a new calorimetric method for the determination of 6-aminopenicillanic acid

8/6/2019 A New Calorimetric Method for the Determination of 6-Aminopenicillanic Acid

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ANALYTICAL BIOCHEMISTRY 86, 11% 126 (1978)

A New Calorimetric Method for the Determination of6-Aminopenicillanic AcidJOSEPH M. KORNFELD

Laboratory Division, Connecticut State Department of Health, 10 Clinton Street,Hartford, Connecticut 06101Received July 13, 1977; accepted November 5, 1977

A new, sensitive calorimetric method for the estimation of 6-aminopenicillanicacid is described. The procedure is based upon formation of a 2,4-pentanedionederivative of 6-aminopenicillanic acid followed by a second reaction withp-dimethylaminobenzaldehyde (Ehrlichs reagent), resulting in a red product whichabsorbs at 538 nm. The absorbance response is linear from 0 to 350 pg of6-aminopenicillanic acid. Penicillins do not interfere with the assay, but6-aminopenicilloic acid does.

Primarily because of its possible importance as an intermediate in thebiosynthesis of naturally occurring penicillins and as a starting material forthe chemical addition of side chains in the manufacture of semisyntheticpenicillins, 6-aminopenicillanic acid (APA) has prompted the introductionof a variety of methods for its estimation, including several calorimetricprocedures. Of these, at least three are dependent upon the reaction ofAPA with p-dimethylaminobenzaldehyde (PDAB) to form a coloredSchiffs base. In the procedure developed by Bomstein and Evans (l), thereaction product, generated in citrate:phosphate buffer at pH 2.5, showedmaximum absorbance at 415 nm and provided a linear concentration:ab-sorbance response in the range of 600-4000 pg of APA/ml. The generationof a similar, if not identical, chromophore by the reaction of APA andPDAB was also reported by Saccani and Pitrolo (2). The system they usedgave rise to a colored product with a similar absorbance maximum(410-415 nm) and differed primarily in the use of methanolic acetic acid inplace of citrate:phosphate buffer. The third of these procedures (3)introduced only minor procedural variations. The colored productabsorbed at 415 nm and was reported to provide a linear concentrationresponse up to approximately 800 pg/ml.

It is the purpose of this report to describe a new calorimetric procedurefor the estimation of APA. It differs from the methods cited above in thatAPA is initially converted to a pentanedione derivative. Reaction of thisderivative with PDAB produces a colored product which absorbs at 5380003-2697/78/0861-0118$02.00/OCopyright 0 1978 by Academic Press, Inc.All rights o f reproduction in any for m reserved.

118


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AS SA Y FOR 6-AMINOPENICILLANIC ACID 119nm. The method is both more sensitive and more rapid than procedureswhich have been described earlier.

MATERIALS AND METHODSApparatus. All absorbance measurements were performed with aBeckman DB-G spectrophotometer using l-cm cuvettes. Kinetic andspectral analyses were simplified by the use of the Beckman IO-in.

recorder.Materials. APA was provided by Dr. J. Stankiewicz of Pfizer, Inc., whoalso supplied most of the penicillins used in this study. All other reagentswere obtained commercially.Standard APA solutions were prepared at a concentration of 1.45 mg/mlin distilled water. The solutions were maintained at refrigerator

temperatures and were stable for at least 1 week.Ehrlichs reagent consisted of p-dimethylaminobenzaldehyde, 4.0 g;95% ethanol, 380 ml; and HCI, 80 ml. The reagent was stored in therefrigerator in a flask wrapped in aluminum foil.Procedure. The procedure generally followed was adapted from the

protocol established by Mauzerall and Granick (4) for the calorimetricestimation of &aminolevulinic acid. Appropriate volumes of APA solutionwere dispensed into screwcapped tubes containing 2.6 ml of 0.5 M sodiumphosphate buffer, pH 6.8. The volume of each tube was adjusted to 2.9 mlwith water. 2,CPentanedione (0.1 ml) was added to each sample andreference tube. All reaction mixtures were agitated briefly with a vortexmixer and placed in a boiling water bath. After 20 min of heating, the tubeswere quickly cooled under running tap water and placed in an ice bath.Color was produced by adding 1.5 ml of Ehrlichs reagent. To ensuremixing, the tubes were vigorously agitated on a vortex mixer. All spectraland kinetic analyses were performed with reagent blanks prepared at thesame time as the samples. For standard curves, a single reagent blank wasprepared for each standard set.

RESULTS AND DISCUSSIONThe spectral characteristics of the colored material formed in thedescribed reaction are shown in Fig. 1. The material describes a clearlydefined, symmetrical peak at 538 nm, rather than the absorbance peak at

415 nm characteristic of the PDAB procedures previously cited. When thereaction was run with 2,4-pentanedione omitted from the reaction mixture,the 538-nm peak did not appear, nor did the reaction mixture turn red.Similarly, the red color was not generated with the complete reactionmixture if Ehrlichs reagent was omitted. It is obvious from these data thatthe reaction described here, although dependent upon PDAB for colorgeneration, is different from those PDAB-dependent systems that producea 415~nm absorbing material. Further, it is clear that the 538-nm material is


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120

0.

1OSEPH M. KORNFELD

00 600WAVELENGTH (Nh4.j

FIG. 1. Spectrum of the colored complex formed following the addition of Ehrlichs reagentto the pentanedione derivative of APA.

not generated from APA itself but rather from the pentanedione derivative.This is indicated by the lack of color development when 2,4-pentanedioneis omitted from the reaction mixture. The nature of the derivative has notbeen determined.

Stability characteristics. During the early stages of this investigation, itwas noted that there was considerable variation in the intensity of the colorproduced among samples of identical APA content. Since this might haveresulted from an inherent instability of the colored material, the kinetics ofcolor production and stability were examined. It was found that the coloredmaterial is produced rapidly upon addition of Ehrlichs reagent, reachingmaximum intensity within 1 min. Although the color fades followingovernight incubation at room temperature, the absorbance remains stablefor periods of at least 1 hr after color development. It was concluded fromthis experiment that any inherent instability of the colored material was nota factor in the observed variation in color intensity.In the original procedure employed, in which the reaction mixture wasbrought to room temperature prior to the addition of PDAB, the time periodbetween removal of the samples from the boiling water bath and theaddition of Ehrlichs reagent represented an uncontrolled variable. Since itwas possible that the pentanedione derivative of APA was unstable, rather


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AS SA Y FOR 6-AMINOPENICILLANIC ACID 121than the entire complex, that question was examined. A series of APAsamples were reacted with pentanedione in the boiling water bath. After the20-min reaction time, all tubes were cooled under the cold water tap andthen set aside to incubate at either 45 or 0C for varying periods of time priorto the addition of the color developer, with t = 0 defined as the time atwhich the boiling period ended. The results ofthat experiment are shown inFig. 2. It is clear that incubation of the derivative at 45C for relatively shortperiods of time prior to color formation resulted in a diminution of coloryield. In contrast, samples maintained at 0C showed almost no change inthe ultimate color yield. When the colored products of both incubationsystems were examined, the characteristic absorbance stability was found.These data were interpreted as indicating that the stability of thepentanedione derivative of APA is temperature dependent and that theformation of the colored complex tends to lessen that dependence. Whenincubation temperatures intermediate between 0 and 45C were examined,it was found that for any incubation time, the color intensity wasintermediate between those obtained at 0 and those at 45C. Consequently,in order to maximize the color intensity for use in an assay system, the use

OOI 20 40 60 60MINUTES

FIG. 2. The effect of hold ing temperature on derivative stab ility. At the end of the 20-minboilin g p eriod, derivative sam ples were incub ated at 0 (0 - 0) or 45C (x - x) for theindicated times. E hrlichs reagent was added and the colored product was measured.


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122 JOSEPH M. KORNFELDof the ice bath following derivative formation was adopted, with a 15minincubation period arbitrarily chosen for use in the assay procedure.Effect ofboiling time. The effect of heating time in the boiling water bathwas examined next, in order to determine the kinetics of derivativeformation. These data are shown in Fig. 3. Maximum color formation wasobtained in those samples heated for 20 min. Increasing the heating timefurther had no appreciable effect on the color yield. Consequently, thistime period was chosen for maximizing the derivative yield.Effect of buffer molarity . The effect of varying the molarity of the bufferwas examined next. The results of that experiment are shown in Fig. 4. It isobvious that the color yield is proportional to the buffer molarity, at least to0.5 M. Although the reasons for this are not clear, it is assumed thatphosphate participates in the formation of the pentanedione derivative.Support for this assumption derives from the observation that thecharacteristic color is not produced when Tris-HCl (at the same molarityand pH) is substituted for the phosphate buffer. Since doubling thephosphate concentration increased the color yield by less than 25%, it wasdecided to retain the phosphate concentration at 0.5 M in all subsequentassays.

0

0.

0

0

/ , I IIO 20 30MINUTESFIG. 3. The ef fect of variations in boiling time on the ultimate color yield.


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ASSAY FOR 6-AM~NOPENICILLANIC ACID 123

0.8-

0.6 -

5,

2s5 04Y

:/ 0

0.2- /D

0 /, , , , ,0 0.2 0.4 0.6 0.8 I.0

MOLARITYFIG. 4. The dependence of ultimate color yield on the molarity of phosphate buffer. pH 6.8.Effect of Ehrlichs reagent concentration. Under the conditions thus far

described, the effect of the concentration of color developer was examined.Reduction of the volume of Ehrlichs reagent by a factor of 2 did not affectcolor yield. However, when the volume of Ehrlichs reagent was reducedthreefold, almost no color was produced. Because of the dependency uponthe chromogenic reagent and the possibility of low color yields when thereagent was limiting, it was decided to retain the arbitrary volume of 1.5 ml,assuming that this would be in excess over the range of APA concentrationsthat might be analyzed by this method.

Effect of the presence of penicillins. In contrast to the red reactionproduct generated from APA, no such material is produced whenpenicillins are used. The penicillins used in this study were ampicillin,carbenicillin, phenethicillin, penicillin V, and penicillin G. Spectra of thereaction products obtained when these penicillins were substituted forAPA in this system described only Aat baselines in the region of 500400nm. When these penicillins were added individually to APA and thereaction run in their presence, the data shown in Table 1 were obtained,


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124 JOSE PH M. KORNFELDindicating a lack of interference in the determination of APA. Con-sequently, it is concluded that initial separation of penicillins isunnecessary for the estimation of APA by this method.

Reaction of 6-aminopenicilloic acid. It has been reported (5) that APA isa substrate for penicillinase, which cleaves the /3-lactam ring to give rise to6-aminopenicilloic acid. It was of interest to determine whether thepenicilloic acid of APA would react in a manner similar to APA.Accordingly, APA (217 pg) in phosphate buffer (0.5 M, pH 6.8) was reactedwith lo6 units of penicillinase (BBL) in a total volume of 2.9 ml for 1 hr at30C. The control contained all reagents but APA. At the conclusion of theincubation period, pentanedione was added to each tube and the assay wasrun as previously described. The results are shown in Table 2. It ap-pears that 6-aminopenicilloic acid is even more reactive than APA interms of color yield. In assays for APA, therefore, the presence of6-aminopenicilloic acid will generate erroneously high values. It should benoted, however, that this is not true if the penicilloic acid contained aside-chain at the 6-position. When the experiment was repeated usingpenicillins as substrate for penicillinase, the colored material was notgenerated.Concentration:absorbance response. Using the optimum conditionsdescribed above, six separate standard sets were prepared and analyzed.The concentration:absorbance relationships are shown in Fig. 5. Adher-ence to Beers Law was noted for all six sets, indicating the value of thesystem as an analytical tool in the estimation of APA. The individualstandard curves varied somewhat in slope, a common characteristic of

TABLE 1THE EFFECT OF VARIOUS PENICILLINS AT SEVE RAL C ONCENTRATIONS

ON TH E COLOR YIELD WIT H 6-AMINOPENICILLANIC ACIDPenicillin (pg)

added toreaction mixture

Recovery of 6-APA (percentage of controW in the presence ofAmp icil l in Phenethicil l in Carbenicill in Pen icil l in G Pen icil l in V

0 (control) 100.0 100.0 100.0 100.0 100.0150 101.8 97.1 100.0 98.0 96.5300 101.1 97.3 91.8 97.0 93.1450 103.1 99.5 99.5 98.0 96.7600 101.6 98.7 101.9 98.4 97.4750 104.2 99.1 104.6 102.0 98. I

u Determined as follows: To a set of tubes each containing a single amount of 6-amino-pen icillanic acid (217 pg), various pen icillins were added in the indicated amounts. Foreach concentration of each pen icillin, six separate determinations were performed. Percent-age control was calculated using the mean value of each of the six determinations in theequation:

OD,,, (+pe nicillin) x 1oo,OD,,, (-penicillin)


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125SSAY FOR 6-AMINOPENICILLANIC ACIDTABLE 2

THE EFFECTOF PENICILLINASE PRETREATMENTO NTHECOLORYIELDOBT AINED FROM 6-AMINOPENICILLANIC ACID

Compound presentAPA (217 j.&APA (217 pg) pretreated withlo6 units of penicillinase

Optical density,,, ,,,0.6730.968

systems in which color is generated, indicating the necessity of an internalstandard when the system is used. The concentration range over which thesystem is useful appears to be from O-350 pg with most laboratoryinstrumentation.The method described here appears to provide significant advantagesover prior methods. Comparison with the method of Balasingham et al. (3)indicates a much greater sensitivity. In the method reported here, a meanmillimolar extinction coefficient has been calculated as 3.24, which isconsiderably higher than the value of 0.1 reported for the prior method.

1.4

I.2

I.(

0.1

4:P 0.1sQ

o.,

0..

0 / I I I I100 200 300 4006-AMINOPENICILLANIC ACID (JJG)FIG. 5. Concentration dependence of absorbance at 538 nm. The curve is the summation ofsix separate sets of standards.


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126 JOSE PH M. KORNFELDSimilarly, it is more sensitive than the method of Bomstein and Evans (1)and, additionally, does not require initial separation from penicillins, sincethose compounds do not interfere under the conditions used. It should bepointed out that in the method described by Shaikh et al. (6) in which APAreacts with D-glucosamine to form a complex which absorbs at 323 nm,initial separation of APA from penicillins is also unnecessary. Thepentanedione derivative method appears to have two advantages over theShaikh ef al. (6) procedure: (i) it is more sensitive, with a millimolarextinction coefficient approximately three times greater, and (ii) it requiresless time to perform the assay, about 30 min compared to 2 hr.As has been indicated, the penicillins which have been tested do notinterfere with the reaction, nor do the penicilloate derivatives of thosepenicillins. In addition, a number of amino acids, including valine,ornithine, cysteine, tryptophan, threonine, arginine, alanine, histidine,phenylalanine, leucine, methionine, lysine, tyrosine, cystine, isoleucine,proline, and glutamic acid have been examined at concentrations of 300pg/3 ml of reaction mixture, with no color production. At an equivalentconcentration, the reaction with 6-APA will produce an absorbance ofabout 1 .O at 538 nm (Fig. 5). Notwithstanding these observations, it must beassumed that in addition to the penicilloic acid formed from APA by thecleavage of the p-lactam ring, other, unidentified molecules may interfere,provided they satisfy the following conditions: (i) that they formpentanedione derivatives under the conditions of the assay, (ii) that thederivatives react with Ehrlichs reagent, and (iii) that the spectrum of theEhrlichs complex absorbs in the 53%nm region. If such molecules are notfound in routine fermentation broths, a question we have not yetaddressed, the value of the assay would obviously be enhanced.As indicated earlier, neither the nature of the pentanedione derivativenor the chemistry of the reaction has been determined. Studies arecurrently in progress which may clarify these points.

ACKNOWLEDGMENTThe technical assistance of John Karolus is acknowledged.

REFERENCES1. Boms tein, J., and Evans, W. G. (1965) Anal. Chem. 37, 576-579.

2. Sa cca ni, F., and Pitrolo, G. (1969) Boll. Chim. Farm. 108, 324-329.3. Balasingham , K., Warburton, D., Dunn ill, P., and Lilly, M. D. (1972)Biochim. Biophys.

Acta 276, 250-256.4. Mauzerall, D., and Granick, S. (1956) J. Bio l. Chem . 219, 435-446.5. Batch elor, F. R., Cameron-Wo od, J., Clair, E . B., and Rolins on, G. N. (196l)Proc. Roy.

Sot. Ser. B 154, 514-521.6. Shaikh, K., Ta lati, P. G., and Gang, D. M. (1973) Antim icrob. Ag. Chemo ther. 3,

194- 197.

a new calorimetric method for the determination of 6-aminopenicillanic acid

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