| Though many HPLC methods have been developed and reported in literature for vitamin analysis for the past two decades, applying certain methods directly from literature more than often fails to reproduce the results reported due to many variables of liquid chromatography. This issue was targeted in this project through the examination of chromatographic behaviors of water-soluble vitamins in order to help the analysts better modify methods from literature or even develop new methods from scratch to fit their analytical need with the resources available (e.g., columns, detectors, etc.) in their lab.;The first part of the project investigated the chromatographic behaviors of five vitamins: thiamine (B1), riboflavin (B2), pyridoxine (B6), cyanocobalamin (B12) and ascorbic acid (C) using different reversed-phase columns. Type-B-silica columns with novel reverse bonded phase compatible with 100% aqueous phase were found to be best suited for the analysis of water-soluble vitamins. With a simple mobile phase system using 0.1% formic acid (A) and acetonitrile (B), the five analytes mentioned above could be conveniently separated in 2 groups. Group 1 with vitamin B1, B6 and C can be eluted under 100% phase A, while group 2 with vitamin B2 and B12 can be eluted under 85% phase A, 15% phase B. Approaches to enhance the retention of the three fast-eluting vitamins (B1, B6 and C) were investigated. Perfluorinated acids such as TFA or HFBA proved to be efficient in improving the retention of B1 and B6 in reversed-phase columns. An alternative is to use buffered mobile phase with pH from 5.0 to 7.0. Ammonium acetate buffer pH 5.8, which is compatible with LCMS, was found to be able to improve B1 and B6 retention significantly. HILIC column was another alternative to enhance the retention of not only B1 and B6 but also C.;The second part of the project was expanded to include the other four water-soluble vitamins (niacinamide B3, pantothenic acid B5, biotin B7 and folic acid B9). The goal was to develop HPLC methods for the analysis of all nine water-soluble vitamins using DAD-ELSD and LCMS. ELSD is a universal detector that responds more or less similar to all vitamins. However, its sensitivity is too low to even allow the analysis of samples with high concentration of target analytes such as dietary supplements. DAD is more sensitive but subject to possible background interferences and noisy baseline at low wavelengths (e.g., 210 nm) that were needed to obtain response from non-chromophoric vitamins like pantothenic and biotin. Therefore, the use of DAD for simultaneous multi-vitamin analysis was limited to simple samples like dietary supplements. LCMS has the highest sensitivity and specificity among the three detectors. It was proven to be effective for the simultaneous analysis of all nine analytes in fortified food products with more complicated matrices like fortified cereals and infant formula powder. |
