OPTIMIZATION OF CHROMATOGRAPHIC CONDITIONS FOR THE DETERMINATION OF FOLATES IN FOODS AND BIOLOGICAL TISSUES FOR NUTRITIONAL AND CLINICAL-WORK

Recent studies implicate folate metabolism in the aetiology of heart d isease, neural tube defects, malignant transformation and affective di sorders. The paper reports a rapid, isocratic HPLC separation of 11 fo lylmonoglutamate compounds which should prove useful when adapted to t he varied needs of analysts researching these and other specific areas . Also reported are the separation, UV spectra including lambda(max) v alues, fluorescence emission scans at an excitation wave-length of 295 nm and electrochemically derived hydrodynamic voltammograms with opti mum oxidation voltages for p-aminobenzoylglutamate, tetrahydrofolate, 5-methyldihydrofolate, 5-methyltetrahydrofolate, 5-formyltetrahydrofol ate, 5,10-methenyltetrahydrofolate, dihydrofolate, pteroylmonoglutamat e and 5,10-methylenetetrahydrofolate. In particular, 5-methyltetrahydr ofolate, the main food folate and form of the vitamin found in plasma, can be measured easily by eiectrochemical detection using a low and h ighly selective voltage of 450 mV. This reduced folate is also readily detected fluorimetrically using an excitation wavelength of 295 nm an d measuring emission at 365 nm. Electrochemical and fluorimetric detec tion offer equal sensitivity for 5-methyltetrahydrofolate measurement (300 pg on column). No other folate studied could be measured down to this level using fluorimetric detection under the described conditions . At pH 3.5, folate coenzyme lambda(max) for UV detection varies betwe en 267 and 300 nm with 5,10-methenyltetrahydrofolate giving maximum ab sorption at 355 nm. UV measurement of 5-methyltetrahydrofolate is appr oaching an order of magnitude less sensitive than the former methods o f detection. However, for in vitro studies, particularly in the form o f a photodiode array, UV detection is a particularly useful tool. For cerebrospinal fluid, plasma, erythrocyte or food measurement of 5CH(3) -H(4)PteGlu, electrochemical or fluorimetric detection is recommended; whilst for pharmacokinetic studies of plasma 5CHO-H(4)PteGlu during m ethotrexate rescue therapy, electrochemical or UV detection is most ap propriate. For analysis of plasma PteGlu following supplementation, or in food stuffs, UV detection offers the best measurement technique. T he information presented should help address the major problem of trac e folate analysis by HPLC, that is the need to combine high sensitivit y with optimum selectivity in studying complex matrices such as physio logical fluids, tissue preparations and food samples.