(LB01-10-25) Comprehensive Analysis of B-vitamins, Water-Soluble Cholines, and Related Compounds in Plasma

Objectives: B-vitamins and related compounds are metabolic co-factors required for human health and normal functions. We developed a LC-MS/MS method for analyzing thiamin, thiamin-monophosphate, thiamin-diphosphate, riboflavin, flavin adenine dinucleotide, flavin mononucleotide, nicotinamide, nicotinic acid, nicotinamide mononucleotide, nicotinamide dinucleotide, nicotinamide riboside, nudifloramide, pantothenic acid, pyridoxal, pyridoxal-5-phosphate, pyridoxamine, pyridoxine, 4-pyridoxic acid, 4-deoxy-pyridoxine, biotin, choline, phospho-choline, glycerol-phospho choline, betaine, carnitine, creatinine, methionine, methylmalonic acid, myo-inositol, and trimethylamine-N-oxide.

Methods: Optimized results without the need for ion-pairing reagents were obtained by injecting 10µL three times, using a Gemini NX-C18 column under alkaline conditions, and a the Synergi Fusion-RP and a Luna Silica (2) Column (all Phenomenex) under acidic conditions. Each analytical run was completed in 5 or 6 min. Only 30µL sample was needed and spiked with an internal standard mix before proteins were precipitated using 180µL chilled methanol. After a centrifugation step, the supernatant was transferred and evaporated to dryness, and the residual reconstituted in 0.2% aqueous acetic acid.

Results: Analyte recoveries over 3 days (3-levels, n=4 each) in pooled plasma ranged from 73-129% (CV: 4.5 to 15%) across analytes; only myo-inositol was recovered at 46% but with satisfactory reproducibility (CV=10.4%). Available plasma samples with certified values for pyridoxal, pyrisoxal-5-phosphate, and methylmalonic acid over 3 days (n=2 each) showed accuracies of 100%,104%, and 99%, respectively.

Conclusions: The new method allows a comprehensive evaluation of 30 B-vitamins and related compounds in a 96-well plate format. Validation for whole blood, red blood cells, and human milk are currently underway, reducing analytical bias when analyzing these different matrices. This platform is a useful tool for assessing effects of interventions, and relationships of mother, milk, and infant nutrient status.

Funding Sources: Bill & Melinda Gates Foundation