Location: Obesity and Metabolism ResearchTitle: Improving LC-MS analysis of human milk B-vitamins by lactose removal
|HAMPEL, DANIELA - University Of California, Davis|
|Allen, Lindsay - A|
Submitted to: Journal of Chromatography B
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/29/2021
Publication Date: 10/4/2021
Citation: Hampel, D., Shahab-Ferdows, S., Newman, J.W., Allen, L.H. 2021. Improving LC-MS analysis of human milk B-vitamins by lactose removal. Journal of Chromatography B. 1183. Article 122968. https://doi.org/10.1016/j.jchromb.2021.122968.
Interpretive Summary: In our previously report on the first method for measuring five B-vitamins simultaneously using UPLC-MS/MS we found that other constituents in human milk severely impact the analysis. High levels of milk sugar (lactose) contaminated the analytical equipment which interfered with the analysis. We evaluated sample purification techniques, namely solid-phase extraction (SPE), liquid-solid extraction (LSE), and protein precipitation (PPT), as well as lactose-removal by chromatographic separation from the target compounds. None of the sample purification approaches provided acceptable vitamin recoveries and lactose removal, but lactose could be separated from the vitamins using an optimized LC-gradient enabling its post-column removal using a switch-valve. Only 40µL milk was used for sample preparation and B-vitamin recoveries were determined (81.9-118.6%; CV=11.9%; precision: 4.9-13.7%) with greatly reduced matrix effects, and imporved process efficiency, and recovery.
Technical Abstract: Our previously reported first validated UPLC-MS/MS-based simultaneous analysis of five human milk B-vitamins revealed severe matrix effects. High levels of endogenous lactose fouled the electrospray ionization source affecting the analysis. We evaluated solid-phase extraction (SPE), liquid-solid extraction (LSE), protein precipitation (PPT), and liquid chromatography effluent diversion for lactose-removal. SPE failed to separate lactose from vitamins; LSE using 2-propanol reduced lactose and vitamin recoveries. PPT-solvent, milk volume, and reconstitution solvent influenced flavin adenine dinucleotide, pyridoxal and nicotinamide recoveries. Using an optimized LC-gradient enabled chromatographic separation of lactose from vitamins and its removal using a post-column switch-valve. Only 40µL milk was subjected to methanol-PPT and non-polar matrix removal by methyl tert-butyl ether. B-vitamin recoveries were established (81.9-118.6%; CV=11.9%; precision: 4.9-13.7%) with greatly improved matrix effects, process efficiency, and recovery.