Pharmaceutical Chemistry Journal

Vol. 42, No. 8, 2008

ION-SELECTIVE ELECTRODES FOR DETERMINING CEPHAZOLIN IN BIOLOGICAL MEDIA O. I. Kulapina,1 V. V. Baraguzina,2 and N. V. Skoblikova2 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 8, pp. 41 – 44, August, 2008. Original article submitted May 11, 2006.

An ion-selective electrode with plasticized polyvinylchloride membrane that is based on a cephazolin—tetradecylammonium ionic associate has been developed. The ratio of components and the solubility product of the ionic associates [(2.2 ± 0.1) ´ 10 – 8] were determined. The compound is thermally stable up to 70°C. The EMF is linearly dependent on the cephazolin concentration in the range from 1 ´ 10 – 5 to 1 ´ 10 – 1 M. The slope of the electrode function is 56 ± 2 mV/pC. The selectivity to inorganic ions allows the proposed system to be used for cephazolin determination in biological media. In particular, a method for rapid determination of cephazolin in complex saliva is developed.

Microbiological, spectroscopic, chromatographic, and electrochemical analytical methods are the principal ones for determining antibiotics, drugs, and biologics [1]. Potentiometry using ion-selective electrodes (ISE) is a simple, rapid, and sensitive method for determining antibiotics in drugs and biological fluids. Cephalosporins exhibit bactericidal activity. The mechanism of action is related to damage to the bacterial cell membrane during multiplication that is due to specific inhibition of cell membrane enzymes [2]. We developed ion-selective electrodes for rapid determination of cephalozin in biological fluids.

by successive dilution of the working solutions. Freshly prepared solutions were used in order to avoid hydrolysis [2]. The electrically active component consisted of ionic associates of cephazolin and tetradecylammonium, [(C10H21)4N]+ × Cef–(TDA+Cef–). Synthesis of the ionic associate TDA+Cef- was carried out using an exchange reaction: A solution of tetradecylammonium bromide in CHCl3 (V = 5 mL, C = 1 ´ 10 – 3 M) and aqueous solutions of cephazolin (V = 2 mL, C = 1.5 ´ 10 – 2 M) were placed in a separatory funnel. The mixture was shaken for 2 h. The CHCl3 layer was separated from the aqueous phase into a tared weighing bottle. The CHCl3 was evaporated on a water bath at 50 – 60°C in order to avoid decomposition of the electrically active component. We investigated liquid electrodes with locally prepared plastified membranes. The reference was an AgCl electrode. The plastified membranes were prepared by dissolving a weighed portion of the electrically active component in dibutylphthalate (DBP) with continuous stirring and adding THF and polyvinylchloride (PVC). The mixture was thoroughly mixed until completely homogeneous (DBP:PVC = 3:1). Film membranes were prepared by pouring the mixture into a Petri dish and storing in air until the THF was completely removed. This produced elastic and transparent membranes of about 0.5 mm thickness. The working solutions of the ISE were sensitive to cephazolin and consisted of a mixture of KCl (C = 10 – 3 M) and antibiotic (C = 10 – 3 M) (1:1 ratio). The composition of the working solution had to be kept constant in order to ob-

EXPERIMENTAL PART We used the pharmaceutical-grade sodium salt of cefazolin (Cef): O

CH2 HC

N

S

NH

C

N

N N

N

N O C

O

CH2

S

C

N S

C

CH3

ONa

Starting aqueous solutions (1 ´ 10 – 1 M) were prepared using accurately weighed portions of the preparation. Working solutions (1 ´ 10 – 2 – 1 ´ 10 – 6 M) were obtained 1 2

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

481 0091-150X/08/4208-0481 © 2008 Springer Science+Business Media, Inc.

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O. I. Kulapina et al.

1.2

A

2.0

A

3 1.0

1.5 1

0.8

1 1.0

0.6

2

2

3

0.4

0.5

0.2 0

0 150

200

250

300

350

400

–0.2

450 l, nm

Fig. 1. Absorption spectra of cephazolin solutions from various manufacturers (CCef = 5 ´ 10 – 5 M): FAO Ferrein (1 ), OOO Hunt-Holding (2 ), OOO Deko (3 ).

tain stable electrode characteristics. Therefore, solutions containing cephazolin were replaced daily with freshly prepared ones. The potentiometric selectivity coefficients (Ksel) of the membrane electrodes were determined using bi-ionic potentials and mixed solutions [3]. The pH of solutions was monitored using a pX-150 M potentiometer with glass and AgCl electrodes. Thermal analytical investigation of the ionic associates was performed on a OD-130 thermal analyzer that was capable of programmed heating in air at room temperature to 1000°C with simultaneous recording of four curves for temperature (T), thermogravimetry (TG), differential thermogravimetry (DTG), and differential thermal analysis (DTA). Temperatures were recorded using a Pt—Rh thermocouple with calcined Al2O3 as the reference. The sample weights were 70 mg. The ratio of components in the ionic associates and the solubility product (Ks) were determined by potentiometric titration [4]. The equivalence point was found graphically using the literature method [3]. Precipitation of proteins and better separation of biological fluids were carried out using a Wirowka MPW-6 centrifuge. Optical density was measured on a SF-201

TABLE 1. Cephazolin Determination in Saliva of Patients (n = 3, p = 0.95) Preparation

Found, mg/mL

Relative standard deviation, Sr

1

ViCef

0.20 ± 0.05

0.10

2

NaCef

0.03 ± 0.005

0.07

3

NaCef

0.81 ± 0.06

0.03

4

ViCef

0.49 ± 0.07

0.06

5

NaCef

0.10 ± 0.05

0.20

Patient

200

250

300

350

400

450 l, nm

–0.5

Fig. 2. Absorption spectra of cephazolin solution (5 ´ 10 – 5 M) (OOO Deko) at various pH values: 2 (1 ), 4 (2 ), and 6 (3 ).

spectrophotometer connected to an IBM PC using a quartz cuvette (l = 10 mm). Cephazolin in saliva was determined by the method of additions. RESULTS AND DISCUSSION Cephazolin batches from various manufacturers were investigated spectrophotometrically. These were OOO Hunt-Holding, FAO Ferrein, and OOO Deko (Fig. 1). Figure 1 shows that the optical density at lmax differed for the different cephazolin batches (DAmax = 0.3). This indicates that the contents of main ingredient in the drugs were different. The state of cephazolin at different pH values was studied (Fig. 2). It was shown that the pH does not affect the conditions of the antibiotic. The lmax value remains constant. However, the optical density decreases significantly with increasing pH. Therefore, the pH of the medium must be controlled for determining cephazolin. The ratio of components in the ionic associates and the solubility product (Ks) were determined by potentiometric titration of tetradecylammonium bromide with cephazolin solution (Fig. 3). It was established that the components reacted is a 1:1 ratio. The Ks value was (2.2 ± 0.1) ´ 10 – 8, i.e., the studied compounds are poorly soluble and can be used as the electrically active membrane components. Thermal analytical studies of the ionic associate Cef—TDA showed that it decomposed in a stepwise manner (Fig. 4). The sample did not undergo changes on heating to 100°C. This was consistent with the lack of weakly bound water. Further heating caused decomposition that was accompanied by a significant exothermic effect at 350°C for DTA because of oxidation of decomposition products. The mass loss as a result of this process was 56%. The next decomposition step (inflection on the TG curve) corresponded to mass loss (67%) and an exothermic effect (max at 460°C) as a result of oxidation by air of the organic part of the molecule (or organic products produced by

Ion-Selective Electrodes for Determining Cephazolin

483

E, mV 180

T, °C T 900 180

280 350

160

DTG

500

700

460

500 DTA

300

140

100 0

120 56%

67%

100 0

0.5

Vtitr, mL

1.0

1.5

Fig. 3. Titration curve of TDA-Br (1 ´ 10 – 3 M) by cephazolin solution (1 ´ 10 – 3 M).

TG

Fig. 4. Thermograms of Cef—DTA ionic associate.

its decomposition). Further heating to 800°C did not produce a detectable mass loss. Thus, it was demonstrated that Cef–TDA is poorly soluble, does not contain water, and should be dried at 70°C. Electroanalytical properties of ion-selective electrodes in cephazolin solutions. The EMF as a function of cephazolin concentration was linear in the range 1 ´ 10 – 1 – 1 ´ 10 – 5 M. The slope of the line was 56 ± 2 mV/pC. This is consistent with the transfer of singly charged cephazolin ions. The potential was determined by an ion-exchange reaction occurring at the membrane—solution interface: Cef-TDAM + Cefs Û CefTDAM + Cefs E = E0 – 0.059log[Cef]. Potentiometric selectivity coefficients (Ksel) for potentiometric sensors based on the Cef—TDA ionic associate were determined for the antibiotics and for anions found in saliva using mixed solutions and bi-ionic potentials (Fig. 5). The Ksel value for cephotaxime in the presence of cephazolin was close to unity. This indicates that the ISE can be used to determine pure antibiotics or their mixtures. The Ksel values for several inorganic ions (Cl–, Br–, HCO3–, H2PO4–, HPO42–, SO42–) enable these electrodes to be used to determine cephazolin in biological fluids. Cephazolin determination method in saliva. The collection of saliva samples had several specific features. One of them was the ability to stimulate salivation. Most researchers stimulate salivation during collection of saliva for studies of the content of drugs because the use of stimulated saliva stabilizes the concentration ratios of the drug to that in blood plasma from various donors [5, 6]. We collected saliva samples by expectorating saliva into clean dry polyethylene tubes. Samples were taken after 1.5 – 2 h after treating patients with cephazolin. The oral cavity was rinsed with water before the collection. Then the sample was centrifuged for 10 min at 3,500 rpm. Precipitant was added (0.2 mL,

C2H5OH). The sample was centrifuged for another 5 min. The supernatant liquid was collected in a cell (3 – 5 mL). Indicator and AgCl electrodes were inserted. The EMF (E1) was measured with stirring. Then, a portion of cephazolin standard solution was added. The EMF (E2) was measured again. The ISE were conditioned beforehand for 20 min by mixing in donor saliva in order to avoid poisoning of the membrane surface by protein. The electrodes were conditioned in distilled water after making measurements in biological fluids. The antibiotic contents were calculated using the formula:

CX

|E1 - E2 | æ V C Vx a a ç = 10 S ç Vx + Va Va + Vx è

-1

ö ÷ , ÷ ø

where CX is the concentration of the determined antibiotic (M); VX, sample volume (mL); Ca, additive concentration Ksel

~ –3

10

Cl

–

Br

2–

–

HPO4

2–

SO4 Ctox

–

– I H2PO4 Interfering ions

–

HCO3

Fig. 5. Potentiometric selectivity coefficients of the ISE for cephotaxime Ctox and several inorganic ions.

484

(M); Va, additive volume (mL); E1 and E2, electrode potentials in the studied solution and in the solution with the additive, respectively (mV); S, slope of the electrode function (mV/pC). Table 1 gives the results for antibiotic determination in patient saliva. Standard amtibiotic solution was added to the donor saliva sample in order to assess the accuracy of the deveoped method. Then, all sample preparation steps were carried out. It was shown that the found values for antibiotic content corresponded to those estimated (relative uncertainty of a determination was 1 – 5%). Thus, the developed electrodes could detect cephazolin in biological fluids.

O. I. Kulapina et al.

REFERENCES 1. E. G. Kulapina, V. V. Baraguzina, O. I. Kulapina, and E. E. Mikhailova, Modern Methods for Determining Antibiotics [in Russian], dep. VINITI, No. 2155-B, 2003. 2. Yu. B. Belousov and E. A. Ushkalova, Antibiot. Khimioter., 46(11), 23 – 35 (2001). 3. B. P. Nikol(skii and E. A. Materova, Ion-Selective Electrodes [in Russian], Khimiya, Leningrad (1980), p. 240. 4. B. M. Mar(yanov, Method of Linearization and Instrumental Titration [in Russian], Tomsk (2001), p. 154. 5. O. A. Gavrilova, Stomatologiya, No. 2, 54 – 56 (2004). 6. E. M. Lakin, E. V. Zoryan, M. M. Kats, et al., Farmakol. Toksikol., 50(4), 93 – 100 (1987).