Research article Received: 27 June 2014,

Revised: 16 December 2014,

Accepted: 6 January 2015

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bmc.3440

A validated sensitive HPLC-MS/MS method for quantification of a potential hypnotic drug MT502 and its application to a pharmacokinetic study in rat Fang-Fang Zhang, Ying Cheng, Ning Wan, Zi-Wei Jing, Jia Ju, Yi-Yang Jia, Si-Yuan Zhou and Bang-Le Zhang* ABSTRACT: A rapid, sensitive HPLC-MS/MS method was established and validated to assay the concentration and pharmacokinetic profile of MT502, a promising hypnotic drug. The plasma sample was treated by a liquid–liquid extraction and separated on a kromasil C18 column at an isocratic flow rate of 0.3 mL/min using methanol and 0.1% formic acid in water (75:25, v/v) as mobile phase. The mass spectrometric detection was carried out using a triple-quadrupole system via positive electrospray ionization. Multiple reaction monitoring was used for quantitation of m/z transitions from 261 to 188 for MT502 and from 247 to 188 for MT501 (internal standard). Good linearity was achieved over the concentration range of 1–1000 ng/mL and 10–5000 ng/mL with lower limit of quantification of 0.30 and 0.80 ng/mL. The intra- and inter-day precisions, accuracy, recovery and stability were satisfactory for the concentration test. The above method can be used for a pharmacokinetic study at doses of 1, 5 and 20 mg/kg. Results indicated that MT502 had rapid absorption, rapid elimination and linear pharmacokinetic properties within the range of the tested intragastric dose. This developed HPLC-MS/MS method was successfully applied to a pharmacokinetic study of MT502 for the first time and was demonstrated to be simple and sensitive. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: melatonin derivatives; HPLC-MS/MS; pharmacokinetics; rat

Introduction Melatonin is a natural neurohormone that was discovered more than 50 years ago (Lerner et al., 1958). It is synthesized primarily in the pineal gland, which plays an important role in regulating several biological functions, such as sleep, mood, tumor growth and aging (Brzezinski, 1997). It has been suggested that melatonin and its derivatives might have a variety of clinical applications as a supplement to combat the delayed sleep phase syndrome (Oldani et al., 1994; Mulchahey et al., 2004; Skocbat et al., 1998; Rodenbeck et al., 1998), jet lag (Arendt et al., 1997) and seasonal disorders (Rosenthal et al., 1986), and as a hypnotic agent in modulation of the sleep–wake cycle (Dollins et al., 1994; Garfinkel et al., 1995; Reid et al., 1996). It can retard the aging process (Bonilla et al., 2002) and prevent the formation of cancers (Reiter et al., 1999; Blask et al., 1999). Owing to its low concentration and the co-existence of many other endogenous compounds in blood, the determination of melatonin and its derivatives has been an analytical challenge (Kane et al., 1994; Dollins et al., 1994; Bechgaard et al., 1997). The dose and circulating level of melatonin or its derivatives are often very low (Lewy et al., 1980; Waldhauser and Dietzel, 1985; Cheung et al., 2006; Mistraletti et al., 2010; Wang et al., 2011). To determine low levels of melatonin and its derivatives in blood to characterize its pharmacokinetics and overcome the existing analytical problems in terms of large sample volume, complex sample preparation procedures and analytical sensitivity, an ultra-high-sensitivity bioanalytical method is required. The analytical method should also have the advantages

Biomed. Chromatogr. 2015

of high speed, low time consumption, better resolution and sensitivity. MT502 {Chemical name N-[1-(5-methoxy-1H-indol-3-yl)propan2-yl]propionamide, Fig. 1} is a promising drug among melatonin derivatives (Zhang et al., 2010; Wan et al., 2013a, b) screened previously by our group that has better pharmacological activities in modulation of the sleep–wake cycle. In light of the potent hypnotic activity, the investigation of the quantitative determination and pharmacokinetic profile of MT502 is very important for the further drug research and development. For this purpose, a rapid and sensitive high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and used to assay the concentration and pharmacokinetic profile of MT502.

Experimental Chemicals and reagents MT502 and MT501 were synthesized by the School of Pharmacy in the 13 Fourth Military Medical University (Xi’an, China) and confirmed by C 1 NMR, H NMR and mass spectrometry (MS). According to 2010 reference substance in the drug registration (standard) relevant technical requirements, the purity of MT502 (batch no. 20130703, C15H20N2O2, MW

* Correspondence to: Bang-Le Zhang, Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi’an, 710032, People’s Republic of China. Email: [email protected] Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi’an, 710032, People’s Republic of China

Copyright © 2015 John Wiley & Sons, Ltd.

F.-F. Zhang et al.

Figure 1. Structure of melatonin and its derivatives.

260.33) is 99.80%. MT501 (batch no. 20120423, C14H18N2O2, MW 246.30, 100% purity by HPLC), an analogue of MT502, was used as an internal standard (IS). Deionized water was obtained from a Milli-Q water purification system (Millipore, Bedford, USA). Methanol of HPLC grade was purchased from Fisher (Fair Lawn, USA). Formic acid of HPLC grade was purchased from TEDIA (Fairfield, USA). Other chemical and reagents were of analytical grades.

Animals Adult male and female Sprague–Dawley rats (weight 200 ± 20 g) were purchased from the Experimental Animal Center of the Fourth Military Medical University (Shaanxi, China) and acclimated in the laboratory for 1 week prior to the experiments, housed in separate cages at a temperature of 23 ± 2°C with a 12 h light–dark cycle and a relative humidity of 50%, free access to standard diet and water. All of the rats were fasted for at least 12 h before the experiments with free access to water. All experiments were conducted according to the institutional guidelines with the protocol approved by the Committee on the Use of Live Animals in Teaching and Research (Fourth Military Medical University).

45°C under a N2 stream. The residue was dissolved in methanol (100 μL) by vortex-mixing for 30 s and centrifuged at 3000 rpm for another 10 min. The supernatant was transferred to an autosampler vial, and then 10 μL of the solution was injected into the analytical column separately for HPLC-MS/MS identification. When a higher concentration calibrator (1000–5000 ng/mL) was further added to the 1–1000 ng/mL calibration curve, it became nonlinear. In addition, it could not meet the requirements of precision, accuracy and recovery by trying to dilute the high concentration samples to the range 1–1000 ng/mL. For preparation of calibration curve at the dose of 20 mg/kg, a linear range of 10–5000 ng/mL was needed in the test of drug concentrations. In order to obtain the calibration curve, appropriate working solutions of MT502 (50 μL) and IS solution (50 μL of 4 μg/mL) were added to the blank plasma. The concentration points were equivalent to 10, 50, 100, 500, 1000 and 5000 ng/mL, respectively. After extraction and evaporation as above, the residue was immediately reconstituted in methanol (200 μL) by vortex-mixing for 30 s and centrifuged at 3000 rpm for another 10 min. The supernatant was transferred to an autosampler vial, and 10 μL was injected into the HPLC-MS/MS system. All validations of accuracy, precision and recovery as well as sample stability were determined at the concentration of low, medium and high quality control (QC) samples. The three different concentrations were 2, 100 and 800 for doses of 1 and 5 mg/kg, or 20, 800 and 4000 ng/mL for the dose of 20 mg/kg. The intra- and inter-day precision were expressed as relative standard deviation (RSD) and the accuracy was expressed as relative error (RE). Intra-day precision and accuracy were calculated using replicate (n = 5) determinations for each concentration of the spiked plasma sample during a single analytical run. Inter-day precision and accuracy were calculated using replicate (n = 3) determinations of each concentration for 5 days. Sample stability was investigated under the conditions of measurement at room temperature for 24 h or immediately.

HPLC-MS/MS instrument and analytical conditions The HPLC-MS/MS system was composed of Waters Quattro-premier mass spectrometer and Waters 2695 Separations Module (Waters, Milford, MA, USA). All data were acquired and processed using Masslynx 4.1 Software. The mass spectrometer was run in ESI-MS mode using multiple reaction monitoring to monitor the mass transitions. The ESI–MS data were acquired in positive mode. In order to improve the sensitivity and response of every compound, the source parameters were optimized. After optimization, the multiple reaction monitoring transitions were chosen to be m/z 261 → m/z 188 for MT502 and m/z 247 → m/z 188 for MT501. The collision energy values set for MT502 and MT501 (IS) were 20 and 12 eV, respectively. Other analytical conditions were presented as follows: the mobile phase consisted of methanol and 0.1% formic acid in water (75:25, v/v); the flow rate was set at 0.3 mL/min; the column temperature was kept at 25°C; the injection volume was 10 μL; and the run time was 10 min.

Pharmacokinetic study The above method was further applied to the pharmacokinetic study of MT502 in Sprague–Dawley rat. The rats were administered intragastrically with MT502 at three different doses of 1, 5 or 20 mg/kg. Blood samples were obtained from the retro-orbital plexus of rats (0.5 mL each) under anesthesia with ether and collected in heparinized tube at 0 (pre-dose baseline), 2, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 360, 480 and 600 min after drug administration. The blood samples were centrifuged immediately at 3000 rpm for 10 min. Then the obtained plasma (100 or 50 μL) was spiked with IS solution (50 μL) and methanol (50 μL). The concentrations of MT502 were measured with the method described above. The maximum plasma concentration (Cmax) and the corresponding time (Tmax) were determined directly from experimental data. The other pharmacokinetic parameters were calculated by using the noncompartmental pharmacokinetics data analysis software of DAS version 2.0.

Preparation of standard solutions and calibration curves

Statistical analysis

MT502 and MT501 were dissolved in methanol to achieve a concentration of 1 mg/mL as stock solutions, which were stored at 4°C and protected from light. All working solutions were freshly prepared by serially diluting stock solutions with mobile phase. Samples were quantified by the ratio of the peak area of the analytes to that of internal standard. Peak area ratios were plotted against analyte concentrations, and calibration curves were calculated using weighted 2 (1/x ) least squares linear regression. For doses of MT502 at 1 and 5 mg/kg in rats, the concentration points on the calibration curve were equivalent to 1, 5, 10, 50, 100, 500 and 1000 ng/mL, respectively. The calibration curve was prepared by mixing appropriate working solutions (50 μL), IS solution (50 μL of 1 μg/mL) and 100 μL of blank plasma. The mixture was added 2 mL of ethyl acetate followed by vortex-mixing for 3 min on a cyclomixer (model TME-21; Toyo Seisakusho Company, Japan) and centrifuging (Hitachi Koki Co. Ltd, Japan) at 1000 rpm for 10 min. The supernatant was transferred to glass tube and evaporated to dryness at

Selected pharmacokinetic parameters were calculated from the concentration of MT502 vs time data using DAS2.0 software. Data are expressed as means ± SD and statistical analysis of the results was performed by Student’s t-test. A p-value of

MS method for quantification of a potential hypnotic drug MT502 and its application to a pharmacokinetic study in rat.

A rapid, sensitive HPLC-MS/MS method was established and validated to assay the concentration and pharmacokinetic profile of MT502, a promising hypnot...
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