Ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) for the rapid, simultaneous analysis of thiamin, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk.

A novel, rapid and sensitive ultra-performance liquid-chromatography tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of several B-vitamins in human milk was developed. Resolution by retention time or multiple reaction monitoring (MRM) for thiamin, riboflavin, flavin adenine dinucleotide (FAD), nicotinamide and pyridoxal (PL) has been optimized within 2 min using a gradient of 10 mM ammonium formate (aq) and acetonitrile. Thiamin-(4-methyl-¹³C-thiazol-5-yl-¹³C₃) hydrochloride, riboflavin-dioxo-pyrimidine-¹³C₄,¹⁵N₂, and pyridoxal-methyl-d₃ hydrochloride were used as internal standards. A sample-like matrix was found to be mandatory for the external standard curve preparation. ¹³C₃-caffeine was added for direct assessment of analyte recovery. Intra- and inter-assay variability for all analytes ranged from 0.4 to 7.9% and from 2.2 to 5.2%, respectively. Samples were subjected to protein precipitation and removal of non-polar constituents by diethyl ether prior to analysis. Quantification was done by ratio response to the stable isotope labeled internal standards. The standard addition method determined recovery rates for each vitamin (73.0-100.2%). The limit of quantitation for all vitamins was between 0.05 and 5 ppb depending on the vitamin. Alternative approaches for sample preparation such as protein removal by centrifugal filter units, acetonitrile or trichloroacetic acid revealed low recovery and a greater coefficient of variation. Matrix effect studies indicated a significant influence by matrix constituents, showing the importance of stable isotope labeled internal standards for analyte quantitation in complex matrices.