Optimisation and validation of a liquid chromatography-tandem mass spectrometry method for folates in rice

An LC-MS/MS method has been developed and validated for the analysis of six monoglutamate folates in rice. Accurate determination of folates in rice seeds required the optimisation of an extraction procedure encompassing homogenisation, heat treatment, tri-enzyme treatment (alpha-amylase, protease and deconjugase), centrifugation and ultrafiltration. The resulting extract was loaded onto a Polaris C 18-A column, followed by gradient elution and detection by tandem mass spectrometry. The validated method is sensitive, reproducible, accurate, and linear between 1 and 400 microg/100g rice (dry weight). The limit of detection and limit of quantification in diluted rice matrix ranged between 0.2 and 1.2 microg/100g and between 0.6 and 4 microg/100g, respectively, depending on the compound. Intra- and inter-day precision varied, respectively, from 1.0 to 9.1% RSD, and from 3.4 to 13.5% RSD, except for one compound (19.1% at low concentrations). The accuracy ranged from 96.9 to 115.4%. Matrix effects were compensated for by use of isotopically labelled internal standards. Most folates in rice extracts were stable when kept for 2 weeks at -20 and -80 degrees C, but degraded after three freeze-thaw cycles or at 4 degrees C, even when stored protected from light. Our validated method offers a sensitive and versatile approach to determine the level of six different monoglutamate folates in a complex matrix as rice.