**3.1 Visible spectrophotometric methods based on redox reactions**

The methods based on the selective oxidation were reported for penam and cephem analogs containing phenolic substitutes at C6 and C7, respectively. These methods permitted also selective determination of β-lactam analogs in the presence of excipients being in their pharmaceutical preparations. The application of oxidation properties of iron ions was used in analysis of a huge number of β-lactam analogs:

 when, as the result of direct reaction with Fe(III) in acidic medium, yellow coloured products (λmax = 397 nm) were produced (cefoperazone sodium, cefadroxil monohydrate, cefprozil anhydrous, amoxicillin trihydrate). A possible mechanism of reaction is presented in Fig. 8 [32].

Fig. 8. The mechanism of reaction of amoxicillin and Fe(III)[32].

 when, as the result of indirect reaction, red complex Fe-(o-phen)2/3 (λmax = 510 nm) was produced. This complex is formed between *o*-phenanthroline and Fe(II) which previously was reduced from Fe(III) as the result of oxidation of β-lactam analogs in alkali medium [33].

The reduction of oxidized quercetin by cephem analogs was used in development of visible spectrophotmetric method for determination of β-lactam analogs. Quercetin is a flavonol (3,5,7,3',4'- pentahydroxyflavone) which is oxidized by *N*-bromosuccinimide giving reddish green colour (λmax = 510 nm). As the result of reduction of oxidized form of quercetin by cephem analog fade colour was observed (Fig. 9). This colour is the result of formation of *o*quinone derivative of quercetin under the mild oxidants [34].

Above-mentioned methods were described only for the determination of penam and cephem analogs. The species, absorbing visible radiation, used in analysis of β-lactam

The methods based on the selective oxidation were reported for penam and cephem analogs containing phenolic substitutes at C6 and C7, respectively. These methods permitted also selective determination of β-lactam analogs in the presence of excipients being in their pharmaceutical preparations. The application of oxidation properties of iron ions was used

 when, as the result of direct reaction with Fe(III) in acidic medium, yellow coloured products (λmax = 397 nm) were produced (cefoperazone sodium, cefadroxil monohydrate, cefprozil anhydrous, amoxicillin trihydrate). A possible mechanism of

> N O

CH3 CH3

+ 2 Ce4+ or 2 Fe3+

+ 2 Ce3+ or 2 Fe2+ - 2 H+

CH3 CH3

COOH

H+

COOH

H S

N O

> H S

 when, as the result of indirect reaction, red complex Fe-(o-phen)2/3 (λmax = 510 nm) was produced. This complex is formed between *o*-phenanthroline and Fe(II) which previously was reduced from Fe(III) as the result of oxidation of β-lactam analogs in

The reduction of oxidized quercetin by cephem analogs was used in development of visible spectrophotmetric method for determination of β-lactam analogs. Quercetin is a flavonol (3,5,7,3',4'- pentahydroxyflavone) which is oxidized by *N*-bromosuccinimide giving reddish green colour (λmax = 510 nm). As the result of reduction of oxidized form of quercetin by cephem analog fade colour was observed (Fig. 9). This colour is the result of formation of *o*-

N H

> N H

O

NH2

O

NH2

degradation product of some β-lactam analog.

redox

complexation of metals

formation of ion pairs

coupling with specified reagents.

analogs were formed as a result of the following reactions:

complexation on the base of charge-tranfer process

in analysis of a huge number of β-lactam analogs:

HO

O

Fig. 8. The mechanism of reaction of amoxicillin and Fe(III)[32].

quinone derivative of quercetin under the mild oxidants [34].

reaction is presented in Fig. 8 [32].

alkali medium [33].

**3.1 Visible spectrophotometric methods based on redox reactions** 

Fig. 9. The mechanism of reduction of quercetin oxidation with *N*-bromosuccinimide [34].

Also, another reagent which is able to oxidize some sulphur atoms present in compounds such as cefotaxime and cefuroxime, is 1-chlorobenzotiazol. As the result of reaction of cephem analogs and 1-chlorobenzotiazol, a product with yellow colour is formed, absorbing radiation at λmax = 298 nm. The suggested possible reaction pathways and absorption spectra are shown in Figure 10 [35].

Fig. 10. The mechanism of reaction of cephem analog and 1-chlorobenzotiazol [35].

In indirect spectrophotometry, redox properties of iodine were used in determination of penam analog (amplicillin, penicillin V, amoxicillin, cloxacillin) and cephem analogs (cefadroxil, ceftezoxime). The method based on formation of hypoiodite, from excess of iodine (which did not react with β-lactam analog) under alkaline conditions. Hypoiodite reduced the intensity of wool fast blue colour (5,9-dianilo-7-phenyl 4,10 disulphbenzo[α]phenazinium hydroxide) by disruption of phenazine chromophore [36].

Especially, for cefadoxil many visible spectrophotometric methods based on redox reactions were proposed (Fig. 11). These methods were based on the reaction with different oxidizing reagents:

Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of β-Lactam Antibiotics 121

cefperazone sodium, cefadroxil monohydrate, cefprozil anhydrous) reacted with nitrous acid forming the nitroso derivatives. They were capable of tautometric interconversions to form colored complex in the presence of copper(II) ions (Fig. 12). The stoichiometric ratios

(nitroso derivative to copper(II)) were determined by the Job's method 2:1 [43].

COOH

S

CH3 CH3

Fig. 12. Scheme of the reaction of nitrous acid, Cu(II) with phenolic β-lactam analogs [43].

The determination of cephalexin, cefixime, ceftriaxone, cefotaxime based on the Bent-French method, in which degradation products of β-lactam analogs with metal ions form the colored complexes, was developed. Hydroxamic acids formed by hydroxiaminolysis of

NH

N H R

O Fe O

O O O O

R

R

**3.3 Visible spectrophotometric methods based on formation of charge-transfer** 

antibiotics were measured depended on what of reagent was used the acceptor:

Some drugs, including penam and cephem analogs, are electron donors. Therefore, they form charge-transfer complexes with compounds that are σ- and π-accepptors of electrons. The wavelengths at which the absorption maxima of charge-transfer complexes of β-lactam

 *p*-chloranilic acid gives coloured complex species at 520–529 nm during analysis of cefotaxime sodium, cefuroxime sodium, ceftazidime pentahydrate, cephalexin monohydrate, cefotaxime sodium, cephradine, cephaloridine sodium, cefoperazone sodium (ratio 1:1); cephalotin sodium, cefixime, cefprozil anhydrous, cefazolin sodium,

 *p*-nitrophenol, 2,4-dinitrophenol, 3,5-dinitrosalycilic acid, picramic acid and picric acid give greenish yellow complexes at 446, 435, 442, 473 and 439 nm, respectively during

 7,7,8,8-tetracyanoquinodimethane (TCNQ) gives coloured complex species at 838–843 nm during analysis of cefotaxime sodium, cefuroxime sodium, cephapirin sodium, cefazoline sodium, cephalexin monohydrate, cefadroxil monohydrate, cefoperazone

 2,3-dichloro-5,6-dicyano-*p*-benzo-quinone (DDQ) gives coloured complex species at 460 nm during analysis of cephapirin sodium, cefazoline sodium, cephalexin monohydrate,

cefadroxil monohydrate, cefoperazone and ceftazidime (ratio 1:1) [47]

HN

NaNO2/HCl Cu2+

R

N Cu O

O

O N

O

R

N O

cephem analogs (1:3), formed complexes with iron (II) ions (Fig. 13) [44].

N H

O

NH2

Fig. 13. The scheme of hydroxamic acid-iron(III) [44].

cephapirin (ratio 1:2); cefaclor (ratio 1:4) [45]

determination of flucloxacillin (ratio 1:1) [46]

and ceftazidime (ratio 1:1)[47]

**complex** 

HO


Fig. 11. Schemes of some oxidation reactions of cefadoxil [39].
