Simultaneous GC–MS Analysis of Melatonin and Its Precursors as Ethoxycarbonyl/ Pentafluoropropionyl Derivatives in Rat Urine M. J. Paik 1,2 , D. T. Nguyen 2 , Y. J. Kim 1,3 , J. Y. Shin 1,3 , W. Shim 1 , E. Y. Cho 1,2 , J. H. Yoon 2 , K. R. Kim 4 , Y. S. Lee 5 , N. Kim 6 , S. W. Park 7 , G. Lee 1,2,8 , Y. H. Ahn 1,3,& 1 Institute for Neuroregeneration and Stem Cell Research, Ajou University School of Medicine, Suwon, Republic of Korea 2 Department of Molecular Science and Technology, Ajou University, Suwon, Republic of Korea 3 Department of Neurosurgery, Ajou University School of Medicine, Suwon, Republic of Korea; E-Mail: yhahn@ajou.ac.kr 4 Biometabolite Analysis Laboratory, College of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea 5 Division of Life Science and Pharmaceuticals, College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea 6 School of Electrical and Computer Engineering, Chungbuk National University, Cheongju 361-763, Republic of Korea 7 Department of Neurosurgery, College of Medicine, Chung-Ang University, Seoul, Republic of Korea 8 Institute for Medical Science, Ajou University School of Medicine, Suwon, Republic of Korea Received: 8 February 2010 / Revised: 14 August 2010 / Accepted: 4 September 2010 Online publication: 7 October 2010 Abstract Melatonin is synthesized from acetylserotonin, which is a deacetylated product of serotoinin. Simultaneous analysis of melatonin and its precursors in rat urine was performed using their (N,O)-ethoxycarbonyl/N-pentafluoropropionyl (EOC/PFP) derivatives by gas chromatog- raphy–mass spectrometry (GC–MS). The mass spectral data of acetylserotonin as mono-O- EOC/mono-N-PFP and serotonin as di-(N,O)-EOC/di-N-PFP derivatives were newly established. This method exhibited good linearity (r  0.996), repeatability (% relative standard deviation = 6.9–9.4), accuracy (% relative error =-8.9 to 8.6) with detection limits of 0.005 to 0.03 ng mL -1 . This method was developed for the simultaneous mea- surement of melatonin and its precursors in rat urine. Keywords Gas chromatography-mass spectrometry Ethoxycarbonyl/pentafluoropropionyl derivatives Melatonin, acetylserotonin and serotonin Introduction Melatonin is the primary hormone syn- thesized in the pineal gland in a cyclic fashion, typically in the dark phase of the circadian cycle. It is converted from acetylserotonin, a deacetylated product of serotonin (5-hydroxytryptamine), is linked to physiologic neuroendocrine function including the regulation of cir- dardian rhythms [1, 2], neuronal differ- entiation and proliferation [3], and the physiologic alteration of the immune system [4]. Furthermore, melatonin and its metabolites act as free radical scav- enger, antioxidant and neuroprotective agent [5]. Serotonin with neurotransmitter ef- fect also regulates various physiological processes including modulation of neu- ral activity, gastrointestinal motility, cardiovascular and bladder function [6]. Thus, measurement of melatonin and its precursors in biological fluids is valuable for the effective monitoring of the phys- iological states. In previous studies, the analysis of melatonin and its precursors using gas chromatography-mass spectrometry (GC–MS) was achieved following pen- tafluoropropionyl (PFP) [7–10] and tri- methylsilyl (TMS) derivatives [11–13]. However, simultaneous analysis of mel- atonin and its precursors [11] have been only reported as TMS derivatives in an anhydrous condition. Disadvantage of 2010, 72, 1213–1217 DOI: 10.1365/s10337-010-1771-y 0009-5893/10/12 Ó 2010 Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH Full Short Communication Chromatographia 2010, 72, December (No. 11/12) 1213