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 extraction, enzymatic deconjugation and a clean-up step with a strong anion exchange column is described. The optimized and rapid HPLC method was carried out on a reverse phase C18 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 interassay 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 conjugase treatment), whole blood and plasma were 45.6 ± 4.6 ng/ml, 25.2 ± 1.8 ng/ ml, 239.9 ± 36.5 nmollitre, and 8.0 ± 1.9 mmollitre. Corresponding 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 nmollitre, and 10.7 ± 3.8 mmollitre, respectively. The average recovery of 5-CH3THF, THF and 5-CHOTHF added to milk samples after heat extraction were 106,70 and 0%, respectively. After deconjugation the recovery for 5-CH3THF decreased while it increased for THF, suggesting that constituents in the biological matrix either degraded or transformed these folates into other forms.