Spectrophotometric Determination of Pyridoxal in the Presence of Pyridoxamine and Pyridoxine FREDERICK
P. SIEGEL
and MARTIN
I. BLAKE
College o f Pharmacy, Universify of Illinois, Chicago, 111.
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.
A
and accurate method has 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, 3-5) 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.
I n 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:
'0' +
2
-
CHO I OH
HOCHn
CH&OCHB
Base
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-
SIMPLE
c
PROCEDURE
pared in 10 X 7 5 mm. cuvettes. To
mixed thoroughly, permitted to stand a t room temperature for 15 minutes, and then read a t 420 mp in a Coleman
concentration was plotted. The pyridoxal content of samples for analysis was readily determined
Table
1.
Determination of Pyridoxal Hydrochloride, Free and in Combination with Other Components
Mg./Ml."
Mg./Ml.
Mg./MI.
%
2.16 1.96
... ... ... ... ...
2.12 1.97 1.59 1.20
102.0 100.5 99.4 100.0
1.20
0.80 0.40 2.00 2.00
2.00
100.0
Sodium a-kkioglutarate 70.0 Sodium pyruvate
0.80 0.39 2.00
2.00
100.0
Pvridoxamine
1.98
99.0
Pvrid oxine
1.98
99.0
2.00
100.0
2.05
102.5
50.0 4.0
2.00 400
440
4 80
Wavelength ( V u )
Figure 1 . Absorption spectrum for condensation product formed when pyridoxal is treated with acetone in presence of base
Pyridoxal HC1 Recovery,
Other Component Added,
1.BO
360
Pyridoxal HCl Found,
Pyridoxal HCl Added,
4.0
Alanine 35.6 Glutamic acid 58.8 Concentration of reaction mixture. 2.00
2.00
97.5 100.0
VOL. 34, NO. 3, MARCH 1962
397
from the calibration curve by using either procedure described above after adjusting the dilution of the sample so that the absorbance fell in the range of the calibration curve.
tures of pyridoxal and these compounds yielded quantitative recovery for pyridoxal. The data are recorded in Table 1.
The proposed procedure proved useful in studying nonenzymatic transamination with vitamin BBof the amino acids and keto acids listed above.
DISCUSSION
The absorption spectrum for the colored condensation product formed when pyridoxal is treated with acetone in the presence of base was obtained with a recording Beckman DU spectrophotometer and is shown in Figure 1. An absorption peak occurs at about 420 mp, Optimum color intensity develops rapidly and increases slo~vlyover a long period of time. Figure 2 shows the effect of time on color intensity over a 3-hour period. Fifteen minutes was selected as convenient for this method. Samples of pure pyridoxal hydrochloride were assayed by the proposed procedure. The recovery of pyridoxal was quantita-
LITERATURE CITED
O01i l
(1) Hochberg, T l m e in M i n u t e s
Figure 2. Effect of time on absorbance of colored condensation product
tive. Alanine, glutamic acid, pyruvic acid, a-ketoglutaric, other similar amino and keto acids, pyridoxine, and pyridoxamine do not produce color with the reagents employed in this assay. Mix-
IT., Melnick, D., Oser, B. L.. J . Bio7. Chem. 155. 109 11914). (2) Metzler, D. E., Snell, E. E.; J . Am. Chem. SOC.74,979 (1952). (3)_Scudi, J. V., J . Biol. Chem. 139, 107 (1941). ( 4 ) Swaminathan, M., Suture 145, 790 (1940). ( 5 ) Sweeney, J. P., Hall, W.L., J . Assoc. Ofic. Agr. Chemzsts 35, 479 (1952). (6) Wada, H., Snell, E. E., J . Biol. Chem. 236, 2089 (1961). RECEIVED for review September 25, 1961. Accepted December 14, 1961.
Determination of Aliphatic Aldehydes by Spectrophotometry M. ALBRECHT,' WILLIAM 1. SCHER, Jr.,2 and HENRY J. VOGEL lnstitute of Microbiology, Rutgers, The State University, New Brunswick, N. J.
ALBERTA
b Aliphatic aldehydes can b e determined b y reaction with methylamine ( 1 .OM, used as hydrochloride) and o-aminobenzaldehyde (0.004M) in aqueous (or aqueous-ethanolic) solution at pH 8.4. The yellow products, obtained within 10 minutes a t 25" C., presumably are lf2-dihydroquinazolinium compounds. With acetaldehyde, the absorbance of the reaction mixtures, measured at 440 mp, is directly proportional to the concentration of this aldehyde up to a t least 0.0004Mf a t which the absorbance is approximately 1.0 (light path, 1 cm.). Quantitatively comparable responses are obtained with formaldehyde, propionaldehyde, butyraldehyde, n-valeraldehyde, or sodium glyoxylate. Inappreciable or slight responses are given b y chloral hydrate, glucose, acetone, a-ketog luta rate, pyruvate, or benzaldehyde.
D
AN enzymological study, it became desirable to find a method to determine aliphatic aldehydes with the aid of relatively mild reagents (adapted for use in aqueous solution a t a p H of approximately 8). I n the method developed, o-aminobenzaldehyde is employed. This com-
398
URISG
ANALYTICAL CHEMISTRY
pound was investigated extensively by Schopf and collaborators (1-3) and was applied by Vogel and Davis (5) to the detection of a n aminoaldehyde, glutamic ysemialdehyde, or its cyclized form, AI-pyrroline-5-carboxylate. It is, however, not necessary for the (aliphatic) amino and aldehyde groups to be part of the same molecule; o-aminobenzaldehyde, under suitable p H conditions, reacts in aqueous (or aqueousethanolic) solution when mixed with any one of several aliphatic primary amines plus a n aliphatic aldehyde. Yellow products are formed which, in line with the work of Schopf and collaborators, appear to be dihydroquinazolinium compounds. The accompanying schematic equation illustrates, a n instance of the presumable reaction involved, namely the formation of a 2 - alkyl - 3 - methyl - 1,2 - dihydroquinazolinium compound from oaminobenzaldehyde, methylamine (used as hydrochloride), and a n aliphatic aldehyde, R ,CHO; no specific reaction mechanism is implied : IVH,
+ 0HC.R
-H20
d
H
H OHEXPERIMENTAL
Reagents. Reagent A is a n aqueous solution, 2.0M in methylamine hydrochloride and 0.20il.1 in sodium pyrophosphate. This solution, which has a p H of approximately 8.4 (without adjustment) remains usable for at least 1 month when stored a t 3" C. It is convenient first to dissolve the requisite amount of finely ground sodium pyrophosphate (Na4P207. 10H20,analytical reagent, Mallinckrodt Chemical Works) and then to dissolve the methylamine hydrochloride (reagent grade, Fisher Scientific Co.). Reagent B is aqueous 0.04M o-aminobenzaldehyde which is dissolved with gentle warming. This reagent is prepared fresh daily and kept a t 3" C. until used. The c-aminobenzaldehyde is readily prepared by the following modification 1 Present address, Sloan-Kettering Institute for Cancer Research, Walker Laboratory, Rye, N. Y. 2 Present address, Kings County Hospital, Brooklyn, N. Y.
