Abstract
The diazotization product from 5-chloroaniline 2,4-disulfonyl chloride was found to be suitable and satisfactorily applicable as a color-producing reagent to an automated colorimetric analysis of pyridoxine (PIN), pyridoxal (PAL) and pyridoxamine (PAM). Optimal conditions for coupling and color development determined and the characteristics of the reaction revealed were as follows : (1) Respective product from PIN, PAM and 4-deoxypyridoxine (DOP) after coupling with the diazotized reagent showed an absorption maximum at 467 nm, and that from PAL at 450 nm. (2) The coupling reaction proceeded fairly rapidly and the color developed was significantly stable when the reaction medium was adjusted to pH 5.4 to 6.0. (3) The coupling reaction with either one of PIN, PAM, PAL and DOP completed within 2 min after mixing when the reaction mixture was adjusted to pH 5.4 and kept at 55℃. (4) Absorbancy of the mixture at absorption maxima was proportional to the amount of vitamin B_6 complex within the range of up to 12.5μg per ml. (5) Coupling capacity of the diazotized reagent with the vitamins was found unchanged after storage for 24 h in a refrigerator. The reagent could be thus stored with perfect impunity for at least 24 h at a refrigerator temperature.
