Stability Indicating RP-HPLC Method Development and Validation for Simultaneous Estimation of Albendazole and Ivermectin in Pharmaceutical Dosage form

 

B. Bhavya, P. Nagaraju*, V. Mounika, G. Indira Priyadarshini

Department of Pharmaceutical Analysis,  Hindu College of Pharmacy, Amaravathi Road, Guntur 522002, Andhra Pradesh, India.

*Corresponding Author E-mail: pappulanagaraju@gmail.com

ABSTRACT:

A new simple, precise, selective, accurate and rapid reverse phase high performance liquid chromatographic stability indicating method had been developed and validated for simultaneous quantitative determination of Albendazole and Ivermectin in pharmaceutical dosage form. The chromatographic separation was achieved with Inertsil ODS 150×4.6 mm, 5μm particle size column. The optimized mobile phase consisting of pH 3.5 sodium phosphate buffer: Methanol (25:75v/v) in isocratic mode. The flow rate was 1.0 mL/min and eluents were detected at 245 nm using DAD (or) UV detector. The retention time of Albendazole and Ivermectin were found to be 1.974 and 2.897 mins respectively. The percentage recoveries for Albendazole and Ivermectin were found to be in the range of 98.67 -100.84%,and 101.46 - 100.26%. The calibration curve was constructed between peak area Vs concentration and demonstrated good linear in the range of 40 -200 μg/ml for Albendazole and 0.6-3 μg/ml for Ivermectin. Degradation studies were studied for Albendazole and Ivermectin under various stress conditions such as acid hydrolysis, base hydrolysis, oxidation, and thermal. All the degradation peaks were resolved effectively using developed method with different retention times. The developed method was validated according to ICH guidelines. As the method could effectively separate the degradation products from active ingredient, it can be used for routine analysis of drug in pharmaceutical dosage form.

 

 

 

INTRODUCTION:

Albendazole¹ (Figure 1) is a anthelmintic. Albendazole is chemically methyl N-[6-(propylsulfanyl)-1H-1,3-benzodiazol-2-yl]carbamate and Ivermectin is chemically Ivermectin² (Figure 2) is chemically 22, 23-dihydroavermectin B1a+ 22, 23-dihydroavermectin B1b is an extremely potent semisynthetic derivative of an antinematodal principle obtained from Streptomyces avermitilis. Ivermectin is a mixture containing at least 90% avermectin B1a and less than 10% avermectin B1b

 

Figure 1: Chemical Structure of Albendazole

 

Figure 2: Chemical Structure of Ivermectin

 

Numerous analytical methods employed for the quantitative determination of individual or multi-component combinations assay in pharmaceutical dosage forms. Various HPLC assay methods were reported in the literature for the estimation of Albendazole, Ivermectin and some other anthelmintic drugs individually and in-combination with other drugs. These methods include HPLC^[3-10]  and^[11-13] UV method On the contrary to the best of our knowledge, there is no official method for the stability-indicating simultaneous estimation of Albendazole and Ivermectin by RP-HPLC in tablet dosage form. Hence, we planned to develop and validate a new method for stability-indicating simultaneous determination of Albendazole and Ivermectin in pharmaceutical dosage form. The new method is capable of separating all active analytes present in the dosage form.

 

MATERIALS AND METHOD:

Chemicals and solvents:

 Water HPLC Grade, Methanol HPLC grade, pH 3.5 sodium phosphate buffer, Albendazole and Ivermectin Working Standards and tablet dosage form. The commercial Pharmaceutical tablets of Averm plus containing 400mg of Albendazole and 6mg of Ivermectin respectively (manufactured by Biotavia labs) were procured from local pharmacy.

 

INSTRUMENTATION:

The chromatographic separations were performed using HPLC-Waters alliance (Model-2695) consisting of an in-built auto sampler, a column oven and DAD (or) UV detector. The data was acquired through Empower-2-software. The column used was Inertsil ODS 150×4.6 mm, 5μm.

 

Chromatographic Conditions:

The mobile phase consists of pH 3.5 sodium phosphate buffer: Methanol 25:75 in isocratic mode. The mobile phase was pumped from solvent reservoir to the column in the flow rate of 1.0 ml/min whereas run time set was 7 min. The separation was performed on Inertsil ODS-3V 150mm x 4.6mm, 5μm column and the column was maintained the ambient temperature and the volume of each injection was 20ml. Prior to injection, the column was equilibrated for at least 30 min with mobile phase flowing through the system. The eluents were monitored at 245 nm.

 

Preparation of Sodium Phosphate buffer:

Weighed 6.8milligrams of Sodium di hydrogen orthophosphate into a 1000ml beaker dissolved and diluted to 1000ml with HPLC water. Adjusted the pH to 3.5 with ortho phosphoric acid.

 

Preparation of mobile phase:

Mix a mixture of above buffer 250 ml (25%) and 750 ml Methanol HPLC (75%) and degas in ultrasonic water bath for 5 minutes. Filter through 4.5 µ filter under vacuum filtration.

 

Diluent Preparation:

The Mobile phase was used as the diluent.

 

Preparation of standard solution:

(400 μg/ml Albendazole and 6 μg/ml Ivermectin).  Accurately Weighed and transferred 400 mg of Albendazole and 6 mg of Ivermectin working Standards into an 10 ml clean dry volumetric flasks, add diluent, sonicated for 10 minutes and make up to the final volume with diluent. From the above stock solution 1 ml was pipetted out in to a 10 ml volumetric flask and then make up to the final volume with diluent. From above stock solution 3ml was pipetted out into a 10 mL volumetric flask and then make up the final volume with diluent and thus we have (120 μg/ml Albendazole and 1.8 μg/ml Ivermectin).

 

Preparation of sample solution:

20 tablets were weighed and calculate the average weight of each tablet then the weight equivalent to 400 mg of Albendazole and 6 mg of Ivermectin was Standards into an 10 ml clean dry volumetric flasks, add diluent, sonicated for 10 minutes and make up to the final volume with diluent. From the above stock solution 1 ml was pipetted out into a 10 ml volumetric flask and then make up to the final volume with diluent. From above stock solution 3ml was pipetted out into a 10 mL volumetric flask and then make up the final volume with diluent  Label Claim: 400mg of Albendazole + 6mg of Ivermectin

 

Optimized Chromatographic conditions and system suitability parameters for proposed HPLC method for Albendazole and Ivermectin Instrument:

Waters 2695, High performance Liquid chromatography

 

Flow rate: 1.0 ml/min

Column: Inertsil ODS-3V, 150 x 4.6 mm, 5µ

Mobile Phase: pH 3.5 sodium phosphate buffer : Methanol (25:75)

Detector wave length: 245nm

Column temperature: ambient

Injection volume: 20μl

Run time: 7 min

Diluent: Mobile phase

Mode of separation: Isocratic mode

 

Fig.3: Standard Chromatogram of Albendazole and Ivermectin

 

Fig.4: Sample Chromatogram of Albendazole and Ivermectin

 

Validation of Proposed method:

The developed method was validated as per the ICH (International Conference on Harmonization) guidelines with respect to System suitability, Precision, Specificity, Forced degradation studies, Linearity, Accuracy, Limit of detection and Limit of quantification ^[14]

 

Linearity:

Aliquots of 1, 2, 3, 4, 5 were taken from stock solution of concentration 400 mg/ml Albendazole, 6 mg/ml Ivermectin into 10ml volumetric flask and then diluted up to mark with diluent. Such that the final concentrations were in the range 40 µg/ml 200 µg/ml for Albendazole and 0.6 µg/ml - 3 µg/ml for Ivermectin. Volume of 20μl of each sample was injected in three times for each concentration level and calibration curve was constructed by plotting the peak area versus drug concentration. A linear relationship between peak areas vs. concentration was observed in the range of study. The observations and calibration curve were shown in Table-1 and Fig. 5 and 6.

 

Table1: Linearity data of Albendazole and Ivermectin

 

 

Fig 5-calibration graph for Albendazole

 

Fig 6-calibration graph for Ivermectin 

PRECISION:

System Precision:

Precision is the measure of closeness of the data values to each other for a number of measurements under the same analytical conditions. Standard solution of Albendazole (120μg/ml) and Ivermectin (1.8μg/ml) were prepared as per procedure and injected for 5 times. Results for responses are shown in Table-2.

 

Method Precision:

Method precision of Albendazole and Ivermectin were carried out by estimating corresponding responses for 5 times on the same day and on consecutive days for the concentration of Albendazole (120μg/ml) and Ivermectin (1.8μg/ml). The percent relative standard deviation (%RSD) was calculated which was within the acceptable criteria of not more than 2.0. The results were shown in Table-3.

 

Table 2: System precision of Albendazole and Ivermectin

S. No	Area of Albendazole	Area of  Ivermectin
1.	7674345	130642
2.	7709409	135377
3.	7709540	135244
4.	7707906	135474
5.	7730011	136945
6.	7763332	133953
Mean	7715757.167	134605.8
SD	29401.0	2161.7
%RSD	0.4	1.6

 

Table 3: Method precision of Albendazole and Ivermectin

 

ACCURACY (Recovery studies):

To determine the accuracy in sample preparation method of standard additions was made for measuring the recovery of the drugs. A fixed amount of sample was taken and standard drug was added at 50%, 100% and 150% levels. The amount added and amount found for Albendazole and Ivermectin were calculated and the individual recovery and mean recovery values were reported. The results were analyzed and the results were found to be within the limits. Table-4: Accuracy

 

%Concentration (at specification Level)	Amount spiked (mg)	Amount Found (mg)	% Recovery	Mean Recovery
50%	20.0	19.92	98.67%	99.53%
100%	40.0	39.63	99.08%
150%	80.0	60.50	100.84%

*Average of three determinations

 

 

%Concentration (at specification Level)	Amount spiked (mg)	Amount Found (mg)	% Recovery	Mean Recovery
50%	3.0	3.04	101.46%	101.22%
100%	6.0	6.02	100.26%
150%	9.0	9.17	101.93%

*Average of three determinations

 

Limit of Detection and Limit of Quantification:

Detection and quantification limit were calculated by the method based on the standard deviation ( s) and slope of the calibration plot, using the formula  

                               σ X 3.3

Limit of detection =----------

                                   S

                                         σ X 10

Limit of quantification =----------

                                              S

 

Where    s = the standard deviation of the response.

S = the slope of the calibration curve (of the analyte).

 

Table 5: LOD and LOQ values

 

Fig.7: Chromatogram at LOD Level

 

Fig.8: Chromatogram at LOQ Level

ROBUSTNESS:

Robustness was carried by varying parameters from the optimized chromatographic conditions such as making small changes in flow rate (±0.1ml/min), and Organic Composition in the Mobile Phase (±10%). It was observed that the small changes in these operational parameters did not lead to drastic changes of retention time of the peak of interest, plate count (NLT 2000), tailing factor (NMT 2.0), resolution (NLT 2.0) and the %RSD for five replicate injections (NMT 2.0) were found to be within the acceptance criteria. The degree of reproducibility of the results proven that the method is robust. The results were shown in Table-6 and Fig 9 to 12.

 

Table-6: Robustness results

S. no	Robustness Condition	Peak Area of Albendazole	Peak Area of Ivermectin
1.	Flow rate 0.8 ml/min	2543.94	3615.88
2.	Flow rate 1.2 ml/min	2555.45	3178
3.	Organic Composition in the Mobile Phase(10% less)	2573.94	2889.76
4.	Organic Composition in the Mobile Phase(10% more)	2526.74	3225.73

Fig.9: Chromatogram for Effect of flow rate 0.8ml/min

 

 

Fig.10: Chromatogram for Effect of flow rate 1.2ml/min

 

Fig.11: Chromatogram showing less organic composition

 

Fig.12: chromatogram showing more organic composition

 

System suitability:

The system suitability was determined by making six replicate injections from freshly prepared standard solutions. The observed RSD values were well within usually accepted limits (≤2%). Theoretical plates, tailing factor and resolution of Albendazole and Ivermectin were determined and found to be well within the acceptable limits. The results summarized in Table-7.

 

Table 7: System suitability parameter results

S. No	Name	Rt(min)	Area (µV sec)	Height (µV)	USP resolution	USP tailing	USP plate count
1	Albendazole	1.974	7702720	587576		1.58	2555.49
2	Ivermectin	2.897	124066	12556	2.96	1.42	3884.11

 

Specificity:

The specificity of the method was performed by injecting blank solution (without any sample) and then a drug solution of 20μl injected into the column, under Optimized chromatographic conditions to demonstrate the separation of two molecules Albendazole and Ivermectin from any of the impurities, if present. As there was no interference of impurities and also no change in the retention time, the method was found to be specific. The chromatogram of blank and sample solution was shown in Fig 13, 14.

 

Fig.13: Chromatogram of blank

 

Fig.14:chromatogram for Sample

FORCED DEGRADATION STUDIES:

Forced degradation studies were performed to demonstrate the optimized method is stability indicating. To prove the method which can be able to measure accurately active pharmaceutical ingredient in presence of degradants which are expected to be formed during different types of degradations applied to the drug sample.  For forced degradation analysis, aliquots of stock were separately treated with 1ml of 0.1N HCl (Acid stability), 1ml of 0.1N NaOH (Alkaline stability), 1ml of 3% H2O2 (Oxidative degradation), exposure of sample drug solution in Hot air oven at 110⁰ C for 24 hours (Thermal degradation). Stability of these samples was compared with fresh sample on the day of analysis. The HPLC chromatograms of degraded products show no interference at the respective analyte peaks and the individual analytes peak purity values found to be within the acceptable limits, hence the method was specific and stability indicating. The chromatograms were shown in figures 15 to 18 and the results were shown in Table-8. The detailed degradation for each condition is as follows:

 

Oxidation:

To sample stock solution of Albendazole and Ivermectin, 1 ml of 3% hydrogen peroxide (H2O2) was added. The solution was kept for 15 min at room temperature and finally made up to volume with diluent. For HPLC study, the resultant solution was diluted to obtain 90μg/ml, 180μg/ml and 15μg/ml of all components and 20 μL of sample solution was injected into the system and the chromatograms were recorded to assess the stability of sample.

 

Acid Degradation Studies:

To sample stock solution of Albendazole andIvermectin, 3mL of 0.1N Hydrochloric acid was added and kept at 60ºC for 6 hours and cooled to room temperature and neutralized with 1 mL of 0.1N sodium hydroxide solution and finally made up to volume with diluent. For HPLC study, the resultant solution was diluted to obtain 90μg/ml, 180μg/ml and 15μg/ml of all components and 20 μL of sample solution was injected into the system and the chromatograms were recorded to assess the stability of sample.

 

Alkali Degradation Studies:

To sample stock solution of Albendazole and Ivermectin, 3mL of 0.1N sodium hydroxide solution was added and kept at 60ºC for 6 hours and cooled to room temperature and neutralized with 1 mL of 0.1N Hydrochloric acid solution and finally made up to volume with diluent. For HPLC study, the resultant solution was diluted to obtain 90μg/ml, 180μg/ml and 15μg/ml of all components and 20 μL of sample solution was injected into the system and the chromatograms were recorded to assess the stability of sample.

 

Thermal induced degradation:

Albendazole and Ivermectin sample was taken in petridish and kept in Hot air oven at 110⁰C for 24 hours. Then the sample was taken and diluted with diluents and injected into HPLC and analyzed.

 

Table 8 : Results of Forced degradation studies

 

 

Fig.15: Chromatogram of Acid Stressed Sample

 

Fig.16: Chromatogram of Base Stressed Sample

 

Fig.17: Chromatogram of Peroxide Stressed Sample

 

Fig.18: Chromatogram of Thermal Stressed Sample

 

 

 

RESULTS AND DISCUSSION:

1.    The estimation of Albendazole and Ivermectin was done by RP-HPLC.

2.    The assay of Albendazole and Ivermectin was performed with tablets and the % assay was found to be 99.83 and 99.98 which shows that the method is useful for routine analysis.

3.    The linearity of Albendazole and Ivermectin was found to be linear with a correlation coefficient of 0.999 and 0.999, which shows that the method is capable of producing good sensitivity.

4.    The acceptance criteria of precision is RSD should be not more than 2.0% and the method show  precision 0.4 and 1.7 for Albendazole  and Ivermectin which shows that the method is precise.

5.    The acceptance criteria of intermediate precision is RSD should be not more than 2.0% and the method show precision 0.4 and 1.6 for Albendazole  and Ivermectin which shows that the method is repeatable when performed in different days also.

6.    The accuracy limit is the percentage recovery should be in the range of 97.0% - 103.0%.  The total recovery was found to be 99.53% and 101.22% for Albendazole and Ivermectin. The validation of developed method shows that the accuracy is well within the limit, which shows that the method is capable of showing good accuracy and reproducibility.

7.    The acceptance criteria for LOD and LOQ are 3 and 10.The LOD and LOQ for Albendazole was found to be 1.62 and 4.91 and LOD and LOQ for Ivermectin was found to be 0.31 and 0.966.

8.    The robustness limit for mobile phase variation and flow rate variation are well within the limit, which shows that the method is having good system suitability and precision under given set of conditions.

 

CONCLUSION:

The estimation of Albendazole and Ivermectin was done by RP-HPLC. The mobile phase consists of sodium phosphate buffer having P^H of 3.5: methanol mixed in the ratio of 25:75 % v/ v. Inertsil C₁₈ (4.6 x 150mm, 5.0mm) or equivalent was used as stationary phase. The detection was carried out using UV detector at 245 nm.

 

The solutions were chromatographed at a constant flow rate of 1.0 ml/min

·      System suitability parameters were calculated which includes efficiency, resolution and tailing factor.

·      The reliability and suitability of the method could be seen from recovery studies. Further there is no interference due to excipients.

·      Precision of the methods were studied by making repeated injections of the samples and values were determined.

·      The method was validated for linearity, accuracy, precision, robustness.

·      The method is simple, specific and easy to perform and requires short to analyze the samples.

·      Low limit of quantification and limit of detection makes this method suitable for Quality control.

·      The method was found to be linear, accurate, precise and robust.

·      Hence it was concluded that the RP-HPLC method developed was very much suitable for routine analysis of tablet formulation containing Albendazole and Ivermectin.

 

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Received on 07.12.2016       Accepted on 12.01.2017     

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