Skip to main content

Hitachi High-Tech
  1. Home
  2. Knowledge
  3. Analytical Systems
  4. Knowledge: High Performance Liquid Chromatographs (HPLC)
  5. Example of Application by Chromaster
  6. Simultaneous Analysis of Water-Soluble Vitamins

Simultaneous Analysis of Water-Soluble Vitamins

Vitamins are essential nutrients, and are classified into water-soluble vitamins and fat soluble vitamins. We have simultaneously analyzed 9 water-soluble vitamin components by separation through a reverse phase column and diode array detection (DAD). By using DAD, detected peaks can be identified from absorption spectra. Thus, food and other samples containing many contaminants can be analyzed particularly effectively. Please take care to note that since vitamin C and erythorbic acid are unstable, they are easily decomposed during sample preparation or with the passage of time, and that it is difficult to obtain linearity and reproducibility. Therefore, these analysis conditions are suitable for qualitative analysis. For quantitative analysis, the use of an individual test method for each vitamin is recommended.

Simultaneous Analysis of Water-Soluble Vitamins

Standard Samples

Concentrations and Structural Formulas


  • Vitamin B1(thiamine)*
  • Vitamin B6(pyridoxine) *
  • Nicotinamide
  • Vitamin B12(cyanocobalamin)
  • Ascorbic glucoside
  • Vitamin C (ascorbic acid)
  • Erythorbic acid
  • Vitamin B2(riboflavin)
  • Nicotine acid

*: hydrochloride salt used.
Concentration of 50 mg/L for each.
Standard stock/standard solution diluted using elute.



Column HITACHI LaChrom C18-PM (5 µm) (4.6 mmI.D. x 250 mm)
Elute Phosphate buffer (pH 5.2)/CH3CN = 90/10 (v/v)
(containing tetrabutylammonium)
Flow rate 0.8 mL/min
Column Temperature 40°C
Detection DAD 260 nm
Injection vol. 10 µL

This analysis uses an ionpair reagent (tetrabutylammonium) as elute. As it is difficult to remove the ion-pair reagent completely from the column, use of a column that is exclusive for watersoluble vitamins is recommended.

System configuration

5110 Pump
5210 AutoSampler
5310 Column Oven
5420 UV-VIS Detector
Empower2 Data Processing System

Measurement examples for standard samples

Contour indication and extraction chromatogram
Water-Soluble Vitamin Spectra


[VB6 Calibration curve]

All of the calibration curves (all components in the range from 0.1 - 50 mg/L) exhibited high linearity, with r2= 0.995 or more. However, regarding Vitamin C, Erythorbic acid, and Vitamin B12, take care to note that it is rather difficult to obtain good linearity.

Analysis Example (1)

Nutritional Supplement Sample

Chromatogram of the Nutritional Supplement
[Peaks and Spectra obtained for the standard sample]

Pretreatment of eye drops

A sample of weight 2 mg was weighed out, dissolved in elute to a volume of 10 mL, and filtered through a 0.45 µm filter.

Analysis Example (2)

Health Drink Sample

Chromatogram and Contour indication of Health Drink
[Peaks and Spectra obtained for the standard sample]

Pretreatment of the Health Drink

The sample was diluted 10-fold with elute and filtered through a 0.45 µm filter.


The peaks of both the Nutritional Supplement and Health Drink obtained at 10.7 min tended to be identified as Erythorbic acid, which has a similar retention time. However, it was found the they were completely different substances via spectral confirmation. Thus, DAD, by which the spectral information was obtained, is effective for food and other samples containing many contaminants.

These data are an example of measurement; the individual values cannot be guaranteed.
The system is for research use only, and is not intended for any animal or human therapeutic or diagnostic use.

In order to read a PDF file, you need to have Adobe® Reader®

Products & Services

Contact Us