用于分离睾酮代谢物的等度高效液相色谱法。
Isocratic high-performance liquid chromatographic method for the separation of testosterone metabolites.
作者信息
Sanwald P, Blankson E A, Duléry B D, Schoun J, Huebert N D, Dow J
机构信息
Department of Pharmacokinetics, Marion Merrell Dow, Strasbourg, France.
出版信息
J Chromatogr B Biomed Appl. 1995 Oct 20;672(2):207-15. doi: 10.1016/0378-4347(95)00216-6.
An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method using an Ultrasphere IP column has been developed for the determination of testosterone and its metabolites after incubation of 4-14C-labelled or unlabelled testosterone with rat liver microsomes. Compounds were eluted with methanol-water-tetrahydrofuran (35:55:10, v/v, pH 4.0) and detected by ultraviolet (UV) absorption at 245 nm. UV or on-line radioactivity detection can be used although, due to differences in detector cell volumes, peak resolution is slightly better with UV detection. Selectivity was validated by collecting HPLC peaks and verifying their identity by gas chromatography-mass spectrometry after derivatization by N,O-bis(trimethylsilyl)trifluoroacetamide-trimethylchlorosilane. A three-day validation was performed to determine the linearity, repeatability, reproducibility and accuracy of the method, using corticosterone as internal standard. The method is applicable to the measurement of cytochrome P-450 isoenzyme activities in rat liver.
已开发出一种使用Ultrasphere IP柱的等度反相高效液相色谱（HPLC）方法，用于在将4-¹⁴C标记或未标记的睾酮与大鼠肝微粒体孵育后测定睾酮及其代谢物。化合物用甲醇 - 水 - 四氢呋喃（35:55:10，v/v，pH 4.0）洗脱，并通过在245 nm处的紫外（UV）吸收进行检测。虽然可以使用UV或在线放射性检测，但由于检测器池体积的差异，UV检测的峰分辨率略好。通过收集HPLC峰并在经N，O-双（三甲基硅基）三氟乙酰胺 - 三甲基氯硅烷衍生化后通过气相色谱 - 质谱法验证其身份来验证选择性。使用皮质酮作为内标进行了为期三天的验证，以确定该方法的线性、重复性、重现性和准确性。该方法适用于大鼠肝脏中细胞色素P-450同工酶活性的测定。
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