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The VitaFast® microbiological assay for vitamin B₆ (pyridoxine) is carried out on microtiter plates and is suitable for the determination of total amount of vitamin B₆ in foods and beverages.

Vitamin B₆ is added as pyridoxine hydrochloride or as pyridoxine-5-phosphate. Since the phosphorylated vitamin B₆ form is not recognized by the microbes in the kit, the beverage is treated with acid phosphatase to determine the amount of supplemental vitamin B₆; if necessary, the beverage is diluted with sterile water from the test kit.

The sample must be digested through enzymatic hydrolysis in order to measure the total content of vitamin B₆ (naturally occurring and added). 

The diluted sample and the vitamin B₆ assay medium are added to the wells of the microtiter plate, which are coated with Saccharomyces cerevisiae. Saccharomyces cerevisiae requires vitamin B₆ to grow. After an addition of vitamin B₆, the microorganism exhibits growth until the vitamin is completely utilized. The plates are incubated at 30 °C for 44–48 h.

The growth of Saccharomyces cerevisiae is dependent upon the concentration of vitamin B₆ and is determined through measuring the turbidity. These results are compared with those obtained from a series of standard concentrations. The measurement is performed with a microtiter plate photometer at 610–630 nm (alternatively at 540–550 nm).

Vitamin B₆ is separated by ion-pair RP-HPLC, detected using UV absorption and quantified through comparison with external standards. If necessary, the identity of the substances can be confirmed via spectra comparison.