Spectrophotometric determination of pyridoxal and pyridoxal 5'-phosphate with 3-methyl-2-benzothiazolone hydrazone hydrochloride, and their selective assay.
A spectrophotometric method with 3-methyl-2-benzothiazolone hydrazone hydrochloride was developed for the determination of pyridoxal and pyridoxal 5'-phosphate, and for the selective determination of each in the presence of the other. Pyridoxal and pyridoxal 5'-phosphate react with the reagent to yield the azine derivatives, which give characteristic absorption spectra. The highest extinction values are obtained when pyridoxal and pyridoxal 5'-phosphate are incubated at pH values of about 3.4 and 8.0 respectively; their maxima are at 430nm. (in 2.74x10(4)) and 380nm. (in 2.24x10(4)) respectively. The azine of pyridoxal is only slightly soluble under the neutral and alkaline conditions, whereas that of pyridoxal 5'-phosphate is substantially insoluble in the acid pH range. This difference in solubility of the azines made possible the selective determination of pyridoxal and pyridoxal 5'-phosphate. alpha-Oxoglutarate and pyruvate are among the substances shown not to interfere with the assay of pyridoxal; their derivatives absorb appreciably only at wavelengths below 420nm. For the assay of pyridoxal 5'-phosphate in the presence of these compounds measurement at 390nm. is necessary.
D001386:Azides; D005977:Glutarates; D006571:Heterocyclic Compounds; D006834:Hydrazines; D006863:Hydrogen-Ion Concentration; D000432:Methanol / Q000032:analysis*; D008722:Methods; D010847:Picolines / Q000032:analysis*; D011732:Pyridoxal Phosphate / Q000032:analysis*; D011773:Pyruvates; D013053:Spectrophotometry; D013844:Thiazoles
D001386:Azides; D005977:Glutarates; D006571:Heterocyclic Compounds; D006834:Hydrazines; D010847:Picolines; D011773:Pyruvates; D013844:Thiazoles; D011732:Pyridoxal Phosphate; D000432:Methanol
Anal Chem. 1968 Jan;40(1):231-2
5634547
Anal Biochem. 1968 Oct 24;25(1):228-35
4387536
J Biol Chem. 1961 Jul;236:2089-95
13782387
Arch Biochem Biophys. 1965 Mar;109:548-59
14320497