**3. Visible spectrophotometric methods for determination of β-lactam antibiotics**

The β-lactam analogs itselfs do not absorb in visible region of radiation. However, many visible spectrophotometric methods were developed for the determination of β-lactam antibiotics using the effect of formation of "species" giving signals in visible region as the result of chemical derivatization (Fig. 7).

Fig. 7. The application of derivatization for detetmination of penam analog.

Formation of "species" absorbing visible radiation can be a result of reactions of chemical reagents with:

β-lactam analog

116 Macro to Nano Spectroscopy

The application of chemometric procedures coupled with derivative spectroscopy permits achievement of higher selectivity in determination of β-lactam antibiotics. Currently, chemometric procedures based on the estimated ratio of spectra derivative for the selective determination of β-lactam analogs are the most common. It was proved that the application of the ratio of different-order spectra derivatives permitted the separation of binary and tertiary mixtures of β-lactam antibiotics [22]. During the determination of concentrations of three components (e.g., penicillin-G sodium, penicillin-G procain and dihydrostreptomycin sulphate salts) in a mixture the equation describing the ratio spectra derivative

�(����/�����)

where �������� is the absorbance of the ternary mixture of *a, b* and *c* at wavelength λ, ����� is the absorbance of pure component at wavelength λ, *Cb* and *Cc* – are the concentrations of *b*  and *c*, *kb,λ* and *kc,λ* are the products of the molar absorption coefficient of *b* at wavelength λ and the thickness of the absorption cell. Equation 2 is divided by Cb while divisor can be any

> = �� ��

J = Cc d(d( �(����/�����)

�(�����/�����)

Finally, after the next derivation J (as the left side of equation 3), is proportional to the Cc value and can be used to determine concentration of component in the ternary mixture

Depending on chemometric procedure, the selective determination of following analogs was

 a separation and determination of carbapenem and degradant (e.g., the determination of ertapenem and its degradant) when the substraction technique was used [24-25] a separation and determination of penam and cephem analogs (e.g., the determination of penicillin-G, penicillin-G procain in the presence of dihydrostreptomycin sulphate salts or the determination of cefotaxime and cefadroxil), cephem analog and inhibitor of β-lactamases (e.g., the determination of cephradine and clavulanic acid) and carbapenem analog and degradation products (e.g., the determination of meropenem and its degradant) when ratio spectra of derivative with all orders were used [26-29] a separation and determination of carbapenem and degradation products (e.g., the

determination of ertapenem) when the substraction technique was used [30]

determination of ertapenem) when the Kraise's method was used [31]

a separation and determination of carbapenem and degradation products (e.g., the

 a separation and determination of penam analogs (e.g., the determination of ampicillin and flucloxacillin) when multivariate methods (classical least squares and principle

�� <sup>+</sup> Cc

� + (Cc<sup>d</sup> �(����/�����)

�(�����/�����)

�(����/�����)

�� (2)

 ) (3)

 ))/dλ (4)

**2.4 Derivative spectrophotometry enriched by chemometric procedures** 

�(������/�����)

�(������/�����) �(�����/�����)

�� = Cb

spetrophotometry is as follows:

component of ternary mixture (Fig. 6):

(when ����� and ����� are fixed) [23].

component regression) were used.

Equation 3 is drawn:

possible:

Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of β-Lactam Antibiotics 119

O O N Br

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

OH HO <sup>O</sup>

OH O

O O

Oxidized quercetin reddish violet colour 510 nm

HO O

O

OH

O OH

O O

> N S

> > N S

> > > O O

N N H N

 + HCl

The schemes of reaction between cefotaxime sodium

+

COONa

COONa

O CH3 O

O CH3 O

N N N Cl

O

S N H2N

S N H2N

and 1-chlorobenzotiazol

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

N H

H2O

O

N H

NOCH3

O

NOCH3

O

OH

HO O

spectra are shown in Figure 10 [35].

Absorption spectra of 65 µg/ml cefotaxime

1-chlorobenzotiazol (0.002 M) (b) and their

sodium (c),

reagents:

reaction product (a).

O O

OH

degradation product of some β-lactam analog.

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 analogs were formed as a result of the following reactions:

