PH stability of individual folates during critical sample preparation steps in prevision of the analysis of plant folates

Abstract

The stability and eventual interconversion of nine mono-glutamate folates (5-methyl-tetrahydrofolate, tetrahydrofolate, 5-formyltetrahydrofolate, 5,10-methenyltetrahydrofolate, 5,10-methylenetetrahydrofolate, dihydrofolate, 10-formylfolic acid, 10-formyltetrahydrofolate and folic acid) during the typical sample preparation steps (heat treatment for 10 min at 100 degrees C and incubation for 2 h at 37 degrees C) at different pH values have been investigated by LC-MS/MS. An LC-MS/MS method with isotopically labelled [(13)C(5)]5-methyltetrahydrofolate and [(13)C(5)] folic acid as internal standards has been developed with enhanced sensitivity using a Chromolith RP-18 column. 5-Methyltetrahydrofolate, folic acid and 10-formylfolic acid are relatively stable at different pHs (from 2 to 10) with and without heat treatment. Tetrahydrofolate shows instability at low pH. 5-Formyltetrahydrofolate and 5,10-methenyltetrahydrofolate can interconvert by changes in pH. Tetrahydrofolate and 5,10-methylenetetrahydrofolate can interconvert with formaldehyde or by changes in pH. Incubation at 37 degrees C for 2 h is much less aggressive for most folates as compared with heat treatment at 100 degrees C. At 37 degrees C most folates are stable at pH values between 4 and 8 except tetrahydrofolate and dihydrofolate, which are degraded at low pH. 10-Formyltetrahydrofolate and 5,10-methylenetetrahydrofolate cannot be quantified in the present method because these compounds are converted to 5,10-methenyltetrahydrofolate and tetrahydrofolate, respectively, in the acidic mobile phase. This study provides useful information for the analysis of folates in the future as well as for the interpretation of quantitative results from earlier work.