Pharmaceutical Chemistry Journal

Vol. 42, No. 3, 2008

STRUCTURE OF CHEMICAL COMPOUNDS, METHODS OF ANALYSIS AND PROCESS CONTROL IONOMETRIC DETERMINATION OF CEFOTAXIME IN BIOLOGICAL MEDIA O. I. Kulapina,1 V. V. Baraguzina,2 and N. V. Skoblikova2 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 3, pp. 48 – 50, March, 2008. Original article submitted November 7, 2006.

Ion-selective electrodes based on ion exchangers of tetradecylammonium (TDA) with cefotaxime (claforan) anions have been developed. The proposed electrodes are sensitive to cephalosporins in the concentration range from 1 ´ 10 – 5 to 1 ´ 10 – 1 M. The time for establishing a steady-state potential is 1 – 2 min. The potential drift does not exceed ± 2 mV/d. The detection threshold for cefotaxime is 3.6 ´ 10 – 5 M in the optimum pH range of 4.3 – 6.5. Comparison of the main electrochemical characteristics of the ion-selective electrodes based on TDA associates with cephalosporins showed that the best parameters are found in electrodes with membranes containing claforan.

Cephalosporins, like penicillins, are â-lactam antibiotics. However, their chemical structure is based on 7-aminocephalosporinic acid. The principal features of cephalosporins compared with penicillins are their high resistence to penicillinases, enzymes that are produced by microorganisms and rather rapidly destroy penicillins, and their broad spectrum of action, including an effect on gram-negative microorganisms [1]. Cephalosporins are currently divided into four groups with respect to their structure, spectrum of action, and resistance to b-lactamases [2]. All cephalosporins exhibit high chemotherapeutic activity. Cefotaxime (claforan) is a third-generation cephalosporin, possesses a broad spectrum of action, and is highly active toward gram-negative bacteria [2]. Cephalosporins differ in pharmacokinetic parameters, degree of assimilation with different pathways of administration, rate of onset of effects and duration of action, and, therefore, required frequency of use, metabolism, and elimination. Therefore, these parameters must be considered when using a particular preparation. Cefotaxime is presently determined using spectroscopic [3], chromatographic [4], and electrochemical [5, 6] meth1 2

ods. We developed ion-selective electrodes (ISE) based on organic ion-exchangers and tetradecylammonium with cefotaxime counterions for rapid determination of cefotaxime in biological media. EXPERIMENTAL PART We used the sodium salt of pharmaceutical-grade cefotaxime (Ctox). O

S

NH

C

N H2N

C N

S

OCH3

N

CH2 O

O C

O

C

O CH3

ONa

Starting aqueous solutions (1 ´ 10 – 1 M) were prepared by dissolving accurately weighed preparation in distilled water; working solutions (1 ´ 10 – 2 – 1 ´ 10 – 6 M), by successive dilution. We used liquid-contact electrodes with plastic membranes based on ion pairs of cefotaxime (claforan) and tetradecylammonium that were produced as before [7]. The ratio polyvinylchloride (PVC):dibutylphthalate (DBP) was 1:3, CAEC = 0.01 mol/kg DBP. The bodies of the film electrodes with liquid filling were PVC tubes with carefully polished ends to which membrane

Saratov State Medical University, Saratov, Russia. Saratov State University, Saratov, Russia

150 0091-150X/08/4203-0150 © 2008 Springer Science+Business Media, Inc.

Ionometric Determination of Cefotaxime in Biological Media

151 À 1.3

À 0.9 0.8 1

3

1.1

2

0.7

2

0.9

0.6

1

0.5 0.7 0.4 200

210

220

230

240

250

260

270 l, nm

Fig. 1. Absorption spectra of cefotaxime (1 ) and claforan (2 ) (Cant = 5 ´ 10 – 5 M).

disks were attached. Glue was prepared by dissolving PVC (0.5 g) in DBP (1 g) and cyclohexanone or THF (5 mL). After the glue dried, the inside of the tube was filled with solutions (1 ´ 10 – 3 M) of KCl and cefotaxime in a 1:1 ratio. The electrodes were stored in cefotaxime solution (1 ´ 10 – 3 M) for 1 d before use. Potentiometric measurements were made using an I-130M universal ionmeter. The uncertainty of the emf measurement was ±1 mV. The reference electrode was an EVL-1MZ AgCl electrode. The acidity of solutions was monitored using a pX-150M ionmeter with glass and AgCl electrodes. Spectrophotometric studies were performed on a SF-201 spectrophotometer in automatic mode. RESULTS AND DISCUSSION Cephalosporins, like penicillins, are unstable when dissolved. The stability of cephalosporin solutions depends on such factors as the temperature, pH, etc. For example, cefotaxime converts to deacetylcefotaxime on storage of biological media (serum) under normal conditions. The antibiotic is stable for three weeks at temperatures of about – 20°C [8]. We faced the task of selecting the optimum acidity at which solutions of antibiotics are stable and the slope of the electrode operation would be as close as possible to the theoretical value. We performed spectrophotometric studies of solutions of cefotaxime from different manufacturers, e.g., Claforan, Laboratories Roussel Diamant (France); Cefotaxime, Green Park, New Delhi (India). Figure 1 shows that lmax = 236 nm is characteristic for cefotaxime and claforan. If the acidity of freshly prepared solutions of cefotaxime is changed, lmax shifts and the optical density changes (Fig. 2). The increase of optical density and lmax upon a change of acidity can be explained by the fact that cephalosporins in acidic medium are quickly hydrolyzed. The b-lactam ring opens to form a derivative of 7-aminocephalosporanic acid. For cefotaxime,

0,5 200

250

300

l, nm

350

Fig. 2. Absorption spectra of cefotaxime (5 ´ 10 – 5 M) solution (Green Park, New Delhi, India) at pH values 2.0 (1 ), 4.0 (2 ), and 6.0 (3 ).

further heterocyclization (lactonization) occurs, forming deacetylcefotaxime. Deacetylcefotaxime is a cefotaxime derivative and metabolite that can be observed in large amounts in biological media. The metabolite exhibits microbiological activity also and is known to be most stable in the pH range 4.3 – 6.5 [8]. All further investigations were conducted with freshly prepared antibiotic solutions. Precipitating potentiometric titration established that tetradecylammonium reacts with cephalosporins in a 1:1 stoichiometry. The solubility product KS = (2.1 ± 0.1) ´ 10 – 8. Ion pairs of tetradecylammonium and cefotaxime and claforan were used as the active electrode components (AEC) of the ISE. Temperature-dependent analytical investigations of the active membrane components found the temperature range for existence of the ion pairs. The samples should be dried at 60 – 70°C. Electroanalytical properties of ISE in cefotaxime solutions. The ISE are operational in cephalosporin solutions in the range 1 ´ 10 – 1 – 1 ´ 10 – 5 M. The angular coefficients of the electrode functions are close to the theoretical values for singly charged ions (Fig. 3). The electroanalytical charac-

TABLE 1. Determination of Cefotaxime in Mixed Patient Saliva (n = 3, p = 0.95) Patient

Found, ìg/mL

Relative standard deviation, S2

1

0.10 ± 0.02

0.08

2

0.12 ± 0.02

0.07

3

0.31 ± 0.20

0.03

4

0.23 ± 0.01

0.02

5

0.40 ± 0.05

0.05

152

O. I. Kulapina et al. E, mV 300

200

100

0 6

4

2

pC

0

Fig. 3. The function E = f(-log Cant) in cefotaxime solution.

teristics remain stable for six months. The time to establish a steady-state potential is 1 – 2 min with a potential drift of ±2 mV/d. The detection limit for cefotaxime is 3.6 ´ 10– 5 M; the optimum pH range, 4.3 – 6.5. A comparison of the main electrochemical characteristics of the ISE based on tetradecylammonium and cephalosporin ion pairs showed that electrodes containing claforan in the membranes had the best operational parameters. The quantity Ksel for cefotaxime in the presence of cefozolin was close to unity, which indicates that the ISE can be used to determine pure antibiotics or their total content. The Ksel values for the inorganic ions Cl–, Br–, HCO3–, H2PO4–, HPO42– and SO42– were in the range n ´ 10 – 2 – n ´ 10 – 3, which enables these electrodes to be used to determine cefotaxime in biological media. We developed previously rapid methods for determining penicillin-type antibiotics in blood serum of patients with tonsillar pathologies [5]. Herein mixed saliva (oral-cavity fluid) was chosen as the study object. Oral-cavity fluid supports ion-exchange reactions between various compartments, tissues, and organs. Datasets for the concentration of drugs in saliva and their free nonbonded fraction in blood plasma may enable more accessible saliva samples to be used in clinical and experimental studies of the pharmacokinetics instead of blood samples [9]. The advantages of analyzing saliva rather than blood are the painless sample collection, simplicity, convenience, low risk of infection, impossibility of skin and blood vessel damage, and the adequacy of the compound concentration for a pharmacotherapeutic effect in the oral cavity. The concentrations of antibiotics in saliva in most instances are tens of times less than those in blood [9]. As a result, a highly sensitive quantitative method is required for their determination. Cefotaxime determination in mixed saliva (oral-cavity fluid). A sample of mixed saliva was collected by spitting oral-cavity fluid into clean dry polyethylene tubes. The sample was collected 1.5 – 2 h after administration of the antibi-

otics. The oral cavity was rinsed with water before collection. The mixed saliva sample was prepared as follows. The mixed saliva sample from patients was centrifuged for 10 min at 3500 rpm, treated with precipitant (0.2 mL, distilled C2H5OH), and centrifuged for another 5 min. The supernatant fluid was collected in a cell (3 – 5 mL). Indicator and AgCl electrodes were inserted. The emf (E1) was measured with stirring. Then, standard cefotaxime solution (Vd = 0.1 mL, Cd = 4.74 mg/mL) was added. The quantity E2 was measured. ISE were conditioned for 20 min in mixed donor saliva beforehand to prevent protein poisoning of the surface. The electrode must be conditioned in distilled water after making the measurements in biological media. The cefotaxime content was calculated using the formula: V C Cx = d d Vx + Vd

æ |E1 - E2 | ç 10 S - V x ç Vd + Vx è

-1

ö ÷ , ÷ ø

where Cx is the cefotaxime concentration, M; Vx, the sample volume, mL; Cd, the additive concentration, M; Vd, the additive volume, mL; E1 and E2, the electrode potentials in the studied solution and the solution with additive, respectively, mV; and S, the slope of the electrode function. Table 1 lists the results of cefotaxime (claforan) determination in mixed patient saliva. The accuracy of the developed method was checked by adding to the sample of mixed donor saliva an aliquot of a standard antibiotic solution and then taking the sample through all sample-preparation steps. It was demonstrated that the found values of antibiotic content corresponded to those calculated (the relative uncertainty of a determination was 1 – 5%). Thus, the developed electrodes can detect cefotaxime in biological media. REFERENCES 1. M. D. Mashkovskii, Medicinal Agents [in Russian], Part 2, Meditsina, Moscow (2005). 2. Yu. B. Belousov and E. A. Ushkalova, Antibiot. Khimioter., 46, No. 11, 23 – 35 (2001). 3. A. Alwartham, S. A. Fattan, and N. Zahran, Talanta, 32, No. 6, 703 – 707 (1992). 4. C. Hendrix, J. Thomas, E. Roets, and J. Hoogmartens, J. Liq. Chromatogr., 16, No. 2, 421 – 445 (1993). 5. E. G. Kulapina, V. V. Baraguzina, and O. I. Kulapina, Zh. Anal. Khim., 59, No. 9, 971 – 975 (2004). 6. A. Abulkibash, S. Sultan, and A. Al-Olyan, Talanta, 61, 239 – 244 (2003). 7. E. G. Kulapina, V. V. Baraguzina, and O. I. Kulapina, Khim.-farm. Zh., 40, No. 3, 97 – 99 (2006). 8. L. I. Sokolova and A. P. Chernyaev, Khim.-farm. Zh., 36, No. 5, 39 – 45 (2002). 9. E. M. Lakin, M. M. Zoryan, M. M. Kats, et al., Farmakol. Toksikol. (Moscow), 50, No. 4, 93 – 100 (1987).