Simultaneous Determination of Underivatized Vitamin B1 and B6 in Whole Blood by Reversed Phase Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry

摘要: Background Vitamin B1 (thiamine-diphosphate) and B6 (pyridoxal-5’phosphate) are micronutrients. Analysis of these micronutrients is important to diagnose potential deficiency which often occurs in elderly people due malnutrition, severe alcoholism gastrointestinal compromise bypass surgery or disease. Existing High Performance Liquid Chromatography (HPLC) based methods include the need for derivatization long analysis time. We developed an Ultra Tandem Mass spectrometry (UHPLC-MS/MS) assay with internal standards simultaneous measurement underivatized thiamine-diphosphate pyridoxal-5’phosphate without use ion pairing reagent. Methods Whole blood, deproteinized perchloric acid, containing deuterium labelled thiamine-diphosphate(thiazole-methyl-D3) pyridoxal-5’phosphate(methyl-D3), was analyzed by UHPLC-MS/MS. The method validated imprecision, linearity, recovery limit quantification. Alternate (quantitative) comparisons new versus currently used routine HPLC were established Deming regression. Results Thiamine-diphosphate measured within 2.5 minutes instrumental run Limits detection 2.8 nmol/L 7.8 respectively. Limit quantification 9.4 25.9 pyridoxal-5’phosphate. total imprecision ranged from 3.5–7.7% (44–157 nmol/L) 6.0–10.4% (30–130 nmol/L). Extraction recoveries 101–102% ± 2.5% 98–100% 5% (pyridoxal-5’phosphate). regression yielded slopes 0.926 0.990 patient samples (n = 282) national proficiency testing 12) respectively, intercepts +3.5 +3 282 n 1.04 0.84, -2.9 +20 376 12). Conclusion The described UHPLC-MS/MS allows determination whole blood intensive sample preparation.