Measurement of Folates in Serum and Conventionally Prepared Whole Blood Lysates: Application of an Automated 96-Well Plate Isotope-Dilution Tandem Mass Spectrometry Method

[摘要] Folate nutriture is associated with neural tube defects (1), vascular diseases (2), certain forms of cancer (3), and cognitive function (4). To reduce the risk for neural tube defects, the US Food and Drug Administration mandated folate fortification of cereal grain products beginning in January 1998 (5). Serum folate and whole blood folate (WBF) are measured to determine folate status. Clinical methods to determine serum folate and WBF, such as the microbiologic assay and various immunoassays, measure only total folate (TF) and present variable results, especially for WBF (6)(7). Few methods have described the measurement of WBF by chromatography-based methods (8)(9)(10)(11)(12)(13)(14)(15). Gas chromatography/mass spectrometry used to analyze WBF after cleavage of folates to p -aminobenzoic acid showed greater sensitivity than previous chromatographic methods and used for the first time an isotope-labeled internal standard (IS). The method, however, requires a complex and lengthy multistep sample preparation including chemical derivatization (11)(13)(14)(15). We describe here the first automated 96-well plate stable-isotope-dilution liquid chromatography–tandem mass spectrometry (LC/MS/MS) method that measures intact folate monoglutamates in conventionally prepared whole blood (WB) lysates and serum.To increase sample throughput and minimize the extent of manual sample preparation, we adapted our recently developed manual solid-phase extraction (SPE) LC/MS/MS method for 5-methyltetrahydrofolic acid (5CH3THF), 5-formyltetrahydrofolic acid (5CHOTHF), and folic acid (FA) in serum (16) to an automated method using 96-well plates. In addition, we extended this method to measure additional folate forms in WB lysates.The folate calibrators, sample extraction, and chromatographic and MS conditions were as described previously (16). 5,10-Methenyltetrahydrofolic acid hydrochloride salt (5,10CH=THF) and 10-formylfolic acid (10CHOFA) were from Merck Eprova AG. We quantitatively isolated folates from 275 μL of serum or WB …

[学科分类] 过敏症与临床免疫学