Monday, August 10, 2009

Microbiological assay for ceftazidime injection

A simple, sensitive, and specific biodiffusion assay for the antibacterial ceftazidime was developed using a strain of Staphylococcus epidermidis (ATCC 12228) as the test organism. Ceftazidime was measured in powder for injection at concentrations ranging from 100 to 400 lag/mL. The calibration graph for ceftazidime was linear ([r.sup.2] =1), and the method validation showed that it was precise (relative standard deviation = 0.415) and accurate. The results obtained by biodiffusion assay were statistically calculated by linear parallel model and by means of regression analysis and were verified using analysis of variance. It was concluded that the microbiological assay is satisfactory for in vitro quantification of the antibacterial activity of ceftazidime in pharmaceuticals.

Ceftazidime is a third-generation cephalosporin that is widely used for the treatment of serious infections caused by Gram-negative bacteria, including Pseudomonas aeruginosa, especially in cystic-fibrosis patients. It is usual to administer this drug by slow intravenous infusion over 24 h. The infusion solutions are prepared in advance and stored in the pharmacy (1). The favorable properties of ceflazidime include efficient penetration of the bacterial cell wall, resistance to bacterial enzyme degradation, a high intrinsic activity against the bacterial cell targets, a broad spectrum of activity, very low toxicity, extensive tissue penetration, metabolic stability, and a low degree of serum protein binding (2).

Assays reported in the literature for determination of ceftazidime in biological fluids include high-performance liquid chromatography (HPLC; 3) and spectrophotometry using batch and flow-injection procedures. For the measurements in pharmaceuticals, several methods are reported for determination of cephalosporins, including HPLC, spectrophotometry, fluorimetry, polarography, colorimetry involving treatment with sodium cobaltinitrite, ninhydrine, molybdophosphoric acid, oxidation with Ce(IV) or Fe (III), hydroxamic acid, ammonium vanadate, iodometric titration, and spectrophotometric UV analysis (4-6).

Although the antimicrobial activity and pharmacokinetics of this drug have been widely researched, few studies in the literature relate to the development of analytical methodology for this cephalosporin. Research involving analytical methods is of basic importance to optimize its analysis in the pharmaceutical industry and to guarantee the quality of the commercialized product.

Parameters specified in assays with other cephalosporins (7) were carried through preliminary tests to standardize the conditions to be used, such as test organism, growth media, diluents, inoculum, and drug concentration. The basic requirement for a test organism is that it should be nonpathogenic, sensitive to the action of the antibiotic under assessment, and capable of rapid growth. The chemical structure of ceflazidime is represented by Figure. 1. Ceftazidime is commercialized in Brazil under the name of Ceftazidon by Ariston Quimica e Farmaceutica Ltda (SAO Paulo, Brazil) and as Fortaz by Glaxo SmithKline (SAO Paulo, Brazil). No microbiological assay for determination of ceflazidime and its formulations has been reported for laboratory quality control. This assay can reveal subtle changes not demonstrated by chemical methods, such as chemical degradation, and allows evaluation of the potency of ceftazidime, which is very important in the analysis of antibiotics.

The objective of the present study was to find a sensitive and reproducible biodiffusion assay to quantify ceftazidime in raw material and powder for injection, and to validate the method by determining the parameters of linearity, precision, and accuracy.


Ceftazidime reference substance (assigned purity 99.98%) as well as ceflazidime powder for injection were generous gifts from Ariston. Ceftazidime powder for injection was claimed to contain 1000 mg of the drug and sodium carbonate as excipient. All chemicals used were of analytical reagent grade.

Preparation of Ceftazidime Reference Substance

The standard solution in water (1 mg/mL) was diluted in potassium phosphate buffer, pH 6.0, and assayed at concentrations of 100, 200, and 400 [micro]g/mL.


Sample Preparation

Twenty samples containing ceftazidime powder for injection were weighed, and the medium weight was determined. An amount of powder equivalent to 100 mg ceftazidime was transferred to a 100 mL volumetric flask with 50 mL water and shaken. This was followed by diluting to volume with water (1000 [micro]g/mL). The dilutions were made with potassium phosphate buffer, pH 6.0, to give final concentrations of 100, 200, and 400 [micro]g/mL. This dilution procedure was performed in triplicate.

Organism and Inoculum

The culture of Staphylococcus epidermidis (ATCC 12228) stored in the freezer was cultivated on Grove Randall No. 1 agar (Merck, Darmstadt, Germany) and transferred to another Grove Randall No. 1 agar (24 h before the assay) that was kept at 35 f 2[degrees]C. The bacteria were suspended in tryptic soy broth (TSB; Sparks, MD) using a glass homogenizer. Diluted culture suspensions of 25 [ or -] 2% light transmission were obtained at 580 nm, using a suitable spectrophotometer (UV-Vis JAS.CO 7800), and a 10 mm diameter test tube of absorption cells against TSB was used as blank. Portions of 0.5 mL of the inoculated TSB were added to 100 mL Grove Randall No. 1 agar at 35 f 2[degrees]C and used as the inoculated layer.

Biodiffusion Assay

The agar was composed of 2 separate layers, and only the upper layer was inoculated; this procedure may afford greater sensitivity than a single-layer plate (8). The Grove Randall No. 2 agar (21 mL) was poured into a 100 x 20 mm Petri dish as a base layer. After solidification, 4 mL portions of inoculated Grove Randall No. 1 agar were poured onto the base layer (9). Six stainless steel cylinders of uniform size (8 od x 6 id x 10 mm high) were placed on the surface of the inoculated medium. Three alternated cylinders were filled with 200 [micro]L of the reference solutions, and the other 3 were filled with the sample solutions. After incubation (35 f 2 [degrees]C for 21 h), the zone diameters (in mm) of the growth inhibition were measured using a caliper (Starret, Chicago, IL).


The percent activity of ceftazidime in powder for injection was calculated by Hewitt equations (10). The assay was statistically calculated by linear parallel model and by means of regression analysis of variance (ANOVA).

Method Validation

The method was validated by determination of linearity, precision, and accuracy (11, 12). In order to assess the validity of the assay, the linearity was determined by using 3 doses of the reference substance and 3 doses of the sample. The calculation of regression line by the method of least squares was used. The accuracy was determined by adding known amounts of ceftazidime reference substance to the samples at the beginning of the process. Amounts of 0.1, 0.2, and 0.4 mL ceftazidime reference solution (500 [micro]g/mL) and 1.0 mL ceftazidime sample solution (1000 [micro]g/mL) were added to 10 mL volumetric flasks, respectively, [R.sub.1], [R.sub.2], and [R.sub.3]. Potassium phosphate buffer, pH 6.0, was added to give final concentrations of 105.0, 110.0, and 120.0 [micro]g/mL, respectively. The solutions were used in the biodiffusion assay described above. The percentage recovery of ceftazidime reference added was calculated using the formula proposed by AOAC (11):

R% = [([P.sub.F] - [P.sub.A])/[P.sub.P]] x 100

where [P.sub.F] = sample standard potency; [P.sub.A] = sample potency; [P.sub.P] = standard added potency. Accuracy and precision of bioassay were determined intraday and interday on the 3 different days.


Results and Discussion

In the development phase of the ceftazidime bioassay, the parameters were evaluated as test microorganism [Staphylococcus aureus (ATCC 6538) or S. epidermidis (ATCC 12228)]; culture medium; buffer pH 6.0 or 8.0; inoculum concentration 0.5, 1.0, or 2.0%; solution concentration (2.0, 4.0, 8.0 dug/mL; 8.0,12.0, 18.0 Pg/mL; and 100, 200,400 [micro]g/mL).

An experimental 3 x 3 design, using 3 dose levels for each standard and sample were used following the procedure described in the Brazilian Pharmacopoeia (7). The calculation procedure normally assumes a direct relationship between the observed zone diameter and logarithm of applied dose. There was a linear relationship between [log.sub.10] of the ceftazidime concentrations and growth inhibition zone diameter (complete diameter) for concentrations 100, 200, and 400 [micro]g/mL. The corresponding mean zone diameters for reference solutions were 12.85 [ or -] 0.04 [coefficient of variation (CV) = 0.31] for low dose, 16.83 [ or -] 0.03 (CV = 0.15) for medium dose, and 20.84 [ or -] 0.03 (CV = 0.12) for high dose (Table 1). The calibration curve for ceftazidime was constructed by plotting log of concentrations (Vg/mL) versus zone diameter (mm) and showed good linearity on the 100-400 [micro]g/mL (Figure 2). The representative linear equation for ceftazidime was y = 13.271x - 13.697, where x is a log of concentration and y is diameter zone inhibition. The coefficient of regression was [r.sup.2] = 1.0.

The experimental values obtained for determination of ceftazidime in samples are presented in Table 2. According to the Brazilian Pharmacopoeia (7), if a parallel-line model is chosen, the 2 log dose-response lines of the preparation to be examined and the reference preparations must be parallel, and they must be linear over the range of doses used in the calculation. These conditions must be verified by validity tests for a given probability, usually P = 0.05. The assays were validated by means of the ANOVA, as described in these official codes. There are no deviations from parallelism and linearity with results obtained here (P <>

The precision and accuracy of the assay were also demonstrated. Precision is usually expressed as the variance, relative standard deviation (RSD), or CV% of a series of measurements (13). The results obtained on different days showed a CV of 0.42 for powder for injection (Table 2). The accuracy is shown by the agreement between the accepted value and the value found to be 98.7% for powder for injection (Table 3).

The quantification of antibiotic components by chemical methods such as HPLC and UV spectrophotometry, although precise, cannot provide a true indication of biological activity. Attempts to correlate antibiotic bioassay results with those from chemical methods have proved disappointing. Therefore, bioassays continue to play an essential role in manufacturing and quality control of antibiotic medicines, and still demand considerable skill and expertise to ensure success. Although the biological assays have a high variability, the analysis of the obtained results demonstrated that the proposed method might be very useful for determination of this drug in pharmaceutical dosage forms.


The results indicated that the biodiffusion assay demonstrated good linearity, precision, and accuracy at concentrations ranging from 100 to 400 [micro]g/mL; therefore, it is an acceptable alternative method for the routine quality control of ceftazidime in raw materials and medicines. The method uses simple reagents, with minimum sample preparation procedures, encouraging its application in routine analysis.


We are grateful to Ariston for providing ceftazidime standard and powder for injection. This work was supported by CNPq-Brazil program and PACD-FCF-UNESP.

Received March 7, 2007. Accepted by Aft May 11, 2007.


(1) Arsene, M., Favetta, E, Favier, B., & Bureau, J. (2002) J. Clin. Pharm. Ther. 27,205-209

(2) Myers, C.M., & Blumer, J.L. (1983) Antimicrob. Agents Chemother. 24,343-346

(3) Samanidou, V.F., Hapeshi, E.A., & Papadoyannis, I.N. (2003) J. Chromatogr. B 788, 147-158

(4) Jamieson, C.E., Lambert, P.A., & Simpson, I.N. (2003) Antimicrob. Agents Chemother. 47,2615-2618

(5) Martinez, L.G, Falco, E.C., & Cabeza, A.S. (2002) J. Pharm. Biomed. Anal. 29,405-423

(6) Salem, H., & Askal, H. (2002) J. Pharm. Biomed. Anal. 29, 347-354

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