Development & Validation of Stability Indicating RP-HPLC Method for Simultaneous Estimation of Mebendazole and Ivermectin in Pharmaceutical Dosage Form

Mebendazole (MBZ) and Ivermectin (IVM) are widely used antiparasitic agents employed in the treatment of various helminthic and parasitic infections. Mebendazole belongs to the benzimidazole class of anthelmintics and exerts its pharmacological action by inhibiting microtubule synthesis in parasitic worms, thereby disrupting glucose uptake and leading to parasite death. Ivermectin, a macrocyclic lactone derivative, acts by binding to glutamate-gated chloride ion channels in parasites, causing paralysis and elimination of the organism. The combination of these two drugs has gained significant therapeutic importance due to their broad-spectrum antiparasitic activity and improved treatment outcomes.

The increasing use of fixed-dose combination (FDC) formulations containing Mebendazole and Ivermectin necessitates the development of reliable analytical methods for their simultaneous quantification. Accurate determination of active pharmaceutical ingredients in combined dosage forms is essential to ensure product quality, safety, efficacy, and regulatory compliance. Analytical methods used for routine quality control must be capable of providing precise, accurate, and reproducible results while effectively separating the analytes from excipients, impurities, and degradation products.

High Performance Liquid Chromatography (HPLC) is one of the most widely accepted analytical techniques for pharmaceutical analysis due to its high sensitivity, selectivity, precision, and versatility. Among various chromatographic approaches, Reverse Phase High Performance Liquid Chromatography (RP-HPLC) has become the method of choice for the analysis of pharmaceutical compounds because of its excellent separation efficiency and suitability for compounds with diverse physicochemical properties. Furthermore, regulatory authorities recommend the use of stability-indicating analytical methods capable of detecting changes in drug quality during storage and handling.

Method validation is an essential requirement in pharmaceutical analysis to demonstrate the suitability of an analytical procedure for its intended purpose. According to International Council for Harmonisation (ICH) guidelines, validation parameters such as specificity, linearity, accuracy, precision, robustness, limit of detection (LOD), and limit of quantification (LOQ) must be evaluated to establish the reliability of the developed method.

Therefore, the present study was undertaken to develop and validate a simple, rapid, accurate, precise, and stability-indicating RP-HPLC method for the simultaneous estimation of Mebendazole and Ivermectin in pharmaceutical dosage forms. The developed method was optimized and validated in accordance with ICH guidelines and is intended for routine quality control analysis and pharmaceutical research applications.

MATERIALS AND METHODS

Materials

Pharmaceutical-grade reference standards of Mebendazole and Ivermectin were obtained as gift samples from reputed pharmaceutical manufacturers. HPLC-grade acetonitrile and methanol were procured from Merck (India) Ltd. Potassium dihydrogen orthophosphate, orthophosphoric acid, hydrochloric acid, sodium hydroxide, and hydrogen peroxide of analytical reagent grade were used throughout the study. Milli-Q water was employed for the preparation of buffers and mobile phases. Commercial tablet formulations containing Mebendazole (100 mg) and Ivermectin (6 mg) were purchased from the local market and used for assay studies.

Instrumentation

Chromatographic analysis was performed using a Shimadzu Prominence RP-HPLC system equipped with a binary pump (LC-20AD), PDA detector (SPD-M20A), autosampler (SIL-20AC), online degasser (DGU-20A5R), and column oven (CTO-20AC). Data acquisition and processing were carried out using LabSolutions software.

Chromatographic Conditions

Separation was achieved on a Phenomenex Luna C18 column (250 mm × 4.6 mm, 5 µm particle size). The mobile phase consisted of 0.1 M potassium dihydrogen orthophosphate buffer (pH 3.5) and acetonitrile in the ratio of 45:55 (v/v). The mobile phase was filtered through a 0.45 µm membrane filter and degassed before use. Chromatographic analysis was carried out at a flow rate of 1.0 mL/min with isocratic elution. Detection was performed at 245 nm using a PDA detector, while the column temperature was maintained at 30 ± 1°C. The injection volume was 20 µL and the total run time was 10 min.

Preparation of Standard Solutions

Standard stock solutions of Mebendazole and Ivermectin were prepared separately by dissolving accurately weighed 10 mg of each drug in methanol and making the volume up to 10 mL to obtain a concentration of 1000 µg/mL. Appropriate aliquots of the stock solutions were further diluted with mobile phase to obtain mixed working standard solutions in the concentration range of 50–150 µg/mL for Mebendazole and 3–9 µg/mL for Ivermectin.

Preparation of Sample Solution

Twenty tablets were accurately weighed and finely powdered. An amount equivalent to 100 mg of Mebendazole and 6 mg of Ivermectin was transferred to a 100 mL volumetric flask, extracted with methanol by sonication for 20 min, and diluted to volume. The resulting solution was filtered and further diluted with mobile phase to obtain concentrations within the analytical range. Prior to injection, all sample solutions were passed through a 0.45 µm syringe filter.

Method Validation

The developed RP-HPLC method was validated according to ICH Q2 guidelines for system suitability, specificity, linearity, accuracy, precision, robustness, limit of detection (LOD), and limit of quantification (LOQ).

System Suitability: Six replicate injections of the mixed standard solution were analyzed and evaluated for retention time, theoretical plates, tailing factor, resolution, and %RSD.

Specificity: Blank, placebo, standard, sample, and degraded sample solutions were analyzed to assess potential interference at the retention times of Mebendazole and Ivermectin.

Linearity: Calibration curves were constructed over the concentration range of 50–150 µg/mL for Mebendazole and 3–9 µg/mL for Ivermectin. Regression analysis was performed to determine correlation coefficients.

Accuracy: Recovery studies were carried out by the standard addition method at 50%, 100%, and 150% concentration levels, and percentage recoveries were calculated.

Precision: Repeatability and intermediate precision studies were performed and expressed as percentage relative standard deviation (%RSD).

Robustness: The robustness of the method was evaluated by introducing small deliberate changes in chromatographic parameters, including flow rate (±0.1 mL/min), mobile phase composition (±2%), pH (±0.2), wavelength (±2 nm), and column temperature (±2°C).

LOD and LOQ: Sensitivity parameters were determined using the equations LOD = 3.3σ/S and LOQ = 10σ/S, where σ represents the standard deviation of the response and S is the slope of the calibration curve.

RESULTS AND DISCUSSION

3.1 Method Development and Optimization

The present study was undertaken to develop a simple, rapid, accurate, precise, robust, and stability-indicating RP-HPLC method for the simultaneous estimation of Mebendazole (MBZ) and Ivermectin (IVM) in pharmaceutical dosage forms. Method development was carried out by systematically optimizing chromatographic variables including detection wavelength, stationary phase, mobile phase composition, pH, flow rate, column temperature, and injection volume. The objective was to achieve satisfactory resolution, acceptable retention time, symmetrical peak shape, and reproducible chromatographic performance.

3.1.1 Selection of Detection Wavelength

Individual standard solutions of Mebendazole and Ivermectin were scanned in the UV region between 200 and 400 nm. The overlay spectra revealed that both analytes exhibited significant absorbance at 245 nm, making it suitable for simultaneous determination. The selected wavelength provided adequate detector response with minimal baseline noise and excellent sensitivity for both drugs.

 

 

Figure 1. UV Spectrum of Mebendazole

 

Figure 2. UV Spectrum of Ivermectin

 

Figure 4. Trial Chromatogram-I

 

Figure 6. Trial Chromatogram-III

 

Figure 8. Trial Chromatogram-V

Figure 9. Standard Chromatogram (Injection 1)

Figure 10. Standard Chromatogram (Injection 6)

 

Figure 11. Blank Chromatogram

Figure 12. Placebo Chromatogram

 

Figure 13. Standard Chromatogram

Figure 14. Sample Chromatogram

Figure 15. Peak Purity Plot of Mebendazole

Figure 16. Peak Purity Plot of Ivermectin

Parameter	Mebendazole	Ivermectin
Regression Equation	y = 18542x + 12456	y = 67284x + 3284
Correlation Coefficient (R²)	0.9997	0.9998
Linearity Range	50–150 µg/mL	3–9 µg/mL

 

Figure 17. Chromatogram of Mebendazole (50 µg/mL)

Figure 18. Chromatogram of Mebendazole (75 µg/mL)

Figure 19. Chromatogram of Mebendazole (100 µg/mL)

Figure 20. Chromatogram of Mebendazole (125 µg/mL)

Figure 21. Chromatogram of Mebendazole (150 µg/mL)

Figure 22. Calibration Curve of Mebendazole

Figure 23. Chromatogram of Ivermectin (3 µg/mL)

Figure 24. Chromatogram of Ivermectin (4.5 µg/mL)

Figure 25. Chromatogram of Ivermectin (6 µg/mL)

 

Figure 26. Chromatogram of Ivermectin (7.5 µg/mL)

Figure 27. Chromatogram of Ivermectin (9 µg/mL)

Figure 28. Calibration Curve of Ivermectin

Figure 29. Recovery Chromatogram at 50% Level

Figure 30. Recovery Chromatogram at 100% Level

Figure 31. Recovery Chromatogram at 150% Level

Figure 32. Overlay Chromatogram for Precision Study

Figure 33. Effect of Flow Rate Variation

Figure 34. Effect of Mobile Phase Variation

Figure 35. Effect of Buffer pH Variation

Figure 36. Effect of Detection Wavelength Variation

Figure 37. Effect of Column Temperature Variation

Figure 38. Sensitivity Comparison of Mebendazole and Ivermectin

The low LOD and LOQ values obtained for both analytes demonstrated the high sensitivity of the developed analytical method. The method was capable of detecting and quantifying very low concentrations of Mebendazole and Ivermectin with acceptable accuracy and precision. Such sensitivity makes the method suitable for routine quality control testing and stability studies.

3.9 Overall Discussion

The present investigation successfully established a simple, accurate, precise, robust, and stability-indicating RP-HPLC method for the simultaneous estimation of Mebendazole and Ivermectin in pharmaceutical dosage forms. Systematic optimization of chromatographic variables resulted in excellent chromatographic performance, with Mebendazole and Ivermectin eluting at retention times of 4.12 min and 6.85 min, respectively. Complete baseline separation was achieved with a resolution value greater than 3.8, demonstrating efficient chromatographic separation.

System suitability studies confirmed the adequacy and reproducibility of the chromatographic system, with theoretical plate counts exceeding 4500 and %RSD values below 1.0%. Validation studies performed according to ICH guidelines demonstrated excellent specificity, linearity, accuracy, precision, robustness, and sensitivity. The developed method exhibited outstanding linearity over the investigated concentration ranges with correlation coefficients greater than 0.999 for both analytes.

Accuracy studies produced recoveries close to 100%, while precision studies demonstrated excellent repeatability and intermediate precision with %RSD values well below the acceptable limit of 2.0%. Robustness testing further confirmed that minor variations in chromatographic conditions did not significantly influence analytical performance. The low LOD and LOQ values highlighted the high sensitivity of the method and its capability to detect trace quantities of the analytes.

The absence of interference from excipients and the excellent peak purity results confirmed the specificity and selectivity of the method. Overall, the developed RP-HPLC method fulfilled all validation requirements and can be successfully employed for routine quality control analysis, method validation studies, and pharmaceutical research involving simultaneous estimation of Mebendazole and Ivermectin in combined dosage forms.

CONCLUSION

The present study successfully developed and validated a simple, rapid, accurate, precise, robust, and stability-indicating RP-HPLC method for the simultaneous estimation of Mebendazole and Ivermectin in pharmaceutical dosage forms. Systematic optimization of chromatographic parameters resulted in efficient separation of both analytes using a Phenomenex Luna C18 column with a mobile phase consisting of 0.1 M potassium dihydrogen orthophosphate buffer (pH 3.5) and acetonitrile (45:55, v/v). Under the optimized chromatographic conditions, Mebendazole and Ivermectin were eluted at retention times of 4.12 min and 6.85 min, respectively, with excellent peak symmetry and satisfactory resolution.

The developed method was validated in accordance with ICH Q2 guidelines and demonstrated excellent performance with respect to system suitability, specificity, linearity, accuracy, precision, robustness, limit of detection, and limit of quantification. The calibration curves exhibited outstanding linearity over the investigated concentration ranges, with correlation coefficient values greater than 0.999 for both analytes. Accuracy studies showed mean recoveries of 99.81% for Mebendazole and 99.92% for Ivermectin, while precision studies produced %RSD values below 2.0%, confirming the reliability and reproducibility of the method.

Specificity studies confirmed the absence of interference from excipients and other formulation components, whereas peak purity analysis demonstrated excellent selectivity of the method. Robustness evaluation revealed that minor deliberate variations in chromatographic conditions did not significantly affect analytical performance. Furthermore, the low LOD and LOQ values indicated high sensitivity of the developed method.

Overall, the validated RP-HPLC method proved to be simple, sensitive, economical, and stability-indicating, making it highly suitable for routine quality control analysis, assay determination, stability studies, and pharmaceutical research involving Mebendazole and Ivermectin in combined dosage forms. The developed method can therefore be confidently employed in pharmaceutical industries and quality control laboratories for reliable simultaneous estimation of these antiparasitic drugs.

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