Vitamin Analysis with an Integrated HPLC System

In the last few years, vitamin supplements have become increasingly important to consumers who are concerned about maintaining the recommended daily allowances of certain vitamins, or who are focusing on specific vitamins that show promise of disease prevention or other health benefits. For example, in the water-soluble family of B vitamins, Vitamin B6 is important for hemoglobin formation, red blood cell metabolism, and synthesis of neurotransmitters; Vitamin B12 maintains healthy nerve and blood cells and is needed to make DNA; and Vitamin B2 aids in the release of energy from food and promotes growth, good vision, and healthy skin. Some examples of fat-soluble vitamins are Vitamin A, which aids in immune system regulation, maintains the surface lining of the eyes, and helps prevent infection in the respiratory and intestinal tracts; Vitamin D which maintains normal blood levels of calcium and phosphorous, and helps with calcium absorption to form and maintain strong bones; and Vitamin E, whose antioxidant properties protect cells against certain free radicals that may contribute to the development of cardiovascular disease and cancer. Any manufacturer of vitamin supplements needs a reliable instrument to test the purity of raw materials and the composition of the final product. Shimadzus LC-2010 is a fully integrated HPLC system with quaternary pump, high-speed autosampler, column oven, and detector in one unit. The following chromatograms illustrate the instruments performance at various flow rates and analysis conditions.

120

120

B6

B6

100 80 80

100

B12
80 60 60

B12

80

mVolts

mVolts

mV olt 60 s

60

mV olt s

40

B2

40

B2
40 40

20

20 20 20

0 2 4 6 8

0 0 5 10 15

Minutes

Minutes

Figure 1 Vitamin B standard eluted at 1.1 mL/min.

Figure 2 Vitamin B standard eluted at 0.5 mL/min.

Not all vitamins have the same absorbance at a particular wavelength, so the LC-2010 can be programmed to change wavelengths during the course of a run.
140 140

120

A Acetate

120

A
100 100

120

120

100

100

D2
80 80
80 ts mVolts

mVolts

mV olts

A
60

A Acetate

80 mV olts 60

60

D3

60

D2

E E Acetate

40

40
40

D3

40

20

20

20

20

E E Acetate
0 5 10 15 20 25 30 0
0 5 10 15 Minute 20 25 0

Minutes
Figure 3 Chromatogram of a fat-soluble vitamin standard acquired at 280 nm.

Minutes
Figure 3 Chromatogram of a fat-soluble vitamin standard acquired at 280 nm from 0-16 min, and 210 nm from 17-30 min.

Experimental Conditions
Water-Soluble Vitamins Mobile Phase: 33:67 A: B (A: MeOH, B: 0.023M H3PO4, pH = 3.54) Flow Rate: 1.1 or 0.5 mL/minute Column Oven: 25oC Fat-Soluble Vitamins Mobile Phase: 95:5 MeOH:H2O Flow Rate: 1.0 mL/min Column Oven: 25oC

Results
The LC-2010 is suitable for the analysis of fat and water-soluble vitamin samples. Wavelength programming allows optimum detection of all components.

System Configuration
228-37000-32 220-91199-06 LC-2010A Shimadzu Premier C-18 column (3 m, 4.6 mm x 150 mm)