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

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


Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of β-Lactam Antibiotics 123

 benzocaine in thrietylamine medium for determination of cefadroxil and amoxicillin. Stoichiometric ratio of formed species was 1:1 with peaking at λ=455 nm and λ=442 nm,

 electron-deficient polinitro derivatives for determination of amoxicillin, cefoperazone, cefadroxil, cefprozil. Complexes of the Meisenheimer type were formed [51].

Suggested mechanisms of coupling reaction of phenol derivative of β-lactam analogs were

N S CH3 CH3

O N H O

> NO2 N2

+ +

OH-

N N N N

H2N

O-

NH2

NO2

HO

COOH

O2N

N <sup>S</sup> CH3 CH3

O HN O

COOH

**3.5 Visible spectrophotometric methods based on coupling with specified reagents**  The analysis of phenolic derivatives of penam and cephem analogs were possible by measurement of absorption species formed as a result of reations with specified reagents.

Diazo coupling of β-lactam analogs was conducted with the following compounds:

respectively [50] .

N S

N S

COOC2H5

COOH

CH3

Fig. 15. Schemes of reactions of coupling of β-lactam analogs [50-51].

Reaction of diazo coupling of cefadroxil Reaction of diazo coupling of amoxicillin

For determination of phenolic derivative of β-lactam analog (cefadroxil) measurement of absorption of formed product in the reaction between it and 4-aminoantipyrine in the presence of alkaline potassium hexacyanoferrate(III) at 510 nm was also proposed (Fig. 16). Potassium hexacyanoferrate(III), being oxidant in this reaction, yielding *N*-substituted quinine imines and in the result was responsible for formation of red-colored antipyrine dye. Additionally, a sequential injection analysis (SIA) spectrophotometric procedure for the

1,2-naphthoquinone-4-sulfonic acid is the reagent permitting the nucleophilic substitution reaction in area of amino group of penem (amoxicillin) and cephem (cephalexin) analogs (Fig. 17). The stoichiometric ratio of these species was 1:1 and they absorb at λ = 463 nm [53]. The extension of the methodology for determination of cephalexin by using the H-point standard additions method (HPSAM) and the generalized H-point standard additions methods (GHPSAM) (after solid phase extraction cartridges) permitted also its analysis in

O

HO CH3

O

NH2

+

N H

+

N N COOC2H5

O

O

NH2

N N O H

determination was reported [52].

urine [53].

N H COOH

presented in Fig. 15.

 iodine gives coloured complex species at 838–843 nm during analysis of cephapirin sodium, cefazoline sodium, cephalexin monohydrate, cefadroxil monohydrate, cefoperazone and ceftazidime (ratio 1:1) [47].

During formation of charge-transfer complexes between *p*-chloranilic acid and d-donors electrons (A) from group of cephem analogs (D) in polar solvents the radical anion is formed.

$$\mathbf{D} + \mathbf{A} \xrightleftharpoons \underbrace{\mathbf{D} \cdots \mathbf{A}}\_{\text{complex}} \\ \left(\mathbf{D} \cdots \mathbf{A}\right) \xrightleftharpoons \mathbf{D} + \mathbf{A} \xrightleftharpoons \mathbf{D} + \mathbf{A} \x$$

Electron transfer from donor to the acceptor moiety occurred with the formation of intensely coloured radical ions with high molar absorptivity value. The formation of charge-transfer complex of some cephem analogs (D) with iodine in 1,2-dichloroethane (J) is observed with the change of colour from violet to lemon yellow:

$$\mathbf{D} + \mathbf{J}\_2 \xrightleftharpoons \mathbf{D} \cdots \mathbf{J}\_\Phi \xrightleftharpoons \mathbf{D} \cdots \mathbf{J}\_\Phi \xrightleftharpoons \mathbf{D} \cdots \mathbf{J}\_\Phi \xrightleftharpoons \mathbf{J}\_\Theta \xrightleftharpoons \mathbf{J}\_\Theta \xrightleftharpoons \mathbf{J}\_\Theta$$

Only in 1,2-dichloroethane, formation of tri-iodide ion pair (inner complex), showing two absorption maxima at 290 nm and 364 nm, was possible. This complex originated from an early intermediate outer complex D---J2. While the interactions of some cephem analogs with DDQ and TCNQ took place according to the following simple relationship:

$$\mathbf{D} + \mathbf{A} \xleftarrow[\mathbf{D} \dots \mathbf{C}]{} \mathbf{D} \overleftarrow{\mathbf{D}} \cdots \mathbf{A} \overleftarrow{\mathbf{D}} \overleftarrow{\mathbf{A}} \mathbf{D} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{D}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}} \overleftarrow{\mathbf{A}}$$

#### **3.4 Visible spectrophotometric methods based on formation of ion pair**

In visible spetrophotometric analysis of penam and cephem analogs, their ability to form ion-pair was also used. The penam analogs contacting the tertiary amine group (ampicillin, dicloxacillin, flucloxacillin, amoxicillin) and Mo(V)-thiocyanate binary complex in hydrochloric acid give coloured ion-pair formation absorbing at λ = 467 nm (Fig. 14) [48].

Fig. 14. Scheme of Mo(V)-thiocyanate-β-lactam ion-pair [48].

Some cephem analogs (cephaprin sodium, cefuroxime sodium, cefotaxime sodium, cefoperazone sodium, cefadroxil, ceftazidime, cefazolin sodium and cefaclor) can be determined spectrophotometrically based on formation of ion-pair complex with ammonium reinecekate. In acidic medium at 25 ± 2oC, as the reaction products, the complex, absorbing at 525 nm, was formed according to the scheme [49]:

cephem analog hydrochloride + ammonium reineckate → cephem reineckate + ammonium chloride

 iodine gives coloured complex species at 838–843 nm during analysis of cephapirin sodium, cefazoline sodium, cephalexin monohydrate, cefadroxil monohydrate,

During formation of charge-transfer complexes between *p*-chloranilic acid and d-donors electrons (A) from group of cephem analogs (D) in polar solvents the radical anion is formed.

polar sovent

outer complex inner complex tri-iodide ion

J2 J3

D + A (D---A) D + A

Electron transfer from donor to the acceptor moiety occurred with the formation of intensely coloured radical ions with high molar absorptivity value. The formation of charge-transfer complex of some cephem analogs (D) with iodine in 1,2-dichloroethane (J) is observed with

Only in 1,2-dichloroethane, formation of tri-iodide ion pair (inner complex), showing two absorption maxima at 290 nm and 364 nm, was possible. This complex originated from an early intermediate outer complex D---J2. While the interactions of some cephem analogs

> D + A (D---A) complex

In visible spetrophotometric analysis of penam and cephem analogs, their ability to form ion-pair was also used. The penam analogs contacting the tertiary amine group (ampicillin, dicloxacillin, flucloxacillin, amoxicillin) and Mo(V)-thiocyanate binary complex in hydrochloric acid give coloured ion-pair formation absorbing at λ = 467 nm (Fig. 14) [48].

Mo(VI) Mo(V) Mo(SCN)-

COOH

CH3 CH3

Some cephem analogs (cephaprin sodium, cefuroxime sodium, cefotaxime sodium, cefoperazone sodium, cefadroxil, ceftazidime, cefazolin sodium and cefaclor) can be determined spectrophotometrically based on formation of ion-pair complex with ammonium reinecekate. In acidic medium at 25 ± 2oC, as the reaction products, the complex,

cephem analog hydrochloride + ammonium reineckate → cephem reineckate + ammonium chloride

6SCN-

+

(Mo(SCN)6) - 6

complex

D + J2 D---J J D---J + J

with DDQ and TCNQ took place according to the following simple relationship:

**3.4 Visible spectrophotometric methods based on formation of ion pair** 

ascrobic acid

O

R

Fig. 14. Scheme of Mo(V)-thiocyanate-β-lactam ion-pair [48].

absorbing at 525 nm, was formed according to the scheme [49]:

N S

cefoperazone and ceftazidime (ratio 1:1) [47].

the change of colour from violet to lemon yellow:
