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A Selective ColorimetricMethod for the Determinationof Penicillins andCephalosporins with α-Aminoacyl FunctionsH.A. El-Obeid , E.A. Gad-Kariem , K.A. Al-Rashood , H.A. Al-Khamees , F.S. El-Shafie &G.A.M. Bawazeera Department of Pharmaceutical Chemistry,College of Pharmacy , King Saud University , P.O.Box 2457, Riyadh-11451, Saudi ArabiaPublished online: 25 Oct 2011.

To cite this article: H.A. El-Obeid , E.A. Gad-Kariem , K.A. Al-Rashood , H.A. Al-Khamees , F.S. El-Shafie & G.A.M. Bawazeer (1999) A Selective Colorimetric Methodfor the Determination of Penicillins and Cephalosporins with α-Aminoacyl Functions,Analytical Letters, 32:14, 2809-2823, DOI: 10.1080/00032719908543008

To link to this article: http://dx.doi.org/10.1080/00032719908543008

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ANALYTICAL LETTERS, 32(14), 2809-2823 (1999)

A SELECTIVE COLORIMETRIC METHOD FOR THE

DETERMINATION OF PENICILLINS AND CEPIIALOSPORINS

WITH a-AMINOACYL FUNCTIONS

Keywords: Colorimetry, penicillins, cephalosporins, p-lactams.

H.A. El-Obeid , E.A. Gad-Kariem, K.A. Al-Rashood, H.A. Al-Khamees,

F.S. El-Shafie and G.A.M. Bawazeer.

Department of Pharmaceutical Chemistry, College of Pharmacy,

King Saud University, P.O. Box 2457, Riyadh-11451, Saudi Arabia.

ABSTRACT

A simple, sensitive and selective colorimetric method is described for the

assay of ampicillin, amoxicillin, cephalexin, cefadroxil and cefaclor in their

pharmaceutical preparations. The method is based on measuring the color

obtained when the alkaline degradation products of these agents are allowed to

react with ascorbic acid. The factors affecting the color generation and

determination were studied and optimized. The reaction is selective to p-lactam

Correspondence

2809

Copyright © 1999 by Marcel Dekker, Inc. www.dekker.com

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2810 EL-OBEID ET AL.

antibiotics having amino acid side-chains with free amino functions and thus

allow interference-free quantitation of some preparations containing these agents

in combination with other p-lactam agents. The procedure is also successfully

adopted as stability-indicating method for cephalosporins. A tentative

mechanism of the color reaction is proposed.

INTRODUCTION

The p-lactam antibiotics encompass the widely used penicillins and

cephalosporins. They demonstrate clinical effective therapy for a large number

of infections and revolutionized the treatment of infectious diseases. Penicillins

and cephalosporins vary widely in their spectra of antimicrobial activities. Much

of the resistance to p-lactam antibiotics is caused by the p-lactamase hydrolytic

enzymes produced by resistant microorganisms. Cephalosporins are more P-

lactamase-stable than are penicillins, in part, because the cephalosporins

structures are more resistant to the hydrolytic effect of the enzyme. One way of

combating the problem of resistance is the use of antibiotic combination therapy

containing more than one agent to achieve synergistic effects e.g. ampicillin and

cloxacillin. Another approach is the use of P-lactamase inhibitors, e.g. clavulanic

acid in combination with amoxicillin. Both approaches demonstrate wide-

spectrum antimicrobial activity against most bacteria including the p-lactam-

resistant pathogenic bacteria and many p-lactamase inhibitors are now available

to limit the hydrolytic deactivation of these antibiotics. The wide use of the

P-lactam antibiotics, in combination or as monotherapy, necessitating the

development of simple, efficient, sensitive and selective procedures for their

quantitation. The literature reported several methods for the determination of

penicillins and cephalosporins. Thus, the methods used for the assay of

ampicillin, amoxicillin, cephalexin, cefadroxil and cefaclor include,

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PENICILLINS AND CEPHALOSPORINS 2811

spectrophotometric,10 electrochemical,6"9 titrimetric,10"13

chromatographic,101114"21 and microbial10'22 assay procedures. Most of these

methods, however, are complicated, lack sensitivity or are nonselective.

Recently, we reported5 that only penicillins having free cc-amino moiety

react, in acid medium, with ascorbic acid in a stoichiometric manner to give a

quantifiable color. All the cephalosporins tested, even those with free a-amino

function failed to give the color reaction under the acidic conditions used.

Cephalosporins, unlike penicillins are acid-stable22'24, accordingly, we suggested

that the color produced is the result of the reaction of ascorbic acid with a

product of hydrolysis of the p-lactam antibiotic. To verify this we investigated

the reaction of some penicillins (ampicillin, amoxicillin) and cephalosporins

(cephalexin, cefadroxil, cefaclor) with ascorbic acid in alkaline medium;

conditions known to cleave the P-lactam rings of both classes of antibiotics."'""6

On the basis of these investigations a simple, sensitive and selective assay

procedure was adopted and applied to determine the antibiotics, under study, in

different commercial pharmaceutical formulations and the results were

compared with those of reported methods.

EXPERIMENTAL

Materials and Equipment

Authentic samples of the penicillins and the cephalosporins used in this

study were supplied by Gist-Brocades, Amsterdam, The Netherlands.

Dimethylformamide (DMF) was obtained from Winlab Ltd., Maidenhead, U.K.

L-ascorbic acid of analytical grade was purchased from Hopkin and Williams,

Ehadwell Heath, England. All other chemicals were from Sigma Chemical

Company, St. Louis, U.S.A. Various pharmaceutical preparations were obtained

locally from different sources. The spectrophotometric measurements were

carried out using a Shimadzu UV-1601 PC spectrophotometer.

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2812 EL-OBEID ET AL.

Reagents

Ascorbic acid reagent was freshly prepared as a 2% w/v solution in DMF.

Stock solutions of ampicillin, amoxicillin, cefadroxil, cephalexin and cefaclor

were prepared by dissolving accurately weiglied amounts equivalent to 20 mg of

the anhydrous authentic material in 100 ml of distilled water. Fifteen ml of

ampicillin, amoxicillin, cefadroxil or cephalexin solutions or 20 ml of cefaclor

solution were transferred to 100-ml volumetric flasks; 3 ml of 0.1N NaOH were

added to each flask before heating at 100°C for 10 min in boiling water. After

cooling to room temperature, 4 ml of 0.1N HC1 was added to each flask before

completion to 100 ml with water.

Calibration Graphs

Different, accurately measured, volumes (1-5 ml) of the stock solutions of

ampicillin, amoxicillin, cefadroxil, cephalexin, or cefaclor were placed in 10 ml

calibrated flasks. Distilled water was added to make a volume of 5 ml in each.

Fresh ascorbic acid reagent (1 ml) was then added to each flask before heating at

100°C for 20 min. After cooling, the volume was completed to 10 ml with water,

mixed well before reading at 410 ran for ampicillin, amoxicillin and cefadroxil

and 406 nm for cephalexin and cefaclor, against a blank prepared by heating 5

ml water with 1 ml ascorbic acid reagent and proceeding as above. The graphs

were constructed by plotting the absorbance values versus drug concentrations.

Assay Procedure

To quantify the specific antibiotic in its various pharmaceutical

formulations, an accurately weighed amount of the preparation, equivalent to 20

mg of the anhydrous drug, was dissolved in 100 ml water and processed as in

Reagents above. A volume of 3 ml of the stock solution of each pharmaceutical

preparation was placed in a 10-ml calibrated flask. After adding 2 ml of water

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PENICILLINS AND CEPHALOSPORINS 2813

and 1 ml of the freshly prepared ascorbic acid reagent, the contents were heated

at I00°C for 20 min. and after cooling the volume was completed to 10 ml with

water and the color developed was measured at the specified wavelength for

each drug.

Difference Spectroscopic Method (AA)

An aliquot of 15 ml of 0.02 % w/v of either of the cephalosporins under

investigation (authentic or in formulation) was treated as in Reagents and

Calibration Graph, the color is read against blanks containing the same

volumes of the cephalosporins but replacing the 3 ml of the 0.1N NaOH with

distilled water and adding 1 ml of 0.1N HC1 instead of 4 ml. The concentration

of the samples can either be calculated from direct sample/standard comparison

as follows:

AA Samp e x C% Standard- — = C% Sample

AA Standard

or from calibration graphs of AA's versus concentrations of the authentic sample

in the same range of the calibration graphs of these drugs.

RESULTS AND DISCUSSION

Ascorbic acid had been used as a reagent for the colorimetric

determination of various amino acids.27 The oxidized form of the vitamin,

dehydroascorbic acid, is believed to react with the amino group of the amino

acid in a manner similar to the reaction of these acids with ninhydrin.2829 The

Schiff s base formed facilitates easy decarboxylation to generate the colored

species which, for the particular reagent used, is common to all a-amino acids.

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2814 EL-OBEID ET AL.

The penicillins or the cephalosporins, each as a group, share a common

structural feature that differs mainly in the side chain attached to the nitrogen of

the 6-aminopenicillanic acid moiety. Some penicillins and cephalosporins have

amino acid residues incorporated as their N-alkyl side chain, i.e. the

N-phenylglycyl of ampicillin, cefaclor and cephalexin and the N-4-

hydroxyphenylglycyl of amoxicillin and cefadroxil. Based on the reported

reaction of ascorbic acid with amino acids, we investigated the behaviour of this

reagent when allowed to react with penicillins and cephalosporins containing

amino acid residues. Ampicillin, amoxicillin, cephalexin, cefadroxil and cefaclor

reacted with ascorbic acid in an alkaline medium and produced a yellow color

absorbing maximally at 410 nm (ampicillin, amoxicillin, cefadroxil) and 406 run

(cephalexin, cefaclor). The factors that influence the color intensity include the

reagent concentration, reaction temperature, heating time and the quantity of the

alkali added. Each of these factors was studied and the optimization of the

reaction conditions was established as that stated under EXPERIMENTAL. It is

found that heating an aqueous solution of these drugs in 0.1 N NaOH at 100°C

for 10 min. is a necessary step prior to color development. The optimum

conditions for color production involve heating this drug solution with ascorbic

acid at 100°C for 20 min. Heating at this temperature greatly enhances the color

production reaction and increases the color intensity. Once produced the color is

stable for at least 24 hours. FIGURE 1 shows typical absorption spectra of

cephalosporins when treated with ascorbic acid reagent at the optimum

conditions set up.

Under these conditions, calibration graphs were constructed using

authentic samples of ampicillin, amoxicillin, cephalexin, cefadroxil or cefaclor.

The graphs were linear within drug concentration ranges of 3 to 15 jig/ml for

ampicillin, amoxicillin, cephalexin and cefadroxil and of 4 to 20 ng/ml for

cefaclor. Data for the linearity of the reaction, correlation coefficients and molar

absorptivities are included in TABLE 1.

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PENICILLINS AND CEPHALOSPORINS

0.900

0.800-

2815

0.000360.0 380.0 400.0 420.0

Wavelength(nm}

450.0

Fig. 1: Absorption spectra of the products of the reaction of ascorbic acid with

cefadroxil (I), cephalexin (II) and cefaclor (III) under alkaline conditions.

The accuracy of the procedure was checked by recovery-testing of added

amounts of the authentic drugs to sample solutions in the ratio of 1:2. The results

showed good recovery (98.98%±1.33). This, together with the excellent

reproducibility (100.00%±0.43) reflected in the relatively low coefficient of

variation (<I%), indicate the good accuracy and precision of the method. The

applicability of the new procedure was assessed by analysing different

pharmaceutical formulations of either drug alone or in combination with others

and statistically comparing the results obtained with those of other methods

using Student T-test. Data of TABLE 2 show the calculated t and F ratios as

compared to the corresponding tabulated values at 95% confidence level. The

results obtained using the proposed colorimetnc method are in good agreement

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2816 EL-OBEID ET AL.

TABLE 1

Spectral data for the reaction of ascorbic acid with the drugs under investigation.

Drug

Ampicillin

Amoxicillin

Cephalexin

Cefadroxil

Cefaclor

Amax

(nm)

410

410

406

410

406

Linearityrange(ug/ml)

3-15

3-15

3-15

3-15

4-20

Intercept

0.0086

0.018

.0.0269

0.01225

0.0225

Slope

0.0441

0.0489

0.0370

0.0393

0.02533

Correlationcoefficient

0.9998

0.9996

0.9996

0.9995

0.9998

Molarabsorptivity(Lmofcnf 1 )

2.40 x 10'

2.10 x 10'

1.30 x 10'

1.42 x 10'

0.93 x 10'

with the declared amounts and confirm that no significant differences exist, in

accuracy and precision, between the new method and the official and other

methods.

The procedure also served as a stability-indicating method for the

determination of the intact cephalosporins tested. On application of the method a

minimum detectable concentration of 0.6 jig/ml was obtained for cephalexin and

cefadroxil; 0.8 ng/ml for cefaclor and of 0.45 jig.ml for ampicillin and

amoxicillin. The minimum detectable concentration is defined here as the

concentration allowing minimum measurable absorbance.

Of interest is the finding that neither cloxacillin (when present in

combination with ampicillin), nor clavulanic acid (added to some amoxicillin

preparations) interferes with the colorimetric assay method for ampicillin and

amoxicillin. Thus, when different concentrations of cloxacillin and clavulanic

acid (up to twice that present in the pharmaceutical formulations) were

intentionally added to ampicillin and amoxicillin samples, respectively, no

change of absorbance was observed. The method, therefore, offers the advantage

of specifically analysing the two drugs in combination with these structurally

related compounds without prior separation. This fact also points to the

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PENICILLINS AND CEPHALOSPORINS 2817

TABLE 2

Results (% ± SD) of assaying penicillins and cephalosporins in differentpharmaceutical fonnulations using the proposed method as compared to official

and other methods.

Preparation

Pcnamoxcapsules

Amoxydarsuspension

Augraentinsuspension

Augmentintablets

Penbritincapsules

Penbritinsuspension

Ampicloxsuspension

Ampicloxcapsules

Ampicloxdrops

Keflexcapsules

KeflexSuspension

Droxilcapsules

Ultracefsuspension

Ceclorcapsules

Ceclorsuspension

Newcolorimetricmethod105.97±0.8n = 5C.V. = 0.76100.210.66n = 4C.V. = 0.66103.4911.58n = 4C.V. = 1.53103.79±0.%n = 4C.V. = 0.93101.6410.43n = 4C.V. = 0.42105.9810.98n = 4C.V. = 0.92103.8410.49n = 4C.V. = 0.47101.810.78n = 4C.V. = 0.77100.4510.93n = 4C.V. = 0.93100.1911.88n = 6C.V. = 1.88105.9711.04n = 5C.V. = 0.98104.5310.66n = 5C.V. = 0.6399.80iO.92n = 5C.V. = 0.93104.6110.86n = 5C.V. = 0.82104.0510.84n = 5C.V. = 0.81

IodometricU.S.P.'" orB.P.0"°"1O4.5311.87n = 3C.V: =1.79U)100.6411.73n = 3C.V. 1.72

l"l0O.2911.25n = 3C.V. = 1.25("104.1811.08n = 3C.V. = 1.04

lbJ101.3411.45n = 3C.V. = 1.43lbl104.6511.22n = 3C.V. = 1.17

HPLC

102.1910.75n = 3C.V. = 0.73102.4311.1n = 3C.V. 1.07

AdoptedHPLCmethod

102.6910.88n = 3C.V. = 0.86102.8211.26n = 3C.V. = 1.23101.2+1.7n = 3C.V. 1.67

(tab)

1.3(2.57)

0.41(2.57)

1.44(2.57)

1.71(2.57)

1.8(2.57)

2.28(2.57)

2.04(2.57)

1.24(2.57)

0.69(2.57)

1.01(2.36)

1.56(2.45)

F«i(tab)

5.46(6.94)

6.87(9.55)

4.42(19.2)

1.31(9.55)

8.45(9.55)

1.21(9.55)

3.22(9.55)

2.6(9.55)

3.34(9.55)

1.68(5.79)

1.38(6.94)

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2818 EL-OBEID ET AL.

importance of a free a-amino group in the chemical species that react with

ascorbic acid to generate the colored product. Furthermore, cefotaxime and

cefuroxime (cephalosponns with substituted amino group) did not produce any

observable color when treated with ascorbic acid under the assay conditions.

This together with the finding that cephalosponns with a-amino acid moiety

failed to give a color reaction under acidic conditions,5 confirm our previous

suggestion that the essential prerequisites for the quantitation of penicillins and

cephalosporins by the ascorbic acid method are the presence of a free amino

group in the alkyl side chain coupled with cleavage of the P-lactam ring

structure. To the best of our knowledge the amino acid moieties of ampicillin,

amoxicillin, cephalexin, cefadroxil and cefaclor remain attached to the

aminopenicillanic acid part of the molecules under the alkaline conditions used

in this study. Therefore, there is no question here of a free amino acid reacting

with ascorbic acid, otherwise the color produced would have been the same as

that obtained when any amino acid is used.

It is well documented that the p-lactam rings of penicillins and

cephalosporins are cleaved under alkaline conditions to form their respective

penilloic acids.22"26 Some reports2426 suggest that p-lactam antibiotics having

free a-amino group form, under alkaline conditions, a cyclic 2,5-

diketopiperazine derivative as exemplified for cefadroxil in SCHEME 1.

The results of this and of our recently reported studies5 do not point to the

presence of such derivative. Indeed, the involvement of a hydrolytic derivative

having a free amino function in the color-producing reaction is established. The

cyclization of penilloic acid to the 2,5-diketopiperazine derivatives24'26 may be

attributed to the high alkali concentration and the long heating time used in these

experiments as compared to the mild conditions employed in the present study.

Investigation of the stoichiometry of the reaction using the molar ratio method30,

revealed a 1:1 ratio of either of the test drugs to ascorbic acid. Furthermore,

when the assay method was conducted in the presence of sodium metabisulfite

the color development was greatly retarded. This confirms the involvement of

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PENICILLINS AND CEPHALOSPORINS 2819

O

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sde

ri

c

X

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/sis

o

•a .so 13

KU

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2820 EL-OBEID ET AL.

R-/ \-(J:H-C-NH-(J;H—CH^ = / ^ NH2 COOH X

R — f \ - C H - C - N H - C H 2 - C H +

/

o-

SCHEME 2: Proposed mechanism for the color-producing reaction.

the oxidized form of ascorbic acid in the color-producing reaction. The results of

these studies tempted us to propose the reaction mechanism outlined in

SCHEME 2. The mechanism involves reaction of the free amino group of

penilloic acid derivative (I) with the carbonyl group of dehydroascorbic acid (II)

to produce (III) which may undergo cyclization to furnish a colored product (IV)

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PENICILLINS AND CEPHALOSPORINS 2821

capable of existing as tautomers. More studies to confirm this mechanism are

now in progress.

It is concluded that the proposed method is simple, accurate and shows

selectivity for specific p-lactam antibiotics and thus allows the quantitation of

such antibiotics in compound preparations, properties that are lacking in many

official and other reported methods. These properties are shared with those of

our previously reported5 procedure although the reactions involved followed

different mechanistic pathways. The new method, however, showed higher

sensitivity and a wider spectrum of application to include quantitative and

stability-indicating analytical techniques for cephalosporins having free amino

function as part of their amino acid side chains.

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Received: March 22, 1999Accepted: July 20, 1999

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