[68] Fluorometric assay of pyridoxal

Publisher Summary This chapter describes the fluorometric assay of pyridoxal. The reductive amination of pyridoxal hydrochloride with methyl anthranilate and sodium cyanoborohydride at pH 4.5–5.0 and 35°–38° yields a highly fluorescent amine, which is used for the fluorometric assay of pyridoxal in ethanol at pH 7. This method can be applied to the determination of pyridoxal phosphate as such or after hydrolyzing it with hydrochloric acid or with acid phosphatase. Pyridoxamine and pyridoxine are readily oxidized to pyridoxal. The chapter also describes the procedure for the preparation of methyl- N -pyridoxylanthranilate. Methyl anthranilate (450 mg in 5 ml of absolute methanol) is added to a solution of pyridoxal hydrochloride (100 mg in 10 ml of absolute methanol), and the pH of the solution is brought to 4.5–5.0 with methanolic HCl. The reaction mixture immediately turns yellow. It is warmed at 35°–38° in a water bath for 7 min, and a freshly prepared solution of sodium cyanoborohydride (20 mg in 5 ml of absolute methanol) is added. The solution turns colorless immediately. It is kept at 350–38 ° for a further period of 15 min while the pH of the reaction mixture is maintained at 4.5–5.0 with methanolic HCl. At the end of this time, the reaction mixture is brought to pH 2–3 with methanolic HCl.