Research article Received: 17 February 2014,

Revised: 2 June 2014,

Accepted: 22 July 2014

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bmc.3316

Development and validation of a highly sensitive LC-MS/MS method for the determination of dexamethasone in nude mice plasma and its application to a pharmacokinetic study Yin Yuanb, Xuan Zhoub, Jian Lib, Suofu Yea, Xiwei Jib, Liang Lib, Tianyan Zhoua,b* and Wei Lua,b ABSTRACT: In the current study, a simple, sensitive and rapid analytical method for the determination of dexamethasone was developed and applied to a pharmacokinetic study in nude mice. Using testosterone as an internal standard, a liquid chromatography–tandem mass spectrometry (LC-MS/MS) approach after one-step precipitation with acetonitrile was validated and used to determine the concentrations of dexamethasone in nude mice plasma. The method utilized a simple isocratic reverse phase separation over a Dionex C18 column with a mobile phase composed of acetonitrile–water (40:60, v/v). The analyte was detected by a triple quadrupole tandem mass spectrometer via electrospray and multiple reaction monitoring was employed to select both dexamethasone at m/z 393.0/147.1 and testosterone at m/z 289.5/97.3 in the positive ion mode. The calibration curves were linear (r >0.99) ranging from 2.5 to 500 ng/mL with a lower limit of quantitation of 2.5 ng/mL. The relative standard deviation ranged from 1.69 to 9.22% while the relative error ranged from 1.92 to 8.46%. This method was successfully applied to a preclinical pharmacokinetic study of dexamethasone and its pharmacokinetics was characterized by a two-compartment model with first-order absorption in female nude mice. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: LC-MS/MS; dexamethasone; nude mice; pharmacokinetics

Introduction Glucocorticoids (GCs) have been found to have potential in the treatment of various diseases, including chronic inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases, diabetic complications and malignant lympocytes (Karmakar et al., 2013; Schweingruber et al., 2011; Zhang et al., 2014). GCs have also been found to be key regulators of various cellular processes, ranging from metabolism and immune response to cell proliferation and apoptosis (Gong et al., 2008). Dexamethasone (DEX, Fig. 1A), one of the most widely used synthetic glucocorticoids, has been demonstrated to have antitumor efficacy in the MCF-7 xenograft nude mice model as well as in other tumor cell lines in vitro and in vivo (Bavaresco et al., 2007; Gong et al., 2008; Lewis et al., 2013; Villeneuve et al., 2008). These findings suggest that dexamethasone may have a promising future as an anticancer drug in both preclinical and clinical studies. The development of a rapid, simple and accurate analytical method for the determination of drug components in vivo is of great importance in pharmacokinetic studies as well as research on its relationship with pharmacodynamics in specific diseases during the process of drug development. Since the variation between different species may exert an influence on the metabolism of DEX and the xenograft nude mice model is generally used in the studies related to cancer, the pharmacokinetic profile of DEX in nude mice has become essential in its further study as an anticancer drug and its possible combination with other drugs in preclinical studies (Gaignage et al., 1991; Grady et al.,

Biomed. Chromatogr. 2014

2010; Watteyn et al., 2013). However, to the best of our knowledge, no analytical method for the determination of dexamethasone in nude mice plasma has been established. In addition, some LC-MS/MS methods previously reported for the determination of dexamethasone in plasma showed a high lower limit of quantitation (LLOQ) of 20–500 ng/mL (Coimbra et al., 2012; Li et al., 2013; Zhang et al., 2011). Recent studies showed that the application of solid-phase extraction and liquid-liquid extraction might further reduce the LLOQ to some extent, but the complicated procedures for sample preparation still limit the widespread use of these methods (Difrancesco et al., 2007; Yang et al., 2008). The aim of this study was to develop a sensitive and rapid LC-MS/MS method with simplicity in both sample preparation and mobile phase constitution for the quantitative detection of DEX in female nude mice plasma, and to apply this method to its pharmacokinetic study in preclinical research.

* Correspondence to: T. Zhou, School of Pharmaceutical science, Peking University, Beijing 100191, China. Email: [email protected] a

School of Pharmaceutical science, Peking University, Beijing 100191, China

b

State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China Abbreviations used: GCs, glucocorticoids; DEX, dexamethasone; ESI, electrospray ionization.

Copyright © 2014 John Wiley & Sons, Ltd.

Y. Yuan et al. was further diluted to a series of working solutions ranging from 50 to 10,000 ng/mL with acetonitrile and water (40:60, v/v) while the testosterone stock solution was diluted to 800 ng/mL as the final internal standard (IS). All these solutions were stored at 4 °C.

Figure 1. The chemical structures of dexamethasone (A) and testosterone (IS, B).

Materials and methods Reagents and chemicals Dexamethasone was purchased from Sigma-Aldrich Co. (Nanjing, China). Testosterone (Fig. 1B) was purchased from Sigma-Aldrich Co. as the internal standard (IS). HPLC-grade acetonitrile was purchased from Merck Co. Inc. Distilled water was bought from Watsons Group Co. Ltd (Beijing, China). The mobile phase consisted of acetonitrile and water.

Preparation of calibration standards and quality controls The calibration standards were prepared by adding 5 μL of dexamethasone standards and 5 μL of IS (800 ng/mL) in 90 μL of blank plasma obtained from nude mice and the final plasma concentrations of dexamethasone were 0, 2.5, 5, 25, 50, 100, 250 and 500 ng/mL. Three quality control (QC) samples were prepared in the same way and the plasma concentrations of dexamethasone were 5 ng/mL (low), 100 ng/mL (medium) and 500 ng/mL (high), respectively. The mixture was vortexed for 1 min and 200 μL of acetonitrile was added to precipitate the protein. After being vortexed for 1 min, the mixture was then centrifuged at 12,000 rpm for 15 min at 4 °C. A 10 μL aliquot of clear supernatant was injected into the LC-MS/MS system for analysis.

Instruments

Preparation of pharmacokinetic samples

The LC-MS/MS system was composed of a Dionex Ultimate 3000 UPLC system interfaced with an API4000 Q TRAP mass spectrometer (Applied Biosystems Inc., USA) equipped with an electrospray ionization (ESI) source system. The system control and data analysis were executed by Analyst software (Applied Biosystems Inc., USA, version 1.6). Dexamethasone and the internal standard were separated by a reversed-phase Dionex Acclaim PA2 C18 column (50 × 3 mm, 3 μm, Dionex Co., USA). Experimental data was further analyzed by NONMEM 7.1.2 (ICON Development Solutions, USA), Microsoft Excel 2013 (Microsoft Inc., USA) and GraphPad Prism (GraphPad Software, Inc., USA, Version 5.01).

Plasma samples were frozen at 20 °C and thawed at 4 °C before analysis. A 5 μL aliquot of IS was added to 95 μL of plasma samples. Then the plasma samples were further prepared as described in ‘Preparation of calibration standards and quality control’.

Analytical conditions HPLC conditions. A simple mobile phase system composed of acetonitrile and water (40:60, v/v) was pumped at an isocratic flow rate of 0.5 mL/min to elute the analyte. The injection volume was 10 μL and the total running time was 6.5 min for each injection. The samples were kept in dark autosampler vials under 4 °C before determination. MS/MS condition. Multiple reaction monitoring mode was applied to detect the analyte, and the detection parameters optimized by the Analyst software ‘Quantitative Optimization wizard’ were as follows: ESI, positive-ion mode, ion spray voltage, 5500 V; temperature, 450 °C, ion source gas 1 (gas 1), 50 psi; ion source gas 2 (gas 2), 55 psi; curtain gas, 15 psi; collision gas, 2. Dexamethasone and testosterone were determined simultaneously under the above mass conditions. The optimized precursor-to-product ion transition monitored for DEX was m/z 393.0/147.1 with declustering potential 54 V and collision energy 35 eV, while testosterone (IS) was monitored at m/z 289.5/97.3 with declustering potential 95 V and collision energy 34 eV. Preparation of stock and working solutions Stock solutions of dexamethasone and testosterone (IS) were prepared by dissolving the respective standards in acetonitrile and water (40:60, v/v) at the concentrations of 256 and 212 μg/mL, respectively. The dexamethasone stock solution

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Method validation According to the US Food and Drug Administration (FDA) guidance for bioanalytical method validation, QC samples were processed in five replicates at each concentration to validate the selectivity, linearity, precision, accuracy, matrix effects, recovery, stability and elution effect of the established analytical method. The relative standard deviation (RSD) determined at each QC concentration should not exceed 15%, except for the LLOQ and QC samples of low concentration, the RSD of which should not exceed 20% (US Food and Drug Administration, 2001). Specificity. The specificity of the method was investigated by making a comparison between blank plasma of six different nude mice, blank plasma spiked with dexamethasone working solution and IS, and a plasma sample (4 h after administration). The retention time of the above samples were compared to ensure the absence of the interference of endogenous substances, metabolites, decomposition products and concomitant medication, according to the FDA guidelines. Linearity and LLOQ. Linearity was validated by constructing a linear regression of 1/x of the standard curve based on the ratio of the peak area responses of calibration standards and internal standard in triplicate on three separate days. The LLOQ was defined as the lowest concentration that could be quantified with a deviation

MS method for the determination of dexamethasone in nude mice plasma and its application to a pharmacokinetic study.

In the current study, a simple, sensitive and rapid analytical method for the determination of dexamethasone was developed and applied to a pharmacoki...
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