Spectrophotometric Determination of Pyridoxal in the Presence of Pyridoxamine and Pyridoxine.

Abstract

b A simple and accurate procedure is presented for the determination of pyridoxal. Pyridoxal is condensed with acetone in the presence of a base. Beer's law is obeyed over a concentration range of 10 to 400 pg. The method is suitable for determining pyridoxal in vitamin Be and for the study of nonenzymatic transamination of certain amino acids and keto acids. SIMPLE and accurate method hasA been developed for determining pyridoxal in the presence of pyridoxamine and pyridoxine. This procedure is suitable for studying nonenzymatic transamination of amino acids and is specific for pyridoxal in the presence of other members of the vitamin Be group.Several chemical methods have been reported (1,(3)(4)(5) for determining vitamin Be, but these do not distinguish among pyridoxal, pyridoxamine, and pyridoxine. Metzler and Snell (2) describe a specific colorimetric assay for pyridoxal which is based on the formation of a yellox Schiff base with an excess of ethanolamine. A nonlinear calibration curve over a rather narroTv concentration range was obtained. More recently Wada and Snell (6) have reported a sensitive and specific procedure for determining pyridoxal involving the formation of the phenylhydrazone. c 360 400 4 4 0 4 80 Figure 1 . Absorption spectrum for condensation product formed when pyridoxal is treated with acetone in presence of base Wavelength ( V u )In the proposed procedure pyridoxal is condensed with acetone in the presence of base to give an intense yellow product. The probable equation for the reaction is: CHO HOCHn I OH Base 2 ' 0 ' + CH&OCHB -Junior spectrophotometer. Water was used as a blank. A calibration curve was prepared by plotting absorbance us. concentration.Beer's law was obeyed over this range of concentrations.An alternate procedure was devised which required smaller over-all amounts of pyridoxal hydrochloride. A series of 1-ml. aliquots containing 20 to 400 pg. of pyridoxal hydrochloride was pre-PROCEDURE pared in 10 X 75 mm. cuvettes. To mixed thoroughly, permitted to stand a t room temperature for 15 minutes,The pyridoxal content of samples and then read a t 420 mp in a Coleman for analysis was readily determined concentration was plotted. Table 1. Determination of Pyridoxal Hydrochloride, Free and in Combination with Other Components

Synthesis of analogs of pyridoxal 5'-phosphate

“…The O-and TV-methylated derivatives do not react with the Gibbs reagent, and no other specific and sensitive color test could be found; therefore, detection relied on the use of general functional group reagents such as phenylhydrazine for the aldehyde group and ninhydrin for the primary amino group. TV-Me derivatives could be detected by their strong fluorescence or by the yellow color produced in alkaline medium in the presence of acetone (Siegel and Blake 1962). This latter procedure could not be applied for the detection of the 3-O-methylated aldehydes because of their poor reactivity.…”

Some pyridoxal analogs and their transamination with amino acids

“…forming an ethanolimlne with maximum absorbancy at 365 np (Metzler and Snell, 1952b;Cennamo, 1961Cennamo, , 1962Cennamo, , I963, 1964Banks _et , 196I;Gregerman and Chrlstensen, 1956) and by forming a red condensation product from two moles of pyrldoxal and one mole of acetone in alkaline solution (Slegel and Blake, 1962;Blake , 1963). Other methods which could be used for analysis of pyrldoxal analogs in nonenzymlc systems include condensation with cyanide (pyrldoxal phosphate was determined as a cyanohydrin derivative at its maximum absorbancy of 385 m)i by Scardi and Bonavlta, 1957), or with phenylhydrazlne (phenylhydrazones of pyrldoxal and pyrldoxal phosphate had identical maximum absorbancy Indices at 4lO mp.,…”

“…Alkaline-acetone method for pyridoxal (Siegel and Blake, 1962) A red condensation product from acetone and pyridoxal in alkaline solution formed slowly, but this method specified the measurement of absorbancy at 420 mp at the point of greatest change in absorbancy with respect to time: fifteen minutes after mixing. This absorbancy increased at a much slower rate after thirty minutes at room temperature.…”

“…This absorbancy increased at a much slower rate after thirty minutes at room temperature. The claims for simplicity, convenience and accuracy of this method were greatly exaggerated (Siegel and Blake, 1962). It has been found that the instability of the colored product, the decreased sensitivity to pyridoxal, and other undesirable features make this method inferior to the ethanolimine method for the analysis of pyridoxal.…”