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High‐throughput adaptation for the quantitation of total folate in human red blood cells by LC‐MS/MS
Author(s) -
Owens Janel Elizabeth,
Holstege Dirk M.,
Clifford Andrew J.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a601-c
Subject(s) - chemistry , chromatography , formic acid , atmospheric pressure chemical ionization , detection limit , whole blood , repeatability , liquid chromatography–mass spectrometry , sample preparation , mass spectrometry , chemical ionization , organic chemistry , ionization , biology , ion , immunology
We adapted an LC‐MS/MS method for measuring whole blood folate ( J. Ag. Food Chem. , 2005; 53 :–7394) to high‐throughput platform for rapid sample processing and analysis in a 96‐well format. Briefly, whole blood was fortified with [ 13 C 6 ] p ABA internal standard and treated with 12.1 N hydrochloric acid at 110 °C for four hours, causing release of the folates from the whole blood and subsequent hydrolysis to para‐ aminobenzoic acid ( p ABA). The hydrolysate was diluted and cleaned‐up using a C18 SPE 96‐well plate; and the p ABA isotopomers were eluted with ethyl acetate/hexane before methylation with methanol and trimethylsilyldiazomethane. The methyl‐ p ABA derivatives were detected and quantified by positive‐ion atmospheric pressure chemical ionization (APCI) LC‐MS/MS. An isocratic mobile phase of acetonitrile‐water (70:30) on a C18 analytical column was used with a post‐column reagent of 0.025% formic acid. The limit of quantitation for folate was 56 nmol/L. Results obtained by this method, an LC‐MS/MS method, and chemiluminescence were compared for fifteen blood samples obtained from healthy volunteers. This method provided enhanced sample throughput (n = 192/day) compared to previously reported GC/MS and LC‐MS/MS methods and is a good candidate for clinical studies where accuracy is needed to assess folate nutritional status. Supported by NIH DK 45939.