Dilute and shoot approach for the screening of stimulants by LC-dynamic-MS/MS

Content Provider	Semantic Scholar
Author	Dong, Yingnan Wang Deng Yang
Copyright Year	2014
Abstract	There is a growing need to improve the sensitivity of determination for multiple chemical constitutes in human urine because the Minimum Required Performance Levels (MRPL) for the detection of prohibited substances is continuously updated by the World Anti-Doping Agency (WADA). The MRPL for stimulants, for instance, was dropped from 500 to 100 ng/mL in early 2013. The conventional multiple reaction monitoring (cMRM) mode, however, is not well suited for multi-component identification due to its low sensitivity. Here we apply a dynamic MRM (dMRM) technique for the screening of 78 stimulants and metabolites in human urine using an Agilent triple-quadrupole 6410B mass spectrometer. By allowing extended dwell times, dMRM provides much higher sensitivity and reproducibility than cMRM. After precipitation of protein, the urine sample was injected into LC-MS/MS system directly without sample pre-concentration. For comparison of the sensitivity, both cMRM and dMRM were performed under same chromatographic conditions in this study. The result showed that both of the sensitivity and peak symmetry of extracted chromatogram for each stimulant improved significantly using dMRM. The LODs for the stimulants under investigation met the requirement set by WADA. The method also provided satisfactory results in terms of intraand inter-day precisions, accuracy, matrix effect and specificity. This approach has been employed for routine analysis in our laboratory and External Quality Assessment Scheme (EQAS), which is designed by WADA to continuously monitor the capabilities of the laboratories, to evaluate laboratory proficiency, and to improve test result uniformity between laboratories. Introduction Detection of stimulants in human urine has been performed by gas chromatography with nitrogen phosphorous detector (NPD) [1], gas chromatography-mass spectrometry (GC-MS) [2] and liquid chromatography-tandem mass spectrometry (LC-MS/MS) [3,4]. Conventional liquid chromatography-tandem mass spectrometry using a triple-quadrupole mass spectrometer has been applied successfully in doping control analysis in sports. Data acquisitions are usually done by multiple reaction monitoring mode allowing multiple targets to be covered in a single run. A major drawback of cMRM is the limited number of target transitions that can be included in a single time segment. This study aims at developing a dynamic MRM approach to screen 78 stimulants and metabolites using low resolution instruments (Agilent triple-quadrupole 6410B mass spectrometer). Unlike cMRM, dMRM automatically associates MRM transitions with retention time and it monitors each MRM transition only around its expected RT instead of monitoring all transitions throughout the entire operation as is the case with cMRM. Thus, dMRM allows more MRM transitions to be monitored in a single acquisition while maintaining high quality, sensitivity, selectivity, and reproducibility of the chromatographic results than with cMRM. Experimental Reference materials of stimulants were purchased from Sigma, Anpu, Alltech, NMI of Australia. Some analytical standards were kind gifts from Canadian and other WADA accredited Laboratories. Lead acetate was of analytical gradeand obtained from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China). Acetonitrile of HPLC grade was obtained from Sigma-Aldrich (St. Louis, USA). Ammonium formate and formic acid of HPLC grade were purchased from Fluka (Pittsburgh, USA) and
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