111 Vol. 7, No. 4, 2016 ISSN 2233-4203/ e-ISSN 2093-8950 LETTER www.msletters.org | Mass Spectrometry Letters Analysis of Glycerol with Isolation of Endogenous Interferences using “Dilute and Shoot” Strategy and High-Resolution Mass Spectrometry in Human Urine for Antidoping Testing Yongseok Kim, 1,2 Hophil Min, 1 Changmin Sung, 1 Ju-hyung Park, 1 Junghyun Son, 1 Kang Mi Lee, 1 Ho Jun Kim, 1 Jaeick Lee, 1 Oh-Seung Kwon, 1 and Ki Hun Kim 1 * 1 Doping Control Center, Korea Institute of Science and Technology, Hwarang-ro 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea 2 Department of Chemistry, Yonsei University, Yonsei-ro 50, Seodaemun-gu, Seoul 03722, Republic of Korea Received November 30, 2016; Revised December 14, 2016; Accepted December 19, 2016 First published on the web December 31, 2016; DOI: 10.5478/MSL.2016.7.4.000 Abstract : Glycerol was identified and isolated from endogenous interferences during analysis of human urine using high-reso- lution mass spectrometry (HRMS) for doping control. Urinary sample preparation was simple; the samples were diluted with an organic solvent and then analyzed using a liquid chromatography-mass spectrometry (“dilute and shoot” method). Although the interfering ion peaks were observed at the similar retention time of glycerol, the inference could be identified by isolation with HRMS and further investigation. Thus, creatinine was identified as the endogenous interference for glycerol analysis and it also caused ion suppression resulting in the decrease of glycerol signal. This study reports the first identification and efficient isola- tion of endogenous interferences in human urine for “dilute and shoot” method. The information about ion suppression could be novel to prevent overestimation or a false result for antidoping analysis. Keywords : Mass spectrometry, Doping control, Dilute and shoot, Orbitrap, Glycerol analysis 1. Introduction The World Anti-Doping Agency (WADA) regulates the list of prohibited substances and requires that an accredited lab should analyze substances with appropriate methods. Various prohibited substances were classified to 11 classes including anabolic steroids, beta-2 agoinsts, diuretics, stimulants, masking agent, etc. 1 Glycerol known to regulate osmosis in human body can induce increment of plasma by osmosis. 2 Therefore, it can lower count of erythrocyte or hemoglobin concentration, which provides a role of ‘masking agent’ to interfere detection of drugs for hematogenesis such as erythropoietin. The oral dosing or intravenous injection of glycerol was prohibited by WADA from 2010 and 4.3 mg/mL of urinary concentration of glycerol is its threshold for a positive result. The analysis of glycerol has been performed by colorimetric test or chromatography-mass spectrometry. The colorimetric test is based on measuring a formaldehyde concentration after reaction of glycerol oxidation with periodate anion. 3 Although this method has cost benefit, some issues remain. It takes a large volume of reagents and sample, and only sugar alcohols can be analyzed by this test. Otherwise gas chromatography-mass spectrometry (GC- MS) method could provide high sensitivity and accuracy but it requires a long reaction time for derivatization. 4 Currently, a method using liquid chromatography-mass spectrometry (LC-MS) has been developed. Dong et al. 5,6 reported a method using derivatization by benzoyl chloride in n-hexane and it showed high selectivity by product ion scanning. Since this method is accompanied by an additional reaction and extraction steps, a simple method is required that can be included in a routine screening method for multi-target analysis covering almost all classes in a single LC-MS sequence for doping control. 7 Recently, a strategy known as the “dilute and shoot” method has been gained much attention as a screening test procedure, which involves direct injection of target analytes in a diluted urine without any extraction or concentration step. This method can be applied successfully in polar compounds or metabolites that are difficult to be extracted or detected. 8 This strategy was *Reprint requests to Ki Hun Kim E-mail: kihun.kim@kist.re.kr All MS Letters content is Open Access, meaning it is accessible online to everyone, without fee and authors’ permission. All MS Letters content is published and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org /licenses/by/3.0/). Under this license, authors reserve the copyright for their content; however, they permit anyone to unrestrictedly use, distribute, and reproduce the content in any medium as far as the original authors and source are cited. For any reuse, redistribution, or reproduction of a work, users must clarify the license terms under which the work was produced.