extraction recoveries of agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine
were
determined
at
corresponding
three
QC
concentrations respectively by comparing the peak areas that were extracted from plasma samples with peak areas of these three analytes reconstituted in blank plasma extracts at the equivalent concentrations. The recovery of the IS was evaluated at 100 ng/mL. To evaluate the matrix effects, blank human plasma samples were processed according to the sample preparation procedure described above and then spiked with analytes and IS at the final concentration after extraction. The matrix effects of the plasma were expressed as the ratio of the mean peak area of analytes spiked postextraction to that of the neat standard solution with mobile phase at the same three concentration levels. When the ratios were within 85-115%, the matrix effects were considered as negligible. 2.5.5 Stability Stability tests for agomelatine, 7-desmethyl-agomelatine and 3-hydroxyagomelatine were performed using analytes-spiked plasma samples under various conditions: ambient temperature storage (at 22°C for 4 h), through
three freeze/thaw cycles (from -40°C to room temperature), and for long-term storage (frozen at -40°C for 90 days), post-preparative storage (at 4°C for 24 h) by analyzing three replicates at low, medium and high QC concentrations, respectively. 2.6 Pharmacokinetic study This method was successfully applied to determine the plasma levels of agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine from a bioequivalence study involving 44 healthy Chinese volunteers aged 18-26 years and weighed 53-72 kg. The study design was a randomized, openlabel, two-treatment and four-period crossover study, and each subject was fasted and orally administered 25 mg test or reference formulation according to the design. The test formulation was agomelatine tablet supplied from Chongqing
Fuke
pharmaceutical
group
Co.,
Ltd
and
VALDOXAN®
manufactured by pharmaceutical company Servier as the reference formulation. The ethics committee approved the protocol and the volunteers provided with informed written consent. A total of 15 blood samples (5mL each) were collected into heparinized polypropylene tubes at 0 (pre-dose), 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7 and 8 h after administration. Plasma was separated by centrifugation at 3000×g for 10 min and stored at -40°C until analysis. The pharmacokinetics parameters were acquired and analyzed by a non-compartmental model performed on Phoenix WinNonlin 6.1 software.
3. Results and discussion 3.1 Method development As to mass spectrometric detection, both positive and negative MRM modes were tested, and positive MRM mode was chosen over negative MRM mode because of its improved signal to noise ratio (S/N) for analytes in extracted samples. In the full scan Q1 mass spectrum, the most abundant ions were the protonated molecular ions [M+H]+ found at m/z = 244.1, 230.1, 260.1 and 180.1 for agomelatine, 7-desmethyl-agomelatine, 3-hydroxyagomelatine and IS, respectively. The corresponding most prominent product ions were shown at m/z = 185.1, 171.1, 201.1 and 110.1, respectively. All mass spectrometric parameters were optimized for optimal intensity of the protonated molecular ions and productions. Subsequently, the mass transitions were monitored at m/z 244.1→185.1, 230.1→171.1, 260.1→201.1 and 180.1→110.1 for agomelatine, 7-desmethyl-agomelatine, 3-hydroxyagomelatine and IS, respectively. The addition of 0.2% formic acid in the mobile phase was found to give higher MS response as it favors ionization. Compared with liquid-liquid extraction, protein precipitation was found to offer satisfactory recovery and desired extraction efficiency in determining the levels of the analytes. There is a phenolic hydroxyl group in both 7-desmethyl-agomelatine and 3-hydroxyagomelatine, thus 20μL sodium metabisulphite solution (20g·L-1) was added during the protein precipitation process to prevent the oxidation of phenolic
hydroxyl group based on our previous bioanalytical experience. Despite using methanol solution (~600µL) as protein-precipitant, the extraction efficiency couldn't be improved. Therefore, stronger precipitating agent like hydrochloric acid was used for harvesting the plasma. Several amounts of hydrochloric acid dissolved in methanol solution were investigated to effectively extract the analytes from plasma, and eventually 600μL 15% hydrochloric acid methanol solution gave the desired extraction efficiency. 3.2 Specificity The typical chromatograms of blank plasma, blank plasma spiked with analytes and IS are shown in the Fig.2A and Fig.2B. The retention times of agomelatine, 7-desmethyl-agomelatine, 3-hydroxy-agomelatine and IS were approximately 5.5 min, 3.4min, 3.1min and 3.1 min, respectively. No obvious interferences from endogenous substance were observed. Other typical chromatograms are presented in Fig. 2C. 3.3 Linearity and lower limit of quantification (LLOQ) The calibration curves for agomelatine, 7-desmethyl-agomelatine and 3hydroxy-agomelatine
were
linear
over
the
concentration
ranges
of
0.0457~100 ng/mL, 0.1372~300 ng/mL and 0.4572~1000 ng/mL in human plasma respectively, and the mean regression equations of the calibration curves for these analytes were Y=0.1188X-0.0005 (r2=0.9962), Y=0.0734X0.0003 (r2=0.9975), and Y=0.0543X-0.0007 (r2=0.9978). The LLOQ for agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in plasma
were 0.0457 ng/mL, 0.1372 ng/mL and 0.4572 ng/mL respectively on the basis that the accuracies were 102.1%, 92.9% and 86.2%, and the precisions were 7.5%, 8.1% and 3.8%, respectively. 3.4 Precision and accuracy The intra- and inter-day precisions and accuracies for agomelatine, 7desmethyl-agomelatine and 3-hydroxy-agomelatine are presented in Table 1. As shown, all the intra- and inter-day precisions were less than 6.6% and accuracies were at the range of 90.2%~105.1%. The data demonstrated that the precisions and accuracies of this method were within the acceptable range. 3.5 Recovery and matrix effect The mean extraction recoveries for the analytes at corresponding low, medium and high QC concentration levels were 88.5%, 87.7% and 90.7% for agomelatine, 99.5%, 91.4% and 95.3% for 7-desmethyl-agomelatine, and 104.6%, 99.9% and 100.3% for 3-hydroxy-agomelatine, while the mean recovery of IS was 93.6%. These results indicated that the analytes showed satisfactory extraction recoveries and not significant concentration dependent. The average matrix effects (%) for the analytes at corresponding low, medium and high QC concentration levels were found to be 112.1%, 102.7% and 107.5% for agomelatine, 107.0%, 103.3% and 108.0% for 7-desmethylagomelatine, and 104.6%, 110.1% and 111.7% for 3-hydroxy-agomelatine. The average matrix effects of IS was 105.3%. These results indicated that
matrix effects were similar and not significant concentration dependent, thus not an issue in the current method. 3.6 Stability In terms of stability, all the results had accuracy values ranged from 89.4% to 111.8% with RSD% ≤ 12.4% (shown in Table 2), indicating that agomelatine,
7-desmethyl-agomelatine and 3-hydroxy-agomelatine were
stable in human plasma and processed samples under the conditions described above. 3.7 Incurred samples reanalysis(ISR) Sixty clinical study samples for agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine were selected for the incurred samples reanalysis study plan. The acceptance criteria specify that the percent differences, i.e. 100×(repeat value−original value)/the mean of original value and repeat value, of at least 2/3 of all the analyzed ISR samples should be within ±20%. The percent differences ranged from −4.72 to 3.23% with the average of −0.37% for agomelatine, from −5.67 to 9.21% with the average of 2.82% for 7-desmethyl-agomelatine and from −6.17 to 5.34% with the average of 2.43% for 3-hydroxy-agomelatine. The ISR results for all analytes demonstrated this method was reproducible and robust. 3.8 Pharmacokinetic study All the subjects completed the study and were incorporated in the pharmacokinetics analysis. The average concentration-time profiles of
agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma are presented in Fig.3. Peak concentrarion (Cmax) and time to peak concentration (Tmax) were directly obtained from experimental observations. The main pharmacokinetics parameters of agomelatine, 7-desmethylagomelatine and 3-hydroxy-agomelatine obtained from 44 Chinese subjects administered 25mg test or reference agomelatine tablet are summarized in Table 3. The mean Tmax and t1/2 of reference tablet (Valdoxan) obtained in this study were similar to those values (1h for Tmax and 0.9h for t1/2) of CHMP Assessment Report for Valdoxan [13]and reported values (1.1h for Tmax and 1.1h for t1/2) in healthy Chinese volunteers by Wang X, et al[11]. The mean Cmax and AUCinf in this study were 1.9-fold and 1.7-fold higher than those values (3.0ng/mL for Cmax and 4.9ng•h/mL for AUCinf) of CHMP Assessment Report for Valdoxan[13], however, the Cmax and AUCinf were much lower compared with the reported values (15.1ng/mL for Cmax and 16.4ng•h/mL for AUCinf) by Wang X, et al[11]. The mean Cmax for agomelatine, 7-desmethyl-agomelatine and 3hydroxy-agomelatine of test formulation were 1.32, 1.15 and 1.17 fold respectively higher than those of reference formulation. The bioequivalence acceptance criteria for Cmax, AUClast and AUCinf of agomelatine are 48.99%~204.13%, 58.48%~170.99% and 61.38%~162.91% respectively based on reference-scaled average bioequivalence approach[14]. The 90%
confidence intervals of the ratios of means Cmax, AUClast and AUCinf for agomelatine
were
104.42%~139.86%,
101.33%~123.83%
and
97.90%~117.94%, respectively, which all falls within the corresponding acceptance range thus the test formulation was considered bioequivalent to the reference formulation.
4. Conclusion A rapid, simple and sensitive LC-MS/MS method for the simultaneous determination of agomelatine, 7-desmethyl-agomelatine and 3-hydroxyagomelatine in human plasma was developed and fully validated for the first time on the basis of specificity, linearity, sensitivity, precision and accuracy, recovery, matrix effect and stability. The method provided satisfactory sensitive, high extraction recoveries, and wide linear ranges, which is suitable for the analysis of this high variability drug. The method was successfully applied to a bioequivalence study in healthy Chinese volunteers.
Acknowledgements This research was supported by National Science and Technology Major Projects for "Major New Drugs Innovation and Development" (No. 2012ZX09303014-001, Beijing, China), and the National Natural Science Foundation of China (Beijing, China) for Project No. 81273585.
Reference [1] S. Yous, J. Andrieux, H.E. Howell, P.J. Morgan, P. Renard, B. Pfeiffer, D. Lesieur, B. Guardiola-Lemaitre, J Med Chem, 35 (1992) 1484-1486. [2] M.J. Millan, A. Gobert, F. Lejeune, A. Dekeyne, A. Newman-Tancredi, V. Pasteau, J.M. Rivet, D. Cussac, J Pharmacol Exp Ther, 306 (2003) 954-964. [3] D. Eser, T.C. Baghai, H.-J.U.R. M O Ller, Core evidence, 4 (2009) 171. [4] B. Green, Current Medical Research \& Opinion, 27 (2011) 745--749. [5] V. Srinivasan, D.P. Cardinali, S.R. Pandi-Perumal, G.M. Brown, Journal of Experimental and Integrative Medicine, 1 (2011) 149--158. [6] J.J.P. Bogaards, E.M. Hissink, M. Briggs, R. Weaver, R. Jochemsen, P. Jackson, M. Bertrand, P.J. van Bladeren, European Journal of Pharmaceutical Sciences, 12 (2000) 117 - 124. [7] S. Conway, S.J. Canning, H.E. Howell, E.S. Mowat, P. Barrett, J.E. Drew, P. Delagrange, D. Lesieur, P.J. Morgan, European Journal of Pharmacology, 390 (2000) 15 - 24. [8]
M.
Fornaro,
D.
Prestia,
S.
Colicchio,
G.
Perugi,
Current
neuropharmacology, 8 (2010) 287-304. [9] Q. Pei, Y. Wang, Z.Y. Hu, S.K. Liu, H.Y. Tan, C.X. Guo, R.R. Zhang, Y.X. Xiang, J. Huang, L. Huang, H. Yuan, G.P. Yang, PLoS One, 9 (2014) e109300. [10] S.R. Patil, K.K. Nerurkar, A.M. Kalamkar, V. Pukale, K.V. Mangaonkar, S.G. Pingale, J Mass Spectrom, 47 (2012) 23-28.
[11] X. Wang, D. Zhang, M. Liu, H. Zhao, D. A, L. Meng, H. Liu, Biomed Chromatogr, 28 (2014) 218-222. [12] T. Ogawa, M. Iwai, H. Hattori, K. Zaitsu, T. Kumazawa, A. Ishii, O. Suzuki, H. Seno, Forensic Toxicology, 32 (2014) 126-131. [13] Agency EM. CHMP Assessment Report for Valdoxan. Document Reference EMEA/655251/2008. [14] B.M. Davit, M.L. Chen, D.P. Conner, S.H. Haidar, S. Kim, C.H. Lee, R.A. Lionberger, F.T. Makhlouf, P.E. Nwakama, D.T. Patel, D.J. Schuirmann, L.X. Yu, The AAPS journal, 14 (2012) 915-924.
Figure captions Fig.1 Chemical structures of agomelatine (A), 7-desmethyl-agomelatine (B), 3-hydroxy-agomelatine (C), 3-hydroxy-7-desmethyl-agomelatine (D) and phenacetin (IS) (E). Fig.2 Typical chromatograms of agomelatine, 7-desmethyl-agomelatine and 3hydroxy-agomelatine in human plasma: (A) blank human plasma from healthy volunteers; (B) blank human plasma spiked with agomelatine (0.0457μg·L-1), 7-desmethyl-agomelatine
metabolite
(0.1372μg·L-1)
and
3-hydroxy-
agomelatine (0.4572μg·L-1) and IS; (C) Plasma sample from a healthy
volunteer 4 h after orally administrated of 25mg agomelatine tablet. For each chromatogram, the panels from top to bottom represent 3-hydroxyagomelatine, agomelatine, 7-desmethyl-agomelatine and IS, respectively. Fig.3
Mean plasma concentration-time curves of agomelatine (A), 7-
desmethyl-agomelatine (B) and 3-hydroxy-agomelatine(C) for test (T) and reference (R) formulations in healthy Chinese subjects after orally administrated of 25mg agomelatine tablet (n=44). Each data represents the mean+SD.
Fig.1
A
B
O
N H HO
OH
C
E
D
Fig.2
A
B
C
Fig.3
A
B
C
Table 1 Inter- and intra-day precisions and accuracies of agomelatine, 7-desmethylagomelatine and 3-hydroxy-agomelatine in QC samples of human plasma. Intra-day(n=5) Mean oncentrati Accuracy on (%) found(ng/ mL)
Inter-day(n=15) Mean concentra Accur RS tion acy D found(ng/ (%) (%) mL)
Analytes
Nominal concentration(n g/mL)
agomelat ine
0.1097
0.1046
95.3
3.4
0.1066
97.2
6.6
3.704 80
3.391 84.01
91.5 105.1
2.9 2.1
3.360 82.59
90.7 103.3
2.9 3.3
0.3292
0.3000
91.1
5.1
0.3066
93.1
4.7
11.11
10.18
91.6
2.7
10.12
91.1
2.5
240
249.0
103.8
2.0
245.5
102.3
2.3
1.097
1.007
91.8
3.0
0.9897
90.2
3.9
37.04
34.79
93.9
2.5
34.61
93.4
2.4
800
825.2
103.2
1.8
813.2
101.7
2.7
7desmeth ylagomelat ine 3hydroxyagomelat ine
RS D (%)
Table 2 Stability studies on QC samples for agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine in human plasma. Storage conditions
agomelatine (ng/mL)
7-desmethyl-agomelatine(ng/mL)
0.1097 3.704 80 0.3292 Ambient temperature storage stability (at 22°C for 4 h)
11.11
240
3-hydroxy-agomelatine(ng/mL) 1.097
37.04
800
Mean(ng/mL) 0.1107 3.388 82.06 0.3308 Accuracy (%) 101.0 91.4 102.6 100.5 RSD (%) 4.9 0.8 3.0 4.4 Post-preparative storage stability (at 4°C for 24 h) Mean(ng/mL) 0.1226 4.001 81.37 0.3513 Accuracy (%) 111.8 108.1 101.7 106.8 RSD (%) 1.6 1.5 4.5 5.3 Freeze/thaw stability (from -40°C to room temperature)
9.938 89.4 1.6
241.7 100.7 2.1
1.075 97.9 1.6
34.35 92.7 0.4
795 99.3 2.1
11.57 104.3 2.4
230.7 96 2.5
1.149 104.8 2.4
39.39 106.4 1.7
762.3 95.2 3.5
Mean(ng/mL) 0.1112 3.836 75.57 0.3358 Accuracy (%) 101.4 103.6 94.4 102 RSD (%) 12.4 5.2 0.8 4.2 Long-term storage stability (at -40°C for 90 days)
11.47 103.2 1.5
227.8 94.8 0.5
1.061 96.7 2.9
38.75 104.7 1.3
752.9 94.1 0.2
10.82 97.3 2.1
224.5 93.5 8.6
1.121 102.2 11.1
35.99 97.1 1.5
764.5 95.5 2.1
Mean(ng/mL) Accuracy (%) RSD (%)
0.1109 101.1 1.9
3.766 101.7 0.9
80.77 101.0 7.2
0.3391 103.0 6.3
Table 3 Main pharmacokinetics parameters of agomelatine, 7-desmethyl-agomelatine and 3-hydroxy-agomelatine obtained from 44 Chinese subjects after orally administered 25mg test or reference agomelatine tablet.
Parameters
Cmax(ng/mL) Tmax(h) AUClast (ng•h/mL) AUCinf (ng•h/mL) t1/2(h)
Reference formulation 73desmethyl agomelat hydroxyine agomelat agomelati ine ne 5.74 ± 43.30 ± 3.77 ± 6.91 22.45 2.44 1.22 ± 1.25 ± 1.23 ± 0.86 0.81 0.80 7.99 ± 84.44 ± 5.34 ± 10.15 32.49 2.38 8.31 ± 86.11 ± 5.76 ± 10.23 33.06 2.51 1.58 ± 1.29 ± 1.47 ± 1.32 0.26 0.92
Test formulation 73desmethyl agomelat hydroxyine agomelat agomelati ine ne 7.55 ± 50.09 ± 4.43 ± 10.01 25.45 3.04 1.14 ± 1.13 ± 1.07 ± 0.75 0.72 0.72 8.59 ± 88.30 ± 5.75 ± 10.17 31.81 2.64 8.72 ± 89.78 ± 6.16 ± 10.16 32.27 2.76 1.24 ± 1.24 ± 1.55 ± 1.40 0.24 1.97