Method for the determination of the concentration of vitamin B6 in a sample

A method for determining the concentration of pyridoxal-5′-phosphate (PLP, vitamin B6) in a sample, comprising the steps of

• a) incubating the sample containing PLP with at least one PLP-dependent tyrosine apo-decarboxylase,
• b) contacting the PLP-dependent enzyme(s) of step a) with tyrosine,
• c) enzymatically converting conversion of the tyrosine of step b) to tyramine using the tyrosine apo-decarboxylase,
• d) enzymatically converting the tyramine resulting from step c) to hydrogen peroxide and at least one other product using a tyramine oxidase,
• e) enzymatically reacting at least one indicator substrate and the hydrogen peroxide of step d) to form a compound detectable using a peroxidase,
• f) measuring the quantity of the detectable compound of step e)
• and
• g) determining the concentration of PLP in the sample based on the quantity of the detectable compound measured in step f) and measurements of the quantity of the detectable compound measured in step f) for known concentrations of PLP,
• wherein
• the enzymes of step a) and d) are produced, expressed and purified as recombinant enzymes and
• the assay time is reduced in comparison with the same assay when using enzymes prepared from crude bacterial extracts in steps a) and c).