Knebel N G, Sharp S R, Madigan M J
Department of Pharmacokinetics and Metabolism, Roche Products Ltd., Hertfordshire, UK.
J Chromatogr B Biomed Appl. 1995 Nov 17;673(2):213-22. doi: 10.1016/0378-4347(95)00270-2.
A rapid, sensitive and specific analytical method has been developed and validated to quantify the collagenase inhibitor N2-(2(5)-[(hydroxycarbamoyl)methyl)-4-methylvaleryl]-N1, 3-dimethyl-L-valin-amide (I) and its major metabolite (II) from plasma and urine. This method employs an automated solid-phase extraction procedure to isolate the analytes and the internal standard from the biological matrix. Reconstituted extracts were analyzed by HPLC-ionspray MS-MS. Chromatography was performed on a 4.6 mm I.D. reversed-phase guard column. The retention times of the analytes and the internal standard were approximately 1.3 min. The assay has a limit of quantification of 5 ng/ml plasma and a limit of detection of 1 ng/ml, based on 100-micro l plasma aliquots. No sample-drying step was required. The standard curves were linear form 5 to 5000 ng/ml using weighted linear regression analysis (1/y2). The intra- and inter-assay precision were better than + or - 10% with intra- and inter-assay accuracies between 95 and 105%. this new HPLC-MS-MS assay procedure for I and II in plasma and urine has proven to be specific, sensitive, accurate and robust, and is being used routinely for the analysis of I in pre-clinical and clinical trial samples. Up to 200 unknowns may be analyzed each 24 h per analyst.
已开发并验证了一种快速、灵敏且特异的分析方法，用于定量测定血浆和尿液中的胶原酶抑制剂N2-(2(5)-[(羟基氨基甲酰基)甲基]-4-甲基戊酰基]-N1,3-二甲基-L-缬氨酰胺（I）及其主要代谢物（II）。该方法采用自动固相萃取程序从生物基质中分离分析物和内标。用HPLC-离子喷雾MS-MS分析重构后的提取物。在4.6 mm内径的反相保护柱上进行色谱分析。分析物和内标的保留时间约为1.3分钟。基于100微升血浆等分试样，该测定法的定量限为5 ng/ml血浆，检测限为1 ng/ml。无需样品干燥步骤。使用加权线性回归分析（1/y2），标准曲线在5至5000 ng/ml范围内呈线性。批内和批间精密度优于±10%，批内和批间准确度在95%至105%之间。这种用于血浆和尿液中I和II的新HPLC-MS-MS测定程序已被证明具有特异性、灵敏性、准确性和稳健性，并且正在常规用于临床前和临床试验样品中I的分析。每位分析人员每24小时最多可分析200个未知样品。
