Simple and Sensitive LC-MS/MS-Based Assay for the Quantiﬁcation of Dimemorfan in Human Plasma for Use in a Pharmacokinetic Study

Research article Received: 13 April 2014, Revised: 17 July 2014, Accepted: 12 August 2014 Published online in Wiley Online Library: 29 September 2014 (wileyonlinelibrary.com) DOI 10.1002/bmc.3325 Simple and sensitive LC-MS/MS-based assay for the quantification of dimemorfan in human plasma for use in a pharmacokinetic study Hongyi Tana†, Jinfu Penga,b†, Qi Peia, Liu Yanga,b, Jun Chena, Chengxian Guoa, Ye Huaa, Hong Yuana and Guoping Yanga* ABSTRACT: Dimemorfan phosphate has been widely used for 40 years throughout the world for the treatment of coughs. This is the first report on the use of an LC-MS/MS-based assay for the determination of dimemorfan in human plasma using estazolam as an internal standard after one-step protein precipitation with acetonitrile. Chromatographic separation was achieved on a Phenomenex Luna C18 column (3 μm, 50 × 2.0 mm) using a fast gradient method, which involves water and methanol as the mobile phase (both containing 0.1% formic acid). Dimemorfan and estazolam were detected with proton adducts at m/z values of 255.8 → 155.1 and 295.0 → 267.0, respectively, in the selected reaction monitoring positive mode. The linear dy- namic range of the assay was 0.04–5.00 ng/mL. The chromatographic run time for each plasma sample was <5 min. The method was proven to be accurate, precise, and repeatable. Finally, the developed method was successfully applied for the determination of dimemorfan in a pharmacokinetic study using healthy Chinese subjects. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: dimemorfan; LC-MS/MS; method validation; human plasma; pharmacokinetics Introduction be costly and time consuming; (b) the LLOQ (lower limit of quan- titation, 1.00–2.50 ng/mL) fails to meet the requirements for use fi Dimemorfan phosphate was rst developed in Japan in 1974 un- in a low-dose pharmacokinetic study; and (c) the long chromato- der the trade name Astomin and then subsequently in Italy and graphic run time (>8.75 min) limits the throughput required for Spain. Dimemorfan phosphate (d-3-methyl-N-methylmorphinan use in a pharmacokinetic study. Therefore, we have developed a phosphate, Fig. 1) directly acts on the cough center in the novel LC-MS/MS-based assay for dimemorfan, which is more medulla, has an antitussive effect and is more potent than dex- sensitive, convenient and efficient than the GC/MS-based assays tromethorphan and codeine. In addition, the adverse reactions (Shigeru et al., 1984; Hasegawa et al., 2012). Finally, the newly associated with the utilization of dimemorfan phosphate are validated LC-MS/MS method was successfully applied to the de- relatively mild, for example, it does not produce drug depen- termination of dimemorfan phosphate in a pharmacokinetic dency (Ida, 1997). In addition, dimemorfan is considered a study on healthy Chinese subjects. sigma-1 receptor antagonist and also affects the NMDA receptor and L-type calcium channels. These cause dimemorfan to have significant anti-inflammatory (Wang et al., 2008), anticonvulsant Materials and experiments (Shin et al., 2004, 2005; Chou et al., 1999) and anti-amnesic (Wang et al., 2003) effects in rodents as well as preventing ische- Materials and reagents mic strokes and having neuroprotective potential (Shen et al., Dimemorfan was obtained from the Sichuan GuoRui Pharma- 2008; Shin et al., 2011). When healthy adult males were orally ceutical Co. Ltd (Sichuan, China). Estazolam (internal standard, given 90 mg of dimemorfan phosphate, peak concentrations of IS) was obtained from the National Institutes for Food and Drug – – dimemorfan (Cmax, 7.00 8.00 ng/mL) were achieved 1 2 h after Control of China. All of the compounds were of 99.9% purity. dosing, 60% of the dosed drug was excreted in the urine in Acetonitrile and methanol of HPLC grade were purchased from 24 h, and <2% was excreted in its intact form (Ida, 1997). Dimemorfan was metabolized by hepatic CYP3A4 and CPYP2D6 to form two major metabolites, including d-3-hydroxymethyl-N- * Correspondence to: G. Yang, Center of Clinical Pharmacology, Third methylmorphinan and d-3-methylmorphinan (Chou et al., 2010). Xiangya Hospital, Central South University, Changsha, Hunan 410013, CYP3A plays a major role in the demethylation of dimemorfan China. Email: [email protected] (Chou et al., 2005). † The first two authors contributed equally to this work. The reported analytical methods available for the determination of dimemorfan in samples are mainly based on GC/MS a Center of Clinical Pharmacology, Third Xiangya Hospital, Central South Uni- (Shigeru et al., 1984; Hasegawa et al., 2012). There are several dis- versity, Changsha, Hunan, 410013, China advantages to these assays, including the following: (a) the utili- b School of Pharmaceutical Sciences, Central South University, Changsha, Hu- 647 zation of solid-phase extraction to treat the samples, which can nan, 410013, China Biomed. Chromatogr. 2015; 29: 647–653 Copyright © 2014 John Wiley & Sons, Ltd. H. Tan et al. modified directly from the method reported by Hu et al. (2011, 2013). The fast gradient method is composed of the following steps: begin with a 95% water mobile phase and hold for 0.5 min; change to a 20% water mobile phase and hold for 1.5 min; increase the percentage of the water in the mobile Figure 1. Chemical structure of dimemorfan phosphate. phase from 20 to 95% in 0.01 min; and finally, stop the gradient at 4.5 min. Merck Co. Ltd (Germany). We purchased HPLC-grade formic acid Mass spectrometry conditions. An AB Sciex API 4000 mass from ROE Scientific Inc. (Newark, USA). Deionized water was pre- spectrometer (Toronto, Canada) with a Turbo V electrospray ion- pared with a SMART ultra-pure water system (Shanghai Canrex ization source was used. Dimemorfan and the IS were detected Analytic Instrument Co. Ltd, Shanghai, China). All of the other in positive ion mode. The selected reaction monitoring transi- chemicals and solvents were of analytical grade. Dimemorfan tions were performed at m/z values of 255.8 → 155.1 for phosphate syrup (100 mL:0.25 g, Sichuan GuoRui Pharmaceutical dimemorfan and 295.0 → 267.0 for the IS. The declustering po- Co. Ltd, China) was used in the pharmacokinetic study. Blank hu- tential (DP), collision energy (CE), entrance potential and colli- man plasma samples were collected from healthy volunteers sion exit potential were 85, 47, 10 and 15 V and 84, 35, 10 and and stored at À80°C. 15 V for dimemorfan and the IS, respectively. The curtain gas, ion source gas 1 and ion source gas 2 pressures were 25, 45 Analytical procedure and 55 psi, respectively. The ion spray voltage was set to 5500 V, and the source temperature was maintained at 500°C. Preparation of stock solutions, calibration standards and AB Sciex Analyst software (version 1.4) was used to control the quality control samples. Two samples of dimemorfan (solid) system and perform the data analysis. were weighed separately by different individuals; one of them was used to prepare the stock standard solutions of the plasma calibration standards, and the other was used to prepare the Method validation stock standard solutions of the quality control (QC) plasma The assay performance was assessed with regard to the specific- samples. Stock standard solutions of dimemorfan and the IS ity, LLOQ (0.04 ng/mL), matrix effects, recovery, linearity, preci- were prepared in methanol to achieve concentrations of sion, accuracy and stability. 1.00 × 106 ng/mL for dimemorfan and 50.00 ng/mL for the IS, and the stock standard solutions were stored at 4°C. Then appro- Specificity. The specificity of the method was measured by priate dilutions of the stock solutions were made to produce analyzing blank human plasma samples from six different do- working stock solutions (0.39–50.00 ng/mL) in methanol–water nors, blank plasma spiked with the IS and a plasma sample col- (1:1, v/v), which were used to prepare plasma calibration stan- lected at 12 h after a single oral administration of dimemorfan dards according to the following method. A 20 μL sample of syrup to a volunteer. μ each working standard solution was mixed with 200 L of blank Matrix effects and extraction recoveries. The matrix effects human plasma to obtain concentrations of 0.04, 0.08, 0.16, 0.31, and extraction recoveries were determined at three QC levels 0.63, 1.25, 2.50 and 5.00 ng/mL. Quality control plasma samples (0.08, 1.25 and 4.00 ng/mL, n = 6) for dimemorfan and at (0.08, 1.25 and 4.00 ng/mL) were also prepared using the 5.00 ng/mL for the IS. The matrix effects were measured by com- same procedure. paring the peak areas of the analyte (A) added to post-protein- Sample preparation. Plasma samples were prepared by pro- precipitated blank plasma (from six different batches) with those tein precipitation. Aliquots of 20 μL of the IS (50.00 ng/mL) were of the pure standard solution containing equivalent amounts of added to 220 μL plasma samples and mixed. Then, a 400 μL vol- the compound (B). The ratio (A/B × 100)% was used to indicate ume of acetonitrile was added. After a thorough vortex mixing the matrix effects. Additionally, according to the European fi for 2 min and centrifugation at 15,700g for 10 min, 20 μL aliquots Medicines Agency, the variable coef cient (CV) of the IS- of the extracted supernatant samples were immediately sub- normalized matrix effects calculated from the six different jected to LC-MS/MS analysis. batches of matrix should not be greater than 15% (European Medicines Agency, 2011). The extraction efficiency was also measured by the equation Instrumentation and LC-MS conditions (A′/B′ × 100)%, where A′ stands for the peak area of the analyte added to blank plasma and then pretreated by protein precipita- Chromatographic conditions.

Load more