**3.5 Visible spectrophotometric methods based on coupling with specified reagents**

The analysis of phenolic derivatives of penam and cephem analogs were possible by measurement of absorption species formed as a result of reations with specified reagents.

Diazo coupling of β-lactam analogs was conducted with the following compounds:


Suggested mechanisms of coupling reaction of phenol derivative of β-lactam analogs were presented in Fig. 15.

Fig. 15. Schemes of reactions of coupling of β-lactam analogs [50-51].

For determination of phenolic derivative of β-lactam analog (cefadroxil) measurement of absorption of formed product in the reaction between it and 4-aminoantipyrine in the presence of alkaline potassium hexacyanoferrate(III) at 510 nm was also proposed (Fig. 16). Potassium hexacyanoferrate(III), being oxidant in this reaction, yielding *N*-substituted quinine imines and in the result was responsible for formation of red-colored antipyrine dye. Additionally, a sequential injection analysis (SIA) spectrophotometric procedure for the determination was reported [52].

1,2-naphthoquinone-4-sulfonic acid is the reagent permitting the nucleophilic substitution reaction in area of amino group of penem (amoxicillin) and cephem (cephalexin) analogs (Fig. 17). The stoichiometric ratio of these species was 1:1 and they absorb at λ = 463 nm [53].

The extension of the methodology for determination of cephalexin by using the H-point standard additions method (HPSAM) and the generalized H-point standard additions methods (GHPSAM) (after solid phase extraction cartridges) permitted also its analysis in urine [53].

Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of β-Lactam Antibiotics 125

The significant expansion of possibilities for the developed analytical method was the usage

Similarly, in the case of determination of penam analysis during acidic hydrolysis (1.0 M HCl), formation of non-absorbing degradation products was the intermediate stage of their analysis. Complex which is necessary for achievement of spectrophotometric signals, was formed between penicillamine and palladium(II) chloride, peak at 334 nm (Fig. 18) [58].

> <sup>H</sup> <sup>N</sup> <sup>+</sup> S

COOH

O

O

NH2


CH3

SH NH2

H3C C C COOH CH3

H

S NH2

HOOC C C CH3 NH2

Fig. 18. Absorption spectra based on reaction of degradation products of penam analogs and

In conditions of acidic hydrolysis, determination of cephem analogs took place. Vanadium(IV) after reduction from vanadium(V), reacted with forming degradation products. Colored complexes of some cephem analogs (cephalexin, cephaprine sodium, cefazolin sodium, cefotaxime) were found, absorbing at 515, 512, 518 and 523 nm,

H CH3

S Pd

PdCl2

N H

H

R

CH3 CH3


R C C NH NH2

O

H

H3C C C COOH CHO

+

The proposal ot hte reaction pathway between the hydrolysed penicillins na palladium(II)

of flow injection analysis (FIA).

Absorption spectrum of the reaction product of palladium(II) chloride with hydrolysis

palladium (II) and suggested mechanism of the reaction [58].

product of ampicillin.

respectively (Fig. 19) [59]

Fig. 16. The reaction mechanism of cefadroxil with 4-aminoantipyrine in the presence of alkaline [Fe(CN)6]3-

Fig. 17. The reaction mechanism of ampicillin sodium and 1,2-naphthoquinone-4-sulfonic acid [53].

#### **3.6 Visible spectrophotometric methods based on degradation products of β-lactam analogs**

The significant instability of β-lactam analogs was also exploited in the spectrophotometrical determination of β-lactam analogs in the visible region.

As the intermediate stages of determination, depending on the affecting factors, the following were present:


The formation of degradation products of β-lactam analogs in conditions of a basic hydrolysis was also the first stage during development of visible spectrophotometric methods. As the result of reactivity of degradation products formed in basic medium with some reagents, the determination of the following β-lactam analogs was possible:


N CH3

OH H3C

O

C6H5

<sup>O</sup> <sup>N</sup>

O O

N N CH3

O

S

CH3 CH3

COONa

R N

O

N H N O

O

C6H5

K3[Fe(CN)6] <sup>N</sup>

R N

Fig. 16. The reaction mechanism of cefadroxil with 4-aminoantipyrine in the presence of

O

Fig. 17. The reaction mechanism of ampicillin sodium and 1,2-naphthoquinone-4-sulfonic

**3.6 Visible spectrophotometric methods based on degradation products of β-lactam** 

The significant instability of β-lactam analogs was also exploited in the spectrophotometrical

As the intermediate stages of determination, depending on the affecting factors, the

The formation of degradation products of β-lactam analogs in conditions of a basic hydrolysis was also the first stage during development of visible spectrophotometric methods. As the result of reactivity of degradation products formed in basic medium with

 cephem analogs (cefadroxil, cefotaxime), when coupling factor were *N,N*-diethyl-*p*phenylenediamine sulfate and Fe(III) (λ = 670 nm) or *p*-phenylenediamine

 cephem analogs (cefotaxime, ceftriaxone, cefradine) when reducing factor was potassium iodate (required acidic medium) and a result of the reaction was colour change of leuco crystal violet under the influence of formed iodine (λ = 588 nm) [55] cephem analogs (cefotaxime sodium) when coupling factor was 1,10-phenanthroline

 penam analogs (amoxicillin, ampicillin) and cephem analogs (cephalexin, cephradine) when reducing factor of formed hydrolyzed products was J2 (required acidic medium)

some reagents, the determination of the following β-lactam analogs was possible:

OH

+

N H

O

NH2

N O

S

H3C

H2N

N N CH3

O

O

O

N H C6H5

N S

determination of β-lactam analogs in the visible region.

 degradation products typical of an acidic environment degradation products characteristic of a basic environment.

dihydrochloride and Fe(III) (λ = 597 nm) [54]

and ferric chloride (λ = 520 nm) [56]

(λ = 460 nm) [57].

CH3 CH3

+

COONa

NH2 SO3H

COOH CH3

R

alkaline [Fe(CN)6]3-

acid [53].

**analogs** 

following were present:

R =

The significant expansion of possibilities for the developed analytical method was the usage of flow injection analysis (FIA).

Similarly, in the case of determination of penam analysis during acidic hydrolysis (1.0 M HCl), formation of non-absorbing degradation products was the intermediate stage of their analysis. Complex which is necessary for achievement of spectrophotometric signals, was formed between penicillamine and palladium(II) chloride, peak at 334 nm (Fig. 18) [58].

Fig. 18. Absorption spectra based on reaction of degradation products of penam analogs and palladium (II) and suggested mechanism of the reaction [58].

In conditions of acidic hydrolysis, determination of cephem analogs took place. Vanadium(IV) after reduction from vanadium(V), reacted with forming degradation products. Colored complexes of some cephem analogs (cephalexin, cephaprine sodium, cefazolin sodium, cefotaxime) were found, absorbing at 515, 512, 518 and 523 nm, respectively (Fig. 19) [59]

Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of β-Lactam Antibiotics 127

[1] Cielecka-Piontek J. Michalska K. Zalewski P. Jelińska A. Recent Advances in stability studies of carbapenems. Current Pharmaceutical Analysis 2011;7 213-227. [2] El-Shaboury S. Saleh G. Mohamed F. Rageh A. Analysis of cephalosporin antibiotics.

[3] Cielecka-Piontek J. Michalska K. Zalewski P. Zasada S. Comparative review of analytical

[4] ICH. Stability testing of new drug substances and products. In: Proceedings of

[5] Parisotto G. Ferrao M. Furtado J. Molz R. Determination of amoxicillin content in

[6] Tabelbpour Z. Tavallaie R. Agmadi S. Abdollahpour A. Simultanesous determination of

[8] Ivama V. Rodrigues L. Guaratini C. Zanoni M. Spectrophotometric determination of cefaclor in pharmaceutical preparations. Quimica Nova 1999;22(2) 1–6. [9] Deshpande A. Baheti K. Chatterjee N. Degradation of β-lactam antibiotics. Current

[10] Rojanarata T. Opanasopit P. Ngawhirunpat T. Saehuan Ch. Wiyakrutta S. Meevootisom

[12] Cantarelli M. Pellerano R. Marchevsky E. Camina J. Simultaneous Determination of

Data and the PLS Chemometric Method. Analytical Sciences 2011;27 73–78. [13] Abdel-Hamid M. FSQ spectrophotometric and HPLC analysis of some cephalosporins

[14] Mahgoub H. Aly F. Uv-spetrophotometric determination of ampicillin sodium and

[15] Murillo J. Lemus J. Garcia L. Simultaneous determination of the binary mixtures of

[16] Rodenas V. Parra A. Garcia-Villanova J. Gomez M. Simultaneous determination of

Journal of Pharmaceutical and Biomedical Analysis 1995;13 1095–1099. [17] Daabees H. Mahrous M. Abdel-Khalek M. Beltagy Y. Emil K. Spectrophotometric

of Pharmaceutical and Biomedical Analysis 1995;13(6) 769–776.

V. A simple sensitive and green bienzymatic UV-spectrophotometric assay for amoxicillin formulations. Enzyme and Microbial Technology 2010;46 292–296. [11] Campins-Falco P. Sevillano-Cabeza A. Gallo-Martinez L. Bosch-Reig F. Monzo-

Mansanet I. Comparative Study on the Determination of Cephalexin in its Dosage Forms by Spectrophotometry and HPLC with UV-vis Detection. Microchimica Acta

Amoxicillin and Diclofenac in Pharmaceutical Formulations Using UV Spectral

in the presence of their alkali-induced degradation products. Il Farmaco 1998;53

sulbactam sodium in two-component mixtures. Journal of Pharmaceutical and

cefsulodin and clavulanic acid by using first-derivative spectrophotometry. Journal

cefepime and L-arginine in injections by second-derivative spectrophotometry.

determination of cefprozil in pharmaceutical dosage forms in urine and in the

techniques for determination of carbapenems. Current Analytical Chemistry 2012; 8

powdered pharmaceutical formulations using DRIFTS and PLS. Brazilian Journal

penicillin G salts by infrared spectroscopy: Evaluation of combining orthogonal signal correction with radial basis function-partial least squares regression.

Journal of Pharmaceutical and Biomedical Analysis.2007;45 1–19.

International conference on Harmonization. Geneva: IFPMA; 2000.

of Pharmaceutical Sciences 2007;43(1) 89-95.

Spectrochimica Acta Part A 2010;76 452–457.

[7] European Pharmacopoeia 7th ed. 2010

1997;126 207–215.

132–138.

Science 2004;78(12) 1684-1695.

Biomedical Analysis 1998;17 1273–1278.

**6. References** 

91-115.
