Method Development and Validation for the Concurrent Analysis of Levosulpiride and Rabeprazole by Liquid Chromatography

Equipments and instruments used in the study:

PREPARATION OF BUFFER AND MOBILE PHASE:

Preparation of 0.1% Formic Acid:

To prepare 0.1% Formic Acid buffer solution by adding 1ml of Formic Acid in 1000ml HPLC water adjust the solution to desired PH.

Preparation of mobile phase:

Mix a mixture of above 0.1% Formic Acid and ACN and degas in ultrasonic water bath for 5 minutes. Filter through 0.45µfilter under vacuum filtration.

2.2 HPLC METHOD DEVELOPMENT:

OPTIMIZED CHROMATOGRAPHIC CONDITIONS

Instrument used : High performance liquid chromatography equipped with Auto Sampler and PDA detector

Temperature              : Ambient

Column : Platisil C18, (250×4.6mm, 5μm)

Buffer : 0.1% Formic Acid (ph-3.5)

Mobile phase : 40% Phosphate buffer: 60% CAN

Flow rate : 1.0 ml per min

Wavelength : 284 nm.

Injection volume : 20 ml

Run time  : 12 min

Chromatogram for system suitability

System Suitability:

Tailing factor for the peaks due to Levosulpiride and Rabeprazole in Standard solution should not be more than 2.0

Theoretical plates for the Levosulpiride and Rabeprazole peaks in Standard solution should not be less than 2000

2.3 METHOD VALIDATION PARAMETERS:

2.3.1 ASSAY:

Standard Solution Preparation:

Accurately weigh and transfer 25mg of Levosulpiride and 6.67mg Rabeprazole working standard into a 25ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents (75ppm Levosulpiride & 20ppm Rabeprazole).

Procedure:

Inject 10 ml of the standard, sample into the chromatographic system and measure the areas for the Levosulpiride and Rabeprazole peaks and calculate the % Assay by using the formulae.

2.3.2 LINEARITY:

Preparation of stock solution:

Accurately weigh and transfer 25mg of Levosulpiride and 6.67mg Rabeprazole working standard into a 25ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Preparation of Level–I (25ppm of Levosulpiride and 6.7ppm Rabeprazole):

0.25 ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with Diluents.

Preparation of Level–II (50ppm of Levosulpiride and     13.3ppm Rabeprazole):

0.5 ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with Diluents.

Preparation of Level–III (75ppm of Levosulpiride and 20ppm Rabeprazole):

0.75 ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with Diluents.

Preparation of Level–IV (100ppm of Levosulpiride and 26.7ppm Rabeprazole):

1.0 ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with Diluents.

Preparation of Level–V (125ppm of Levosulpiride and 33.3ppm Rabeprazole):

1.25 ml of stock solution has taken in 10ml of volumetric flask dilute up to the mark with Diluents.

Procedure:

Inject each level into the chromatographic system and measure the peak area.

Plot a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and calculate the correlation coefficient.

2.3.3 PRECISION:

Preparation of standard Solution

Accurately weigh and transfer 25mg of Levosulpiride and 6.67mg Rabeprazole working standard into a 25ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents (75ppm Levosulpiride &20ppm Rabeprazole).

Procedure:

The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of six replicate injections was found to be within the specified limits.

2.3.4 INTERMEDIATE PRECISION/ RUGGEDNESS:

To evaluate the intermediate precision (also known as Ruggedness) of the method, Precision was performed on different day within the laboratory.

Preparation of standard solution:

Accurately weigh and transfer 25mg of Levosulpiride and 6.67mg Rabeprazole working standard into a 25ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents (75ppm Levosulpiride &20ppm Rabeprazole).

Procedure:

The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of six replicate injections was found to be within the specified limits.

2.3.5 ACCURACY:

For accuracy determination, three different concentrations were prepared separately i.e. 50%, 100% and 150% for the analyte and chromatograms are recorded for the same.

Preparation of Standard solution:

Accurately weigh and transfer 25mg of Levosulpiride and 6.67mg Rabeprazole working standard into a 25ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents (75ppm Levosulpiride &20ppm Rabeprazole).

Preparation Sample solutions:

For preparation of 50% solution (With respect to target Assay concentration):

Accurately weigh and transfer 12.5mg of Levosulpiride and 3.33mg Rabeprazole working standard into a 25ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents (75ppm Levosulpiride &20ppm Rabeprazole)..

For preparation of 100% solution (With respect to target Assay concentration):

Accurately weigh and transfer 25mg of Levosulpiride and 6.67mg Rabeprazole working standard into a 25ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents (75ppm Levosulpiride &20ppm Rabeprazole).

For preparation of 150% solution (With respect to target Assay concentration):

Accurately weigh and transfer 37.5mg of Levosulpiride and 10mg Rabeprazole working standard into a 25ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents (75ppm Levosulpiride &20ppm Rabeprazole).

Procedure:

Inject the standard solution, Accuracy -50%, Accuracy -100% and Accuracy -150% solutions.

Calculate the Amount found and Amount added for Levosulpiride and Rabeprazole and calculate the individual recovery and mean recovery values.

2.4 LIMIT OF DETECTION:

2.4.1 Preparation of Levosulpiride and Rabeprazole solution:

Preparation of 0.02µg/ml solution:

Accurately weigh and transfer 25mg of Levosulpiride working standard into a 25ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents

Further pipette 0.1 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent.

Further pipette 0.25 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent.

Preparation of 0.20µg/ml solution:

Accurately weigh and transfer 6.67mg Rabeprazole working standard into a 25ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents

Further pipette 0.1 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent.

2.4.2 LIMIT OF QUANTIFICATION:

Preparation of Levosulpiride and Rabeprazole solution:

Preparation of 0.06µg/ml solution:

Accurately weigh and transfer 25mg of Levosulpiride working standard into a 25ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents

Further pipette 0.1 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent.

Further pipette 0.8 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent.

Preparation of 0.66µg/ml solution:

Accurately weigh and transfer 6.67mg Rabeprazole working standard into a 25ml clean dry volumetric flask add Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette 0.75ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluents

Further pipette 0.33 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent.

2.5 ROBUSTNESS:

As part of the Robustness, deliberate change in the Flow rate, Mobile Phase composition, Temperature Variation was made to evaluate the impact on the method.

A) The flow rate was varied at 0.9 ml/min to 1.1 ml/min.

Standard solution 75 µg/ml of Levosulpiride and 20 µg/ml Rabeprazole prepared and analysed using the varied flow rates along with method flow rate.

B) The Organic composition in the Mobile phase was varied from 54% to 66%

Standard solution 75µg/ml of Levosulpiride and 20 µg/ml Rabeprazole was prepared and analysed using the varied Mobile phase composition along with the actual mobile phase composition in the method.

3. RESULTS AND DISCUSSIONS

TRIAL 1

TRIAL2

Acceptance criteria:

• Resolution between two drugs must be not less than 2.
• Theoretical plates must be not less than 2000.
• Tailing factor must be not more than 2.
• It was found from above data that all the system suitability parameters for developed method were within the limit.

3.1 VALIDATION PARAMETERS:

3.1.1 ASSAY:

Standard and sample solution injected as described under experimental work. The corresponding chromatograms and results are shown below.

Figure 1 Chromatogram for Standard

Figure 2: Chromatogram for Sample

Results of Assay for Levosulpirideand Rabeprazole

3.1.2 LINEARITY:

The linearity range was found to lie from 10µg/ml to 50µg/ml of Levosulpirideand Rabeprazole and chromatograms are shown below.

Acceptance criteria:

Correlation coefficient (R2) should not be less than 0.999

The correlation coefficient obtained was 0.999 which is in the acceptance limit.

3.1.3 PRECISION:

Precision of the method was carried out for both sample solutions as described under experimental work. The corresponding chromatograms and results are shown below.

Results of Precision for Levosulpirideand Rabeprazole

Acceptance criteria:

%RSD for sample should be NMT 2

The %RSD for the standard solution is below 2, which is within the limits hence method is precise.

3.1.4 INTERMEDIATE PRECISION (ruggedness)

There was no significant change in assay content and system suitability parameters at different conditions of ruggedness like day to day and system to system variation.

Results of Intermediate precision for Levosulpiride and Rabeprazole

Acceptance criteria:

%RSD of five different sample solutions should not more than 2

The %RSD obtained is within the limit, hence the method is rugged.

3.1.5 ACCURACY:

Sample solutions at different concentrations (50%, 100%, and 150%) were prepared and the% recovery was calculated.

Accuracy (recovery) data for Levosulpiride and Rabeprazole

3.1.6 ROBUSTNESS:

The standard and samples of Levosulpiride and Rabeprazole were injected by changing the conditions of chromatography. There was no significant change in the parameters like resolution, tailing factor, asymmetric factor, and plate count.

Results for variation in flow for Levosulpiride and Rabeprazole