Development of a Validated Stability Indicating Method for Quantification of Amoxicillin, Clarithromycin and Lansoprazole in Bulk and Pharmaceutical Dosage form by RP-HPLC

Objective: The main objective of the present work is to develop an efficient, unique, reliable Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method for the simultaneous quantification of Amoxicillin (AMX), Clarithromycin (CTM) and Lansoprazole (LPZ) in bulk and pharmaceutical formulations. Methods: The chromatographic separation was achieved by using Kinetex column C18 (100 x 4.6 mm, 2.6 μm) with Buffer (2.5 g of hexane sulphonic acid and 1ml of Triethylamine which are added to 1000 ml of HPLC water and adjusted its pH at 5.0 with Ortho phosphoric acid) and acetonitrile in the ratio of 70: 30 (%v/v) as a mobile phase at flow rate of 1.0 ml/min. The column effluents were monitored by a photodiode array detector at wavelength predetermined at 240 nm. Results: The method produced reliable results at optimized chromatographic conditions. The method was linear at concentration range of 15-225 μg/ml of AMX, 15-225 μg/ml of CTM and Original Research Article Sushma et al.; JPRI, 33(45B): 207-220, 2021; Article no.JPRI.73910 208 0.9-13.5 μg/ml of LPZ with regression coefficients of 0.9999, 0.9999, and 0.9999 respectively. The retention times of AMX, CTM, LPZ were obtained as 1.513, 3.124, 3.770 min respectively. Results obtained for system suitability, precision, LOD and LOQ were in acceptable range and were validated according to the guidelines of the International Council for Harmonization (ICH). Conclusion: The proposed method was validated in accordance with ICH and all the obtained results were found satisfactory and were successfully applicable to the analysis of the bulk and the pharmaceutical formulations.


INTRODUCTION
Amoxicillin is chemically (2S, 5R, 6R)-6-[(2R)-2amino-2-(4-hydroxyphenyl) acetyl] amino]-3, 3dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid with a molecular formula C 16  . It is an antibiotic [1,2] used to treat a number of bacterial infections [3] which include middle ear infection [4], strep throat [5], pneumonia [6], skin infections and urinary tract infections [7]. Common adverse effects include nausea and rash. It may also increase the risk of yeast infections [8] and, when used in combination with clavulanic acid [9], it causes adverse effects like diarrohea. It should not be used in those who are allergic to penicillin [10]. When it is to be used in patients with kidney problems, the dose may need to be decreased. Amoxicillin belongs to beta-lactam family [11] of antibiotics. It is one of the most commonly prescribed antibiotics in children.

Chemicals
Analytical grade reagents like HPLC grade acetonitrile, orthophosphoric acid, hexane sulphonic acid, triethylamine and water (HPLC grade), were purchased from Merck Ltd., Mumbai, India. APIs of amoxicillin, clarithromycin and lansoprazole as reference standards were procured from Spectrum Pharma research solutions pvt. Ltd, Hyderabad.

Instrumentation
The HPLC system used for the method development and validation consisted of Waters Alliance e-2695 chromatographic system equipped with quaternary pump and waters 2996 PDA detector. Data acquisition, recording and chromatographic integration was performed by software Empower-2.0.

Chromatographic Conditions
The chromatographic separation was performed on Kinetex column C18(100 x 4.6mm, 2.6µm) with Buffer (2.5g of hexane sulphonic acid and 1ml of triethylamine were added to 1000 ml of HPLC water and adjusted its pH at 5.0 with ortho phosphoric acid) and acetonitrile in the ratio of 70:30 (%v/v) as a mobile phase at flow rate of 1.0 ml/min in an isocratic mode with injection volume of 10 µl for all samples. The buffer was filtered through 0.45µ filter paper and degassed in sonicator before its use.

Preparation of Standard Solution
Accurately weighed and transferred 150mg of AMX, 150mg of CTM and 9mg of LPZ working standards into 100ml volumetric flask and added approximately 70ml of diluents. This solution was sonicated for 30min to dissolve it. The final volume was made up to the mark with diluents; which was used as stock solution.
The above stock solution of about 5 ml was transferred into 50ml volumetric flask and made up to the mark with diluents to obtain the solutions of concentrations respectively. (AMX-150 µg/ml, CTM-150 µg/ml and LPZ-9µg/ml).

Preparation of Sample Solution
Ten Capsules of LPZ and AMX and CTM tablets were taken into a mortar and powdered. The powdered sample equivalent to the weight of 150mg of AMX, 150mg of CTM and 9mg of LPZ were transferred into 100ml volumetric flask. 70ml of diluents was added and sonicated for 30min to dissolve it and final volume was made up to the mark with diluent. 5ml of the above sample stock solution was transferred into 50ml volumetric flask and made up to the mark with diluent. The resultant solutions were of concentrations (AMX-150 µg/ml, CTM-150 µg/ml and LPZ-9µg/ml) respectively.

Method Development and Optimization
The significant difference in the physical and chemical properties of APIs in proposed method leads the selection of a suitable mobile phase a critical step. Several ratios of components of mobile phases and columns were trailed to achieve a separation of API's with good resolution. The suitability of the column and the mobile phase used in the optimized method have been decided based upon the basis of the selectivity, sensitivity as well as acceptable chromatographic parameters of the produced peaks in terms of peak sharpness, peak symmetry, tailing factor and resolution between the two peaks. We used the mobile phase as a diluent for all samples to ensure minimum noise and to eliminate any unwanted solvent peaks.

Validation
The validation of any method demonstrates that the method is suitable for its intended purpose, as stated in ICH Q2(R1) guidelines [29]. The method was validated for linearity, precision (method precision and intermediate precision), accuracy, selectivity and specificity.

System Suitability
System suitability test is performed to ensure that the resolution and reproducibility of the chromatographic systems are adequate for the analysis to be carried out.
The limits were set for No. of theoretical plates, tailing factor, and resolution. The HPLC system was stabilized for 60 min to get a stable baseline. Six replicate injections of standard solution were injected.

Specificity
The specificity of the analytical method was established by injecting the 100 μg/mL concentration solutions of diluent (blank), placebo, working standards and sample solution individually to investigate interference from the representative peaks.

Linearity
The area of the linearity peak versus various concentrations has been evaluated for AMX, CTM, LPZ. Linear regression analysis was plotted using peak area against concentration data. Correlation coefficients were calculated for the percent regression, Y-intercept and slope of the calibration curves. The linearity was observed in the concentration range of 15-225 µg/ml of amoxicillin, 15-225 µg/ml of Clarithromycin and 0.9-13.5 µg/ml of lansoprazole with the triplicate injections (n=3) for each concentration.

Method Precision
Method precision was investigated by the analysis of six separately prepared samples of the same batch. From these six separate sample solutions were injected and the peak areas obtained were used to calculate mean and percentage RSD values.

Intermediate Precision
Ruggedness of the method was studied and showed that chromatographic parameters did not significantly change when different HPLC system, analyst, column were applied.

Accuracy
Accuracy was evaluated in triplicate, at three different concentration levels equivalent to 50%, 100% and 150% of the target concentration of active ingredient, by adding a known amount of each of the Standard to a pre-analysed concentration of all drugs (AMX, CTM and LPZ) and calculating the % of recovery.

Limit of Detection (LOD) and Limit of Quantification (LOQ)
Limits for the identification and quantification of the drug were calculated from the calibration curves of the drugs. The standard deviation and slope were considered from the calibration plots.

Robustness
The conditions of the experiment were designed to test the robustness of established system intentionally altered, such as flow rate, organic percentage in movable phase; all these varied conditions.

Stock solution preparation
Accurately weighed 37.4 mg of sample was taken. in 10 ml vacuum flask and added 7 ml of diluents. This solution is sonicated for 30 minutes and diluted to the final volume with diluents.

Acid degradation
The above sample stock solution of 1ml was transferred to a 10ml volumetric flask. To the flask 1 ml of 1N HCl was added and kept for 15 minutes. After 15 minutes, 1ml of 1N NaOH was added and the final volume was made up to the mark with diluents.

Alkali degradation
The above sample stock solution of 1ml was transferred to a 10ml volumetric flask. To the flask 1 ml of 0.1N NaOH was added and kept for 15 minutes. After 15 minutes 1ml of the 1N HCl was added and the final volume was made up to the mark with diluents.

Peroxide degradation
The above sample stock solution of 1ml was transferred to a 10ml volumetric flask. To the flask 0.3 ml 30 percent hydrogen peroxide was added and the final volume was made up to the mark with diluents.

Reduction degradation
The above sample stock solution of 1ml was transferred to a 10 ml volumetric flask. To the flask 1 ml of 30 percent sodium bisulphate solution was added and the final volume was made up to the mark with diluents.

Thermal degradation
The sample solution was set in an oven at 105º for 6 hours.

Hydrolysis degradation
The above sample stock solution of 1ml was transferred to a 10 ml volumetric flask. To the flask 1ml of water was added and made up to the mark with diluents.
All the above final solutions were injected into system and % degradation was observed.
Forced degradation studies were conducted on the basis of ICH requirements including acid, base, oxidation, reduction, thermal and hydrolysis degradation.

System Suitability
The system suitability was estimated by the mentioned parameters and all the obtained values were within the specified limits in accordance with ICH guidelines. The results were summarized in below Table 2.

Specificity
The obtained chromatograms in Figs. 5 to 11 it can be inferred that there were no co-eluting peaks at the retention time of AMX, CTM, LPZ which shows that peak of analyte was pure and the excipients in the formulation did not interfere with the analyte of interest.

Linearity
The Linearity plots of the drugs executed a honest linearity which were confirmed by the correlation coefficients for all drugs achieved are greater than 0.999. The slope, Y-intercept values were presented in Table 3.

Method precision (or) repeatability
The calculated mean and percentage RSD values of the three drugs obtained have inferred that the method was precise.

Intermediate precision
The value of percentage RSD was below 2% exhibits the ruggedness of the developed method. The results of both method precision and intermediate precision were present in Table  4.

Accuracy
Accuracy was evaluated in triplicate, at three different concentration levels equivalent to 50, 100 and 150% of the target. concentration of active ingredient, by adding a known amount of each of the Standard to a preanalysed concentration of all drugs (AMX, CTM and LPZ) and calculating the % of recovery. The recovery results should be not less than 98% and not more than 102%.

Limit of detection (LOD) and limit of quantification (LOQ)
The calculation of LOD and LOQ were proceeded using the following equation in compliance with the ICH guidelines.

Robustness
The resolution between active ingredients from impurities was not significantly affected and there was no significant influence on the time of retention, plate count and tailing factor. Hence this method was robust. Robustness of the method was found to be % RSD should be less than 2%. Slightly variations were done in the optimized method parameters like flow rate (±20%), organic content in mobile phase (±10%). The results are present in Table 7.

Forced Degradation Studies
It is evident from the obtained data that the selected drugs were stable under the applied stress conditions although the degraded peaks were observed the percentage degradation was with in the acceptable limits.

CONCLUSION
In this study a novel, simple, rapid, economical, sensitive and easily available HPLC method was developed for the simultaneous determination of AMX, CTM and LPZ in bulk and pharmaceutical dosage form. The shorter run time, low price, accessibility, sensitivity, reliability and reproducibility of the method proves in applicability in rapid quantification of many samples in routine and quality control analysis of tablets. The validation of all the parameters like linearity, accuracy, precision, robustness was performed and found to be within the acceptance criteria. The RSD values for all parameters were found to be less than 2%, which indicates the validity of method and results obtained by this method are in fair agreement. So, the proposed method could be easily applied for the routine analysis and pharmaceutical formulations of amoxicillin, clarithromycin and lansoprazole in quality control laboratories without any preliminary separation.