Lensmeyer G L, Wiebe D A, Carlson I H
Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison 53792.
Clin Chem. 1987 Oct;33(10):1841-50.
We describe an extraction and an isocratic "high-performance" liquid-chromatographic (HPLC) separation of cyclosporine (CsA) and nine metabolites (M1, M8, M17, M18, M21, M25, M26, M203-218, and MUNDF1) from whole blood. Metabolites (for standards) were purified from human bile with liquid-liquid and solid-phase extractions, chromatographed on a cyanopropyl (CN) semipreparative HPLC column, and further purified on octyl, CN, and silica columns. The identity of each metabolite was verified with authentic standards on three chemically different HPLC columns and on the basis of cross-reactivity data from radioimmunoassay. For the routine analytical method, 1 mL of whole blood is diluted, hemolyzed, and applied to a Bond Elut CN (500 mg) cartridge to extract CsA, metabolites, and cyclosporin C, the internal standard. Interferences are removed by using four wash solutions and an additional cartridge of octyldecyl sorbent introduced prior to elution. Analytes are separated on a Zorbax CN analytical column maintained at 58 degrees C, with detection at 214 nm. Analytical recovery, as tested with three lots of CN sorbent, ranged from 47% to 95% for the 10 cyclosporines. Between-run CVs are less than 10% at 200 micrograms/L (concentration of each compound) and the standard curves are linear to 1500 micrograms/L. We also report a study of the separation mechanisms.
我们描述了一种从全血中提取和采用等度“高效”液相色谱（HPLC）分离环孢素（CsA）及其九种代谢物（M1、M8、M17、M18、M21、M25、M26、M203 - 218和MUNDF1）的方法。代谢物（作为标准品）通过液 - 液萃取和固相萃取从人胆汁中纯化，在氰丙基（CN）半制备HPLC柱上进行色谱分离，然后在辛基、CN和硅胶柱上进一步纯化。每种代谢物的身份通过在三种化学性质不同的HPLC柱上使用标准品以及基于放射免疫分析的交叉反应数据进行验证。对于常规分析方法，将1 mL全血稀释、溶血，然后应用于Bond Elut CN（500 mg）柱以提取CsA、代谢物和内标环孢菌素C。通过使用四种洗涤溶液和在洗脱前引入的额外的十八烷基吸附剂柱去除干扰物。分析物在保持在58℃的Zorbax CN分析柱上进行分离，在214 nm处检测。用三批CN吸附剂测试的分析回收率，对于这10种环孢素而言，范围为47%至95%。在200μg/L（每种化合物的浓度）时，批间变异系数小于10%，标准曲线在1500μg/L范围内呈线性。我们还报告了一项关于分离机制的研究。
