Optimized liquid chromatography–tandem mass spectrometry for Otaplimastat quantification in rat plasma and brain tissue

Journal of Chromatographic Science, 2019, Vol. 57, No. 3, 258–264 doi: 10.1093/chromsci/bmy109 Advance Access Publication Date: 19 December 2018 Article Article Seolhee Lee1, Miri Kim1, Ju-Hee Oh2, Joo Hyun Lee2, Naree Shin1, Taehoon Park1, Ji Hyeon Lee1, Min Chang Kim1, and Young-Joo Lee1,2,* 1 Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemungu, Seoul, 02453, South Korea and 2Division of Biopharmaceutics, College of Pharmacy, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul, 02453, South Korea * Author to whom correspondence should be addressed. Email: Received 29 November 2017; Revised 16 October 2018; Editorial Decision 20 November 2018 Abstract An optimized liquid chromatography–tandem mass spectrometry method for simple and sensitive quantification of Otaplimastat in rat plasma and brain tissue was developed and validated. Protein precipitation with acetonitrile was selected for sample preparation method based on recovery and matrix effect. The chromatographic separation of the sample was performed on a reverse-phase AQ column with an isocratic mobile phase consisting of 10 mM ammonium acetate (pH 4.0) and acetonitrile (50:50, v/v). The analyte was quantified by multiple reaction monitoring with a Waters Quattro micro™ API mass spectrometer. The lower limits of quantification were 20 ng/mL in plasma and 2 ng/g in brain, with the relative standard deviation % of 7.6 and 8.0% for plasma and brain samples, respectively. Acceptable intra-day and inter-day precisions and accuracies were obtained. Otaplimastat was sufficiently stable under all relevant analytical conditions, including a temperature of 4°C for 24 hr, room temperature 20°C for 24 hr, −80°C for 10 days and three freezethaw cycles (each at −80°C for 24 hr), for rat plasma and brain tissue. The validated method was successfully used to measure Otaplimastat concentrations in rat plasma and brain samples. Introduction Otaplimastat (SP-8203, N-[3-(2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)propyl]-N-{4-[3-(2,4-diozo-1,4-dihydro-2H-quinazolin-3-yl) propylamino]butyl}acetamide, Figure 1), discovered in the purified coelomic fluid of live earth worms (Eisenia andrei), is a potential agent for the treatment of cerebral ischemia and is currently under clinical development (clinicaltrials.gov ID: NCT01757795, NCT02787278) (1). Otaplimastat reduced the infarction volume in cerebral ischemia model rat significantly and improved memory deficits, suggesting that Otaplimastat may play be effective treating in brain ischemic injuries (2). To conduct a pharmacokinetic study for drug development, it is important to develop a simple and validated analytical method for bioanalysis using blood plasma and/or organ tissues. However, to date, a validated and sensitive analytical method for Otaplimastat in plasma and its target organ, the brain, has not been reported. Only limited information has been published on a partially validated analytical method for quantification of Otaplimastat in plasma with limited sensitivity using high-performance liquid chromatography (HPLC) coupled with ultraviolet detection (3). With, the lower limit of quantitation (LLOQ) in plasma was reported as 50 ng/mL from 100-μL rat plasma. Also, the sample preparation required excessive organic solvent (dichloromethane) for a rather complicated liquidliquid extraction (LLE) (3, 4). Another researcher has briefly described a HPLC–tandem mass spectrometry (LC–MS/MS) method for Otaplimastat determination in plasma, but adequate information including validation, such as LLOQ and a calibration curve, was not reported (5). © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: 258 Optimized liquid chromatography–tandem mass spectrometry for Otaplimastat quantification in rat plasma and brain tissue 259 Optimized LC-MS/MS method for Otaplimastat Methods such as protein precipitation (PPT), LLE and solidphase extraction (SPE) are commonly used for sample pretreatment. Matrix effect (ME) may suppress or enhance the analyte signal, thereby leading to an inaccurate quantification of the analyte. However, appropriate sample pretreatment methods can reduce MEs and significantly increase the likelihood of obtaining more accurate bioanalytical data (6). In this study, we investigated and compared the reduction of MEs and the improvement of recovery by PPT using acetonitrile (ACN) and trichloroacetic acid (TCA) and by LLE using dichloromethane that was reported previously (3). Consequently, a simple and sensitive LC–MS/MS method for quantitative analysis of Otaplimastat in rat plasma and brain was developed and validated, which was necessary for pharmacokinetic studies of Otaplimastat. Experimental Animals Male Sprague-Dawley rats 8 weeks old, weighing 270 ± 20 g purchased from Orient Bio (Seongnam, South Korea) were used for pharmacokinetic study. Animal studies were performed in accordance with the guidelines set by the animal care and use committee of Kyung Hee University, Seoul, South Korea. Chemicals Otaplimastat hydrochloride and SP-8205 ([3-(2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-propyl]-{4-[3-(2,4-dioxo-1,4-dihydro-2H- quinazolin-3-yl)-propylamino]-butyl}-carbamic acid ethyl ester hydrochloride, Figure 1) were donated by Central Research Institute, Shin Poong Pharmaceutical Company, Ltd. (Ansan, South Korea). ACN, TCA and dichloromethane were purchased from SigmaAldrich (St. Louis, MO, USA). Ammonium acetate was purchased from Merck Company (Darmstadt, Germany). A Direct-Q (Millipore, Billerica, MA, USA) water purification system was used to prepare deionized distilled water. The distilled water was further filtered with a 0.22-μm filter prior to use. All other chemicals and solvents were of analytical grade and were obtained commercially. Standard solutions As an internal standard (IS), SP-8205, a structural analog of Otaplimastat, was used (Figure 1). Stock solutions of Otaplimastat and SP-8205 were prepared at 1 mg/mL in methanol and stored at −80°C. Working standard solutions of Otaplimastat for calibration were prepared by appropriate dilution in methanol. Calibration samples were prepared by spiking blank rat plasma and brain samples with the working standard solution to obtain final concentrations of 20, 60, 100, 500, 2,000 and 5,000 ng/mL for plasma and of 1, 2, 4, 20, 100 and 200 ng/g brain for brain. QC samples at three concentration levels 50, 500 and 1,000 ng/mL for Otaplimastat in rat plasma; 4, 20 and 100 ng/g brain for Otaplimastat in rat brain tissue were prepared from a different primary stock solution in the same manner. Three concentration levels 80, 800 and 4,000 ng/mL for Otaplimastat were used to estimate MEs in rat plasma. A working solution of SP-8205 100 ng/mL was prepared by diluting its primary stock solution with ACN. Figure 1. Full scan mass spectrum and product ion mass spectrum in electrospray ioniz (...truncated)