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United States Department of Agriculture

Agricultural Research Service

Title: 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

Authors
item Hampel, Daniela -
item York, Emily -
item Allen, Lindsay

Submitted to: Journal of Chromatography B
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 21, 2012
Publication Date: June 27, 2012
Citation: Hampel, D., York, E.R., Allen, L.H. 2012. 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. Journal of Chromatography B. 903 p. 7-13.

Interpretive Summary: Poor maternal intake of B-vitamins during lactation can result in maternal depletion, lower concentrations in breast milk and subsequent infant deficiency. Little is known about infant requirement; recommended intakes of B-vitamins are generally set as Adequate Intakes (AI), which have been based on analyses of only a few breast milk samples. Protocols applied include microbiological, chromatographic, and spectroscopic methods and are generally optimized for analyzing each B-vitamin individually. Some require laborious techniques and up to several mL of sample. Using Ultra Performance Liquid-Chromatography tandem Mass-Spectrometry (UPLC-MS/MS) a new, rapid and sensitive method for the simultaneous determination of thiamin, riboflavin, flavin adenine dinucleotide (FAD), nicotinamide and pyridoxal (PL) in human milk was developed. Proteins and non-polar compounds of the samples were removed prior to analysis. Quantification was done by ratio response to the stable isotope labeled internal standards using a matrix-like standard curve. Intra- and inter-assay variability for all analytes ranged from 0.4 to 7.9% and from 2.2 to 5.2% respectively; recovery rates for each vitamin ranged from 73.0 to 100.2%. The limit of quantitation for all vitamins was between 0.05 and 5 ppb depending on the vitamin. 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.

Technical Abstract: 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 two minutes using a gradient of 10mM 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.

Last Modified: 10/23/2014