Wang Ganfeng, Hsieh Yunsheng, Cui Xiaoming, Cheng Kuo-Chi, Korfmacher Walter A
Department of Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.
Rapid Commun Mass Spectrom. 2006;20(14):2215-21. doi: 10.1002/rcm.2580.
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.
本文描述了一种快速、灵敏且特异的超高效液相色谱/串联质谱（UPLC/MS/MS）方法的开发及部分验证，该方法用于测定体外样品中的睾酮（T）及其四种代谢物，即6β-羟基睾酮（6beta-OH-T）、16α-羟基睾酮（16alpha-OH-T）、16β-羟基睾酮（16beta-OH-T）和2α-羟基睾酮（2alpha-OH-T）。该分析方法包括用含有内标的乙腈直接稀释样品，然后在Acquity UPLC商标C(18)柱上分离睾酮和四种代谢物，并采用涡轮离子喷雾电离在正离子模式下通过选择反应监测（SRM）进行检测。在所研究的母化合物及其代谢物在梯度条件下4分钟的运行时间内得到了良好的分离（Rs>1.5）。该方法进行了部分验证。所有目标化合物的线性浓度范围为0.01至5 microM。批间平均偏差和相对标准偏差（RSD）分别在-12%至8%和4.1%至8.5%的范围内。批内平均偏差和RSD分别在-8.0%至5.2%和3.4%至9.6%的范围内。该测定方法的定量下限为0.01 microM。通过将UPLC与另一种先前针对基于HPLC的睾酮及其代谢物的分离和定量进行优化的方法进行比较，研究了LC/MS性能的差异。
