Analysis Of Water-soluble Vitamins By Titania-based RP-HPLC

With the increasingly wide application of high performance liquid chromatography（HPLC） in the analytical field, the development of chromatographic packings therefore has attracted more and more attention. Silica is the most popular material for chromatographic columns, whose main disadvantages are narrow p H range（p H 3-10） and irreversible adsorption for basic compounds. Compared with silica, titania shows higher chemical, mechanical and thermal stabilities. For example, titania is quite stable within the range of p H 1-14. Titania could separate basic compounds well, and bind reversibly with hydroxyl, phosphoric and carboxylic groups and thus good for analysis of compounds with these groups. In addition, titania column could keep low pressure under increasing temperature, which is benificial to analysis of compounds. Owing to these unique properties, titania has appeared as an attractive alternative to silica in recent years.Vitamins play important roles in maintenance of human health, prevention and treatment of various diseases. Although food such as cereal, fruits and vegetables, contain vitamins, there are more and more emerging food and health care products with vitamin supplements recently. Consequently, analysis and determination of vitamin content would be significant. To our best knowledge, analysis of vitamins by titania-based reversed phase chromatography has not been reported. The present study therefore aimed to apply titania-based reversed phase column to analysis of water-soluble vitamins.The present study included three parts:（1） A rapid method for the determination of vitamin B₁ by reversed phase high performance liquid chromatography（RP-HPLC） on titania has been developed. The effects of methanol percentage, temperature and flow rate on the retention behavior of vitamin B₁ have been investigated. The optimized chromatographic conditions were as follows: The maximum wavelength of vitamin B₁, the p H value and concentration of phosphate buffer, the percentage of methanl, the column temperature and flow rate were 265 nm, 7.0, 5.0 mmol/L, 65%, 30 ℃ and 0.8 m L/min, respectively. The present method showed good linearity（R₂=0.9998） within the range of 0.083-125 μg/m L. The limit of detection for vitamin B₁ was 0.025 μg/m L.The relative standard deviation（RSD） of eight replicates was 0.56%. The recovery of spiked vitamin B₁ ranged from 95.46 to 105.40%.（2） The present study has also developed a new method for simultaneous determination of vitamins B₂ and B₅ by HPLC on a titania-based column. The relationships of buffer concentration, column temperature and flow rate withthe separation of two vitamins have been investigated. The optimized chromatographic conditions were as follows: Vitamin B₂ was detected at 270 nm, while vitamin B₅ was observed at 210 nm. The p H value and concentration of phosphate buffer, the column temperature and flow rate were 2.5, 80 mmol/L, 50 and 0.8 m L℃/min, respectively. The thermodynamic parameters of the two vitamins including enthalpy, entropy and Gibbs free energy were also calculated. The method presented good linearity toward vitamin B₂ in the range of 1.0-50.0 μg/m L（R₂=0.9999） and vitamin B₅ in the range of 3.0-150.0 μg/m L（R₂=0.9998）, respectively. The limit of detection for vitamins B₂ and B₅ were 0.3 and 0.9 μg/m L, respectively. Both precisions（RSD） for the two vitamins were less than 0.22%, and both recoveries were between 94.55%-104.06%. The precision and accuracy of the present method could meet the requirements of HPLC.（3） Another orignial method for simultaneous separation and determination of five vitamins including biotin（VB₇）, folic acid（VB₉）, cyanocobalamin(VB₁₂), nicotinic acid and nicotinamide（vitamin PP） by HPLC on a titania-based column has been developed. The influences of buffer p H, buffer concentration, column temperature and flow rate on separation have been investigated. The optimized chromatographic conditions were as follows: Biotin and cyanocobalamin were detected at 210 nm, while nicotinic acid, nicotinamide and folic acid were observed at 270 nm. The p H value and concentration of phosphate buffer solution were at 7.0 and 5.0 mmol/L, respectively. The column temperature and flow rate were 50 ℃and 0.8 m L/min, respectively. The thermodynamic parameters of eachanalytes including enthalpy, entropy and Gibbs free energy were calculated. Good linearity（R₂>0.9990） were found for all five vitamins. The limit of detection for nicotinic acid, biotin, nicotinamide, folic acid and cyanocobalamin were 0.006 μg/m L, 0.010 μg/m L, 0.020 μg/m L, 0.022 μg/m L and 0.008 μg/m L, respectively. The precisions（RSD） for all five vitamins were less than 1.36%, and all the recoveries were between 92.30-107.20%. The precision and accuracy of the present method could perfectly fit the requirements of HPLC.