Ultra-performance liquid chromatography/tandem mass spectrometric determination of testosterone and its metabolites in in vitro samples.
This paper describes the development and partial validation of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method for the determination of testosterone (T) and its four metabolites, 6beta-OH-T, 16alpha-OH-T, 16beta-OH-T and 2alpha-OH-T, in in vitro samples. The analytical method involves direct dilution of samples with acetonitrile containing an internal standard, followed by separation of testosterone and the four metabolites on an Acquity UPLCtrade mark C(18) column and detected by selected reaction monitoring (SRM) in positive ionization mode using turbo ionspray ionization. The parent compound and its metabolites investigated were well separated (Rs >1.5) with a run time of 4 min under a gradient condition. The method was partially validated. The linear concentration range was 0.01 to 5 microM for all the compounds of interest. Inter-assay mean bias and relative standard deviation (RSD) were in the range of -12% to 8% and 4.1% to 8.5%, respectively. Intra-assay mean bias and RSD were in the range of -8.0% to 5.2% and 3.4% to 9.6%, respectively. The lower limit of quantitation for this assay was 0.01 microM. The differences in LC/MS performance were investigated by conducting a comparison of UPLC with another method previously optimized for HPLC-based separation and quantification of testosterone and its metabolites.
Wang
Ganfeng
G
Department of Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA. ganfeng.wang@spcorp.com
Hsieh
Yunsheng
Y
Cui
Xiaoming
X
Cheng
Kuo-Chi
KC
Korfmacher
Walter A
WA
D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D013739:Testosterone / Q000031:analogs & derivatives / Q000032:analysis* / Q000378:metabolism; D015262:Xenobiotics / Q000494:pharmacology
D015262:Xenobiotics; D013739:Testosterone