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Determination of Water Soluble Vitamins

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Analysis of Water-Soluble Vitamins by HPLC Vitamins are compounds, which are necessary to maintain a healthy and properly functioning human organism. A few milligrams a day are enough to regulate the utilization of nutrients, such as carbohydrates, fats, proteins and minerals. Usually vitamins are supplied in food. Figure 1 shows the HPLC chromatogram of the eleven water-soluble vitamins pyridoxamine (B 6 ), thiamine (B 1 ), niacinamide, pyridoxal (B 6 ), pyridoxine (B 6 ), aminobenzoic acid,
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  Analysis of Water-SolubleVitamins by HPLC Vitamins are compounds, which are necessary to maintain a healthy and properly functioninghuman organism. A few milligrams a day are enough to regulate the utilization of nutrients, suchas carbohydrates, fats, proteins and minerals. Usually vitamins are supplied in food.Figure 1 shows the HPLC chromatogram of the eleven water-soluble vitamins pyridoxamine (B 6 ),thiamine (B 1 ), niacinamide, pyridoxal (B 6 ), pyridoxine (B 6 ), aminobenzoic acid, pantothenic acid,folic acid, riboflavin (B 2 ), biotin and thiotic acid using gradient analysis on a reversed phasecolumn and UV detection. The autosampler temperature was set to 4 ºC to avoid decompositionof the samples. Column 4.6 x 75 mm Zorbax SB-C18, 3.5 µm Mobile phase A = 0.05M KH 2 P0 4 in water (pH = 2.5), B = acetonitrile Flow rate 1.0 ml/min Gradient at 0 min 0.6 % Bat 0.5 min 0.6 % Bat 4 min 6 % Bat 12 min 30 % Bat 17 min 60 % B Column wash at 19 min 60 % Bat 20 min 0.6 % B UV detector variable wavelength detector204 nm, standard cell Column compartment temperature 15 °C Stop time 20 min Post time 10 min Injection volume 5 µl Conditions Udo HuberPharmaceutical Agilent Technologies Innovating the HP Way Figure 1Analysis of water-soluble vitamins  1 Pyridoxamine 7 Pantothenic acid 2 Thiamine 8 Folic acid 3 Niacinamide 9 Riboflavine 4 Pyridoxal 10 Biotin 5 Pyridoxine 11 Thiotic acid 6 Aminobenzoic acid1234Time [min]024681012141618Absorbance[mAU]050100150200250300567891011  Equipment Agilent 1100 Series ãQuaternary pump (includesvaccum degasser)ãThermostatted autosamplerãThermostatted column compartmentãVariable wavelength detector, standard flow cell10-mm path length,13-µl cell volume Alternative:  ãBinary pump Vacuum degasserãDiode array detectorstandard flow cell 10-mm path length,13-µl cell volumeãAgilent ChemStation +3D software Columns ãZorbax SB-C18, 3. 5 µm, 4.6 x 75 mm (Agilent part number 866953-902)ãRecommended:Guard cartridges Zorbax SB-C18, 5 µm, 4.6 x 12.5 mm(Agilent part number 820950-920, 4/pk) Note:  Since the method was speci-fically developed on theAgilent 1100 Series systemyou might not be able toreproduce this analysis onan older system or even ona new system with lowerperformance. To avoidsample decomposition it isnecessary to use a cooledautosampler, for example,the Agilent 1100 Seriesthermostatted autosampler. © Copyright 1998 Agilent TechnologiesReleased 12/98Publication Number 5968-2971E  The performance of the HPLC method is shown in the table above.  The HPLC method presented here shows an easy but reliable andprecise analysis of the water-soluble vitamins pyridoxamine (B 6 ),thiamine (B 1 ), niacinamide, pyridoxal (B 6 ), pyridoxine (B 6 ),aminobenzoic acid, pantothenic acid, folic acid, riboflavin (B 2 ), biotinand thiotic acid. The values for LOD, precision of RT and precision of area show the good performance of the analysis. Agilent Technologies Innovating the HP Way HPLC PerformanceCompoundLOD for Precision of RTPrecision of AreaS/N=2(RSD of 10 runs)(RSD of 10 runs)(mg/l)*(250 mg/l)*(250 mg/l)* Pyridoxamine0.30.090.17Thiamine0.70.230.90Niacinamide0.70.220.28Pyridoxale0.30.360.28Pyridoxine0.30.390.13Aminobenzoic acid0.40.130.14Pantothenic acid2.50.070.34Folic acid0.70.080.15Riboflavin0.70.051.04Biotin6.00.050.33Thiotic acid6.00.041.50* Injection volume: 5 µl

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