Simple HPLC Method with Internal Standard for Evaluation of Vitamin B1 Status By Use of Whole Blood

Background: Two primary assays are routinely used for evaluating a patient's vitamin B1 status: plasma free thiamine and whole blood thiamine diphosphate (TDP). TDP is the bioactive form of vitamin B1 and best reflects body stores. Plasma free thiamine levels are driven by recent dietary intake. The objective of this study was to develop a simple HPLC method with an internal standard (IS) that simultaneously measures TDP and thiamine in whole blood, and to assess the use of this single-tube assay to provide comprehensive evaluation of vitamin B1 status.

Methods: The final assay used amprolium thiochrome as an IS, and the sample preparation procedure takes approximately 1 h. Whole blood thiamine and plasma thiamine were concurrently measured for 126 subjects.

Results: The analytical measurement range was 1.7 to 442.3 nmol/L (TDP) and 1.7 to 375.4 nmol/L (thiamine), with interassay precisions of 4.0% to 4.8% (TDP) and 2.9% to 8.0% (thiamine), respectively. Method comparison with a reference laboratory HPLC method showed r = 0.9625, slope = 1.021, and intercept = 0.982 (n = 53) for TDP quantification. Whole blood thiamine correlated closely with plasma thiamine levels but were slightly higher with a mean difference of 1.0 nmol/L (range: -3.0 to 5.0 nmol/L). The reference interval for whole blood TDP and thiamine was 84.3 to 213.3 nmol/L and 1.7 to 21.9 nmol/L, respectively.

Conclusions: This assay provides a simple and reliable HPLC method with a suitable IS for quantification of both TDP and thiamine from whole blood. It also eliminates the need for separate samples for TDP and thiamine measurement, which will allow both short-term and long-term vitamin B1 status to be assessed from a single sample.