Daniela Hampel - 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

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      Publication Details (including relevant citation   information):

      Journal of Chromatpgraphy B 2012, 903: 7-13

      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 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.

      Address (URL): http://http://www.sciencedirect.com/science/journal/15700232