**2.2 Direct spectrophotometry enriched by chemometric procedures**

112 Macro to Nano Spectroscopy

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].

cefaclor.

Fig. 2. Chemical structures of degradation products of cefaclor (1.0 mmol/l) formed at pH

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

The spectrum of piperazine-2,5-dione derivative of

O H N

O OH H NH2

D-4-hydroxyphenylglycine

O OH

Enzymatic degradation of amoxicillin using

O

HO

HO

HO

D-4-hydroxyphenylglycine

HO

bienzymatic factors

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

H H H NH2

Amoxicillin

N <sup>S</sup> CH3 CH3

COOH <sup>O</sup>

H H H2N

N <sup>S</sup> CH3 CH3 COOH

O OH O

O OH OH O

H NH2

2-oxoglutaric acid

O

6-aminopenicillinic acid

O

D-PhgAT

Penicilin acylase

+

O OH OH H NH2

+

4-hydroxybenzoylformic acid L-glutamic acid

+

'

2,5-dione derivative.

R O H N

R O H N

R O H N HN

conditions

O

N <sup>S</sup> CH3 CH3

Nu-

N <sup>S</sup> CH3 CH3 - O

COOH Nu

H H

Penicilloic acid

H H

COOH

<sup>S</sup> CH3 CH3

COOH OH

H H

O

11.0 and its spectrum [8].

HN

NH

O

O

products The degradation products of cefactor - piperazine-

strongly absorb UV light at 335 nm [10].


N <sup>S</sup> CH3 CH3

HN

O

H2O

Penicillenic acid

H H

COOH

<sup>S</sup> CH3 CH3

COOH

<sup>3</sup> O COOH OH Penamaldic acid

H H

O-

N O R

N O R

> R O H N HN HS CH 3 CH

H+

N <sup>S</sup> CH3 CH3 COOH

Penillic acid

Chemical degradation of penicillin in acidic

N R

HOOC

HN

S

COO-

Cl

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 preparation, were used:


Each chemometric method relies on different tools of regression analysis of multicomponent system permitting simultaneous determination of two or more components.

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 calculated from the following equation:

$$\frac{\text{(A}\_0\text{-}\text{b}\_0\text{)} + \text{(A'-b)}}{\text{M}(\text{A}\_1) \cdot \text{M}(\text{A}\_2)} = \text{-C}\_X + \frac{\text{(A'-b)}}{\text{M}(\text{A}\_1) \cdot \text{M}(\text{A}\_2)} \tag{1}$$

where b0 and A0 are the absorbance values for β-lactam analyte, b and A, ones for the interferent, at λ1 and λ2 and M(λ1), M(λ2) are slopes of plots at selected wavelengths.

In PLS technique, analytical sensibility was defined as γ = SENk ‖σr‖ where SENk = <sup>1</sup> ‖bk‖ , *σ<sup>r</sup>* is a 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.

#### **2.3 Derivative spectrophotometry**

A derivative spectrophotometry using derivatives of absorbance with respect to wavelength (first �� �� = f(λ)', second ��� ��� = f(λ)��; third ��� ��� = f(λ)���; respectively) is a suitable tool for 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 developed for cephem analogs including the same nuclei (Fig. 4).

Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of β-Lactam Antibiotics 115

M)

NH2 O

First-derivative of spectrum of cephalothin CL (20.0 µg/ml) and cefoxitin CX (20.0 µg/ml) and mixtures of each component

First derivative spectrum of

(0.03 µg/ml).

triethylammonium salt of cefotaxime A (40 µg/ml) , 7-aminocephalosporanic acid B (40 µg/ml), S-(2-benzothazolyl)2-amino-α- (methoxyimino-4-thiazoleethanethioate) C

First-derivative spectra of biapenem during

degradation at 313 K: in HCl

Chemical structures of components of mixture Derivative spectra

N O

H3CO H

H N S O

The separation of β-lactam analog and impurties from synthesis

mercaptobenzothiazole

S

The separation of β-lactam analog and degradation products

+ N

HOOC OH <sup>H</sup>

O

H

products of biapenem

O O

<sup>N</sup> <sup>S</sup> <sup>N</sup> <sup>N</sup>

Fig. 5. Separation of some β-lactam analogs using derivative spectra [18, 20-21].

SH

N

2-

S

COONa

The separation of β-lactam analog with the same nuclei

Cephalothin Cefoxitin

N O

H

Triethylammonium salt of cefotaxime

> N O

H N N O CH3

O N S H2N

> N H3C

H

OH S H CH3 OH O O

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

S COONa

CH3 O

Biapenem Open-ring hydrolysis

H N O

S

S

COOH

O O CH3

The application of derivative spectrophotometry for determination of β-lactam antibiotics was used in the following areas:


The separation of often structurally very similar species (e.g., two analogs of cephem, cephem analog and its impurities from synthesis or carbapenem analog and its degradation products) was possible by using derivative spectrophotometry (Fig. 5).

It was proved that the derivative spectrophotometry can be recommended as a method for routine control analysis of pharmaceutical preparation of β-lactam antibiotics. Derivative spectrophotometry ensured the rapid analysis of parenteral dosage forms and also removed a "*background*" excipients in oral pharmaceutical dosage forms.

The special potency of derivative spectrophotometry was possibility of its usage in determination of β-lactam analogs in biological matrix. In this case, to meet the requirements of analytical methods, the selectivity had to be extended in regard with interference of biological endogenous components. It was noticed that the selective determination of penam/cephem/carbapenem in the presence of metabolites (open-ring degradation product) and endogenous substance of urine was possible to achieve.

Second-derivative spectrum of cefepime (20

µg/ml) in water.

Fig. 4. The application of derivative spectrophotometry for analysis of cephem analogs [15-16].

The application of derivative spectrophotometry for determination of β-lactam antibiotics

 a separation and determination of penam/cephem analogs and inhibitors of βlactamase in aqueous solution (e.g., determination of ampicillin sodium in the presence of sulbactam sodium; determination of cefsulodin in the presence of clavulanic acid)

 a separation and determination of cephem/carbapenem analog and excipients used in parenteral pharmaceutical dosage forms (e.g., determination of cefepime in the

a separation and determination of cephem analog and its degradation products (e.g.,

 a separation and determination of cephem analog and related compounds from the synthesis (e.g., determination of triethylammonium salt of cefotaxime in the presence of

 a separation and determination of penam/cephem/carbapenem analogs in biological matrix (e.g., determination of amoxicillin, cefuroxime, imipenem in urine) [19].

The separation of often structurally very similar species (e.g., two analogs of cephem, cephem analog and its impurities from synthesis or carbapenem analog and its degradation

It was proved that the derivative spectrophotometry can be recommended as a method for routine control analysis of pharmaceutical preparation of β-lactam antibiotics. Derivative spectrophotometry ensured the rapid analysis of parenteral dosage forms and also removed

The special potency of derivative spectrophotometry was possibility of its usage in determination of β-lactam analogs in biological matrix. In this case, to meet the requirements of analytical methods, the selectivity had to be extended in regard with interference of biological endogenous components. It was noticed that the selective determination of penam/cephem/carbapenem in the presence of metabolites (open-ring

degradation product) and endogenous substance of urine was possible to achieve.

determination of cefprozil in the presence of its degradation products) [17]

products) was possible by using derivative spectrophotometry (Fig. 5).

a "*background*" excipients in oral pharmaceutical dosage forms.

First-derivative spectrum of cefprozil (1 mg%) in

was used in the following areas:

presence of *L*-argininie) [16]

2-mercaptobenzothiazole) [18]

0.1 M HCl.

[14-15]

Fig. 5. Separation of some β-lactam analogs using derivative spectra [18, 20-21].

Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of β-Lactam Antibiotics 117

First order derivative ratio spectra of dicloxacillin sodium 50.0–400.0 mg/l using of 68.47 µg/ml ampicillin sodium as a divisor [26]

First order derivative ratio spectra of meropenem 4.0–60.0 µg/l using

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

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

Fig. 6. The application of ratio spectra of derivative spectrophotometry in analysis of β-

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

Absorption spectra of amoxicillin trihydrate (a) 30 µg/ml; (b) after reaction with Fe (III) [31] Fig. 7. The application of derivatization for detetmination of penam analog.

of 32 µg/ml degradate as a divisor [29]

lactam antibiotics.

result of chemical derivatization (Fig. 7).

**antibiotics** 

reagents with:

β-lactam analog

#### **2.4 Derivative spectrophotometry enriched by chemometric procedures**

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 spetrophotometry is as follows:

$$\frac{\mathrm{d}(\mathrm{A}\_{\mathrm{a}+\mathrm{b},\lambda}/\mathrm{A}\_{\mathrm{a},\lambda})}{\mathrm{d}\lambda} = \mathrm{C}\_{\mathrm{b}} \frac{\mathrm{d}(\mathrm{k}\_{\mathrm{b},\lambda}/\mathrm{A}\_{\mathrm{a},\lambda}0)}{\mathrm{d}\lambda} + \mathrm{C}\_{\mathrm{c}} \frac{\mathrm{d}(\mathrm{k}\_{\mathrm{c},\lambda}/\mathrm{A}\_{\mathrm{a},\lambda}0)}{\mathrm{d}\lambda} \tag{2}$$

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 component of ternary mixture (Fig. 6):

$$\frac{\mathrm{d}(\mathrm{A}\_{\mathrm{a}\mapsto\mathrm{b},\lambda}/\mathrm{A}\_{\mathrm{a}\lambda^{\mathrm{o}}})}{\mathrm{d}(\mathrm{A}\_{\mathrm{b},\lambda^{\mathrm{o}}}/\mathrm{A}\_{\mathrm{a}\lambda^{\mathrm{o}}})} = \frac{\mathrm{c}\_{\mathrm{b}}}{\mathrm{c}\_{\mathrm{b}}^{\mathrm{o}}} + \left(\mathrm{C}\_{\mathrm{c}}\mathrm{d}\frac{\mathrm{d}(\mathrm{k}\_{\mathrm{c},\lambda}/\mathrm{A}\_{\mathrm{a},\lambda^{\mathrm{o}}})}{\mathrm{d}(\mathrm{A}\_{\mathrm{b},\lambda^{\mathrm{o}}}/\mathrm{A}\_{\mathrm{a},\lambda^{\mathrm{o}}})}\right) \tag{3}$$

Equation 3 is drawn:

$$\mathbf{J} = \mathbf{C}\_{\mathbf{c}} \, \mathrm{d} (\mathrm{d} (\mathrm{d} (\_{\mathrm{d} (\mathrm{A}\_{\mathrm{b} \lambda^{0}}/ \mathrm{A}\_{\mathrm{a} \lambda^{0}})}))) / \mathrm{d} \lambda \tag{4}$$

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

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


Fig. 6. The application of ratio spectra of derivative spectrophotometry in analysis of βlactam antibiotics.
