Purpose

Water-soluble vitamins in cell culture and fermentation media can be separated, identified, and quantified via HPLC. This method outlines the accurate determination and quantification of water-soluble vitamins.

Summary

A portion of liquid sample is placed in an amber HPLC vial after undergoing the appropriate preparation methods. The separation uses a reversed phase C-18 column with a combination of aqueous buffer and organic solvents using an HPLC analyzer.  Peak detection uses a combination of in-line, programmable, UV (diode array) and scanning fluorescence detectors. All vitamins are spectrally verified. An external standard technique is applied to develop a 5 point calibration curve for each run. The calibration curve is then applied to samples to calculate concentrations for the analytes. A set media control sample is run on each sequence using a standard addition method to ensure that the sample matrix will not be affecting results of unknown samples. 

Vitamins quantified

Pyridoxamine 2 HCl (B6)

Thiamine HCl (B1)

Niacinamide (B3)

Pyridoxal HCl (B6)

Pyridoxine HCl (B6)

Folic Acid (B9)

Cyanocobalamin (B12)

Riboflavin (B2)