**2.1 Direct spectrophotometry**

Spectra of β-lactam antibiotics recorded by using direct spectrophotometry do not have desired selectivity due to the presence of related products. A comparison of sharp zeroorder spectra and/or value of absorption maxima for some β-lactam analogs with ones obtained for CRS (*chemical reference substance*) is recommended by pharmacopeias for an their identification [7] . Lack of desired absorbing species in chemical structure of penam analog often do not allow to apply direct spectrophotometry even for qualitative studies of substance of high purity.

Paradoxically, the significant instability of analogs can sometimes solve this problem due to formation of degradation products that can absorb ultraviolet radiation permitting determination of parental substance.

Significant susceptibility of β-lactam analogs to degradation in basic medium was reported during analysis of cephem analogs. It was confirmed that formation of piperazine-2,5-dione

Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of β-Lactam Antibiotics 113

The other way of improving the selectivity of direct spectrophotometry for the determination of β-lactam antibiotics is the enrichment of data analysis by chemometric procedures. A literature review revealed the application of the following determinations of β-lactam antibiotics enriched by chemometric procedures that solved the problem of spectral overlap without additional separation techniques at the stage of sample

 a separation of analog of cephem in the presence of impurities originating from synthesis (e.g., cephalexin in the presence of 7-aminocephalosporanic acid and acid-induced

Each chemometric method relies on different tools of regression analysis of multicomponent

The determination of β-lactam analyte in the presence of known and unknown inferences was possible by the application of HPSAM procedure, where analyte concentration is

where b0 and A0 are the absorbance values for β-lactam analyte, b and A, ones for the

value estimated from standard deviation of blank samples, *bk* value is a vector of the regression coefficient for the *k* analytes and *k* is a number of components in a mixture.

The FSQ technique during a determination of β-lactam antibiotics applies Fourier preprocessing of the entire absorption spectra of the individual β-lactam analogs with their degradation products at variable concentration to calculate matrix calibration coefficients.

A derivative spectrophotometry using derivatives of absorbance with respect to wavelength

overcoming the overlapping spectra problem in analysis of many β-lactam analogs. Possibility of application of derivative spectrophotometry with zero-crossing point is widely used in analysis of all β-lactam analogs. The direct correlation between order of used derivative spectrophotometry and similarities of chemical structures of nuclei of β-lactam analogs has not been observed, e.g., both second-derivative and first-derivative were


(1)

‖bk‖

, *σ<sup>r</sup>* is a

‖σr‖ where SENk = <sup>1</sup>

 ��� = f(λ)���; respectively) is a suitable tool for

degradation products) using H-point standard additions method (HPSAM) [11] determination of analog of penam in the presence of other drugs (e.g., amoxicillin in the presence of diclofenac) using partial least squares (PLS) regression analysis [12] determination of analog of cephem in the presence of alkali-induced degradation products using full spectrum quantitation (FSQ) (e.g., cefotaxime, ceftazidine,

**2.2 Direct spectrophotometry enriched by chemometric procedures** 

ceftiaxome, in the presence of degradation products) [13].

system permitting simultaneous determination of two or more components.

�A0-b0�+ (A,

In PLS technique, analytical sensibility was defined as γ = SENk

��� = f(λ)��; third ���

developed for cephem analogs including the same nuclei (Fig. 4).


interferent, at λ1 and λ2 and M(λ1), M(λ2) are slopes of plots at selected wavelengths.

preparation, were used:

calculated from the following equation:

**2.3 Derivative spectrophotometry** 

�� = f(λ)', second ���

(first ��

derivative, peak at 340 nm, was possible via intra-molecular nucleophilic attack of the primary amine from the side chain on β-lactam ring (pH = 11 was required) (Fig. 2) [8].

Fig. 2. Chemical structures of degradation products of cefaclor (1.0 mmol/l) formed at pH 11.0 and its spectrum [8].

The degradation of penam analogs in acidic conditions was also a base for spectrophotometric determination. As it is shown in Fig. 3, different pathways of degradation (including enzymatic one) can lead to obtaining absorbing species in the range of ultraviolet radiation. As a results of chemical degradation of penam analog in acidic conditions, the penicilloic acid, penillic acid and penicillenic acid are formed and absorb the ultraviolet radiation in the range 320–360 nm, respectively [9]. While during the enzymatic degradation under the influence of penicillin acylase, D-4-hydroxyphenylglycine (D-HPhG) and 6-aminopenicillanic acid are formed. Then the D-HPhG was catalyzed by Dphenylglycine aminotransferase to form L-glutamate and hydroxybenzoylformate which strongly absorb UV light at 335 nm [10].

Fig. 3. The pathways of obtaining of absorbing degradation products of penam analog [9-10].
