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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.

HPLC methods have been successfully used for the analysis of vitamin content in non-alcoholic beverages. However, for the most part, these methods cannot be employed with a wort or beer matrix due to the vitamin concentrations present.

The reagent kit for the determination of vitamins B₁ and B₂ in blood from Chromsystems GmbH in Munich, Germany was used for the analysis. This company specializes in the manufacture of reagent kits for clinical applications. The kits are easy to use and do not require much additional equipment; a simple isocratic HPLC system is sufficient. In cooperation with the company, this kit was modified to accommodate a different sample matrix (mash, wort and beer).

A reliable sampling method for whole hops and hop products is a prerequisite for obtaining suitable material for analysis. The effort involved in collecting the sample depends upon how uniform the hop product is.

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).

The VitaFast® microbiological assay for vitamin B₂ (riboflavin) is carried out on microtiter plates and is suitable for the determination of total amount of vitamin B₂ in foods and beverages.

The beverage is first treated enzymatically and subsequently extracted hot; 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 B₂ (naturally occurring and added). 

The diluted sample and the vitamin B₂ assay medium are added to the wells of the microtiter plate, which are coated with Lactobacillus rhamnosus. Lactobacillus rhamnosus requires vitamin B₂ to grow. After an addition of vitamin B₂, the microorganism exhibits growth until the vitamin is completely utilized. The plates are incubated at 37 °C for 44–48 h.

The growth of Lactobacillus rhamnosus is dependent upon the concentration of vitamin B₂ 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).

The VitaFast® microbiological assay for vitamin B₁ (thiamine) is carried out on microtiter plates and is suitable for the determination of total amount of vitamin B₁ in foods and beverages.

The beverage is sterile-filtered and diluted with sterile water for the determination of added vitamin B₁.

The sample must be digested through enzymatic hydrolysis in order to measure the total content of vitamin B1 (naturally occurring and added). 

The diluted sample and the vitamin B₁ assay medium are added to the wells of the microtiter plate, which are coated with Lactobacillus fermentum. Lactobacillus fermentum requires vitamin B₁ to grow. After an addition of vitamin B₁, the microorganism exhibits growth until the vitamin is completely utilized. The plates are incubated at 37 °C for 44–48 h.

The growth of Lactobacillus fermentum is dependent upon the concentration of vitamin B₁ and is determined through 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).