Stability Indicating RP-HPLC Method Development and Validation for Ceftriaxone and Sulbactam

Ceftriaxone Sodium is a third-generation cephalosporin antibiotic widely used for the treatment of various bacterial infections due to its broad-spectrum antibacterial activity and high therapeutic efficacy. It acts by inhibiting bacterial cell wall synthesis, leading to bacterial cell death. Sulbactam Sodium is a β-lactamase inhibitor that enhances the antibacterial activity of β-lactam antibiotics by preventing enzymatic degradation caused by β-lactamase-producing microorganisms. The combination of Ceftriaxone Sodium and Sulbactam Sodium is extensively used in the treatment of severe respiratory tract infections, urinary tract infections, skin infections, septicemia, meningitis, and other bacterial diseases caused by resistant pathogens.

The increasing therapeutic use of combined injectable formulations containing Ceftriaxone Sodium and Sulbactam Sodium necessitates the development of reliable analytical methods for their simultaneous estimation in pharmaceutical dosage forms. Analytical methods employed for routine quality control should be accurate, precise, specific, rapid, and capable of distinguishing the active pharmaceutical ingredients from degradation products and formulation excipients. Stability-indicating analytical methods are particularly important because they provide information regarding the stability profile of drugs under various stress conditions and ensure the safety, efficacy, and quality of pharmaceutical formulations throughout their shelf life.

Reverse Phase High Performance Liquid Chromatography (RP-HPLC) is one of the most widely used analytical techniques in pharmaceutical analysis owing to its high sensitivity, selectivity, reproducibility, and suitability for simultaneous estimation of multiple analytes. Although several analytical methods have been reported for individual estimation of Ceftriaxone Sodium and Sulbactam Sodium, limited studies are available on simple and validated stability-indicating RP-HPLC methods for their combined estimation in pharmaceutical dosage forms.

Therefore, the present study was undertaken to develop and validate a simple, precise, accurate, robust, and stability-indicating RP-HPLC method for the simultaneous estimation of Ceftriaxone Sodium and Sulbactam Sodium in bulk and marketed formulations in accordance with ICH guidelines. The developed method was further evaluated for various validation parameters and applied for routine pharmaceutical analysis and stability studies.

MATERIALS AND METHODS:

Materials and Reagents:

The materials and instruments used in the present study were selected to ensure accuracy and reproducibility of the developed stability indicating RP-HPLC method for Ceftriaxone and Sulbactam. All solvents and chemicals employed during the analysis were of HPLC or Analytical Reagent (AR) grade. Ceftriaxone was procured from SM Pharma & Company Ltd., Pune, while Sulbactam was obtained from Swapnroop Drugs & Pharmaceuticals. Methanol and acetonitrile of HPLC grade were purchased from Advent, whereas potassium dihydrogen phosphate, orthophosphoric acid, and hydrogen peroxide were obtained from Merck. Hydrochloric acid and sodium hydroxide were procured from Dipa Chemical Industry. Milli-Q water was used throughout the analytical work. All analytical instruments were calibrated prior to use to ensure reliability and precision of the experimental results.

METHODOLOGY:

Characterization of Drugs

Ceftriaxone sodium and Sulbactam sodium were characterized prior to method development to confirm their identity, purity, and physicochemical properties. The characterization studies included organoleptic evaluation, melting point determination, solubility studies, FT-IR analysis, DSC analysis, and UV spectroscopic studies.

Organoleptic Evaluation

The drugs were visually examined for color, appearance, texture, and odor under normal laboratory conditions. The observed characteristics were compared with standard pharmacopeial descriptions to confirm the identity and quality of the drugs.

Melting Point Determination

The melting point of Ceftriaxone sodium and Sulbactam sodium was determined using the capillary tube method. Finely powdered drug samples were filled in sealed capillary tubes and analyzed using a digital melting point apparatus. The observed melting points were compared with reported values to assess purity and identity.

Solubility Studies

Solubility studies were carried out in various solvents including distilled water, methanol, ethanol, acetonitrile, and phosphate buffer (pH 6.8). Excess quantity of each drug was added to the solvents and allowed to equilibrate for 24 h at room temperature. The solubility behavior was visually observed and recorded.

FT-IR Spectroscopic Analysis

FT-IR spectroscopy was performed to identify characteristic functional groups of Ceftriaxone sodium and Sulbactam sodium. Drug samples were mixed with potassium bromide and compressed into pellets, and the spectra were recorded in the range of 4000–400 cm⁻¹. The obtained spectra were compared with standard reference spectra for confirmation of drug identity.

Differential Scanning Calorimetry (DSC)

DSC analysis was carried out to study the thermal behavior and crystallinity of both drugs. Accurately weighed samples were sealed in aluminum pans and heated over a temperature range of 30 °C to 300 °C under nitrogen atmosphere. Thermograms were analyzed for thermal transitions and drug stability.

UV–Visible Spectroscopic Analysis

UV spectroscopic analysis was performed to determine the absorption characteristics of Ceftriaxone sodium and Sulbactam sodium. Drug solutions were prepared and scanned in the wavelength range of 200–400 nm using a UV–Visible spectrophotometer to identify the wavelength of maximum absorbance (λmax).

Determination of λmax

Standard stock solutions of Ceftriaxone sodium and Sulbactam sodium were prepared separately and further diluted to obtain suitable concentrations. The prepared solutions were scanned between 200–400 nm against distilled water as blank, and the wavelength showing maximum absorbance was selected as λmax for further analytical studies.

Preparation of Calibration Curve

Calibration curves for both drugs were prepared using suitable serial dilutions from the stock solutions. The absorbance of each concentration was measured at the respective λmax, and calibration curves were plotted between concentration and absorbance. Regression equations and correlation coefficients were calculated to evaluate method linearity.

RP-HPLC Analysis

Chromatographic analysis was performed using a Shimadzu RP-HPLC system equipped with a quaternary pump, manual injector, UV detector, and LC-solution software. Separation was achieved using an Inertsil ODS C18 column (150 mm × 4.6 mm, 3.5 µm). Auxiliary instruments including a digital pH meter, ultrasonic bath sonicator, vacuum filtration assembly, and 0.45 µm membrane filters were used during analysis.

Method Development

The RP-HPLC method was developed by optimizing chromatographic parameters such as mobile phase composition, pH, flow rate, detection wavelength, and injection volume to obtain satisfactory separation and peak symmetry for Ceftriaxone sodium and Sulbactam sodium. Different combinations of aqueous buffers and organic solvents were evaluated during optimization trials.

Selection of Wavelength

The analytical wavelength was selected by scanning standard solutions of Ceftriaxone sodium and Sulbactam sodium in the range of 200–400 nm using a UV–Visible spectrophotometer. Based on overlay spectra, 254 nm was selected as the detection wavelength as both drugs exhibited appreciable absorbance at this wavelength.

Optimization of Mobile Phase

Various combinations of phosphate buffer with methanol and acetonitrile were evaluated to achieve good resolution and peak symmetry. The optimized mobile phase consisted of phosphate buffer (pH 3.5) and acetonitrile in the ratio of 60:40 v/v, which provided satisfactory chromatographic performance and shorter retention time.

Preparation of Mobile Phase

The phosphate buffer was prepared using potassium dihydrogen phosphate in Milli-Q water and the pH was adjusted to 3.5 with orthophosphoric acid. The buffer was mixed with acetonitrile in the ratio of 60:40 v/v, filtered through a 0.45 µm membrane filter, and sonicated prior to use.

Preparation of Standard Stock Solution

Standard stock solutions were prepared separately by dissolving 10 mg of Ceftriaxone sodium and Sulbactam sodium in 10 mL of mobile phase to obtain concentrations of 1000 µg/mL. Further dilutions were prepared using the mobile phase to obtain working standard solutions for analysis.

Degassing and Filtration of Mobile Phase

The prepared mobile phase was degassed using an ultrasonic bath sonicator for 15 min to remove dissolved gases. The mobile phase was further filtered through a 0.45 µm membrane filter under vacuum to remove particulate matter before chromatographic analysis.

Optimized Chromatographic Conditions

The optimized chromatographic separation was achieved using an Inertsil ODS C18 column with phosphate buffer (pH 3.5) and acetonitrile (60:40 v/v) as the mobile phase under isocratic elution mode. The flow rate was maintained at 1.0 mL/min with detection at 254 nm and injection volume of 20 µL. The total run time was 10 min at ambient temperature.

Chromatographic conditions were optimized to develop a simple, accurate, precise, and stability-indicating RP-HPLC method for simultaneous estimation of Ceftriaxone sodium and Sulbactam sodium. Different mobile phase compositions, pH conditions, flow rates, and detection wavelengths were evaluated to achieve satisfactory resolution, peak symmetry, and reproducibility. The optimized chromatographic conditions were selected based on system suitability parameters.

Method Validation

System Suitability Study

System suitability testing was carried out before analysis to verify the performance of the chromatographic system. Mixed standard solutions were injected repeatedly under optimized chromatographic conditions, and parameters such as retention time, peak area, theoretical plates, tailing factor, and resolution were evaluated to ensure acceptable chromatographic performance.

Specificity

Specificity of the developed method was evaluated by analyzing blank, placebo, standard, and sample solutions individually. The chromatograms were examined for interference at the retention times of Ceftriaxone sodium and Sulbactam sodium. The absence of interfering peaks confirmed the specificity of the method.

Precision

Precision of the developed RP-HPLC method was evaluated in terms of system precision, method precision, intraday precision, and inter-day precision. Repeated injections of standard and sample solutions were analyzed, and the percentage relative standard deviation (% RSD) values were calculated to assess repeatability and reproducibility of the method under the same and varied analytical conditions.

Accuracy

Accuracy of the method was assessed by recovery studies using the standard addition technique at different concentration levels. Known quantities of standard drugs were added to pre-analyzed sample solutions and analyzed in triplicate. The percentage recovery values obtained confirmed the accuracy of the developed method.

Linearity and Range

Linearity of the method was established by preparing standard solutions at different concentration levels for both drugs. The prepared solutions were analyzed under optimized chromatographic conditions, and calibration curves were plotted between concentration and peak area. The correlation coefficient values demonstrated good linearity within the selected concentration range.

Stability in Analytical Solution

The stability of analytical solutions was evaluated by analyzing prepared sample solutions immediately after preparation and after storage under room temperature and refrigerated conditions. The assay values obtained confirmed the stability of Ceftriaxone sodium and Sulbactam sodium in solution during the analysis period.

Limit of Detection and Limit of Quantification

The sensitivity of the developed RP-HPLC method was determined by evaluating the limit of detection (LOD) and limit of quantification (LOQ) based on calibration data and analytical response. The obtained values indicated the suitability of the method for detection and quantification of both drugs at low concentrations.

Robustness

Robustness of the method was evaluated by introducing small deliberate variations in chromatographic conditions such as flow rate. The effect of these changes on system suitability parameters and assay values was studied to assess the reliability of the method under varied analytical conditions.

Ruggedness

Ruggedness studies were carried out by performing the analysis under different laboratory conditions and temperature variations. The obtained chromatographic responses and % RSD values confirmed the reproducibility of the developed method under varied environmental conditions.

Forced Degradation Studies

Forced degradation studies were performed under acidic, alkaline, oxidative, thermal, and photolytic stress conditions to evaluate the stability-indicating capability of the developed RP-HPLC method. The stressed samples were analyzed chromatographically, and degradation behavior was assessed by observing degradation peaks and peak purity.

Photolytic Degradation Study

Photolytic degradation studies were conducted by exposing mixed standard drug solutions to UV light and direct sunlight for a specified period. The exposed samples were analyzed under optimized chromatographic conditions to evaluate the degradation pattern and stability of the drugs under light exposure.

Analysis of Marketed Formulation

The developed RP-HPLC method was applied for analysis of a marketed injectable formulation containing Ceftriaxone sodium and Sulbactam sodium. The formulation sample was prepared using the mobile phase, filtered, diluted appropriately, and analyzed under optimized chromatographic conditions to evaluate the applicability of the method for routine quality control analysis.

RESULTS AND DISCUSSION:

Characterization of Drugs

RP-HPLC method development was carried out for simultaneous estimation of ceftriaxone sodium and sulbactam sodium. Preliminary trials evaluated different mobile phases, column conditions, flow rates and wavelengths. The ultimate chromatographic parameters were determined based on peak symmetry, resolution, theoretical plates, and retention time reproducibility.

The drugs were characterized before chromatographic method development to verify identity, purity, and suitability for analysis. Organoleptic examination, melting point, solubility, Fourier-transform infrared spectroscopy, differential scanning calorimetry, ultraviolet-visible spectroscopy, and calibration studies were performed for ceftriaxone sodium and sulbactam sodium.

Organoleptic Characteristics

The organoleptic properties of ceftriaxone sodium and sulbactam sodium were examined and compared with reported descriptions. Both samples were white to yellowish or white to off-white powders, odourless, and slightly bitter. These observations supported preliminary identification and showed that the samples were free from visible contamination.

Melting Point Determination

The melting point determination was carried out to assess the purity and thermal behavior of Ceftriaxone sodium and Sulbactam sodium. The observed melting point ranges were sharp and consistent, indicating the absence of significant impurities. The experimentally determined values were found to be in close agreement with reported literature values, thereby confirming the identity and purity of both drugs.

Solubility Studies

The melting point determination was carried out to assess the purity and thermal behavior of Ceftriaxone sodium and Sulbactam sodium. The observed melting point ranges were sharp and consistent, indicating the absence of significant impurities. The experimentally determined values were found to be in close agreement with reported literature values, thereby confirming the identity and purity of both drugs.

Figure 1: FTIR spectrum of Ceftriaxone Sodium

Figure 2: FTIR spectrum of Sulbactam Sodium

Figure 3: Maximum wavelength detection of Ceftriaxone

Figure 4: Maximum wavelength detection of Sulbactam

Figure 5: Calibration Curve of Ceftriaxone Sodium

Figure 6: Calibration Curve of Sulbactam Sodium

Figure 7: Optimized RP-HPLC chromatogram of Ceftriaxone Sodium and Sulbactam Sodium

Figure 9.14: Calibration curve of Ceftriaxone Sodium

Figure 9.15: Calibration curve of Sulbactam Sodium

CONCLUSION:

In conclusion, a new stability-indicating RP-HPLC method was developed and validated for concurrent assessment of ceftriaxone sodium and sulbactam sodium in bulk and combined injectable dosage form. The technique is straightforward, swift, exact, and meticulous, sensitive, robust, and rugged, and it fulfils ICH Q2(R1) validation requirements. Validation results show that this procedure is appropriate for standard quality control, stability assessment, and regulatory compliance. analysis of ceftriaxone sodium and sulbactam sodium pharmaceutical products. Its successful application to a marketed formulation confirms practical and industrial usefulness. Overall, the study provides a scientifically sound and pharmaceutically relevant analytical approach for concurrent quantification of ceftriaxone sodium and sulbactam sodium in quality-assurance laboratories, improving analytical efficiency and supporting dependable therapeutic quality control.

CONFLICT OF INTERESTS:

The authors declare that there are no conflicts of interest.

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