Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 85–89 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis j o ur na l ho mepage: www.elsevier.com/locate/jpba Short communication Simultaneous determination of 7-O-succinyl macrolactin A and its metabolite macrolactin A in rat plasma using liquid chromatography coupled to tandem mass spectrometry Keumhan Noh a , Dong Hee Kim b , Beom Soo Shin c , Hwi-yeol Yun d , Eunyoung Kim e,∗∗ , Wonku Kang e,∗ a College of Pharmacy, Yeungnam University, Kyoungbuk 712-749, South Korea b Research and Development Center, Daewoo Pharm. Co. Ltd., Busan 604-836, South Korea c College of Pharmacy, Catholic University of Daegu, Kyoungbuk 712-702, South Korea d College of Pharmacy, Chungnam National University, Daejeon 305-764, South Korea e College of Pharmacy, Chung-Ang University, Seoul 156-756, South Korea a r t i c l e i n f o Article history: Received 11 February 2014 Received in revised form 7 May 2014 Accepted 9 May 2014 Available online 17 May 2014 Keywords: 7-O-Succinyl macrolactin A Macrolactin A LC–MS/MS Rat plasma Stability a b s t r a c t 7-O-Succinyl macrolactin A (SMA) and its major metabolite macrolactin A (MA) are generated from Bacillus polyfermenticus KJS-2. Both substances show inhibitory effects on angiogenesis and cancer cell invasion. SMA in rat plasma is known to be relatively stable at room temperature, but MA was not detected due to its instability. Therefore, a stabilizer is required to accurately measure the substance in biological rat samples. In this study, NaF and eserine were examined to determine whether they could stabilize MA to allow for accurate measurement in rat plasma. We also developed a rapid and simple chromato- graphic method using tandem mass spectrometry (MS/MS) for the simultaneous determination of these compounds in rat plasma. After simple protein precipitation with acetonitrile including methaqualone (internal standard), the analytes were chromatographed on a Hilic column with a mobile phase of 10 mM formic acid aqueous solution, methanol, and acetonitrile (15:15:70, v/v). The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods. This ana- lytical method was successfully applied to monitor plasma concentrations of both compounds over time following intravenous administration of a salt form of SMA in rats. © 2014 Elsevier B.V. All rights reserved. 1. Introduction 7-O-Succinyl macrolactin A (SMA) and its major metabolite macrolactin A (MA) are generated from Bacillus polyfermen- ticus KJS-2 [1]. Both substances have antibacterial activities against vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus [1,2], and show inhibitory effects on angio- genesis and cancer cell invasion [3]. During the drug development process, pharmacokinetic studies are required to evaluate drug clearance and metabolism in the body. However, an appropriate analytical method is required prior to performing pharmacokinetic studies. At this time, SMA has been measured in rat plasma and urine using high-performance liquid chromatography (HPLC) with UV detection by Kim et al. [4]. ∗ Corresponding author. Tel.: +82 28205601; fax: +82 28167338. ∗∗ Corresponding author. E-mail addresses: eykimjcb777@cau.ac.kr (E. Kim), wkang@cau.ac.kr (W. Kang). They reported that SMA in rat plasma is stable for only 2 h at room temperature, and that MA was not detected due to its instability. As shown in Fig. 1, the ester in the ring structure of MA seems to be easily hydrolyzed by esterases in biological fluids. Therefore, a stabilizer is required to accurately measure the substance in biological rat samples. In general, rat plasma contains four esterases: acetyl- cholinesterase (AChE), arylesterase (ArE), butyrylcholinesterase (BChE), and carboxylesterase (CES) [5]. Eserine can selectively inhibit AChE and BChE, sodium fluoride (NaF) inhibits AchE, BChE, and CES, and metal chlorides inhibit ArE [6,7]. In this study, Naf and eserine were examined to determine whether they could stabilize both SMA and MA to allow for accu- rate measurement in rat plasma. We also developed a rapid and simple chromatographic method using tandem mass spectrometry (MS/MS) for the simultaneous determination of these compounds in rat plasma. This analytical method was successfully applied to monitor plasma concentrations of both compounds over time fol- lowing intravenous administration of a salt form of SMA in rats. http://dx.doi.org/10.1016/j.jpba.2014.05.009 0731-7085/© 2014 Elsevier B.V. All rights reserved.