**1. Introduction**

108 Macro to Nano Spectroscopy

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Following the discovery of the first analog of penicillin by A. Fleming (1929), the β-lactam antibiotics are still a developing group of chemotherapeutics and are used in treatment of majority of diseases with bacterial etiology. β-lactam antibiotics have a broad spectrum of antibacterial activity, favourable pharmacokinetic parameters and low side effects. In βlactam therapy two main problems are still current. The increasing resistance of some bacterial strains which implicates necessity to combine the therapy with inhibitors of βlactamases and other chemotherapeutics. The second problem of therapy of β-lactam antibiotics is their significant instability [1-3]. The analogs from that group are easily degraded in aqueous solutions and in solid state. They are a special group of drugs because parallel to losing the antibacterial efficiency, the strong allergic properties can also appear as a results of their degradation. Therefore in terms of quality control, the stability of β-lactam antibiotics in solutions was widely studied. The evaluation of stability concerned also the studies of their metabolites and intravenous solutions after preparations of pharmaceutical dosage forms. Moreover, the evaluation of concentration changes during storage of substance in solid state was also conducted. As problem of the instability of some β-lactam analogs has been solved their oral administration is possible. An intake of oral formulations is connected with appearance of excipients, which can influence rate of degradation and cause formation of different degradation products.

The common element of chemical structure of all β-lactam antibiotics is five-membered βlactam ring. Currently, higher significance in treatment have derivatives in which the βlactam ring is fused to:


These connections implicate the different intra-ring stress. The presence of sulphur atom and/or double bonds influence on length of bond and intra-molecular angle in molecule of β-lactam analog. Finally for some derivatives, the differences in stability are noticeable. Additionally, the factor distinguishing a stability of derivatives of β-lactam analogs are

Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of β-Lactam Antibiotics 111

Derivative Ultraviolet region Visible region

Analog of penam

I generation

II generation

III generation

1.\* measurement of absorption of species being a result of reaction between analyte and

2.\* measurement of absorption of species being a result of reaction between degradation

Table 1. Possibilities of application of visible and ultraviolet spectrophotometric determinations for analysis of β-lactam antibiotics in the period of time 1994–2011.

**2. Spectrophotometric methods for determination of β-lactam antibiotics** 

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

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

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

IV generation

Analog of penam

1. direct spectrophotometry enriched by chemometric procedures

3. derivative spectrotometry enriched by chemometric procedures

Analog of carbapenem

Analog of cephem

2. derivative spetrophotometry

**2.1 Direct spectrophotometry** 

substance of high purity.

determination of parental substance.

products of analyte and derivatizating reagent

derivatizating reagent

1 2 3 1\* 2\*

Fig. 1. Chemical structure of penam, cephem, carbapenem and thiopenem nuclei.

chemical structures of substituents at C2, C3, C5, C6, and C7. The amount and type of degradation products of β-lactam antibiotics often depend on affecting factors (solvents, concentration of substance and hydrogen ions, temperature). Moreover, most of the βlactam antibiotics obtained by chemical synthesis or fermentation contain impurities being remnants of the process. In the development of analytical methods for the determination of β-lactam antibiotics, selectivity is a fundamental validation parameter. A reliable, selective method is expected to allow separation and determination of parental substance in the presence of related ones. Current International Conference on Harmonization (ICH) guidelines require the development of analytical methods permitting analysis in the presence of related products (Q1A–R2) [4]. These requirements are restrictions but also challenges during the development of analytical methods for the determination of β-lactam antibiotics. The problem of the overlapping of the "background" originating from related products (impurities, degradation products and metabolites) and/or the presence of other active substances in a sample (inhibitors of β-lactamases, other drugs) was solved during the determination of β-lactam antibiotics by using chromatographic techniques (highperformance liquid chromatography, thin layer chromatography). On the other hand, search of new solutions and analytical methods, especially being in accordance with the "*green chemistry*" concept, is very important and up-to-date. Analytical methods based on determination of spectrophotometric properties of β-lactam analogs are a developing tools in their analysis. Non-destructive investigations of β-lactam analogs, did not producing residues, were reported in fields of many spectrophotometric methods. A few methods of determination of β-lactam analogs were developed by using infrared spectrophotometry enriched by chemometric procedures [5-6]. Most of all analytical methods for the determination of β-lactam analogs were developed in range of visible and ultraviolet radiations. Desired, selective signals were possible to obtain by application of following techniques:

	- direct spectrophotometry
	- direct spectrophotometry enriched by chemometric procedures
	- derivative spetrophotometry
	- derivative spectrotometry enriched by chemometric procedures
	- measurement of absorption of species being a result of reaction between analyte and derivatizating reagent
	- measurement of absorption of species being a result of reaction between degradation products of analyte and derivatizating reagent (Table 1).


1. direct spectrophotometry enriched by chemometric procedures

2. derivative spetrophotometry

110 Macro to Nano Spectroscopy

N O

N

O

chemical structures of substituents at C2, C3, C5, C6, and C7. The amount and type of degradation products of β-lactam antibiotics often depend on affecting factors (solvents, concentration of substance and hydrogen ions, temperature). Moreover, most of the βlactam antibiotics obtained by chemical synthesis or fermentation contain impurities being remnants of the process. In the development of analytical methods for the determination of β-lactam antibiotics, selectivity is a fundamental validation parameter. A reliable, selective method is expected to allow separation and determination of parental substance in the presence of related ones. Current International Conference on Harmonization (ICH) guidelines require the development of analytical methods permitting analysis in the presence of related products (Q1A–R2) [4]. These requirements are restrictions but also challenges during the development of analytical methods for the determination of β-lactam antibiotics. The problem of the overlapping of the "background" originating from related products (impurities, degradation products and metabolites) and/or the presence of other active substances in a sample (inhibitors of β-lactamases, other drugs) was solved during the determination of β-lactam antibiotics by using chromatographic techniques (highperformance liquid chromatography, thin layer chromatography). On the other hand, search of new solutions and analytical methods, especially being in accordance with the "*green chemistry*" concept, is very important and up-to-date. Analytical methods based on determination of spectrophotometric properties of β-lactam analogs are a developing tools in their analysis. Non-destructive investigations of β-lactam analogs, did not producing residues, were reported in fields of many spectrophotometric methods. A few methods of determination of β-lactam analogs were developed by using infrared spectrophotometry enriched by chemometric procedures [5-6]. Most of all analytical methods for the determination of β-lactam analogs were developed in range of visible and ultraviolet radiations. Desired, selective signals

S

S

R COOR

R Cephem analog

COOH

R

R Thiopenem analog

N S COOR CH3 CH3

R

were possible to obtain by application of following techniques:

direct spectrophotometry enriched by chemometric procedures

derivative spectrotometry enriched by chemometric procedures

measurement of absorption of species being a result of reaction between analyte

measurement of absorption of species being a result of reaction between

degradation products of analyte and derivatizating reagent (Table 1).

 as spectrophotometric methods direct spectrophotometry

derivative spetrophotometry

as visible spectrophotometric methods

and derivatizating reagent

COOH

Penam analog

Fig. 1. Chemical structure of penam, cephem, carbapenem and thiopenem nuclei.

R Carbapenem analog

O

R

O

R

N

3. derivative spectrotometry enriched by chemometric procedures

1.\* measurement of absorption of species being a result of reaction between analyte and derivatizating reagent

2.\* measurement of absorption of species being a result of reaction between degradation products of analyte and derivatizating reagent

Table 1. Possibilities of application of visible and ultraviolet spectrophotometric determinations for analysis of β-lactam antibiotics in the period of time 1994–2011.
