COMPARISON OF A HPLC AND RADIOPROTEIN-BINDING ASSAY FOR THE DETERMINATION OF FOLATES IN MILK AND BLOOD-SAMPLES

A high-performance liquid chromatographic method for the determination of folates in milk, whole blood and plasma following thermal extracti on, enzymatic deconjugation and a clean-up step with a strong anion ex change column is described. The optimized and rapid HPLC method was ca rried out on a reverse phase CII column where the native fluorescence was monitored at excitation and emission wavelengths of 310 and 352 nm , respectively. The method is sensitive with low detection limits for 5-CH3THF, THF and 5-CHOTHF, below 3 pmol/injection. The intra-and inte rassay CV for the individual folate standards is in the range of 4.0 - 8.8%. 5-CH3THF was the dominant form found in all samples. The mean /-SD, concentrations of total folate in milk (with and without conjuga se treatment), whole blood and plasma were 45.6 +/- 4.6 ng/ml, 25.2 +/ - 1.8 ng/ml, 239.9 +/- 36.5 nmol/litre, and 8.0 +/- 1.9 mmol/litre. Co rresponding figures using a commercial protein-binding assay kit were 47.7 +/- 9.8 ng/ml, 79.6 +/- 8.8 ng/ml, 522.2 +/- 90.3 nmol/litre, and 10.7 +/- 3.8 mmol/litre, respectively. The average recovery of 5-CH3T HF, THF and 5-CHOTHF added to milk samples after heat extraction were 106, 70 and 0%, respectively. After deconjugation the recovery for 5-C H3THF decreased while it increased for THF, suggesting that constituen ts in the biological matrix either degraded or transformed these folat es into other forms.