TECHNICAL REPORT. Supelco Supelco Park Bellefonte, PA USA

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1 TECHNICAL REPORT Supelco Supelco Park 68 USA Title: Separations of Water-Soluble and Fat-Soluble Vitamins by Reversed-Phase HPLC Using Discovery TM Columns Authors: Gang Huang Carmen Santasania Research Scientist Applications Lab Manager Tel: Tel: Fax: Fax: Date: 6 May, 999 Abstract: Water-soluble and fat-soluble vitamins were separated by reversed-phase HPLC on Discovery TM C8, Discovery C8, and Discovery RP-AmideC6 columns. These columns offer excellent resolution and unique selectivity. 999 Sigma-Aldrich Co. T9908 CII

2 Introduction Vitamins are an extremely diverse range of organic compounds present in minute amounts in natural foodstuffs which are essential to normal metabolism. They play a vital role in enzyme reactions which are necessary for carbohydrate, fat, and protein metabolism. Lack of vitamins in the diet can cause diseases. Vitamins can be classified into two groups: water-soluble and fat-soluble. Water-soluble vitamins are thiamine (B ), riboflavin (B ), pyridoxine (B 6 ), pantothenic acid (B ), cyanocobalamin (B ), biotin, niacin, folic acid, and ascorbic acid (C). Fat-soluble vitamins are vitamins A, D, E, and K. Vitamins can act as either coenzymes or prohormones. Vitamins are relatively unstable and are affected by a number of factors such as heat, light, air, other food components, as well as food processing conditions (, ). Because of their critical role in nutrition and their relative instability, qualitative and quantitative analyses of vitamins are an important issue as well as a challenging task. Food manufacturers routinely perform analyses of vitamins in their products to determine quantitative levels and to monitor changes that have occurred in food processing. HPLC is the preferred technique for separation of vitamins due to its high selectivity. Many studies up until the 980s on vitamins using HPLC technique are collected in reference. Recent studies showed various applications in determining water-soluble and fat-soluble vitamins in different sample sources (-6). In this application, standard vitamin mixtures and a real sample Centrum Multivitamin Liquid were analyzed by HPLC using Discovery C8, Discovery C8, and Discovery RP-AmideC6 columns. Experimental Section Instrumentation Chromatographic separations were performed on a Waters 600E HPLC system. All injections were controlled by a Waters 77 autosampler. A Waters 8 tunable absorbance detector was used to monitor the UV absorbance of samples. The wavelength was set at 0nm for water-soluble vitamins and at 90nm for fat-soluble vitamins. The columns used in the experiments were Discovery C8, Discovery C8, and Discovery RP-AmideC6 columns. All columns were cm x.6mm and packed with µm particles. The column temperature was controlled at either 0 0 C or 0 C. No guard columns or filters were used. The column pressure was well below 00psi in all cases. Materials Water-soluble vitamins niacin, niacinamide, thiamine HCl (B ), riboflavin (B ), pantothenic acid (B ), ascorbic acid (C), and folic acid were obtained from Sigma Chemical Co. Fat-soluble vitamins retinol acetate (A), cholecalciferol (D ), ergocalciferol (D ), α- tocopherol (E), α-tocopherol acetate, δ-tocopherol, and γ-tocopherol were obtained from Sigma as well. Biotin, cyanocobalamin (B ), and retinol palmitate were obtained from Supelco. Water-soluble vitamins were dissolved in deionized water. Fat-soluble vitamins were dissolved in methanol. Centrum Multivitamin Liquid was purchased from a drug store. It was passed through a PVDF syringe filter for sample pre-treatment. Run Conditions Water-soluble and fat-soluble vitamins were separated by reversed-phase HPLC on Discovery C8, Discovery C8, and Discovery RP-AmideC6 columns using either isocratic elution or gradient elution. Run conditions for each experiment are explained in detail along with each chromatogram. Results and Discussion Many of the vitamins are light-sensitive and unstable. The sample must be freshly prepared each time and kept in the dark. The concentration of each vitamin chosen was first based on information from some previous work done at Supelco. Then some experiments were conducted to find the appropriate concentration and injection quantity. Because each vitamin has its own maximum absorbance at a different wavelength, a compromise must be made to detect them all together. For water-soluble vitamins, 0nm was selected, while 90nm was used for fat-soluble vitamins. In the mobile phase, we attempted to use simple buffers without addition of any kind of ion-pairing agents or competing amine modifiers. Water-Soluble Vitamins Water-soluble vitamins can be eluted from various types of reversed-phase columns with low concentrations of aqueous methanol or acetonitrile. Five of the water-soluble vitamins ascorbic acid, niacin, thiamine, niacinamide, and pantothenic acid were separated isocratically using % methanol in acidic buffer (Figure ). Four other vitamins folic acid, biotin, cyanocobalamin, and riboflavin were resolved and eluted within 8 minutes using 0% methanol (Figure ). Pantothenic acid, which eluted last in Figure A, eluted much earlier when buffer ph was raised to neutral (Figure ). All nine vitamins were very well separated in a single run using a gradient elution (Figure ). We used the same conditions to analyze Centrum multivitamin liquid sample (Figure ), and identified the peaks through a comparison with those in Figure. Figure 6 illustrates the chromatogram of these nine vitamins obtained on a Discovery C8 column. A similar elution pattern was obtained using the same conditions on a Discovery C8 column.

3 Fat-Soluble Vitamins Fat-soluble vitamins are very hydrophobic and must be dissolved in organic solvents. In this experiment, methanol was used to dissolve each fat-soluble vitamin. In reversed-phase HPLC, very high concentration aqueous methanol or acetonitrile must be used in order to elute fat-soluble vitamins, sometimes even using pure methanol or acetonitrile. Figure 7 shows the reversed-phase analysis of vitamin A acetate, three vitamin E isomers, and vitamin E acetate performed on a Discovery C8 column using 9% methanol. An almost identical result is illustrated in Figure 8 for a reversed-phase analysis of the same mixture on a Discovery C8 column using 90% acetonitrile. In this analysis, a mixture of vitamin A acetate, vitamin D, three vitamin E isomers, and vitamin E acetate was completely separated by reversed-phase HPLC using the Discovery C8 column (Figure 9). All chromatograms exhibit excellent resolution and peak shape. The fat-soluble vitamins in the real sample Centrum Multivitamin Liquid were separated on the Discovery C8 column (Figure 0) and studied. Vitamin E acetate and vitamin A palmitate were clearly identified, while vitamin D was barely seen on the baseline. This sample spiked with vitamin D generated the chromatogram shown in Figure. A Discovery RP-AmideC6 column also was examined for separation of different mixtures of fat-soluble vitamins. The two main forms of vitamin D, ergocalciferol (D ) and cholecalciferol (D ), were separated on this column using 90% acetonitrile (Figure ). Figure shows the reversed-phase separation of a mixture of vitamin A acetate, vitamin D, vitamin D, δ-tocopherol, γ-tocopherol, vitamin E acetate, and vitamin E by 88% methanol. Conclusion Various Mixtures of water-soluble vitamins and fat-soluble vitamins in A, D, and E groups were studied by reversed-phase HPLC using Discovery C8, Discovery C8, and Discovery RP-AmideC6 columns. Feasibility of these columns for separation of real samples was demonstrated as well. Excellent resolution and unique selectivity are achieved in separations of these compounds. Peak shape and reproducibility were very good as well. References. Machlin, L.J. ed. Handbook of Vitamins, Marcel Dekker, Inc., New York, 99.. Ottaway, P.B. ed. The Technology of Vitamins in Food, Chapman & Hall, Inc., New York, 99.. De Leenheer, A.P., Lambert, W.E., De Ruyter, M.G., M Ed. Modern Chromatographic Analysis of the Vitamins, Chromatographic Science Series, Vol. 0, Marcel Dekker, Inc., New York, 98.. Burri, B.J., Neidlinger, T.R., Lo, A.O., Kwan, C., Wong, M.R. J. Chromatogr. A. 997, 76: 0.. Qian, H., Sheng, M. J. Chromatogr. A. 998, 8: Gratzfeld-Huesgen, A., Schuster, R., Haecker, W. Tec. Lab. 99, : 77. References not available from Supelco. Trademarks Centrum Lederle Consumer Health Discovery Sigma-Aldrich Co.

4 s Figure. Five Water-Soluble Vitamins Column: Discovery C8, cm x.6mm ID, µm particles Cat. No.: 09 Mobile Phase: 0mM KH PO, ph.:methanol, 97: Flow Rate: ml/min Pressure: 00psi Temp.: 0 C Det.: UV, 0nm Inj.: µl. Ascorbic acid (Vitamin C), 0µg/mL. Niacin, 0µg/mL. Thiamine (Vitamin B ), 0µg/mL. Niacinamide, 0µg/mL. Pantothenic acid (Vitamin B ), 00µg/mL G00 Figure. Four Water-Soluble Vitamins Column: Discovery C8, cm x.6mm ID, µm particles Cat. No.: 09 Mobile Phase: 0mM KH PO, ph.:methanol, 80:0 Flow Rate: ml/min Pressure: 00psi Temp.: 0 C Det.: UV, 0nm Inj.: µl. Folic acid, 0µg/mL. Biotin, 00µg/mL. Cyanocobalamin (Vitamin B ), 0µg/mL. Riboflavin (Vitamin B ), 0µg/mL G006

5 Figure. Buffer ph Effect on Elution Order Column: Discovery C8, cm x.6mm ID, µm particles Cat. No.: 09 Mobile Phase: mm K HPO, ph 7:methanol, 97: Flow Rate: ml/min Pressure: 00psi Temp.: 0 C Det.: UV, 0nm Inj.: µl. Ascorbic acid (Vitamin C), 0µg/mL. Niacin, 0µg/mL. Pantothenic acid (Vitamin B ), 00µg/mL. Thiamine (Vitamin B ), 0µg/mL. Niacinamide, 0µg/mL G007 Figure. Gradient Elution of Nine Water-Soluble Vitamins Column: Discovery C8, cm x.6mm ID, µm particles Cat. No.: 09 Mobile Phase: A = 0mM KH PO, ph. B = methanol Flow Rate: ml/min Pressure: 00psi Temp.: 0 C Det.: UV, 0nm Inj.: µl 8. Ascorbic acid (Vitamin C), 0µg/mL. Niacin, 0µg/mL. Thiamine (Vitamin B ), 0µg/mL. Niacinamide, 0µg/mL. Pantothenic acid (Vitamin B ), 00µg/mL 6. Folic acid, 0µg/mL 7. Biotin, 00µg/mL 8. Cyanocobalamin (Vitamin B ), 0µg/mL 9. Riboflavin (Vitamin B ), 0µg/mL 6 Gradient Program %B G008

6 Figure. Vitamins in Centrum Multivitamin Liquid Column: Discovery C8, cm x.6mm ID, µm particles Cat. No.: 09 Mobile Phase: A = 0mM KH PO, ph. B = methanol Flow Rate: ml/min Pressure: 00psi Temp.: 0 C Det.: UV, 0nm Inj.: µl 6. Ascorbic acid (Vitamin C), 0µg/mL. Niacin, 0µg/mL. Thiamine (Vitamin B ), 0µg/mL. Niacinamide, 0µg/mL. Pantothenic acid (Vitamin B ), 00µg/mL 6. Biotin, 00µg/mL 7. Cyanocobalamin (Vitamin B ), 0µg/mL 8. Riboflavin (Vitamin B ), 0µg/mL Gradient Program %B G009 Figure 6. Gradient Elution of Nine Water-Soluble Vitamins Column: Discovery C8, cm x.6mm ID, µm particles Cat. No.: 9-U Mobile Phase: A = 0mM KH PO, ph. B = methanol Flow Rate: ml/min Pressure: 00psi Temp.: 0 C Det.: UV, 0nm Inj.: µl 8. Ascorbic acid (Vitamin C), 0µg/mL. Niacin, 0µg/mL. Thiamine (Vitamin B ), 0µg/mL. Niacinamide, 0µg/mL. Pantothenic acid (Vitamin B ), 00µg/mL 6. Folic acid, 0µg/mL 7. Biotin, 00µg/mL 8. Cyanocobalamin (Vitamin B ), 0µg/mL 9. Riboflavin (Vitamin B ), 0µg/mL 6 Gradient Program %B G000 6

7 Figure 7. Chromatogram of Fat-Soluble Vitamins, A & E, Discovery C8 Column: Discovery C8, cm x.6mm ID, µm particles Cat. No.: 09 Mobile Phase: methanol:water (9:) Flow Rate: ml/min Pressure: 00psi Temp.: 0 0 C Det.: UV, 90nm Inj.: 0µL. Retinol acetate (Vitamin A acetate), 0µg/mL. δ-tocopherol, 6µg/mL. γ-tocopherol, 00µg/mL. α-tocopherol (Vitamin E), 9µg/mL. α-tocopherol acetate (Vitamin E acetate), 0µg/mL G0009 Figure 8. Chromatogram of Fat-Soluble Vitamins, A & E, Discovery C8 Column: Discovery C8, cm x.6mm ID, µm particles Cat. No.: 9-U Mobile Phase: acetonitrile:water (90:0) Flow Rate: ml/min Pressure: 900psi Temp.: 0 0 C Det.: UV, 90nm Inj.: 0µL. Retinol acetate (Vitamin A acetate), 0µg/mL. δ-tocopherol, 6µg/mL. γ-tocopherol, 00µg/mL. α-tocopherol (Vitamin E), 9µg/mL. α-tocopherol acetate (Vitamin E acetate), 0µg/mL G0009 7

8 Figure 9. Chromatogram of Fat-Soluble Vitamins, A, D, & E, Discovery C8 Column: Discovery C8, cm x.6mm ID, µm particles Cat. No.: 9-U Mobile Phase: acetonitrile:water (90:0) Flow Rate: ml/min Pressure: 900psi Temp.: 0 0 C Det.: UV, 90nm Inj.: 0µL. Retinol acetate (Vitamin A acetate), 0µg/mL. Vitamin D, 00µg/mL. δ-tocopherol, 6µg/mL. γ-tocopherol, 00µg/mL. α-tocopherol (Vitamin E), 9µg/mL 6. α-tocopherol acetate (Vitamin E acetate), 0µg/mL G0009 Figure 0. Chromatogram of Fat-Soluble Vitamins in Centrum Multivitamin Liquid, Discovery C8 Column: Discovery C8, cm x.6mm ID, µm particles Cat. No.: 9-U Mobile Phase: acetonitrile:water (90:0) Flow Rate: ml/min Pressure: 900psi Temp.: 0 0 C Det.: UV, 90nm Inj.: 0µL Sample: Centrum Multivitamin Liquid Vitamin E acetate Vitamin A palmitate Vitamin D G0009 8

9 Figure. Chromatogram of Fat-Soluble Vitamins in Centrum Multivitamin Liquid Spiked with Vitamin D, Discovery C8 Column: Discovery C8, cm x.6mm ID, µm particles Cat. No.: 9-U Mobile Phase: acetonitrile:water (90:0) Flow Rate: ml/min Pressure: 900psi Temp.: 0 0 C Det.: UV, 90nm Inj.: 0µL Sample: Centrum Multivitamin Liquid spiked with vitamin D Vitamin E acetate Vitamin D spiked Vitamin A palmitate G0009 Figure. Chromatogram of Fat-Soluble Vitamins, D & D, Discovery RP-AmideC6 Column: Discovery RP-AmideC6, cm x.6mm ID, µm particles Cat. No.: 00 Mobile Phase: acetonitrile:water (90:0) Flow Rate:.mL/min Pressure: 00psi Temp.: 0 0 C Det.: UV, 90nm Inj.: 0µL. Ergocalciferol (Vitamin D ), 0µg/mL. Cholecalciferol (Vitamin D ), 0µg/mL G

10 Figure. Chromatogram of Fat-Soluble Vitamins, A, D, & E, Discovery RP-AmideC6 Column: Discovery RP-AmideC6, cm x.6mm ID, µm particles Cat. No.: 00 Mobile Phase: methanol:water (88:) Flow Rate: ml/min Pressure: 00psi Temp.: 0 0 C Det.: UV, 90nm Inj.: 0µL. Retinol acetate (Vitamin A acetate), 0µg/mL. Ergocalciferol (Vitamin D ), 0µg/mL. Cholecalciferol (Vitamin D ), 0µg/mL. δ-tocopherol, 6µg/mL. γ-tocopherol, 00µg/mL 6. α-tocopherol acetate (Vitamin E acetate), 0µg/mL 7. α-tocopherol (Vitamin E), 9µg/mL G