Development and validation of an LC-MS/MS method for determination of B vitamins and some its derivatives in whole blood

Abstract

AbstractObligate symbiotic bacteria associated with the insects feeding exclusively on vertebrate blood are supposed to complement B vitamins presumably lacking in their diet, vertebrate blood. Recent genomic analyses revealed considerable differences in biosynthetic capacities across different symbionts, indicating that levels of B vitamins vary across different vertebrate hosts. However, a rigorous determination of B vitamins content in blood of various vertebrates has not yet been approached. A reliable analytical method focused on B vitamin complex in blood and hemolymph can provide valuable informative background and understanding of general principles of insect symbiosis.In this work chromatographic separation of a mixture of eight B vitamins (B1 – thiamine, B2 – riboflavin, B3 – niacin, B5 – pantothenic acid, B6 – pyridoxine, B7 – biotin, B9 – folic acid and B12 – cyanocobalamine), four B vitamin derivatives (B3 – niacinamide, B6 – pyridoxal-5-phosphate, B6 – 4-pyridoxic acid and B9 – tetrahydrofolic acid) and 3 stable isotope labelled internal standards (B3 – niacin-13C6, B5 – pantothenic acid (di-β-alanine-13C6,15N2) and B7 – biotin-(ring-6,6-d2)) on C30 column was developed. Detection was carried out using dual-pressure linear ion trap mass spectrometer in FullScan MS/MS and SIM mode. Matching internal standards with analytes was done according to the results of linearity, accuracy and precision. Except for vitamin B9 (tetrahydrofolic acid) instrument quantitation limits of all analytes were ranging from 0.42 to 5.0 μg/L, correlation coefficients from 0.9997 to 1.0000 and QC coefficients from 0.53 to 3.2 %.Optimization of whole blood sample preparation step was focused especially on evaluation of two types of protein-precipitation agents: trichloroacetic acid and zinc sulphate in methanol. Samples of whole blood prepared in six independent replicates were spiked at 10 μg/L and 100 μg/L level. The best results were obtained for zinc sulphate in methanol, but only nine analytes (B1 – thiamine, B2 – riboflavin, B3 – niacin, B3 – niacinamide, B5 – pantothenic acid, B6 – pyridoxine, B6 – 4-pyridoxic acid, B7 – biotin and B12 – cyanocobalamine) were successfully validated. Accuracy of the procedure using this protein-precipitating agent was ranging from 89 to 120 %, precision from 0.5 to 13 % and process efficiency from 65 to 108 %.HighlightsLC-MS/MS method for quantitation of eight B vitamins and four B vitamin derivatives was developed.Deproteinization agents trichloroacetic acid and ZnSO4/methanol were tested for protein precipitation of whole blood.Accuracy, precision and process efficiency were evaluated.Successful method validation for seven B vitamins and two B vitamin derivatives in whole blood.