The interest of researchers on this vitamin has increased because of the recent importance granted to folic acid, in view of its healthy action towards man, and consequently the concern of analysts to develop appropriate methodologies for folic acid determination and control in enriched or non-enriched foods. In this landscape, the objective of this research was to evaluate some experimental conditions in the determination of folic acid (FA), by high performance liquid chromatography (HPLC), in enriched milks. Using a reverse-phase column (C18), 35 different sujstenis isocratic and 11gradient elution. Detection was at four different wavelengths. Studies were carried out about the stability of the FA standard solutions employed in the quantification. In relations a pre-chromatographic step, were available 15 solutions of extraction. The best results were obtained using an extraction procedure with alkaline solution, pH above of 7.0. The best conditions of analysis were obtained with gradient elution, flow rate of 0.5mL/min, using 10% acetronitrile plus 90% buffer aqueous phase (acetic acid 0.166mol/L; potassium hydroxide 0.01mol/L; pH 2.8) in the beginning, changing to 24% acetonitrile plus 76% buffer aqueous phase after 8.5minutes, remaining this conditions until nine minutes (9.0min) of run. Detection of folic acid was obtained in the ultraviolet region, at 290nm, due to low presence of interfering substances from matrix. Quantification by means of an external standard curve. The folic acid standard dissolved in phosphate buffer (pH 6.5), under refrigeration (4°C), can to be used during 30 days. The limits of detection, quantification being respectively 1.3ng/mL; 2.6ng/mL.
folic acid; high performance liquid chromatography; methodology
