You Youwen, Uboh Cornelius E, Soma Lawrence R, Guan Fuyu, Li Xiaoqing, Rudy Jeffrey A, Liu Ying, Chen Jinwen
University of Pennsylvania, School of Veterinary Medicine, Department of Clinical Studies, New Bolton Center Campus, Kennett Square, PA 19348, USA.
Rapid Commun Mass Spectrom. 2009 Jul;23(13):2035-44. doi: 10.1002/rcm.4114.
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 microm 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.
报道了一种用于快速高通量分析马血浆中8种合成代谢和雄性激素类固醇（AAS）的超高效液相色谱/串联质谱（UPLC/MS/MS）方法。分析物通过使用甲基叔丁基醚的液液萃取进行回收，在1.9微米的C(18)反相柱上分离，并在正电喷雾电离模式下在具有选择反应监测（SRM）和全产物离子扫描的三重四极杆质谱仪上进行分析。在筛选过程中，对每种AAS监测两个SRM离子跃迁以获得高度选择性的筛选结果。在血浆中100 pg/0.5 mL的浓度下获得了质量优异的AAS全产物离子光谱，且没有来自血浆中杂质的干扰光谱。据我们所知，这是首次使用三重四极杆仪器在如此低的分析物浓度和血浆体积下获得全产物离子光谱的报道。除了从三次SRM扫描获得的产物离子强度比用于鉴定马血浆中的AAS外，全产物离子光谱还用作确认的辅助证据。为了进行定量，使用氘代睾酮和司坦唑醇作为内标（IS）。检测限、定量限和确认限分别为6.25 - 12.5 pg/0.5 mL、25 pg/0.5 mL和50 - 100 pg/0.5 mL。对所有8种AAS的分析均无显著基质效应。日内精密度和准确度分别为2 - 15%和91 - 107%。日间精密度和准确度分别为1 - 21%和94 - 110%。总分析时间为5分钟。迄今为止，该方法已成功用于分析宾夕法尼亚州参赛赛马血浆样本中超过12,000个AAS样本。该方法快速、选择性好、可重现且可靠。
