Quantitative analysis of total thiamine in human blood, milk and cerebrospinal fluid by reversed-phase ion-pair high-performance liquid chromatography

Abstract

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.