Development of novel liquid chromatography method for clinical monitoring of vitamin B1 metabolites and B6 status in the whole blood
Abstrakt
A method for the simultaneous determination of vitamin B1 thiamine, and its phosphorylated biologically active derivatives thiamine diphosphate and thiamine monophosphate, together with the active form of vitamin B6 pyridoxal-5-phosphate in human whole blood has been developed and optimized using high performance liquid chromatography with pre-column derivatization coupled with fluorescence detection. The meteoric core column packed with core-shell stationary phase enabling separation using reversed phase mechanism was used for the analysis of real sample extracts and separation without the application of ion-pairing reagents was achieved.
Recoveries were between 96.3% and 113.8% for all analytes. The analysis has quantitation limits suitable for clinical monitoring with LOQ values for pyridoxal-5-phosphate of 8.32 nmol/L, 4.35 nmol/L for thiamine diphosphate, 5.17 nmol/L for thiamine monophosphate, and 4.27 nmol/L for thiamine.
Our method allowed determination and monitoring of trace levels of vitamins B1 and B6 in whole blood of patients receiving vitamin supplementation treatment while being in intensive care. Monitoring of their status was incorporated in clinical guidelines as an useful predictor of possible complications during treatment.