Jongen M J, Kuiper S, van der Vijgh W J, Lips P, Netelenbos J C
Department of Internal Medicine, Academisch Ziekenhuis Vrije Universiteit, Amsterdam, The Netherlands.
J Clin Chem Clin Biochem. 1988 Jan;26(1):25-8. doi: 10.1515/cclm.1988.26.1.25.
Technical improvements have been applied to reduce the time required for the determination of calcidiol and calcitriol in serum or plasma. The modifications include the use of Sep-Pak C18 cartridges for the extraction of calciol metabolites from serum instead of a classical liquid/liquid extraction, a considerable shortening of the HPLC purification time compared with our previously described method, and the application of HPLC with UV detection at 254 nm of calcidiol as an alternative to the usual competitive protein binding methods. In none of the 199 samples where calcidiol was determined by HPLC did we observe a detectable peak of ercalcidiol. Quantitation of calcidiol by HPLC and competitive protein binding was compared in 5 series of assays. The correlation between the two methods was 0.99. The average slope of the linear regression line when the HPLC values were plotted versus the competitive protein binding values was 1.14. The average intercept was 0.19 nmol/l. The mean within-run coefficient of variation for calcidiol in these series was 5% for the competitive protein binding method, and 4% for HPLC method. Between-run coefficients of variation were 6% and 12% for the competitive protein binding and for the HPLC method, respectively. Within-run and between-run coefficients of variation for calcitriol were 6% and 15%, respectively.
已采用技术改进措施来缩短血清或血浆中骨化二醇和骨化三醇测定所需的时间。这些改进包括使用Sep - Pak C18柱从血清中提取骨化二醇代谢物，取代传统的液/液萃取法；与我们之前描述的方法相比，高效液相色谱（HPLC）纯化时间大幅缩短；以及采用在254 nm处进行紫外检测的HPLC法来替代常用的竞争性蛋白结合法。在通过HPLC测定骨化二醇的199个样本中，均未观察到骨化二醇的可检测峰。在5组测定中比较了HPLC法和竞争性蛋白结合法对骨化二醇的定量结果。两种方法的相关性为0.99。将HPLC值与竞争性蛋白结合值绘制线性回归线时，平均斜率为1.14，平均截距为0.19 nmol/l。在这些系列中，竞争性蛋白结合法测定骨化二醇的批内变异系数平均为5%，HPLC法为4%。竞争性蛋白结合法和HPLC法的批间变异系数分别为6%和12%。骨化三醇的批内和批间变异系数分别为6%和15%。
