RP-HPLC Method Development and Validation for The Estimation of Metronidazole and Ciprofloxacin in Tablets

Metronidazole and Ciprofloxacin are widely used antimicrobial agents employed either alone or in combination for the treatment of various bacterial and protozoal infections. Metronidazole, a nitroimidazole derivative, exhibits potent activity against anaerobic bacteria and protozoa by disrupting microbial DNA synthesis. Ciprofloxacin, a second-generation fluoroquinolone antibiotic, exerts its antibacterial effect through inhibition of bacterial DNA gyrase and topoisomerase IV, resulting in the suppression of DNA replication and cell division. The combination of these drugs is frequently prescribed for the management of mixed aerobic and anaerobic infections, gastrointestinal infections, intra-abdominal infections, and gynecological infections.^1-3

The increasing use of combination pharmaceutical formulations necessitates the development of reliable analytical methods for their routine quality control and regulatory compliance. Accurate quantification of active pharmaceutical ingredients in combined dosage forms is essential to ensure product efficacy, safety, and consistency. Various analytical techniques, including spectrophotometry, HPLC, HPTLC, and LC-MS, have been reported for the estimation of Metronidazole and Ciprofloxacin individually or in combination with other drugs. However, there remains a need for simple, rapid, precise, and cost-effective methods suitable for routine analysis in quality control laboratories.^4-5

Reverse-phase high-performance liquid chromatography (RP-HPLC) is one of the most widely employed analytical techniques in pharmaceutical analysis due to its high sensitivity, selectivity, accuracy, and reproducibility. Method validation according to the International Council for Harmonisation (ICH) guidelines is essential to demonstrate the suitability of the developed analytical procedure for its intended purpose.^6-9

Therefore, the present study was undertaken to develop and validate a simple, accurate, precise, robust, and economical RP-HPLC method for the simultaneous estimation of Metronidazole and Ciprofloxacin in tablet dosage forms. The developed method was validated in accordance with ICH guidelines and successfully applied to the analysis of marketed pharmaceutical formulations.

MATERIALS AND METHODS:

MATERIALS:

Metronidazole and Ciprofloxacin reference standards were procured from Yarrow Chem Products and Trimurti Drug Supplier, Surat, Gujarat, respectively. HPLC-grade methanol and acetonitrile used as mobile phase components were obtained from Advent. Milli-Q water of HPLC grade was used throughout the study. Hydrochloric acid (HCl) and sodium hydroxide (NaOH), employed for pH adjustment and stress degradation studies, were procured from Dipa Chemical Industry and were of EP grade. All chemicals and reagents utilized in the present investigation were of analytical or HPLC grade and were used without further purification.

Characterization of Drugs

Organoleptic Evaluation

The obtained samples of Metronidazole and Ciprofloxacin were subjected to organoleptic evaluation. The drugs were examined visually for their color, odor, appearance, and other physical characteristics to confirm their identity and purity.¹⁰

Physical Characterization

The melting points of Metronidazole and Ciprofloxacin were determined using the open capillary method and recorded as uncorrected values. Solubility studies were performed in various solvents, including water, phosphate buffer, ethanol, methanol, and acetonitrile, to identify suitable solvents for analytical method development and ensure drug stability.^11-12

Fourier Transform Infrared (FT-IR) Spectroscopy

The identity and purity of Metronidazole and Ciprofloxacin were confirmed using Fourier Transform Infrared (FT-IR) spectroscopy. The infrared spectra of both drugs were recorded using an FTIR-4600 spectrophotometer (Jasco, Japan), and the characteristic absorption peaks were compared with reported reference values.^13-15

Differential Scanning Calorimetry (DSC)

Thermal analysis of Metronidazole and Ciprofloxacin was carried out using Differential Scanning Calorimetry (DSC) (PerkinElmer, San Jose, CA, USA). Samples were scanned over an appropriate temperature range under a nitrogen atmosphere at a controlled heating rate to evaluate their thermal behavior and melting characteristics.^16-17

Spectroscopic Analysis

Determination of λmax

Prior to RP-HPLC method development, UV spectroscopic studies were performed to determine the maximum absorbance wavelength (λmax) of Metronidazole and Ciprofloxacin. Standard solutions of both drugs were scanned in the wavelength range of 200–400 nm using a UV-Visible spectrophotometer, and the wavelengths showing maximum absorbance were selected for further analysis.^18-20

Preparation of Calibration Curve

Standard stock solutions of Metronidazole and Ciprofloxacin were prepared by accurately weighing 10 mg of each drug and dissolving separately in Methanol:Acetonitrile (70:30 v/v). The solutions were sonicated, filtered through a 0.45 µm membrane filter, and further diluted to obtain concentration ranges of 5–25 µg/mL for Metronidazole and 4–20 µg/mL for Ciprofloxacin. The absorbance of these solutions was measured, and calibration curves were constructed by plotting absorbance against concentration.^21-22

Instrumentation

UV spectroscopic measurements were carried out using a Lasany LI-2702 double-beam UV-Visible spectrophotometer equipped with matched 1 cm quartz cells. Drug samples were accurately weighed using an Elder digital analytical balance, and solution preparation was facilitated using a Prama ultrasonic sonicator.²³

UV Method Development and Validation

A suitable solvent system for UV spectrophotometric analysis was selected based on the solubility of Metronidazole and Ciprofloxacin in various solvents, including distilled water, ethanol, methanol, acetonitrile, phosphate buffer, and their combinations. Among the tested systems, Methanol:Acetonitrile (70:30 v/v) provided satisfactory solubility and stability for both drugs and was therefore selected as the common solvent for further studies.

The developed UV method was validated according to ICH guidelines with respect to linearity, accuracy, precision, sensitivity, robustness, and ruggedness. Linearity was evaluated by preparing standard solutions in the concentration range of 5–25 µg/mL for Metronidazole and 4–20 µg/mL for Ciprofloxacin, and calibration curves were constructed by plotting absorbance against concentration.

The sensitivity of the method was determined by calculating the limit of detection (LOD) and limit of quantification (LOQ) using the standard deviation of the response and the slope of the calibration curve. Accuracy was assessed by recovery studies using the standard addition method at 80%, 100%, and 120% levels, and the percentage recovery was calculated.

Precision was evaluated in terms of repeatability (intra-day precision) and intermediate precision (inter-day precision) by analyzing selected concentrations of both drugs on the same day and on different days. The results were expressed as percentage relative standard deviation (%RSD). Robustness of the method was examined by introducing small deliberate variations in analytical conditions, while ruggedness was assessed by evaluating the reproducibility of the method under different operating conditions. The obtained results demonstrated the reliability and suitability of the developed UV spectrophotometric method for the simultaneous estimation of Metronidazole and Ciprofloxacin.^23-25

RP-HPLC Method Development

RP-HPLC analysis was carried out using a Shimadzu LC-2010 series HPLC system equipped with an autosampler and UV-Visible detector. Chromatographic separation was performed on an Inertsil ODS-3V C18 column (250 × 4.6 mm, 5 μm particle size). Standard and sample solutions were prepared using HPLC-grade solvents and filtered before analysis.

The detection wavelength was selected by scanning individual solutions of Metronidazole and Ciprofloxacin in the UV range of 200–400 nm. Based on the overlay spectra, 250 nm was chosen as the optimum wavelength for simultaneous estimation of both drugs. Several mobile phase compositions comprising methanol, acetonitrile, and water were evaluated to achieve satisfactory peak resolution and system suitability.

After optimization, the chromatographic separation was achieved using a mobile phase consisting of Methanol:Acetonitrile:Water (50:30:20, v/v/v) at a flow rate of 1.0 mL/min under isocratic conditions. The mobile phase was filtered through a 0.45 μm membrane filter and degassed by ultrasonication before use. Prior to sample analysis, the HPLC system was primed and the column was conditioned to ensure stable chromatographic performance and a consistent baseline.^26-28

Standard stock solutions

of Metronidazole and Ciprofloxacin were prepared in the mobile phase and subsequently diluted to the required concentrations. The prepared solutions were filtered through a 0.20 μm nylon membrane filter, sonicated, and injected into the HPLC system. Chromatograms were recorded under the optimized chromatographic conditions and evaluated for peak characteristics, retention time, and system suitability parameters.^29-30

RP-HPLC Method Validation^31-38

System Suitability and Specificity

The suitability of the RP-HPLC system was evaluated by analyzing replicate injections of standard solutions and assessing chromatographic parameters including retention time, peak area, theoretical plates, tailing factor, and resolution. Specificity of the method was established by demonstrating complete separation of Metronidazole and Ciprofloxacin without interference from tablet excipients or other components present in the sample matrix.

Linearity and Sensitivity

The linearity of the method was assessed over concentration ranges of 50–150 µg/mL for Metronidazole and 20–60 µg/mL for Ciprofloxacin. Calibration curves were constructed by plotting peak area against concentration, and the corresponding regression equations and correlation coefficients were determined. The sensitivity of the method was evaluated by calculating the limit of detection (LOD) and limit of quantification (LOQ) using the standard deviation of the response and the slope of the calibration curve.

Accuracy and Precision

The accuracy of the developed method was determined by recovery studies using the standard addition technique at 80%, 100%, and 120% concentration levels. Precision was evaluated in terms of repeatability (intra-day precision) and intermediate precision (inter-day precision) by analyzing replicate samples and expressing the results as percentage relative standard deviation (%RSD). The obtained results demonstrated the reliability and reproducibility of the method.

Robustness and Ruggedness

Robustness was assessed by introducing small deliberate variations in chromatographic conditions, such as flow rate, detection wavelength, and mobile phase composition, and evaluating their effect on chromatographic performance. Ruggedness was determined by examining the reproducibility of the analytical results under different operating conditions. The validation results confirmed that the developed RP-HPLC method was accurate, precise, sensitive, robust, and suitable for the routine quantitative estimation of Metronidazole and Ciprofloxacin in tablet dosage forms.

Stability Studies       

Accelerated stability studies were conducted in accordance with ICH Q1A(R2) guidelines to evaluate the stability of Metronidazole and Ciprofloxacin tablets and to establish the stability-indicating nature of the developed RP-HPLC method. The marketed tablet formulation was stored under accelerated conditions of 40 ± 2°C and 75 ± 5% relative humidity for a period of three months. Samples were withdrawn at predetermined intervals and analyzed for physical appearance and drug content.

The collected samples were prepared using the optimized analytical procedure and analyzed under the established chromatographic conditions. Assay values and chromatographic characteristics were monitored throughout the study period. No significant changes in drug content or physical appearance were observed, indicating adequate stability of the formulation. The results demonstrated the suitability of the developed RP-HPLC method for routine stability testing and quality control applications.

Analysis of Marketed Tablet Formulation

The validated RP-HPLC method was successfully applied to the quantitative estimation of Metronidazole and Ciprofloxacin in a marketed tablet formulation containing 500 mg of each drug. Tablet samples were accurately weighed, powdered, and extracted using the optimized mobile phase. The resulting solution was sonicated, filtered, and suitably diluted to obtain concentrations within the analytical range of the method.

The prepared sample solutions were injected into the RP-HPLC system under the optimized chromatographic conditions. Peak areas of Metronidazole and Ciprofloxacin were measured and compared with those obtained from corresponding standard solutions. The assay results confirmed satisfactory drug content and demonstrated the applicability of the developed method for routine quality control analysis of combined tablet dosage forms containing Metronidazole and Ciprofloxacin.

RESULTS AND DISCUSSION:

Organoleptic Evaluation

The organoleptic properties of Metronidazole and Ciprofloxacin were evaluated for preliminary identification and quality assessment. Both drugs appeared as pale yellow, fine crystalline powders, were odorless, and exhibited a characteristic bitter taste. The observed properties were found to be in agreement with the reported specifications, confirming the authenticity and purity of the drug samples.

Table 1. Organoleptic Properties of Metronidazole and Ciprofloxacin

Physical Characterization

Melting Point Determination

The observed melting points of Metronidazole and Ciprofloxacin were found to be in close agreement with the reported values, indicating the purity and identity of the drug samples.

Table 2. Melting Point of Metronidazole and Ciprofloxacin

Solubility Studies

Solubility studies were performed in various solvents to identify a suitable solvent system for analytical method development. Both drugs exhibited good solubility in methanol and poor solubility in water, supporting the selection of organic solvents for further RP-HPLC studies.

Table 3. Solubility Profile of Metronidazole and Ciprofloxacin

The results of organoleptic evaluation, melting point determination, and solubility studies confirmed the identity, purity, and suitability of Metronidazole and Ciprofloxacin for subsequent method development and validation studies.

FT-IR spectroscopy study:

Identification of Metronidazole and Ciprofloxacin was confirmed by FTIR Spectra. All peaks was found in Metronidazole and Ciprofloxacin drugs.

 

 

Figure 1: FTIR spectrum of Metronidazole

 

 

Figure 2: FTIR spectrum of Ciprofloxacin

 

 

Figure 3: DSC Thermogram of MNZ and CFC

UV analysis:

 

 

Figure 4: λmax of drug MNZ and CFC

 

 

Figure 5: Mobile phase optimization (trial 1) - Acetonitrile: Water (80:20 v/v)

 

 

Figure 6: Mobile phase optimization (optimized trial: Acetonitrile: Methanol: Water (50:30:20 v/v)

 

 

Figure 7: Standard Chromatogram of Ciprofloxacin

 

 

Figure 8: Standard Chromatogram of Metronidazole

 

 

Figure 9: Standard Chromatogram of Mixture of Ciprofloxacin and Metronidazole

 

 

Figure 10 : Blank Chromatogram for Specificity

 

 

Figure 11: Purity chromatogram of Standard CFC and MNZ

 

 

Figure 12: Purity chromatogram of Sample of CFC and MNZ

Table 14. Linearity and Regression Data

Parameter	Ciprofloxacin	Metronidazole
Linearity Range (μg/mL)	20–60	50–150
Slope	45701	246911
Intercept	73433	96879
Correlation Coefficient (R²)	0.9997	0.9992

 

 

Figure 13: Plot of linearity study for Metronidazole

 

 

Figure 14: Plot of linearity study for Ciprofloxacin

 

 

Figure 15: Analysis of Marketed formulation (Ciprodiazole tablets, Minapharm Pharmaceuticals)

CONCLUSION

A simple, rapid, accurate, and stability-indicating RP-HPLC method was successfully developed and validated for the simultaneous estimation of Metronidazole and Ciprofloxacin in tablet dosage forms. The optimized chromatographic conditions provided satisfactory separation with good peak symmetry, resolution, and reproducibility. Validation studies performed according to ICH guidelines confirmed that the method is specific, linear, precise, accurate, sensitive, robust, and rugged. The developed method exhibited excellent recovery values and low %RSD values, demonstrating its reliability for quantitative analysis.

The method was effectively applied to the analysis of marketed Ciprodiazole tablets, and the assay results were found to be within acceptable pharmacopoeial limits. Accelerated stability studies further established the stability-indicating capability of the method, as no significant degradation or changes in chromatographic performance were observed during the study period. Therefore, the proposed RP-HPLC method can be successfully employed for routine quality control, assay determination, and stability testing of pharmaceutical formulations containing Metronidazole and Ciprofloxacin.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest regarding the publication of this research work.

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