Rhodes C J, Claridge P A, Trafford D J, Makin H L
J Steroid Biochem. 1983 Sep;19(3):1349-54. doi: 10.1016/0022-4731(83)90162-0.
The use of Sep-Pak C18 cartridges for the extraction of vitamin D and some of its metabolites from plasma and urine has been evaluated by studying the recovery of added tritiated secosteroids. The preparation of the cartridges, recoveries, extraction and elution with a number of solvents, effect of varying flow rates for application and elution, and the effect of increasing volumes of plasma and urine have been investigated. Two methods for the application of secosteroids present in plasma to Sep-Pak C18 cartridges have been examined, using methyl cyanide extracts removing precipitated protein by centrifugation, and using acidified methanolic plasma. Methyl cyanide extracts applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave the cleanest extracts suitable for direct HPLC. Acidified methanolic plasma, applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave extracts which could not be applied directly to an HPLC--further fractionation using Sep-Pak SIL cartridges was necessary. Recoveries of added tritiated secosteroids using both methods were greater than 80% with the exception of vitamin D itself which was poorly recovered--methyl cyanide extraction giving only 30% recovery and use of acidified methanolic plasma giving 66% recovery.
通过研究添加的氚标记甾醇的回收率，评估了使用Sep-Pak C18柱从血浆和尿液中提取维生素D及其一些代谢物的方法。研究了柱的制备、回收率、用多种溶剂进行提取和洗脱、不同进样和洗脱流速的影响以及血浆和尿液体积增加的影响。考察了将血浆中存在的甾醇应用于Sep-Pak C18柱的两种方法，一种是使用甲基氰提取物，通过离心去除沉淀的蛋白质，另一种是使用酸化的甲醇血浆。应用于Sep-Pak C18柱并用甲醇或甲基氰洗脱的甲基氰提取物得到了最纯净的提取物，适合直接进行高效液相色谱分析。应用于Sep-Pak C18柱并用甲醇或甲基氰洗脱的酸化甲醇血浆得到的提取物不能直接应用于高效液相色谱——需要使用Sep-Pak SIL柱进行进一步分离。除维生素D本身回收率较低外，两种方法对添加的氚标记甾醇的回收率均大于80%——甲基氰提取法的回收率仅为30%，酸化甲醇血浆法的回收率为66%。
