A simple, rapid, and sensitive method is described whereby 6-aminopenicillaiic acid can be spectrophotometrically determined in the presence of penicillins and their degradation products without prior separation. D-(+)-Glucosamine is used as reagent. The effect of such parameters as pH, temperature, and time of heating on the formation of the chromophore is described. The recommended range is from 25 to 250 pug of 6-aminopenicillanic acid.There has been considerable interest in the development of analytical methods for the determination of 6-aminopenicillanic acid (6-APA) because of the new vistas this compound has opened for the preparation of semisynthetic penicillins. Among the common methods employed for the estimation of 6-APA are the iodometric method (1) and the hydroxylamine method (4). In both these methods, interfering penicillins are removed by preliminary extraction.Bomstein and Evans (3) The standard 6-APA solution of 2.5 mg per ml was prepared in pH 8.0 barbiturate buffer, and 0.5% D-(+)-glucosamine hydrochloride was prepared in distilled water. After 2 to 3 days, fresh reagent solution was prepared.Barbiturate buffer (0.05 M), pH 8.0, contained 10.3 g of sodium barbiturate and 87.5 ml of 0.2 M hydrochloric acid per liter.Procedure. Samples of exactly 0.02, 0.04, 0.06, 0.08, and 0.1 ml were taken from the 6-APA standard solution, and the volume brought to 4.0 ml with pH 8.0 barbiturate buffer. To this, 1.0 ml of 0.5% D-glucosamine reagent was added. The tubes were kept at 70 C in a constant-temperature water bath for 2 hr and then allowed to cool. A reagent blank was run with each standard set, and the absorbance neasurements were made at 324 nm.The sample blanks were prepared by adding penicillinase and incubating for a length of time sufficient to degrade the 6-APA or penicillin present; alkali hydrolysis was used with samples containing penicillinase-resistant penicillins before buffer and reagent were added. Table 1 summarizes the recoveries of 6-APA. Ultraviolet absorption of 6-APA and D-(+)-glucosamine reaction product adhered to the Beer-Lambert law over a wide range, as indicated both by recoveries in Table 1 and by the plots of absorbance in Fig. 1. Linearity extended at least over a rainge of 25 to 250 ug of 6-APA.
RESULTS AND DISCUSSION