INTRODUCTION:

Cefotaxime sodium is a broad-spectrum, semi-synthetic, third-generation cephalosporin antibiotic that kills bacteria by inhibiting cell wall synthesis^1,2,3. It is administered via intramuscular or intravenous injection to treat infections caused by gram-positive and gram-negative bacteria, offering enhanced effectiveness against Gram-negative bacteria compared to first- and second-generation cephalosporins⁴. Cefotaxime sodium also demonstrates high stability against most beta-lactamase enzymes, penetrates well into the cerebrospinal fluid (CSF), and is used to treat meningitis^2,4.

Chemically, cefotaxime sodium is described as 5- THIA-1-Azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid, 3-[(acetyloxy) methyl]-7-[[(2-amino-4-thiazolyl) (methoxyimino)acetyl] amino] -8-OXO-, monosodium salt, [6R - [6,7 (Z)]]-; Sodium (6R,7R)-7-[2-(2-amino-4-thiazolyl) Glyoxylamido]-3-(hydroxymethyl)-8- OXO -5-THIA-1-Azabicyclo [4.2.0] oct-2-ene-2-Carboxylate 72 -(Z)-(O-methyloxime), Acetate (ester). The empirical formula of cefotaxime sodium is C₁₆H₁₆N₅NaO₇S₂ with a molecular weight of 477.45g/mol. It is a white or slightly yellow powder, freely soluble in water, sparingly soluble in methanol, and practically insoluble in organic solvents^4,5.

Multiple analytical techniques, including physicochemical and microbiological methods, have been employed to assess antibiotic effectiveness. Physicochemical methods, such as High-Performance Liquid Chromatography (HPLC) and Ultraviolet spectroscopy (UV)^6,7, provide precise and rapid quantification. However, microbiological assays remain essential in pharmacopeial standards and are crucial for the quality control of antibiotics^8,9.

Both the American and British Pharmacopoeias recommend chromatographic methods for cefotaxime sodium quantification^5,10. Numerous analytical approaches for its determination in biological fluids and pharmaceutical formulations have been documented, including chromatography^11-18, spectrophotometry^19-25, and spectrofluorimetry²⁶. These methods assess cefotaxime sodium’s potency but do not directly evaluate its biological activity⁸. Microbiological assays, in contrast, are uniquely suited for antibiotic analysis because they assess both potency and biological activity without interference from degradation products or impurities, and they avoid the need for toxic chemicals or complex equipment^27,28.

On the other hand, there is ongoing debate in the medical community regarding microbiological equivalence across different antibiotic formulations, which raises questions about their interchangeability. Additionally, there is a perception among some Syrian healthcare providers and patients that brand pharmaceutical preparations of antibiotics are better than locally manufactured ones. Microbiological methods provide an objective measure of efficacy, enabling precise determination of an antibiotic’s therapeutic effectiveness. Therefore, we will use the results of the developed microbiological method for cefotaxime sodium to study the microbiological equivalence between its local commercial preparations and the brand commercial preparation through a statistical method for evaluating in vitro equivalence.

Many previous studies have shown the use of microbiological assay in determining the potency and microbiological activity of many antibiotics^29-36, in addition to its use in determining the microbiological equivalence between commercial antibiotic preparations in vitro^37-39.

OBJECTIVE:

To our knowledge, a microbiological assay method for cefotaxime sodium has not been previously reported. This research aimed to develop and validate a simple, precise, accurate, sensitive, and low- cost one-level agar diffusion (5+1) microbiological assay to quantify cefotaxime sodium potency and biological activity in pharmaceutical preparations. This validated method will be applied to evaluate the microbiological equivalence of cefotaxime sodium formulations, contributing to more informed therapeutic choices.

MATERIALS AND METHODS:

Materials, Test microorganisms, and Equipment:

Materials: Mueller-Hinton agar (HiMedia, Mumbai, India), Cefotaxime sodium working standard was provided by ALPHA Pharmaceutical Industries (Aleppo, Syria), and 5 locally manufactured commercial preparations and one brand preparation, which is a powder for injection of cefotaxime sodium, were obtained from the local Syrian market.

Test microorganisms: Klebsiella pneumoniae (ATCC-13885), and Staphylococcus aureus (ATCC-43300) were used in microbiological assay.

Equipment: Sterilized glassware, including petri plates, test tubes, volumetric flasks, graduated cylinder, and pipettes, was used.

Phosphate buffer preparation:

Phosphate buffer solution (pH 7.0) was prepared according to the United States Pharmacopoeia 46 (USP 46) by adding 50mL of potassium dihydrogen phosphate solution (0.2M) to 29.1mL of sodium hydroxide solution (0.2M), then making up the volume to 200mL with double-distilled water, and the solution was sterilized in an autoclave at 121°C for 15 minutes.

Culture media preparation:

Mueller-Hinton agar was prepared as instructed on the label by suspending 38g of it in 1000ml of distilled water and heating the medium until completely dissolved, then sterilizing it using an autoclave at 121°C for 15 minutes. After the sterilization period was over, the medium was stirred well and 25ml of it was poured into each sterile Petri plate using a sterile graduated cylinder. Then the plates were left until the agar solidified and gave an equal height in all plates, which was 4 mm.

Preparation of stock suspensions of test microorganisms:

Bacterial isolates were cultured on Mueller-Hinton agar and incubated at 37°C for 18 to 24h, then collected using sterile NaCl 0.9%. The concentration of the bacterial suspension was adjusted to 0.5 McFarland⁴⁰by measuring the absorbance at a wavelength of 625 nm, using a spectrophotometer (the absorbance value should range between 0.08 - 0.12).

Preparation of standard solutions:

50 mg of standard cefotaxime sodium was weighed accurately and placed in a 100ml volumetric flask. It was dissolved in a quantity of double-distilled water, then the volume was completed to the mark in the same solvent, so we got a solution with a concentration of 500 μg/ml. Several aliquots were taken from this solution and transferred to five 10mL volumetric flasks. The volume in each flask was then made up to the specified mark using a phosphate buffer (pH 7.0), resulting in five standard solutions with concentrations increasing in a 1:1.25 ratio, which were (S₁= 0.416, S₂= 0.52, S₃= 0.65, S₄= 0.8125, S₅= 1.02)μg/mL, where S₃ was considered the median reference standard solution.

Preparation of sample solution:

A 500 µg/mL stock solution of cefotaxime sodium was prepared from a commercial preparation, following the procedure for the sodium cefotaxime standard solution. This stock solution was then diluted with phosphate buffer (pH 7.0) to create sample solution T, with a concentration of 0.65μg/ml, equivalent to the median reference standard solution S₃.

Microbiological assay (5+1) method:

Microbiological assay was performed using the (5+1) method by following the steps below:

1. 150μl of freshly prepared bacterial suspension was inoculated onto the surface of the poured culture medium within the petri plate.

2. Six wells were made in the agar, spaced equally apart, using a sterile tool with a diameter of 6 mm.

3. Three of the wells, positioned alternately, were filled with 50 μl of the S₃ standard solution, and the remaining three wells were filled with the same volume of one of the four standard solutions S₁, S₂, S₄, and S₅, or the diluted sample solution T, as shown in the figure (1).

4. The plates were placed in the refrigerator for about 1-2 hours until the antibiotic solutions spread throughout the agar, then they were transferred to the incubator and incubated at 37°C for 18 hours.

5. Following incubation, growth inhibition zone diameters were measured, and mathematical relationships were applied to correct the diameters of the growth inhibition zones and calculate the effectiveness of the antibiotic sample solution.

Calculations:

After recording the diameters (in mm) of the resulting inhibition zones, a correction was made using the following mathematical relationship:

Xc= Xs – ( Xg- P) …….. (1)

Where:

X_C: The average diameters of the inhibition zones corrected for one of the standard solutions (S₁, S₂, S₄, and S₅).

X_S: The average diameters of the actual inhibition zones for one of the standard solutions (S₁, S₂, S₄, and S₅).

X_R: The average diameters of the inhibition zones for the median reference standard solution S₃ in the same Petri dishes with one of the standard solutions (S₁, S₂, S₄, and S₅).

P: The correction point (calculated by finding the overall arithmetic mean of the reference standard solution S₃), where X_Cfor S₃ = P.

The logarithm of standard solution concentrations was then plotted against corrected inhibition zone diameters.

A linear equation was generated by plotting the logarithm of standard solution concentrations against corrected inhibition zone diameters

Then the relationship between the logarithm of the standard solutions concentrations and the corrected inhibition zone diameters was plotted, resulting in a linear equation of the curve, which was as follows:

Z = b x log (C) + a …….. (2)

Where:

Z: equal to Xc for each standard solution.

C: concentration of the standard solution.

b: slope of the regression line.

a: intercept of the regression line.

Then, the logarithm of the antibiotic concentration in the sample was calculated from the following equation:

(U - a)

L_U= ------------- …….. (3)

Where:

L_U: logarithm of sample solution concentration.

U: The average diameters of the inhibition zones corrected for the sample solution.

Then, the sample concentration is calculated from the following relationship:

Cu = 10^Lu …….. (4)

Then, the recovery of the antibiotic was calculated, which expresses its potency and biological activity:

Percentage = (Cu / Cs₃) x 100 …….. (5)

Where:

C_S3: concentration of the median reference standard solution S₃.

In-vitro microbiological equivalent study:

One-Way ANOVA followed by Tukey test was performed on the results of the developed microbiological assay of cefotaxime sodium to study the microbiological equivalence between its local commercial preparations and its brand preparation using the SPSS software (version 25.0, IBM).

Figure 1. Shows the method of filling the wells with standard antibiotic solutions and sample solution.

RESULTS AND DISCUSSION:

Development of microbiological assay method:

Selection of antibiotic-sensitive microorganisms:

Susceptibility testing was performed for Klebsiella neumonia (ATCC-13885) and Staphylococcus aureus (ATCC-43300). Both strains showed sensitivity to sodium cefotaxime, but Klebsiella neumonia was selected due to its clear, well-defined zones of inhibition.

Determining the optimal antibiotic concentration range:

From a 500 µg/mL stock solution of cefotaxime sodium, several increasing standard series were prepared with a 1:1.25 dilution ratio. These dilutions were tested, and the series ranging from 0.416–1.02µg/mL was selected because it provided higher sensitivity at low concentrations, better linearity, and a zone of inhibition diameter for the median standard solution (S₃) of 11–19 mm, as recommended by the United States Pharmacopeia. Table (1) shows the zone of inhibition diameters resulting from the concentrations of the selected dilution series.

Establishing other conditions affecting microbiological assay

Several conditions affecting the zones of inhibition were standardized as follows:

a) The thickness of the agar medium: it was unified in all plates at 4 mm, as a thick agar medium leads to small zones of inhibition and a thin agar medium leads to large zones of inhibition. b) The incubation temperature and duration were set at 37°C±0.5 for 18 hours. c) The plates were transferred to the refrigerator for about 1-2 hours after filling the wells with antibiotic solutions, in order to minimize the time difference between the application of the solutions and to allow the solutions to diffuse in the agar before the growth of the microorganisms. Incubating the plates before the antibiotic solution had diffused would result in small zones of inhibition. d) A phosphate buffer (pH 7.0) was used as a diluent. e) The concentration of the bacterial suspension was adjusted to 0.5 McFarland, with an absorbance of 0.10 – 0.11, resulting in a bacterial concentration of 1.5 × 10⁸ CFU/ml. Then, 150 μl was taken from it to be inoculated on the surface of the culture medium. Significant variations in the concentration of microorganisms lead to differences in the diameters of the inhibition zones, thus causing errors in the microbiological assay results.

Table 1. The diameters of the inhibition zones resulting from the selected dilution series concentrations.

Standard Concentration (µg/ml)	Mean diameters of the inhibition zones (mm)
0.416	14.6
0.52	15.6
0.65	17.1
0.8125	18.8
1.02	19.8

Method validation⁴¹

Linearity

The linearity of the developed microbiological assay was assessed by applying this method to five standard solutions with increasing concentrations at a ratio of 1:1.25 (0.416, 0.52, 0.65, 0.8125, 1.02)µg/mL. Three plates were used for each standard solution (each plate containing three wells for one of the four standard solutions S₁, S₂, S₄, S₅, and the remaining three wells for the median standard solution S₃). The assay was repeated three times at different time points. The average diameters of the resulting inhibition zones were then calculated and corrected for each corresponding concentration using equation (1) mentioned previously. The relationship between the logarithm of the standard solution concentrations (µg/mL) and the average corrected diameters of the inhibition zones (mm) was plotted. This yielded the standard curve equation Z = 13.448x + 19.748, with a correlation coefficient R² = 0.996. This confirms the linearity of the method within the studied concentration range, as shown in Figure (2). The results of the linearity assessment of the developed microbiological method for sodium cefotaxime are summarized in Table (2).

Figure 2. Standard curve for cefotaxime sodium assay.

Table 2. The results of the linearity assessment of the developed microbiological method for cefotaxime sodium.

Parameters	Proposed method
Linearity range (μg/ml)	0.416 – 1.02
Slope	13.448
Intercept	19.748
Correlation coefficient (R²)	0.996

Accuracy:

The accuracy of the developed microbiological assay was validated through a standard addition method. A sample solution was prepared with a concentration of 0.416µg/mL, and standard additions were performed at three levels (80%, 100%, and 120%) using a 50 µg/mL standard solution of cefotaxime sodium. The volume was then adjusted to the specified mark using phosphate buffer (pH 7.0). The three mixtures were analyzed by the developed microbiological method three times, and the Potency was calculated. The mean Potency was 99.4%, which is within the acceptable range for accuracy (96-102%), confirming the accuracy of the developed method (Table 3).

Precision:

The precision of the developed microbiological assay method was evaluated for repeatability (intra-day) and intermediate precision (inter-day). The repeatability was determined by analyzing six replicates of a 0.65 µg/ml cefotaxime sodium solution (100% concentration) on the same day. In the same way, the intermediate precision was evaluated over two consecutive days, and then the relative standard deviation values were calculated. The method showed good precision, as the relative standard deviation value for repeatability and the relative standard deviation value for intermediate precision were less than 2%, as shown in Table (4) and Table (5).

Table 4. A repeatability study for determining cefotaxime sodium using the developed microbiological assay.

Sample No.	Theoretical concentration (μg/ml)	Potency %	*Mean ±SD	RSD%
1	0.65	98.7	99.8 ± 1.48	1.47
2		98.4
3		101
4		101.5
5		98.4
6		101

*Average of six assays (n = 6).

Table 5. Intermediate precision study for determining cefotaxime sodium using the developed microbiological assay.

Proposed method	Potency%	*Mean ±SD	*RSD%
Day 1	100.01	100.27±1.79	1.79
Day 2	100.53	100.27±1.79	1.79

* Average of 12 assays.

Robustness:

The robustness of the proposed method was assessed by making some modifications to the following specified experimental conditions: diluent (double-distilled water), incubation temperature (34°C), and incubation duration (14 h). The results obtained showed no significant differences in efficacy, and all relative standard deviation values were less than 2% as shown in Table (6).

Table 6. Robustness results for the developed microbiological assay of cefotaxime sodium.

Factors	Parameters	*Potency%	RSD%
Diluent	Distilled water	99.2	1.5
Incubation tempreature	34˚C	100.1	1.3
Incubation duration	14 h	100.5	0.8

* The value is the Average of three assays.

Application to pharmaceutical formulations for quality control:

Five local commercial preparations and one brand preparation of cefotaxime sodium were assayed using the developed microbiological assay. Each assay used three plates, and the assay was repeated three times. Equations (1), (3), (4), and (5), previously mentioned, were then applied to correct the mean of inhibition zone and calculate the potency of the cefotaxime sodium. The potency in all studied samples was within the range specified by the United States Pharmacopeia (90-115%), as shown in Table (7).

Table 7. Results of assay of cefotaxime sodium in its pharmaceutical preparations using the developed microbiological assay.

*Commercial preparation	Assay No.	Corrected mean of inhibition zone (mm)	C Found (µg/ml)	Potency %	Mean Potency% ±SD	RSD%
A	1	16.897	0.613	94.3	94.93±0.554	0.58
	2	16.93	0.618	95.1
	3	16.964	0.62	95.4
B	1	17.364	0.665	102.3	101.73±0.621	0.61
	2	17.331	0.662	101.8
	3	17.297	0.657	101.1
C	1	17	0.628	96.6	96.66±0.54	0.55
	2	17.063	0.625	96.2
	3	17.097	0.632	97.2
D	1	17.097	0.635	97.7	97.9±0.789	0.8
	2	17.07	0.632	97.2
	3	17.164	0.642	98.8
E	1	17.131	0.638	98.2	99.16±1.194	1.2
	2	17.161	0.642	98.8
	3	17.263	0.653	100.5
F	1	17.397	0.668	102.8	103.03±0.32	0.31
	2	17.403	0.669	102.9
	3	17.431	0.672	103.4

* A, B, C, D, E are local commercial preparations, and F is brand preparation.

Study the microbiological equivalence:

Statistical analysis showed a significant difference (sig < 0.05) in efficacy between the brand-name preparation and the local preparations. The brand-name preparation showed the highest efficacy (103.03%), while local preparation A showed the lowest (94.93%). Therefore, the brand-name preparation is preferred. However, the local preparations can be used as acceptable alternatives because they are cheaper, and the efficacy of all preparations fell within the acceptable range (90-115%) defined by the United States Pharmacopeia.

CONCLUSION:

Selecting an appropriate analytical method for quality control of pharmaceutical preparations is crucial. This depends on several factors, including the drug substance to be analyzed and the available resources in terms of equipment, reagents, and materials required for the analysis. Since the drug to be analyzed in this research, within its pharmaceutical preparations, is cefotaxime, a bactericidal antibiotic, one of the best methods to determine its efficacy is microbiological assay. This method is based on the principle of measuring the ability of the antibiotic to inhibit the microorganisms growth, which is manifested by the formation of zones of inhibition under well-controlled experimental conditions.

What distinguishes microbiological assay from physicochemical methods is its ability to determine both the quantity and biological activity of the antibiotic, whereas physiochemical methods determine only the quantity. Since none of the pharmacopeias or previous studies mentioned a microbiological assay method for cefotaxime sodium, we developed a simple, low-cost microbiological assay and verified its validity following ICH guidelines.

When the developed method was applied to the branded preparation and the local preparations, it was found that all possessed acceptable efficacy according to the United States Pharmacopeia. Therefore, the local preparations can be used as a cost-effective and acceptably effective alternative. According to the statistical analysis, the branded preparation showed higher efficacy than the local preparations. Thus, local pharmaceutical companies must implement stricter manufacturing practices to reach the quality level of the branded preparation.

INTEREST CONFLICTS:

The authors declare no conflicts of interest.

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Received on 30.05.2025 Revised on 11.10.2025

Accepted on 18.12.2025 Published on 05.06.2026

Available online from June 06, 2026

Research J. Pharmacy and Technology. 2026;19(6):2636-2642.

DOI: 10.52711/0974-360X.2026.00377

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Dosage form	C taken (µg/ml)	C added%	C found ±SD*	Potency%	Mean Potency%
Cefotaxime vial	0.416	80	0.741±0.033	98.9	99.4
		100	0.813±0.035	97.7
		120	0.93±0.046	101.6