A MASS SPECTROMETRIC VALIDATED HIGH=PERFORMACE LIQUID CHROMATOGRAPHY PROCEDURE FOR THE DETERMINATION OF FOLATES IN FOODS

Authors: PAWLOSKY, ROBERT - NIH; FLANAGAN, VINCENT; DOHERTY, ROBERT

Submitted to: Journal of Agriculture and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/25/2003
Publication Date: 6/30/2003
Citation: Pawlosky, R., Flanagan, V.P., Doherty, R.F. 2003. A mass spectrometric validated high=performace liquid chromatography procedure for the determination of folates in foods. Journal of Agriculture and Food Chemistry. 51:3726-3730.

Interpretive Summary: Folates, a family of water soluble vitamins, are consumed in foods and from supplements and have been increasingly recognized as important in many aspects of human health. From the prevention of neural tube defects in infants, to the prevention of coronary artery disease and cancer, folates are recognized as a key nutrient in the human diet. In order to judge how adequate the consumption of folates is, methods must exist to analyze folates in our foods. Methods have existed for many years to perform these analyses. They varied widely in the physical methods used and the results many times didn¿t agree very well. In addition, for many of these methods the individual members of the folate family weren¿t measured ¿ only the total amount of folate. With recent actions taken by the Food and Nutrition Board of the National Academy of Science, with respect to folate intake and requirements, it has become important that the results of the analysis of folate content in foods be independent of the method used for the analysis. In addition, these actions require the individual folate members to be independently reported. The present study uses an easily implemented, and relatively inexpensive, method alongside an expensive but definitive method and compares the results from the two methods. The inexpensive method uses high performance liquid chromatography (HPLC) with fluorimetric detection while the expensive method used HPLC with electrospray ionization mass spectrometric detection. Both methods report the separate amounts of each folate member and the definitive method assures that the value reported by the inexpensive method is correct. A representative sample of common foods as well as standard reference materials were analyzed and the results show that the values measured by the two methods agree, and thus analyses reported by the inexpensive method are accurate. This paves the way for an inexpensive method to be used to provide definitive values for this important family of nutrients.

Technical Abstract: A series of five food reference materials (RM) that had certified values of folate concentrations and five frozen food samples were analyzed for 5-methyltetrahydrofolic acid (5-MTHFA) and folic acid (FA) using a High Performance Liquid Chromatography (HPLC) method with fluorescence detection that was validated using an HPLC mass spectrometry (MS) method with electrospray ionization (ESI). Identical sample specimens were extracted and analyzed in triplicate using both instrumental methods and a comparison was made of the mean values of 5-MTHFA and FA resulting from these determinations. The analytes were isolated on either a high capacity strong anion exchange solid-phase extraction (SPE) column (HPLC method) or a phenyl Bond Elut column (MS method) prior to analyses. For quantification of the analytes by mass spectrometry, 13C-labeled 5-MTHFA and FA were added to samples as internal standards prior to enzymatic digestion and conversion of the poly-glutamate forms of 5-MTHFA to the mono-glutamic acid. Quantification of FA and 5-MTHFA using the HPLC analysis was carried out using external standards. With the exception of one RM (pig liver) the values established for 5-MTHFA using these methods were highly comparable. In determining the variance associated with these procedures, it was observed that the mean relative standard error (RSE) for 5-MTHFA was 12% (range: 2-27%) and 11% (range: 5-25%) for the HPLC and MS methods, respectively. Folic acid was detected in only three of the samples and the values obtained for it by either method were similar. This is the first report that describes a mass spectrometric method used in the validation of an HPLC determination of food folates across a wide range of sample matrices. The comparable values for 5-MTHFA and FA suggest that HPLC analysis with fluorescent detection may be used to accurately quantify folates present in a variety of food matrices.