Wielders J P, Mink C J
J Chromatogr. 1983 Oct 14;277:145-56. doi: 10.1016/s0378-4347(00)84831-8.
Whole blood hemolysed by freezing, serum, cerebrospinal fluid, and milk of human origin were deproteinized by perchloric acid. Thiamine pyrophosphate and thiamine monophosphate were hydrolysed to thiamine by acid phosphatase. Chromatography was performed on C18-coated silica using an n-octanesulfonate containing mobile phase methanol-aqueous citrate buffer pH 4.0 (45:55, v/v). In a post-column reaction K3Fe(CN)6 is used to oxidise thiamine to thiochrome, which is detected by fluorometry. Two ml blood is needed. The minimum detectable amount is 60 femtomol of thiamine. The intra-assay coefficient of variation (C.V.) is 2.3% and the inter-assay C.V. is 3.9%. The recovery of added thiamine pyrophosphate to blood samples was 98.7%. The reference range was found to be 88-157 nmol/1 whole blood. Examples of the analysis of cerebrospinal fluid, serum and milk are given.
通过冷冻使全血溶血，人源血清、脑脊液和牛奶用高氯酸进行脱蛋白处理。硫胺素焦磷酸和硫胺素单磷酸通过酸性磷酸酶水解为硫胺素。使用含正辛烷磺酸盐的流动相甲醇 - 柠檬酸水溶液缓冲液（pH 4.0，45:55，v/v）在C18涂覆的硅胶上进行色谱分析。在柱后反应中，使用K3Fe(CN)6将硫胺素氧化为硫色素，通过荧光法进行检测。需要2毫升血液。硫胺素的最低检测量为60飞摩尔。批内变异系数（C.V.）为2.3%，批间C.V.为3.9%。向血样中添加硫胺素焦磷酸的回收率为98.7%。参考范围为88 - 157 nmol/1全血。给出了脑脊液、血清和牛奶分析的示例。
