A Novel LC-MS/MS Assay for Vitamin B1, B2 and B6 Determination in Driedblood Spots and Its Application in Children

Abstract

Abstract Objectives Several HPLC assays can be used to determine vitamin B1 (thiamin), B2 (riboflavin) and B6 (PLP) in biological matrix. However, no existing LC-MS/MS methods have been developed for quantifying thiamin, riboflavin and PLP in dried blood spots (DBSs), which are often used as a sampling/storage vessel for finger blood from infants and children. This study firstly used vacuum package in DBS cards storage, and evaluated the validity, reliability and stability of an LC-MS/MS assay for measuring thiamin, riboflavin and PLP in DBS cards. Methods DBS samples were deproteinized by adding trichloroacetic acid containing thiamine-[13C4], riboflavin-[13C4,15N2] and pyridoxal-d3 as internal standards. Thiamin, riboflavin and PLP were separated on a C8 column with a 5-min run time. Results Thiamin, riboflavin and PLP were stable on the DBS card during at least 15 days of room-temperature storage under vacuum in the dark. Both the between-run and within-run variable coefficients (CV% values) were <8.56%. The accuracies (relative errors RE% values) were from −7.40% to 3.12%. Lower limits of quantifications (LLOQs) ranged from 0.2 to 0.5 ng/mL, and the recoveries were from 81.49% to 112.23% for all 3 analytes. Measurements of thiamin, riboflavin and PLP in the DBSs showed good agreement with the corresponding concentrations determined from liquid blood (R2 values >0.96). Conclusions This validated method can be applied to the nutritional assessment of vitamins B1, B2, and B6. Funding Sources This work was supported by the National Natural Science Foundation of China (Grant No. 81,400,848), Beijing Municipal