A Novel Method for the Analysis of Water-Soluble Vitamins by Uplc
A NOVEL METHOD FOR THE ANALYSIS OF WATER-SOLUBLE VITAMINS BY UPLC
M. Rossi and A. Regine Industria Farmaceutica Serono, Ardea, Italy
INTRODUCTION EXPERIMENTAL
Vitamins are essential substances for health and LC Conditions growth, and have to be supplied by food in sufficient LC System: Waters® ACQUITY UPLC® System amounts. If the intake is not adequate, or special with an ACQUITY UPLC PDA dietary requirements exist, multivitamin preparations Detector should be taken in order to prevent deficiencies. Column: ACQUITY UPLC BEH C18, Accurate quantitative analysis are needed to measure 2.1 x 100 mm 1.7 µm vitamins in food, in pharmaceutical preparations and Mobile Phase A: 25 mM KH2PO4, 5 mM other matrices where vitamins are present. Hexanesulphonic Acid, pH 4.0 Mobile Phase B: 5 mM Hexanesulphonic Acid in Vitamins can be divided in two major groups, water- Methanol and fat-soluble. Several separation techniques, mainly Flow Rate: 0.4 mL/min. based on capillary zone electrophoresis and HPLC Column Temp: 35 °C (detected by UV or Fluorescence), are well known for Sample Temp: 4 °C quantification of water-soluble vitamins. Niacinamide, Wavelength: 205 nm (UV) Thiamine, Riboflavin, Pyridoxine, Folic acid, Gradient: Cyanocobalamin, Pantothenic acid and Pyridoxal Time %A %B Curve were investigated in this application note. A typical 0.00 98 2 Initial separation obtained by RP-HPLC/UV is shown in 0.50 98 2 6 Figure 1. 10.0 70 30 6 10.5 20 80 6 The quality of the profile is satisfactory in terms of 14.5 20 80 6 resolution, even if amongst 8 species, some are not 15.0 98 2 6 completely separated. Analysis time (including column regeneration and equilibration) is 50 minutes. The Sample Preparation improved separation capabilities of UltraPerformance A pool of water-soluble vitamins was dissolved in water LC® (UPLC®) Technology was tested on water-soluble or in mobile phase A. The average concentration was vitamins for the development of a new application. 2–4 µg/mL and µL amounts were injected.
2.00 1. Thiamine 6 2. Pyridoxal HCl 3 21.95 3. Pyridoxine 7 4. Niacinamide 6.177 22.70 5. Pantothenic acid 6. Folic acid 1.00 AU 7. Cyanocobalamin Figure 1. Resolution of water-soluble 4 8. d-Biotin vitamins by RP-HPLC. 11.07 9 9. Riboflavin 1 2 8 25.85 5.881 5 4.511 15.97 22.92
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0.00 10.00 20.00 30.00 40.00 48.00 Minutes RESULTS AND DISCUSSION
The separation achieved by UPLC is shown in Figure 2. The profile shows the vitamins were very well separated with superior resolution with respect to that obtained by RP-HPLC (see Figure 1). The peaks are well shaped and symmetric, and all nine species were completely separated. This allowed for the unequivocal identification and accurate quantification of water-soluble vitamins in different biological and chemical matrices. The analysis time was reduced three-fold, thus making this UPLC method both an accurate and robust high-throughput methodology.
0.70 1. Niacinamide 0.65 2. Pantothenic acid 0.60 3. Pyridoxine 0.55 4. Pyridoxal HCl 0.50 5.11 1 5. Folic acid 0.45 4 6. Thiamine 0.40 1 7. d-Biotin
AU 0.35 5.62 8. Cyanocobalamin 3 0.30 5 3.74 9. Riboflavin 9 0.25 3 6.99 0.20 9 9 3.16 67.25 8 1 0.15 2 8 7 0.10
0.05
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0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 Minutes Figure 2. Resolution of water-soluble vitamins by UPLC.
CONCLUSIONS
The analysis of water-soluble vitamins by ACQUITY UPLC is a further demonstration of the wide versatility of this system, already well-known for the superior analysis of amino acids, hydrolyzed peptides and proteins. These results ensure the use of UPLC for testing water-soluble vitamins in all matrices where they are present (tablets, food biological fluids, etc), across a wide number of applications and laboratories.
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