Determination and Validation of Folic Acid Content in Fortified Wheat Flour by Triple Quadrupole Liquid Chromatography Tandem Mass Spectrometry

Quantification of Folic acid (FA) in fortified wheat flour was carried out using a triple quadrupole liquid chromatography tandem mass spectrometry method. The method includes FA extraction with 0.03 mM dibasic potassium phosphate buffer containing trifluoroacetic acid, mercaptoethanol, and 0.1% ascorbic acid at pH 9.2. A reverse phase separation on a C18 column by isocratic elution using ammonium acetate and acetonitrile as the mobile phase separated FA during the 5 minute run. The retention time of FA was 1.82 min. Tandem MS-MS analysis was performed in selective reaction monitoring mode (SRM). Product-ion traces at m/z 120.141, 176.055, and 295.074 were used for quantitation of FA, and traces at m/z 442.236 were used as parent mass quantified using positive heated electrospray ionization (HESI).The validated method showed linearity ranging from 1.25 to 640 ng/ml of FA and a correlation coefficient of 0.996. The limit of detection (LOD) was 0.02µg/g and the limit of quantification (LOQ) was 0.05µg/g. The recovery ranged from 72%-99% for the wheat flour spiked at different LOQ levels. No matrix effect was noted. The precision as coefficient of variation ranged from 2.34%-6.07% for spiked wheat flour samples at different LOQ levels. Extraction and quantification of total folate using a microbiological assay (MA) with Lactobacillus casei as the assay organism and Enzyme Linked Immuno Sorbent Assay (ELISA) by competitive binding rapid ELISA kit was also performed. UHPLC-MS/MS is the most sensitive method for determination of even trace level FA and hence can efficiently determine the FA fortification level.