A novel LC-MS/MS assay for vitamin B1, B2 and B6 determination in dried blood spots and its application in children

Thiamin, riboflavin and pyridoxal phosphate (PLP) concentrations are useful indices for evaluating vitamin B1, B2 and B6 status. Several HPLC assays have been developed for determining thiamin, riboflavin and PLP in biological matrix. However, no existing LC-MS/MS methods can be used to quantify thiamin, riboflavin and PLP in dried blood spots (DBSs), which are often used as a sampling/storage vessel for blood from infants and children. This study evaluated the validity, reliability and stability of an LC-MS/MS assay for measuring thiamin, riboflavin and PLP in DBS cards. 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. Both the between-run and within-run variable coefficients (CV% values) were < 8.56%. The accuracies were good and showed relative errors (RE% values) from -7.40% to 3.12%. The lower limits of quantification (LLOQs) ranged from 0.2 to 0.5 ng/mL, and the recoveries were from 81.49% to 112.23% for all 3 analytes. The matrix effects (ME% values) were acceptable, and the CV% values of the internal standard-normalized matrix factors were <15% (n = 6). Thiamin, riboflavin and PLP were stable on the DBS card during at least 15 days of room-temperature storage under vacuum in the dark, and the measurements of thiamin, riboflavin and PLP in the DBSs showed good agreement with the corresponding concentrations determined from liquid blood (R2 values >0.96). The validated method was successfully applied to the nutritional assessment of vitamins B1, B2, and B6 in 48 Chinese children.