Optimisation of an analytical method for the analysis of folic acid derivatives in biological materials.

Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2007.Folic acid is a water-soluble, B-complex vitamin influencing a number of biological processes in humans and particularly important in the prevention of neural tube defects (associated with spinal bifida) in unborn children. Reliable analytical methods are therefore needed for quantisation of the amount of total folic acid (FA) in biological materials of quality assurance and regulatory purposes. What is particularly needed are rapid and reliable methods for ensuring that the correct amount of FA is consumed and the degradation rates of these compound is kept at minimum during the extraction process. Analytical methods for determination of folic acid in biological materials have been around for decades and the most common procedures include microbiological assay; biomolecular interaction analysis (BIA); immunoassay; conventional chromatographic procedure such as thin-layer column chromatography (TLC) and high performance liquid chromatography (HPLC). These procedures were replaced by HPLC, which is more rapid and in many instances yields a better resolution. Current HPLC methods uses C-18 column and reverse phase conditions in combination with ion-pair or ion suppression techniques; fluorescence or electrochemical detector, unfortunately, excitation and emission of folic acid is found not sufficiently to allow physiological levels of the form of the vitamin to be detected. In addition, ion-pair reagent nullifies the mobile phase and interferes with the absorption! fluoresce spectrum resulting in poor separation. Therefore this study was carried out to address and improve the problems that are in the existing HPLC methods. Currently scarce information is available on the determination of folic acid in biological materials by HPLC with UV detection. Serum samples were spiked with folic acid standard to check the efficiency of the method. Other wavelengths from 200 nm to 300 nm were attempted for detection of folic acid, in which the wavelength 250 nm was found to have better absorbance compared to other wavelengths. Folic acid was detected at 250 nm wavelength under isocratic elution using a mobile phase consisting of citrate phosphate buffer: acetic acid: methanol. Folic acid in maize meal was detected at 290 nm using mobile phase containing potassium phosphate containing ascorbic acid/sodium ascorbate mixture and 2-mercaptoethanol under gradient elution. The mobile phase used for gradient and isocratic elution was suitable for separation of folic acid from other compounds with flow rate of 3 ml/min modified to Iml/mim to avoid overloading of the column under isocratic elution. For good separation of folic acid under gradient elution the flow rate was set at 0.8ml/min with pH of mobile phase modified from pH 2.2 to pH 2.5. The recovery of folic acid added to human serum was 91% -100% and recovery of folic acid added in unfermented maize meal and fermented maize meal ranged from 55% - 73%. Folic acid level from unfermented maize meal and fermented maize meal ranged between 1.29 - 1.3 [!g/g and 1 - 2.1 [!g/g respectively. In conclusion the optimised method in this study gives better analytical results when compared with earlier HPLC method in terms of efficiency, reproducibility and sensitivity for folic acid in human serum and maize meal. However, there is a need to minimise the loss of folic acid during the sample treatment. The outcome of this work indicated that more work has to be done to improve extraction procedure for specific foods with minimum time preparation to sample analysis