Hydrophilic interaction liquid-chromatography (HILIC) in conjunction with diode array detection has been applied for the separation of selected-water-soluble vitamins using an end-capped HILIC-diol column. Vitamins with significant biological importance, such as thiamine (B(1)), riboflavin (B(2)), nicotinic acid (B(3)), nicotinamide (B(3)), pyridoxine (B(6)), folic acid (B(9)), cyanocobalamin (B(12)) and ascorbic acid (vitamin C) were simultaneously separated. Chromatographic conditions including type and percentage of organic modifier in the mobile phase, pH, type and concentration of buffer salt and flow rate were investigated. ACN was shown to offer superior separation for the compounds tested as compared to methanol, isopropanol and THF. Isocratic separation and analysis were achieved for six vitamins (B(1), B(2), nicotinic acid/nicotinamide, B(6) and C) at ACN-H(2)O 90:10, containing ammonium acetate 10 mM, triethylamine 20 mM, pH 5.0, using a flow rate of 0.8 mL/min, while a gradient was necessary to resolve a mixture of all eight water-soluble vitamins. The HILIC method was validated and successfully applied to the analysis of a pharmaceutical formulation and an energy drink negating the need for time consuming clean-up steps.

HILIC separation and quantitation of water‐soluble vitamins using diol column

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Cobalamin speciation using reversed-phase micro-high-performance liquid chromatography interfaced to inductively coupled plasma mass spectrometry

The analytical procedure of thiamine quantification in premixtures and compound feedingstuffs by HPLC method is presented. Thiamine was extracted from feed samples with 0.1M hydrochloric acid at 100°C for 30 min. In the case of compound feedingstuffs, 10% Taka-diastase solution was added to the samples, and then the samples were incubated at 37°C for 17 h. Afterwards, thiamine was oxidised to thiochrom, using 1% alkaline potassium ferricyanide (III). The filtrated solution was analysed by HPLC with isocratic flow of eluent chloroform-methanol 90:10 (v/v). The measurement was done by a fluorescence detector. This method has been applied for the quantification of the total content of thiamine in compound feedingstuffs, as well as added thiamine in the form of hydrochloride or nitrate salt. The limit of the quantification of this method was determined on the level of 1 mg/kg. The coefficient variation of thiamine quantification results in premixtures samples was 3.7%, and in compound feedingstuffs was 5.56%. The Horrat value for premixture samples reached a value of 0.60, whereas in the case of compound feedingstuffs it was 0.63, thus confirming acceptable precision of the procedure. The recovery rate for thiamine added in the form of hydrochloride was 102.3% in premixes and 98.9% in compound feedingstuffs. The recovery rate for thiamine added in the form of nitrate salt to compound feedingstuffs was similar and reached a value equal to 98.7%.

C.J. Bates

Papers

VITAMINS | Water-Soluble