Department of Bio-analytical Chemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Akiha-ku, Niigata, Niigata 956-8603, Japan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Dec 15;878(32):3358-62. doi: 10.1016/j.jchromb.2010.10.020. Epub 2010 Oct 30.
A method for the simultaneous determination of pregnenolone and 17α-hydroxypregnenolone by high-performance liquid chromatography with an immobilized cholesterol oxidation enzyme reactor was developed. Pregnenolone and 17α-hydroxypregnenolone were converted to progesterone and 17α-hydroxyprogesterone, respectively, by the immobilized enzyme packed into the reactor column, and could thus be monitored by UV absorption at 240 nm. The calibration curves for pregnenolone and 17α-hydroxypregnenolone were linear in the range of 0.4-10 and 0.3-10 μg/ml with a correlation coefficient of 0.9993 and 0.9998, respectively. The detection limit at a signal-to-noise ratio of 3 was 0.12 and 0.08 μg/ml for pregnenolone and 17α-hydroxypregnenolone, respectively. The conversion rate of pregnenolone to progesterone and 17α-hydroxypregnenolone to 17α-hydroxyprogesterone was 90.6% and 99.3%, respectively. Intra-day and inter-day precision (in terms of percentage coefficient of variation) were less than 9.3%, with accuracy greater than 94.8%. This method was successfully applied to the simultaneous determination of pregnenolone and 17α-hydroxypregnenolone secreted into the culture medium of bovine adrenal fasciculata cells and of both analytes produced within the cells.
采用固定化胆固醇氧化酶反应器的高效液相色谱法同时测定孕烯醇酮和 17α-羟孕烯醇酮。将固定化酶填充到反应器柱中，将孕烯醇酮和 17α-羟孕烯醇酮分别转化为孕酮和 17α-羟孕酮，然后可以在 240nm 处通过紫外吸收进行监测。孕烯醇酮和 17α-羟孕烯醇酮的校准曲线在 0.4-10μg/ml 和 0.3-10μg/ml 范围内呈线性，相关系数分别为 0.9993 和 0.9998。以信噪比 3 计算，孕烯醇酮和 17α-羟孕烯醇酮的检测限分别为 0.12μg/ml 和 0.08μg/ml。孕烯醇酮转化为孕酮和 17α-羟孕烯醇酮转化为 17α-羟孕酮的转化率分别为 90.6%和 99.3%。日内和日间精密度（以变异系数百分比表示）均小于 9.3%，准确度大于 94.8%。该方法成功应用于同时测定牛肾上腺束状带细胞培养上清液中分泌的孕烯醇酮和 17α-羟孕烯醇酮，以及细胞内产生的两种分析物。
