Chromatographic assays for folic acid.

TLC and high-pressure liquid chromatographic (HPLC) assays for folic acid were developed. In the TLC procedure, the folic acid band was extracted from the silica gel after development and determined spectrophotometrically by an oxidation and Bratton-Marshall sequence. A column packed with octadecylsilane chemically bonded to microparticulate silica gel was used for the HPLC assay. Potentiometric determination of water in folic acid samples was necessary to obtain accurate purity values. In a comparison of four assay methods, a direct colorimetric method gave precise, but unspecific, results, while the USP XIX colorimetric method lacked both precision and specificity. The two chromatographic methods were both precise and specific and gave the same assay results. Both methods separated p-aminobenzoic acid, N-(p-amino-benzoyl)-L-glutamic acid, 2-amino-1,4-dihydro-4-oxo-6-pteridinecarboxylic acid, 2-amino-4(1H)-pteridinone, and several unidentified impurities from folic acid.