Discrimination and Direct Determination of Cephalosporins by Circular Dichroism

RESEARCH ARTlCLES

Discrimination and Direct Determination of Cephalosporins by Circular Dichroism PllAR

GORTAZAR AND JESUST. VAZQUEZ'

Received January 11, 1993, from the Centro de Productos Naturales Organicos Antonio Gonzalez, Universidad de La Laguna-C.S.I.C., Accepted for publication May 2, 1994@. Carrefera de La fsperanza 2, 38206 La Laguna, Tenerife, Spain. Abstract 0 The circular dichroism and ultraviolet spectra of 15 commercial cephalosporins in common clinical use are analyzed. Distinguishing between the p-lactam antibiotics (penicillins, cephalosporins, and cephamycins) on the basis of their CD spectral data has been found to be straightforward. Furthermore, sufficient CD spectral dissimilarities are observed to discriminate among the cephalosporin homologues and to classify these antibiotics in five spectroscopic groups, on the basis of the wavelengths of their Cotton effects. In addition, some chemical structural characteristics for these spectroscopic groups are discussed. Besides molar absorptivity and CD data, the slopes and the intercepts of the equations of the regression line are calculated for each of these antibiotics, the correlation coefficients being higher than 0.9993. The validity of the proposed model is confirmed by analysis of the variance. The results demonstrated that the proposed method is accurate and precise. The method was successfully applied to the direct determination of these drugs in commercial oral suspensions, injections, and capsules. The principal advantages of this method are quickness and simplicity, no derivatization or chromatographic separation steps being needed.

Circular dichroism (CD) is only recently being applied to pharmaceutical ~hemistry.l-~Its high degree of analytical selectivity allows an optically active absorbing compound in a mixture t o be analyzed directly, as a result of the fact that two physical requirements (ellipticity and absorbance) are measured simultaneously. Thus, CD has been applied to the direct determination of p-lactam antibiotics in pharmaceutical preparations, additives not interfering with the CD measurement.4 The remarkable progress made in the study and production of semisynthetic cephalosporins with an expanded spectrum of activity5 prompted a detailed analytical study of these important compounds, CD being a valuable technique for this purpose. Therefore, 15 commercial cephalosporins in common clinical use were characterized. Unlike penicillins: they exhibited sufficiently characteristic CD spectra to allow a more accurate analytical determination to be performed, as well as to discriminate among the cephalosporin homologues.

Experimental Section Materials-All the cephalosporins analyzed in this study were obtained from Sigma Chemical Co. and used without further purification: cefadroxil, cefoperazone sodium, cephalexin, cephradine, cefotaxime sodium, cephalotin sodium, cefuroxime sodium, cefaclor, cefapirin sodium, cefsulodin sodium, cefamandole nafate, cephazolin sodium, cefoxitin sodium, moxalactam diammonium, and ceftriaxone

@

Abstract published in Advance ACS Abstracts, July 1, 1994.

1204 / Journal of Pharmaceutical Sciences Vol. 83, No. 9, September 1994

sodium. Oral suspensions and capsules of cefadroxil and injectable dosage forms of cefoxitin sodium were obtained from a local pharmacy. Standard solutions for each antibiotic were prepared by dissolving the accurately weighed compound in calibrated flasks with distilled water to obtain solutions containing 2 mg/mL. Procedures-For calibration, measured volumes of the standard solutions were diluted into calibrated flasks with distilled water to cover the calibration range given for each antibiotic (Table 4 and text). At least a volume of 3 mL is required when cylindrical quartz cells of 10 mm length are used. CD spectra of these solutions were recorded, and the ellipticity angle a t the selected wavelength (Tables 4 and 5) was measured (millidegree). For the analysis of pharmaceutical formulations, the same procedure followed for the standard solutions was applied, the concentration of the CD samples containing 40 ,ug/mL of antibiotic. Containers of cefadroxil for oral suspension were constituted as directed in the labeling. Suitable aliquots of these suspensions were diluted in calibrated flasks with distilled water to obtain the above concentration. For cefadroxil capsules, the contents of at least six capsules per assay were finely ground and a portion of the powder was weighed and dissolved in a calibrated flask with distilled water. Containers of cefoxitin injection were constituted, and accurately measured volumes of injection were diluted in calibrated flasks with distilled water. A p p a r a t u s - W absorbance and CD data were recorded in the range 400-200 nm by using 10-mm cells. UV absorption measurements were made on a Perkin-Elmer Model 550D spectrophotometer. CD spectra were obtained on a JASCO 5-600 spectropolarimeter, instrument parameters being selected to give the optimum signalto-noise ( S N ) ratio. Prior to measurements, the spectropolarimeter was calibrated with a standard solution of ammonium d-camphor10-sulfonate in distilled water.

Results and Discussion Spectroscopic Classification-The UV and CD studies of a number of model cephalosporins have confirmed that the 3-cephem chromophore, the basic skeleton of cephalosporin antibiotics, has two transitions, one at 260 nm and the other a t 230 nm.6 However, the cephalosporins of pharmaceutical interest (Table 1)having at least one extra chromophore in the substituents attached to the 3-cephem moiety exhibit important shifts in the wavelengths of these absorptions, and present new ones (Tables 2 and 3). This observation prompted us to classify the cephalosporins in five spectroscopic groups, based on the wavelengths of their positive and negative Cotton effects in the regions centered around 260 and 230 nm, respectively, as well as the points where Ac changes sign. A gradually increasing bathochromic shift of the first and/or the second Cotton effect can be observed in the CD spectra shown in Figure 1for a member of each group: cephradine (2551221nm), cephalotin (258/228), cephazolin (263/228),cefoxitin (262/238),and ceftriaxone (284/ 236). This behavior comes from red shifts of the transitions of the 3-cephem chromophore produced by the substituents

0022-3549/94/1200-1204$04.50/0

0 1994, American Chemical Society and American Pharmaceutical Association

Table 1-Structures of the Cephalosporins Examined

Table 2-UV Data of the Cephalosporins I

u

'

, I

R3H

BASIC CEPHALOSPORIN STRUCTURE

R I T l y y $ R2 COOH

Name (CD group)

R1

R2

Cefadroxil (1)

H O a f , ; H NH2

CH3

Cefadroxil Cefoperazone Cephalexin Cephradine Cefotaxime Cephalotin Cefuroxime Cefaclor Cefapirin Cefsulodin Cefamandole Cephazolin Cefoxitin Moxalactam Ceftriaxone

-

H"-(c&f.; H

NH

Cefoperazone (1 1

nm (4

UV A,,

Name 261 (8 600) 267 sh (13 100) 261 (8 600) 260 (7 900) 300 sh (6 500) 266 sh (8 400) 274 (17 200) 263 (9 200) 257 (18 500) 261 (13 800) 267 (10 000) 270 (14 500) 260 (8 800) 268 (12 700) 270 (27 600)

228 (13 500) 225 (25 000) 254 (17 500) 236 (14 000) 225 sh (12 900)

233 (19 500)

234 ( 1 4 500) 224 (18 700) 239 (30 700)

Table 3-CD Data of the Cephalosporins Cephalexin (1 1

Group

&*/A€

Name

Ac=O

First

Cefadroxil 25519.2 2111-16.3 235,202 Cefoperazone 25513.1 2231-17.4 2071-17.7 238 Cephalexin 255112.1 2231-18.3 237 Cephradine 255110.6 2211-14.4 2021-15.2 237 Second Cefotaxime 3061-0.6 258110.5 2281-1 8.3 289,242,206 Cephalotin 258112.4 2281-17.4 242 Cefuroxime 2961-1.1 258111.4 2271-16.4 296,242,207 Cefaclor 259111.O 2281-15.8 242 Cefapirin 259114.6 2291-17.5 242,208 Third Cefsulodin 26316.3 2311-16.9 247,210 Cefamandole 26316.6 2301-16.9 248 Cephazolin 2961-1.6 26317.4 2281-16.6 285,247 Fourth Cefoxitin 262/17.2 2381-18.7 21513.5 250,222,208 Moxalactam 26518.0 2391-28.8 255,215 Fifth Ceftriaxone 2841-5.2 2361-17.4 206

Cephradine (1 1

Cefotaxime

0

(2)

-CH20-C-CH3

Cephalotin (2)

CH2 -

0

-CH2O- C - CH3

CI

20.00L

'

'

'

'

'

'

1

"

t

Cefapirin (2)

a SCH2 -

N

.

'

'

'

'

'

'

1

j

fl

-CH2O-C -CH3 AE

-

N-N

0

Cefamandole nafate (3)

Cephazolin (3)

CH,-

-CH?S

*),,I

N-N

Moxalactamaib (4)

~

Ho

-CH2S

H COOH

I

CH-a " H

~

Rg = H. aR3 = OCH3 bS atom replaced by 0 on p-lactarn ring

I

-20.001

200

'

240

'

'

h(m)

' 300

'

'

330

Figure 1-CD spectra for one member of each group: cephradine (first group), cephalotin (second group), cephazolin (third group), cefoxitin (fourth group), and ceftriaxone (fifth group).

directly attached to it (R2and Rs),and/or the overlapping of new transitions to the one existent at 260 and 230 nm originating from the chromophores present in R1 and R2. The different origin of these shiRs makes the relationship between the chemical structural characteristics and the CD spectral Journal of Pharmaceutical Sciences 11205 Vol. 83, No. 9, September 1994

1 0 . 0 0 ~ " " " ' "

' ' 1

1

4

cephalexin cephradine cefadroxil cefoperazone

-_--20.001

'

'

200

'

230

.

'

'

'

h(m)

'

290

'

'

1

320

Figure 2-CD spectra for the cephalosporins of the first group. 18.00;

1

.

'

'

'

1

'

'

1

'

-cefsulodin cefamandole -------cephazolin

----

:

1

-20.00 200

l

"

.

"

"

"

l

.

.

.

'

1 (nm) 300 Figure 4-CD spectra for the cephalosporins of the third group. 240

330

25.00

4

0 0

A&

-cefapirin

cefuroxime .-.cefaclor

\

F

1-

-20.001 . 200

'

'

230

.

'

'

'

h(nm)

'

'

290

'

'

1

320

-35.001 . 200

I

230

\/

'

'

I

'

L(nm)

'

'

I

290

I

320

Figure 3-CD spectra for the cephalosporins of the second group

Figure 5-CD spectra for the cephalosporins of the fourth group.

pattern difficult to study, but some structural features can be observed. The first group comprises cefadroxil, cefoperazone, cephalexin, and cephradine. The observation of the presence of one or two Cotton effects a t wavelengths below 225 nm together with the corresponding intensities and wavelengths of their Cotton effects (Table 3, Figure 2) make these cephalosporins easy to discriminate. The methyl group at Rz seems to be a chemical structural characteristic of this spectroscopic group, cefoperazone being an exception (Table l h 7 At first sight, the spectral characteristics of the members of the second group, cefotaxime, cephalotin, cefuroxime, cefaclor, and cefapirin, are similar (Table 3), although they can be discriminated by the intensities of their Cotton effects and by the shape of the CD spectrum a t short wavelengths (200-220 nm) (Figure 3). As can be observed in Table 1,the replacement of the methyl group a t position 3 by the acetoxymethyl group, the (carbamoy1oxy)methyl group, or the chlorine atom is indicated by the small bathochromic shift observed for the two transitions of the 3-cephem chromophore. The characteristic negative Cotton effects observed a t longer wavelengths in cefotaxime and cefuroxime may be due to the conjugated methoximino group in the a-position of the N-acyl side chain (R1).

The components of the third group, cefsulosin, cefamandole, and cephazolin, can be readily distinguished by their CD spectra (Table 3, Figure 41, cephazolin showing a negative Cotton effect a t 296 nm. The cephamycins, cefoxitin and moxalactam,8 which are characterized by an a-methoxy group a t the 7-position (R3) that confers great P-lactamase stability to these compounds, constitute the fourth group (Table 3, Figure 5). The methoxy group is responsible for the red shift observed for the 230-nm transition6 and makes these compounds easy to recognize. The only cephalosporin analyzed that resisted classification in any of the above groups was ceftriaxone (Figure 11, which exhibited only two negative Cotton effects. Chemical structural comparison with cefotaxime reveals that the first negative Cotton effect (284 nm) of ceftriaxone is due to negative Cotton effects arising from the [(2-methyl-5,6-dioxy1,2,4-triazinyl)thiolmethyl group (Rz) and from the conjugated methoximino group in R1 which cancel the positive Cotton effect of the 3-cephem chromophore. We believe that the present classification facilitates the discrimination of cephalosporin homologues, which possess sufficient spectral dissimilarities in most cases for their correct identification.

1206 / Journal of Pharmaceutical Sciences Vol. 83, No. 9, September 1994

Table 4-Parameters of Calibration Graphs. I

Compound Cefadroxil Cefapirin Cefamandole Cefoxitin Ceftriaxone

Range (pg/mL)

n

CD ,Iefl (nm)

Slope,rn

Intercept, z

Correlation Coefficient, r

S,

1.O-80.0 1.0-60.0 5.0-80.0 1.O-80.0 5.0-40.0

30 25 24 28 24

255 259 263 262 236

0.8575 10.0016 0.9960 ? 0.0047 0.4228 f 0.020 1.3209 k 0.0051 -0.9968 k 0.0065

-0.0656 & 0.060 0.2014 k 0.1583 -0.0521 10.0912 -0.1915 10.2281 0.3867 k 0.1642

0.9999 0.9998 0.9997 0.9998 0.9995

0.265 0.537 0.277 0.806 0.410

+

Table 5-Parameters of Calibration Graphs. II

Compound

CD I,, (nm)

m

Z

Cefoperazone Cephalexin Cephradine Cefotaxime Cephalotin Cefuroxime Cefaclor Cefsulodin Cephazolin Moxalactam

255 255 255 258 258 258 259 263 263 265

0.1397 1.1538 0.9761 0.6896 0.9792 0.8084 1.0345 0.3682 0.5055 0.4582

0.0070 0.0332 0.0699 -0.1131 -0.1226 0.0558 -0.0744 0.0015 -0.0969 0.0303

Validation of the Method-The analysis of variance of the linear regression of the calibration line (ANOVA), carried out with one member of each spectroscopic group, confirms the linearity of the present method. Cefadroxil, cefapirin, cefamandole, cefoxitin, and ceftriaxone were the selected cephalosporins, since they cover the range of ellipticities and absorbances observed. Table 4 shows the root mean square of the ANOVA error term, Soe, for these antibiotics, which demonstrate the validity of the model. Linearity-The regression line equations of the linear relationship between the ellipticity angle and the concentra-

tion of the antibiotic were defined as I9 = mc z, where 8 is the ellipticity angle (millidegree, mdeg) at the selected wavelength, c is the concentration (uglmL), m is the slope of the fitted line, and z is the I9 intercept of the regression line. Beer’s law is followed for concentrations up to 80 pg1mL for cefadroxil, cefamandole, and cefoxitin, up to 60 pg/mL for cefapirin, and up to 40 pglmL for ceftriaxone, the correlation coefficient, r, being equal or higher than 0.9995 for each drug. Table 4 summarizes these results. For the 10 other cephalosporins, linear calibration graphs between 5 and 60 pglmL were obtained from eight working solutions (Table 5). The observed correlation coefficientswere in all cases greater than 0.9993. Reproducibility, Accuracy, and Precision-The statistical analyses indicate that the obtained assay values were reproducible, with very satisfactory mean average coefficients of variations (CV).Based on the precision and accuracy values shown in Table 6 , the acceptable limit of quantitation is 5 pgl mL, the overall accuracies for cefadroxil, cefapirin, cefamandole, cefoxitin, and ceftriaxone being 99.9,100.1,101.1,100.1, and 100.1%,with precisions of 1.10, 1.46,1.69, 1.19, and 1.66, respectively. Applications-The method described herein allows the correct identification and determination of cephalosporins in pure form, in an accurate and precise way. It is an economical

Table 6-Data for Precision and Accuracy of Calibration Curve Standards

Compound Cefadroxil

Cefapirin

Cefamandole

Cefoxitin

Ceftriaxone

Concentration of Calibration Standard, pg/mL 1.oo 5.00 10.00 20.00 40.00 80.00 1.oo 5.00 20.00 40.00 60.00 5.00 10.00 20.00 40.00 60.00 80.00 1.oo 5.00 10.00 20.00 40.00 60.00 80.00 5.00 10.00 20.00 25.00 30.00 40.00

n 5

5 5 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

Mean Determined Value, pg/mL

Standard Deviation (SD)

Mean Accuracy, % of Nominal

Precision CV, %

1.20 5.06 10.03 19.80 39.78 79.84 0.86 4.94 20.27 40.26 59.80 5.17 10.28 20.23 39.58 60.20 80.26 1.14 5.11 10.02 19.84 39.28 60.30 80.05 5.13 10.05 19.61 24.88 29.98 40.14

0.022 0.053 0.196 0.183 0.305 0.653 0.080 0.131 0.103 0.455 0.923 0.191 0.141 0.259 0.488 0.735 1.069 0.028 0.087 0.096 0.1 94 0.420 0.819 0.863 0.127 0.155 0.249 0.465 0.479 0.476

120.0 101.2 100.3 99.0 99.4 99.8 86.0 98.8 101.3 100.6 99.7 103.4 102.8 101.1 98.9 100.3 100.3 114.0 102.2 100.2 99.2 98.2 100.5 100.1 102.6 100.5 98.1 99.5 99.9 100.3

1.83 1.05 1.95 0.92 0.77 0.82 9.30 2.65 0.51 1.13 1.54 3.69 1.37 1.28 1.23 1.22 1.33 2.46 1.70 0.96 0.98 1.07 1.36 1.08 2.47 1.54 1.27 1.87 1.60 1.19

Journal of Pharmaceutical Sciences / 1207 Vol. 83, No. 9, September 1994

Table 7-Determination of Cephalosporins in Pharmaceutical Formulations by the Proposed Method

Dosage Form Oral suspension (n = 3)

Capsules (n = 4) Injection (n = 5)

Amount Claimed on Label, glunit 3.0 (Cefadroxil) 35.0 (Saccharose) 0.5 (Cefadroxil) 1.O(Cefoxitin)

Mean Recovery k SD,% 104.5 k 2.13 104.8 k 1.40 103.1 k 1.24

and very simple method, no derivatization or addition of any standard is needed, and the measurement can be achieved in a few minutes, these properties being very practical for quality control laboratories. In addition, enantiomeric differentiation and/or optical purity can be readily achieved by means of CD.'s2 The method proposed was applied to the direct determination of these drugs in pharmaceutical preparations (oral suspensions, capsules, and injections). Table 7 shows the results of these determinations, which are in good agreement with the amounts of label claim, and establishes an excellent basis for further study and development. The CD spectra of these assays were superimposable with those of the standard solutions, by multiplying one of them by a factor t o achieve identical concentrations, even for the one from the oral suspension assay. The double requirement of optical activity and absorption for a compound to exhibit CD activity is responsible for the high degree of analytical selectivity observed. Therefore, no chromatographic separation steps, derivatization, or addition of any standard are needed.

Conclusions The method proposed discriminates successfully among the cephalosporin homologues, the accomplished spectroscopic

1208 /Journal of Pharmaceutical Sciences Vol. 83, No. 9, September 1994

classification facilitating their correct identification. In addition, the method is simple, economical, and rapid and confirms that circular dichroism is a useful technique for the direct, accurate, and precise determination of drugs.

References and Notes 1. Purdie, N.;Swallows, K. A.Anal. Chem. 1989,61, 77A-89A. 2. (a) Purdie, N.; Swallows, K. A,; Murphy, L. H.; Purdie, R. B. J . Pharm. Biomed. Anal. 1989,7 , 1519-1526. (b) Purdie, N.; Swallows. K. A,: Murahv, L. H.; Purdie, R. B. Trends Anal. Chem. 1990,9,136-142: 3. Gergely, A.J . Pharm. Biomed. Anal. 1989,7,523-541. 4. Purdie, N.;Swallows, K. A.Anal. Chem. 1987,59,1349-1351. 5 . Williams, J. D., Ed. Drugs 1987,34 (Suppl. 2), 1-258. 6. The absorption at 260 nm has been attributed to the interaction between the sulfur, the ring nitrogen, and the double bond. The transition at 230 nm comes from the inherently dissymmetric chromoDhore constituted bv the double bond and the 8-lactam carbon$\. Nagarajan, R.; Spry, D. 0. J . Am. Chem. Sbc. 1971, 93,2310-2312. 7. The chromophorically complex substituent RI in cefoperazone might be the responsible for the observed wavelengths, especially due to the presence of the dioxopiperazine group in its ureido side chain. 8. Although moxalactam is not, strictly speaking, a cephamycin, or indeed a cephalosporin since the sulfur atom is replaced by an oxygen, it is so chemically and biologically similar to a cephamycin that it has been considered as such. '

Acknowledgments The authors are grateful to Dr. Manuel Ravina, for his cgllaboration to carry out the present work, and to Drs. Julio D. Martin and Jose Fariiia, for their interest and useful suggestions.