It is recognized that some years ago at the time of submission marketing authorization dossiers for medicinal products containing folic acid, there were no part of test procedure for its impurities determination.
In most cases, in the single-component preparations, the spectrophotometric or spectrofluorometric methods are used for folic acid determination. In multivitamin preparations the microbiological method was the method of choice for folic acid determination. However it was impossible, using these methods, to determine folic acid impurities at the same time.
Development of modern instrumental methods such as HPLC, GC, EC makes possible folic acid impurities determination. But it isn't possible to determine, in the same chromatographic conditions, both: folic acid and its impurities simultaneously.
The aim of this work was to determine the conditions for HPLC procedure for identification and quantitative determination of folic acid impurities: p-aminobenzoic acid and N-4-aminobenzoyl-L-glutaminic acid.
Chromatographic separation was achieved on Waters Spherisorb S5ODS1 column (250 x 4.6 mm). Detection was performed using UV/VIS detector at wavelength of 269 nm. A solution of: potassium dihydrogenphosphate water solution: tetrabutyloammoniumhydroxide methanol solution : methanol as a mobil phase was used (pH = 5.0). The injection volume was 25 ml.
The concentration of reference substances mixture in mobile phase was: 0.5 mg/ml of p-aminobenzoic acid and 2mg/ml of N-4- aminobenzoyl-L-glutaminic acid.
Analysed samples were alkalized with 12% ammonium hydroxide prior to extraction with mobile phase solution.
The proposed HPLC method, owing to its satisfactory precision (low relative standard deviation) and accuracy as well as short retention times (4.1 min for p-aminobenzoic acid and 5.3 min for N-4- aminobenzoyl-L-glutaminic acid), can be applied to determine the impurities of folic acid in pharmaceutical preparations. |