A single-step method for simultaneous quantification of thiamine and its phosphate esters in whole blood sample by...

Thiamine and its phosphate esters play vital physiological roles and thiamine deficiency causes deleterious effects on human body. It is important to quantify accurately the thiamine metabolites in body fluids. However, due to the lack of appropriate internal standards, poor inter-laboratory standardization and time-consuming pretreatment procedure, the existing methods are limited in clinical applications. Hence, we developed a single-step HPLC-MS/MS method for accurate and precise measurement of thiamine and its phosphate esters in human whole blood. Whole blood samples were deproteinized and the supernatants were collected. The levels of thiamine diphosphate (TDP), thiamine monophosphate (TMP), and thiamine were determined by HPLC-MS/MS method after adding isotopic internal standards. The method was linear from 15.625-3.125-1.563 nmol/L to 1000-200-100 nmol/L for TDP-TMP-thiamine. The lower limit of quantification was 15.625-3.125-1.563 nmol/L. The intra-day and inter-day precisions and accuracy for all QCs samples were ≤15.9% and ≤11.1%, respectively. The matrix effect was not significant. Recoveries were 103.7% for TDP, 102.7% for TMP, and 105.3% for thiamine. All QCs were stable for three freeze-thaw cycles, or at room temperature for 3 h, or at -80 °C for 15 days. We compared this new method with an established HPLC method based on derivatization of thiamine metabolites. It is found that this method correlated well with HPLC method for TDP determination (R² = 0.93). However, the correlation was not ideal for TMP (R² = 0.40) or thiamine (R² = 0.72) determination. Subject's diet was shown to have no significant effect on the concentrations of thiamine metabolites in their blood samples. To conclude, we developed a single-step, non-derivatization HPLC-MS/MS method that can detect thiamine and its phosphate esters in human whole blood accurately and quickly.