ANALYTICAL

BIOCHEMISTRY

86,

11%

126 (1978)

A New Calorimetric Method for the Determination 6-Aminopenicillanic Acid
JOSEPH M. KORNFELD

of

Laboratory Division, Connecticut State Department of Health, 10 Clinton Street, Hartford, Connecticut 06101 Received July 13, 1977; accepted November 5, 1977 A new, sensitive calorimetric method for the estimation of 6-aminopenicillanic acid is described. The procedure is based upon formation of a 2,4-pentanedione derivative of 6-aminopenicillanic acid followed by a second reaction with p-dimethylaminobenzaldehyde (Ehrlich’s reagent), resulting in a red product which absorbs at 538 nm. The absorbance response is linear from 0 to 350 pg of 6-aminopenicillanic acid. Penicillins do not interfere with the assay, but 6-aminopenicilloic acid does.

Primarily because of its possible importance as an intermediate in the biosynthesis of naturally occurring penicillins and as a starting material for the chemical addition of side chains in the manufacture of “semisynthetic” penicillins, 6-aminopenicillanic acid (APA) has prompted the introduction of a variety of methods for its estimation, including several calorimetric procedures. Of these, at least three are dependent upon the reaction of APA with p-dimethylaminobenzaldehyde (PDAB) to form a colored Schiff’s base. In the procedure developed by Bomstein and Evans (l), the reaction product, generated in citrate:phosphate buffer at pH 2.5, showed maximum absorbance at 415 nm and provided a linear concentration:absorbance response in the range of 600-4000 pg of APA/ml. The generation of a similar, if not identical, chromophore by the reaction of APA and PDAB was also reported by Saccani and Pitrolo (2). The system they used gave rise to a colored product with a similar absorbance maximum (410-415 nm) and differed primarily in the use of methanolic acetic acid in place of citrate:phosphate buffer. The third of these procedures (3) introduced only minor procedural variations. The colored product absorbed at 415 nm and was reported to provide a linear concentration response up to approximately 800 pg/ml. It is the purpose of this report to describe a new calorimetric procedure for the estimation of APA. It differs from the methods cited above in that APA is initially converted to a pentanedione derivative. Reaction of this derivative with PDAB produces a colored product which absorbs at 538
0003-2697/78/0861-0118$02.00/O
Copyright All rights 0 1978 by Academic Press, Inc. of reproduction in any form reserved.

118

1.6 ml of 0.1 ml) was added to each sample and reference tube. the tubes were vigorously agitated on a vortex mixer. Similarly.. Materials.ASSAY FOR 6-AMINOPENICILLANIC ACID 119 nm. Color was produced by adding 1. Procedure. symmetrical peak at 538 nm. RESULTS AND DISCUSSION The spectral characteristics of the colored material formed in the described reaction are shown in Fig. Appropriate volumes of APA solution were dispensed into screwcapped tubes containing 2. the red color was not generated with the complete reaction mixture if Ehrlich’s reagent was omitted.8. 4. All absorbance measurements were performed with a Beckman DB-G spectrophotometer using l-cm cuvettes. 380 ml. it is clear that the 538-nm material is . The solutions were maintained at refrigerator temperatures and were stable for at least 1 week. 95% ethanol.45 mg/ml in distilled water. The material describes a clearly defined. Standard APA solutions were prepared at a concentration of 1. Ehrlich’s reagent consisted of p-dimethylaminobenzaldehyde. APA was provided by Dr. The reagent was stored in the refrigerator in a flask wrapped in aluminum foil. J. The procedure generally followed was adapted from the protocol established by Mauzerall and Granick (4) for the calorimetric estimation of &aminolevulinic acid. To ensure mixing. Inc. After 20 min of heating. and HCI.CPentanedione (0. All other reagents were obtained commercially. who also supplied most of the penicillins used in this study.5 ml of Ehrlich’s reagent. pH 6.9 ml with water. Stankiewicz of Pfizer. All reaction mixtures were agitated briefly with a vortex mixer and placed in a boiling water bath. For standard curves. a single reagent blank was prepared for each standard set. The method is both more sensitive and more rapid than procedures which have been described earlier.5 M sodium phosphate buffer. The volume of each tube was adjusted to 2.0 g. 80 ml. the tubes were quickly cooled under running tap water and placed in an ice bath. Kinetic and spectral analyses were simplified by the use of the Beckman IO-in. rather than the absorbance peak at 415 nm characteristic of the PDAB procedures previously cited. recorder. MATERIALS AND METHODS Apparatus. although dependent upon PDAB for color generation. All spectral and kinetic analyses were performed with reagent blanks prepared at the same time as the samples.4-pentanedione omitted from the reaction mixture. is different from those PDAB-dependent systems that produce a 415~nm absorbing material. It is obvious from these data that the reaction described here. the 538-nm peak did not appear. nor did the reaction mixture turn red. Further. 2. When the reaction was run with 2.

It was found that the colored material is produced rapidly upon addition of Ehrlich’s reagent. in which the reaction mixture was brought to room temperature prior to the addition of PDAB. It was concluded from this experiment that any inherent instability of the colored material was not a factor in the observed variation in color intensity. The nature of the derivative has not been determined. In the original procedure employed. KORNFELD 0. This is indicated by the lack of color development when 2. Stability characteristics. Since it was possible that the pentanedione derivative of APA was unstable.4-pentanedione is omitted from the reaction mixture. rather . the absorbance remains stable for periods of at least 1 hr after color development. 00 WAVELENGTH (Nh4. 1. Spectrum of the colored complex formed following the addition of Ehrlich’s reagent to the pentanedione derivative of APA. During the early stages of this investigation. it was noted that there was considerable variation in the intensity of the color produced among samples of identical APA content. Although the color fades following overnight incubation at room temperature. the kinetics of color production and stability were examined. Since this might have resulted from an inherent instability of the colored material. not generated from APA itself but rather from the pentanedione derivative. the time period between removal of the samples from the boiling water bath and the addition of Ehrlich’s reagent represented an uncontrolled variable.120 1OSEPH M. reaching maximum intensity within 1 min.j 600 FIG.

derivative samples were incubated at 0 (0 0) or 45°C (x x) for the indicated times. all tubes were cooled under the cold water tap and then set aside to incubate at either 45 or 0°C for varying periods of time prior to the addition of the color developer. . At the end of the 20-min boiling period. samples maintained at 0°C showed almost no change in the ultimate color yield. It is clear that incubation of the derivative at 45°C for relatively short periods of time prior to color formation resulted in a diminution of color yield. When incubation temperatures intermediate between 0 and 45°C were examined. Consequently. the color intensity was intermediate between those obtained at 0 and those at 45°C. with t = 0 defined as the time at which the boiling period ended. The results ofthat experiment are shown in Fig. Ehrlich’s reagent was added and the colored product was measured. that question was examined.ASSAY FOR 6-AMINOPENICILLANIC ACID 121 than the entire complex. After the 20-min reaction time. 2. These data were interpreted as indicating that the stability of the pentanedione derivative of APA is temperature dependent and that the formation of the colored complex tends to lessen that dependence. in order to maximize the color intensity for use in an assay system. the use OOI 20 40 MINUTES 60 60 FIG. A series of APA samples were reacted with pentanedione in the boiling water bath. In contrast. 2. The effect of holding temperature on derivative stability. When the colored products of both incubation systems were examined. it was found that for any incubation time. the characteristic absorbance stability was found.

with a 15min incubation period arbitrarily chosen for use in the assay procedure. it was decided to retain the phosphate concentration at 0. It is obvious that the color yield is proportional to the buffer molarity. Maximum color formation was obtained in those samples heated for 20 min.5 M. Support for this assumption derives from the observation that the characteristic color is not produced when Tris-HCl (at the same molarity and pH) is substituted for the phosphate buffer.122 JOSEPH M. The results of that experiment are shown in Fig. 0 0. 20 30 I 3. Since doubling the phosphate concentration increased the color yield by less than 25%. The effect of varying the molarity of the buffer was examined next. Effect ofboiling time.5 M in all subsequent assays. The effect of heating time in the boiling water bath was examined next. in order to determine the kinetics of derivative formation. KORNFELD of the ice bath following derivative formation was adopted. I IO MINUTES FIG. it is assumed that phosphate participates in the formation of the pentanedione derivative. Consequently. Effect of buffer molarity . this time period was chosen for maximizing the derivative yield. . Although the reasons for this are not clear. 4. at least to 0. The effect of variations in boiling time on the ultimate color yield. These data are shown in Fig. 0 0 / . Increasing the heating time further had no appreciable effect on the color yield. 3.

6 - 2 s 5 Y 5. 0. Under the conditions thus far described.5 ml.6 .8- 0. when the volume of Ehrlich’s reagent was reduced threefold. the effect of the concentration of color developer was examined. . the data shown in Table 1 were obtained. FIG. almost no color was produced. pH 6. Spectra of the reaction products obtained when these penicillins were substituted for APA in this system described only Aat baselines in the region of 500400 nm.2- / D 0 /. Effect of Ehrlich’s reagent concentration. it was decided to retain the arbitrary volume of 1. Because of the dependency upon the chromogenic reagent and the possibility of low color yields when the reagent was limiting. When these penicillins were added individually to APA and the reaction run in their presence. product generated from APA. 4.8. In contrast to the red reaction Effect of the presence of penicillins. 0. 0. phenethicillin. no such material is produced when penicillins are used. 0 0. penicillin V.8 I. The dependence of ultimate color yield on the molarity of phosphate buffer. However. assuming that this would be in excess over the range of APA concentrations that might be analyzed by this method. carbenicillin. The penicillins used in this study were ampicillin.ASSAY FOR 6-AM~NOPENICILLANIC ACID 123 0. and penicillin G.0 .2 . Reduction of the volume of Ehrlich’s reagent by a factor of 2 did not affect color yield.4 MOLARITY . :/ 0 04 0.

.5 101.0 97.5 98. a common characteristic of TABLE 1 THE EFFECT OF VARIOUS PENICILLINS AT SEVERAL CONCENTRATIONS ON THE COLOR YIELD WITH 6-AMINOPENICILLANIC ACID Penicillin (pg) added to reaction mixture 0 (control) 150 300 450 600 750 Recovery Ampicillin 100. various penicillins were added in the indicated amounts. which cleaves the /3-lactam ring to give rise to 6-aminopenicilloic acid.8 99. (+penicillin) (-penicillin) x 1oo.124 JOSEPH M.1 96. pentanedione was added to each tube and the assay was run as previously described. Using the optimum conditions described above.0 u Determined as follows: To a set of tubes each containing a single amount of 6-aminopenicillanic acid (217 pg). It should be noted.5 M. the presence of 6-aminopenicilloic acid will generate erroneously high values. the colored material was not generated.2 of 6-APA Phenethicillin 100.. Adherence to Beer’s Law was noted for all six sets.0 91.1 (percentage of controW in the presence G Penicillin 100. six separate determinations were performed.6 104. It has been reported (5) that APA is a substrate for penicillinase. six separate standard sets were prepared and analyzed.8) was reacted with lo6 units of penicillinase (BBL) in a total volume of 2.1 97. Concentration:absorbance response.7 97.0 96.3 99. The concentration:absorbance relationships are shown in Fig.0 98. indicating the value of the system as an analytical tool in the estimation of APA.6 Penicillin 100. .0 100.1 103. Reaction of 6-aminopenicilloic acid.1 101.8 101. Percentage control was calculated using the mean value of each of the six determinations in the equation: OD. it is concluded that initial separation of penicillins is unnecessary for the estimation of APA by this method. APA (217 pg) in phosphate buffer (0. KORNFELD indicating a lack of interference in the determination of APA.0 98. At the conclusion of the incubation period. that this is not true if the penicilloic ‘acid contained a side-chain at the 6-position. For each concentration of each penicillin. therefore.7 99. pH 6.4 102. Accordingly.0 98.9 104. The control contained all reagents but APA. It was of interest to determine whether the penicilloic acid of APA would react in a manner similar to APA. The results are shown in Table 2. however. The individual standard curves varied somewhat in slope.9 ml for 1 hr at 30°C.0 97. 5. When the experiment was repeated using penicillins as substrate for penicillinase. I of V Carbenicillin 100. OD... It appears that 6-aminopenicilloic acid is even more reactive than APA in terms of color yield.4 98.5 93. In assays for APA. Consequently.0 101.

.673 0. Comparison with the method of Balasingham et al. The method described here appears to provide significant advantages over prior methods. (3) indicates a much greater sensitivity. 0. . “. Concentration dependence of absorbance at 538 nm.2 I.ASSAY FOR 6-AMINOPENICILLANIC TABLE 2 ACID 125 THE EFFECTOF PENICILLINASE PRETREATMENTONTHECOLORYIELD OBTAINED FROM 6-AMINOPENICILLANIC ACID Compound present APA (217 j. In the method reported here. 5..24.1 reported for the prior method. The curve is the summation of six separate sets of standards.. 1.4 I.968 systems in which color is generated..1 4 : P 0. a mean millimolar extinction coefficient has been calculated as 3.& APA (217 pg) pretreated with lo6 units of penicillinase Optical density.( 0. / 0 I I 200 100 6-AMINOPENICILLANIC I 300 ACID (JJG) I 400 FIG.. 0..1 s Q o. The concentration range over which the system is useful appears to be from O-350 pg with most laboratory instrumentation. indicating the necessity of an internal standard when the system is used. which is considerably higher than the value of 0.

Chemother. F. (1969) Boll.126 JOSEPH M. 4. initial separation of APA from penicillins is also unnecessary. . and Granick.. D. S. F. Biol. nor do the penicilloate derivatives of those penicillins.Oat 538 nm (Fig. As has been indicated. 108. and glutamic acid have been examined at concentrations of 300 pg/3 ml of reaction mixture. threonine. N.. with no color production. Ser. 576-579. 3. 194. 6. As indicated earlier. G. The pentanedione derivative method appears to have two advantages over the Shaikh ef al. and (iii) that the spectrum of the Ehrlich’s complex absorbs in the 53%nm region. Chem. isoleucine. (ii) that the derivatives react with Ehrlich’s reagent. since those compounds do not interfere under the conditions used. additionally. 5. 324-329. Talati. Warburton. P. Bomstein. Cameron-Wood.. and (ii) it requires less time to perform the assay.. proline.. cysteine. other. Chem. Clair. methionine. It should be pointed out that in the method described by Shaikh et al. REFERENCES 1.. (1973) Antimicrob. K. J. W. leucine. Dunnill.. and Gang. a question we have not yet addressed. and Pitrolo. Biophys. 435-446. G. about 30 min compared to 2 hr. 3. 5).. ornithine. it is more sensitive than the method of Bomstein and Evans (1) and. Roy. Sot. provided they satisfy the following conditions: (i) that they form pentanedione derivatives under the conditions of the assay. cystine. E. Batchelor. (1972)Biochim. the penicillins which have been tested do not interfere with the reaction. Ag. 514-521. Balasingham. Notwithstanding these observations. and Rolinson. does not require initial separation from penicillins. histidine. and Evans. arginine. M. D. (1965) Anal. ACKNOWLEDGMENT The technical assistance of John Karolus is acknowledged. M. lysine. B 154. with a millimolar extinction coefficient approximately three times greater. KORNFELD Similarly. Acta 276. the reaction with 6-APA will produce an absorbance of about 1 . If such molecules are not found in routine fermentation broths. D. In addition. Saccani. G. (196l)Proc. G. Farm. 250-256. tyrosine. J. Shaikh.. (6) procedure: (i) it is more sensitive. it must be assumed that in addition to the penicilloic acid formed from APA by the cleavage of the p-lactam ring. K. R. the value of the assay would obviously be enhanced.197.. and Lilly. neither the nature of the pentanedione derivative nor the chemistry of the reaction has been determined. Chim. tryptophan. (1956) J. 2. (6) in which APA reacts with D-glucosamine to form a complex which absorbs at 323 nm. alanine. Studies are currently in progress which may clarify these points. P. phenylalanine. B. including valine. a number of amino acids. unidentified molecules may interfere. At an equivalent concentration. 37.. D. Mauzerall. 219.

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