Optimization of Conditions for the Determination of Folic Acid by Spectrophotometric, Fluorimetric and Chromatographic Methods of Analysis

Purpose. The aim of the work was to assess the possibility to use a folic acid drug as a standard sample and to choose the conditions of determination of folic acid by spectrophotometric, fluorimetric and chromatographic methods of analysis with the best metrological parameters.

Methods. For spectrophotometric measurements we used a two-beam scanning spectrophotometer UV-1800 Shimadzu (Japan), for evaluation of fluorescence - liquid analyzer "Fluorat-02-Panorama" (Russia), For chromatographic studies - liquid chromatograph LC-20AD from Shimadzu (Japan), equipped with a spectrophotometric detector SPDM20A and a column Nucleosil C18 (octadecyl), size 25 x 4,6 mm, firm Supelcosil. The method of spectrophotometric determination of folic acid is based on the reaction of obtaining an azo dye with a steady violet color, the intensity of which is proportional to the content of the analyte. The fluorimetric determination of folic acid is based on the oxidation of the analyte with potassium permanganate, which produces reaction products with fluorescence. Chromatographic analysis is based on a directly proportional relationship between peak area and folic acid concentration.

Results. In this work we studied the influence of the nature and pH of four buffer solutions on the degree of extraction of folic acid from dosage forms. The most complete extraction of the analyte (R = 96.83%) was detected in case of using phosphate buffer solution with pH 6.8. A fluorimetric method of determination of folic acid with higher sensitivity as compared to the state methods was proposed, the detection limit was 0.02 µg/ml. Due to a break in the concentration range of the analyte, the sensitivity of the colorimetric method was increased, the detection limit was reduced to  0.13 µg/ml. A chromatographic method of folic acid determination with a linear range of 0.1 - 10 µg/ml, and a detection limit of 0.1 µg/ml were developed.

Conclusion. The proposed methods for determining folic acid have been validated in the analysis of samples of cow's milk and white beans. The use of Fisher's test and a modified Student's test showed the absence of systematic error.

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