Song H, Gu X, Riffel K, Yan K, Lo M W
Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA.
J Chromatogr B Biomed Sci Appl. 2000 Jan 28;738(1):83-91. doi: 10.1016/s0378-4347(99)00491-0.
An LC-MS-MS method and an HPLC-UV method have been developed for the assay of a novel thrombin inhibitor in human fluids. The LC-MS-MS method is developed for plasma, which usually requires maximum sensitivity. The HPLC-UV method is for urine. In both methods, analytes are extracted using liquid-liquid extraction, and analyzed by reversed-phase high-performance liquid chromatography. A tandem mass spectrometer in the multiple reaction monitoring (MRM) mode is used for detection of the analytes in the plasma method. UV is the detector for the urine method. The plasma method has a lower limit of quantitation (LOQ) of 0.1 ng/ml with a linearity range of 0.1-100 ng/ml. The urine method has an LOQ of 8.12 ng/ml (20 nM) and the linear dynamic range is 8.12-8120 ng/ml (20-20000 nM). Both methods are fast, specific and sensitive. Various validation procedures have proven that both methods are rugged, robust and reproducible. The research also suggested that, while LC-MS-MS provides superior sensitivity and selectivity for the determination of drugs and their metabolites at very low concentrations, HPLC with a conventional detector such as UV is still useful in the analysis when the sensitivity requirement is not crucial.
已开发出一种液相色谱-串联质谱法（LC-MS-MS）和一种高效液相色谱-紫外检测法（HPLC-UV）用于测定人体体液中的一种新型凝血酶抑制剂。液相色谱-串联质谱法用于血浆检测，血浆检测通常需要最高灵敏度。高效液相色谱-紫外检测法用于尿液检测。在这两种方法中，分析物均采用液液萃取法进行提取，然后通过反相高效液相色谱法进行分析。血浆检测方法中使用处于多反应监测（MRM）模式的串联质谱仪来检测分析物。尿液检测方法中使用紫外检测器。血浆检测方法的定量下限（LOQ）为0.1 ng/ml，线性范围为0.1 - 100 ng/ml。尿液检测方法的定量下限为8.12 ng/ml（20 nM），线性动态范围为8.12 - 8120 ng/ml（20 - 20000 nM）。两种方法均快速、特异且灵敏。各种验证程序证明这两种方法耐用、稳健且可重复。该研究还表明，虽然液相色谱-串联质谱法在测定极低浓度的药物及其代谢物时具有卓越的灵敏度和选择性，但当灵敏度要求不高时，配备常规检测器（如紫外检测器）的高效液相色谱法在分析中仍有用处。
