Stability Indicating Method Development and Validation for the simultaneous Determination of Levosulpiride and Esomeprazole in Bulk and Formulation

Present study deals with the development and validation of a rapid, simple and efficient method for the simultaneous determination as well as stability studies of Esomeprazole and Levosulpiride in bulk and formulations. The method involves reverse phase High Performance Liquid Chromatography (HPLC) using stationery Phase ODS C18 column (250mm x 4.6 mm, 5m), Mobile phase as Phosphate buffer, Acetonitrile and methanol at the ratio of 65:30:5, subjected to isocratic elution, observed the peaks with PDA detector wavelength 254nm, maintaining the mobile phase flow rate at 1ml/minute, keeping the run time fixed for 8 minutes. Column temperature was maintained at 300C, pH of the mobile phase was 3.0; complete separation of both the compounds took place within four minutes. Retention time was found 2.41 minutes and 3.56 minutes for Esomeprazole and Levosulpiride respectively. The developed new method was validated as per ICH guideline taking the parameters like accuracy, precision, linearity, limit of detection, limit of quantification, intermediate precision and robustness. In the linearity test Correlation Coefficient was found to be 0.999 for both the molecules, percentage relative standard deviation results from precision studies were 0.34 and 0.44; mean percentage recoveries in accuracy studies were found to be 100.35% and 100.14% for Esomeprazole and Levosulpiride respectively. Very low concentrations of LOD and LOQ indicate the method was highly sensitive enough. The designed validated method can be used effectively in the laboratory for regular determination of Levosulpiride and Esomeprazole in formulation and bulk form.


Preparation of Solutions Diluent
First the compounds were dissolved in small amount of solution of water and Acetonitrile in the ratio of 1:1 and then made up the volume with Buffer.

Buffer
(0.1% Ortho phosphoric acid): Transferred 1ml of Concentrated Ortho phosphoric acid in a 1000ml volumetric flask, added about 900ml of milli-Q water and sonicated for 15 minutes and finally made up the volume with water.

Standard Preparation Stock solution Preparation
(800 µg/ml Esomeprazole, 1500 µg/ml Levosulpiride): Transferred 8 mg of Esomeprazole, 15 mg of Levosulpiride Standards into a 10 ml clean dry volumetric flask, added 7 ml of diluent, sonicated for 30 minutes and made up to the final volume with mobile phase.

Standard solution Preparation
(64 µg/ml Esomeprazole, 120µg/ml Levosulpiride) From the above stock solution, 0.8 ml was pipetted out in to a 10 ml volumetric flask and then made up to the final volume with mobile phase.

Sample Preparation Stock sample solution Preparation
( 800 µg/ml Esomeprazole, 1500 µg/ ml Levosulpiride) 10 tablets were weighed and calculated the average weight of each tablet, then the weight equivalent to 1 tablet (380 mg) was transferred into a 50 ml volumetric flask, 30 ml of diluent was added and sonicated for 30 min, further the volume was made up with mobile phase and filtered off.

Standard sample solution Preparation
( 64 µg/ml Esomeprazole, 120 µg/ml Levosulpiride) From the filtered stock solution 0.8 ml was pipetted out into a 10 ml volumetric flask and made up to 10 ml with mobile phase.

Method development
To develop a new method 10 for estimation and degradation studies several trials were conducted so that we can achieve most suitable chromatographic condition.The initial attempt was to employ as much low proportion of organic solvents for elution of the compounds.More part of aqueous solvents in mobile phase resulted in prolonging of retention time of both the compounds.Reasonable retention time, number of theoretical plates, value of tailing factors and all were found within the validation limit by using optimized Chromatographic condition.

Method Validation
The developed stability-indicating HPLC analytical method was validated following ICH guidelines 11

Accuracy
It was conducted by recovery studies, using spiking method.50%, 100%, and 150% of standard of Levosulpiride and Esomeprazole were spiked to pre-quantified sample solutions and the quantity recovered was estimated.

Test for Precision
It was checked by applying same concentration of compounds repeatedly for six times

Test for Linearity
For Esomeprazole the range was 16ppm to 96ppm and Levosulpiride 30ppm to 180ppm.Each Concentration was injected thrice and calculated correlation coefficient.

Intermediate precisions
The test for Intermediate precisions of this method was determined by experimenting the results in different days keeping a gap of 24 hours.

Test for LOD and LOQ
The limit of detection, the limit of quantification was determined by considering standard deviation of y intercept and slope of regression line and using them into the formula-LOD = 3.3 × SD/Slope LOQ= 10 × SD/Slope

Test for Robustness
It was conducted by maling small change in the mobile phase composition (aqueous phase 10% ±), temperature (±5 0 C) and flow rate (± 0.2 ml per minute).

Test for Stability: Oxidation
To 1 ml of stock solution 1 ml of 20% hydrogen peroxide (H 2 O 2 ) was added.The solutions were kept for 30 min at 60 0 c.For HPLC study, the resultant solution was diluted to obtain 64µg/ml and 120µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Acid Degradation Studies
To 1 ml of s tock solution 1ml of 2N Hydrochloric acid was added and refluxed for 30mins at 60 0 c, diluted, and 10 µl was injected into the system.

Alkali Degradation Studies
To 1 ml of stock solution 1 ml of 2N sodium hydroxide was added and refluxed for 30mins at 60 0 c. diluted, 10 µl was injected into the system.

Dry Heat Degradation Studies
The standard drug solution was placed in oven at 105 0 c for 6 hours to study dry heat degradation, diluted, 10µl was injected into the system.

Photo Stability studies
It was conducted by exposing the solution to UV Light by keeping the beaker in UV Chamber for 7days, solution was diluted, 10 µl was injected into the system.

Neutral Degradation Studies
It was studied by refluxing the drug in water for 6hrs at a temperature of 60°C, diluted, 10 µl was injected into the system.

Method development
Optimization of chromatographic condition was achieved after several trials.Reasonable retention time for Esomeprazole was 2.4 minutes and for Levosulpiride was 3.5 minutes.Resolution, tailing factor and number of theoretical plates were acceptable enough for quantitative analysis and stability studies.

Optimized Chromatographic conditions
Mobile phase was as Buffer, Acetonitrile and Methanol at the ratio of 65:30:5, Flow rate 1 ml/minute, Column ODS 250 mm x 4.6 mm, 5 m, Detector wave length 254 nm, Column temperature 30°C, Injection volume 10 mL, run time 8 minutes, Diluent: First drug dissolved in water and Acetonitrile at the ratio of 1:1 and then made up the volume with Buffer.

Validation Results: Accuracy Results
Number of replicates were three for each trial.Mean percentage recovery was found to be 100.48%and 100.0 % for Esomeprazole and Levosulpiride respectively.Table 1

Precision Results
SD was found to be 6568.0and 1325.8;percentage relative standard deviation 0.38 and 0.60 for Esomeprazole Levosulpiride respectively.Table 2 contains details of precision/System suitability results.

Linearity Result
Linearity test was conducted by applying Levosulpiride (30ppm-180ppm) and Esomeprazole (16ppm-96ppm), calculated the value of Correlation Coefficient, and was found to be 0.999 for both the compounds.Details of Linearity results are given in Table 3.

Intermediate precision Results
The test for Intermediate precisions of this method was determined by conducting the trials in

LOD
The limit of detection was found to be 0.04µg/ml and 0.02µg/ml for Esomeprazole and Levosulpiride respectively.

LOQ
The limits of quantification were found to be 0.12µg/ml and 0.05µg/ml for Esomeprazole and Levosulpiride respectively.

Robustness Study
The test for robustness was performed by taking the parameters like flowrate-1.0 ± 0.2 ml per minute, Mobile phase composition -aqueous phase 10% ± and temperature ±5 0 C. Result was found like change in retention time from 2.138 minutes to 2.439 minutes for Esomeprazole and from 3.144 minutes to 3.666 minutes for Levosulpiride, other parameters like theoretical plates, tailing factors etc. were not affected significantly.Table 5 contains details of robustness results.

Results of Stability test
Stability studies indicate that both the compounds were prone to peroxide, acid and alkali degradation.Thermal, UV or Water degradation was not significant.Figure IV to IX represent chromatogram due to force degradation and Table 6 contains details of degradation results.

CONCLUSION
In this research work the method developed for assay and stability studies was found to be rapid, simple, accurate, precise and robust for regular analysis of the drugs simultaneously.The present method has certainly beneficial edges analytically as compared to available methods which makes the method quiet advantageous and unique.
Figure I. represents the structure of Esomeprazole.Levosulpiride 2 a substituted derivative of benzamide, GI motilator and anti-psychotic drug, was reported to be a selective antagonist of central dopamine (D-2, D-3 and D-4) receptors, Levosulpiride also has shown to have mood elevating properties.Chemically the molecule is named as N-(((2s)-1-Ethylpyrrolidin-2-yl) methyl)-2-methoxy-5suifamoylbenzamide. Figure II.Represents structure of the molecule Levosulpiride.

Table 1 : Accuracy Results
Note: number of replicates for each spiking = 3.