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This Article Full Text Full Text (PDF) Data Supplements All Versions of this Article: clinchem.2005.053801v1 51/12/2318    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (9) Citing Articles via Google Scholar Google Scholar Articles by Fazili, Z. Articles by Jain, R. Search for Related Content PubMed PubMed Citation Articles by Fazili, Z. Articles by Jain, R. Related Collections General Clinical Chemistry

Erythrocyte Folate Extraction and Quantitative Determination by Liquid Chromatography–Tandem Mass Spectrometry: Comparison of Results with Microbiologic Assay

Inorganic Toxicology and Nutrition Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA.

^aAddress correspondence to this author at: Centers for Disease Control and Prevention, 4770 Buford Hwy, Atlanta, GA 30341. Fax 770-488-4139; e-mail CPfeiffer{at}cdc.gov.

Background: Erythrocyte folate analysis is an important diagnostic tool to establish folate status or screen for folate deficiency.

Methods: We evaluated conditions that influence the complete hemolysis and deconjugation of folate polyglutamates to folate monoglutamates (FMGs) from whole blood (WB). WB samples were hemolyzed in 10 g/L ascorbic acid at various temperatures (room temperature, 30 °C, and 37 °C; n = 15) or hemolysate pH values (pH 4.0, 4.7, 5.2; n = 11) and incubated up to 6 h. FMGs and folate diglutamates (FDGs) were analyzed by liquid chromatography–tandem mass spectrometry (LC/MS/MS) and total folate (TF) by microbiologic assay. We investigated delaying hemolysis by freezing WB for 10 days (n = 20).

Results: Hemolysates frozen immediately after preparation contained 22%–27% FDGs, depending on hemolysate pH. The proportion of FDGs decreased to <3% after incubation at pH 4.7/37 °C for 3 h and did not significantly change on extended incubation up to 5 h. Short-term delayed hemolysis of WB produced results indistinguishable from those of immediate hemolysis. TF results obtained by the microbiologic assay were not different across incubation conditions and agreed with the sum of FMGs and FDGs by LC/MS/MS. The difference between the 2 methods was an insignificant 3% for pH 4.7/37 °C for 3 h.

Conclusions: Hemolysate incubation up to 2 h at 37 °C is not adequate for full polyglutamate deconjugation. We obtained the highest yield of FMGs with lowest FDG concentrations at pH 4.7/37 °C for 3 h. Delaying hemolysis of WB for several days had no negative effect on measurable folate for presumed MTHFR C/C genotype samples.

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