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Ultra‐performance liquid chromatography/tandem mass spectrometry in high‐throughput detection, quantification and confirmation of anabolic steroids in equine plasma
Author(s) -
You Youwen,
Uboh Cornelius E.,
Soma Lawrence R.,
Guan Fuyu,
Li Xiaoqing,
Rudy Jeffrey A.,
Liu Ying,
Chen Jinwen
Publication year - 2009
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4114
Subject(s) - chemistry , chromatography , triple quadrupole mass spectrometer , mass spectrometry , analyte , selected reaction monitoring , analytical chemistry (journal) , tandem mass spectrometry , liquid chromatography–mass spectrometry , electrospray ionization , electrospray , detection limit
An ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method for fast‐throughput analysis of eight anabolic and androgenic steroids (AAS) in equine plasma is reported. Analytes were recovered by liquid‐liquid extraction using methyl tert ‐butyl ether, separated on a 1.9 µm C 18 reversed‐phase column, and analyzed in positive electrospray ionization mode on a triple quadrupole mass spectrometer with selected reaction monitoring (SRM) and full product ion scans. Two SRM ion transitions were monitored for each AAS during screening to obtain highly selective screening results. Full product ion spectra of excellent quality for AAS, at 100 pg/0.5 mL in plasma, devoid of interfering spectra from impurities in plasma, were obtained. To our knowledge, this is the first report on the acquisition of full product ion spectra at such a low analyte concentration and plasma volume using a triple quadrupole instrument. In addition to product ion intensity ratios obtained from three SRM scans for identifying AAS in equine plasma, full product ion spectra were used as supporting evidence for confirmation. For quantification, deuterium‐labeled testosterone and stanozolol were used as internal standards (ISs). The limits of detection, quantification and confirmation were 6.25–12.5 pg/0.5 mL, 25 pg/0.5 mL and 50–100 pg/0.5 mL, respectively. There was no significant matrix effect on the analysis of all eight AAS. Intra‐day precision and accuracy were 2–15% and 91–107%, respectively. Inter‐day precision and accuracy were 1–21% and 94–110%, respectively. Total analysis time was 5 min. To date, the method has been successfully used in the analysis of >12 000 samples for AAS in plasma samples from racehorses competing in the State of Pennsylvania. The method is fast, selective, reproducible, and reliable. Copyright © 2009 John Wiley & Sons, Ltd.