Development and Validation of Analytical Method for Assay of Carbimazole Tablet

Introduction to chromatography: High-performance liquid chromatography (or High-pressure liquid chromatography, HPLC) is a specific form of column chromatography generally used in biochemistry and analysis to separate, identify, and quantify the active compounds. HPLC mainly utilizes a column that holds packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent(s) used. The sample to be analyzed is introduced in small volume to the stream of mobile phase and is retarded by specific chemical or physical interactions with the stationary phase. The amount of retardation depends on the nature of the analyte and composition of both stationary and mobile phase The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time. Common solvents used include any miscible combinations of water or organic liquids (the most common are methanol and acetonitrile). Separation has been done to vary the mobile phase composition during the analysis; this is known as gradient elution. The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase. The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte.

Origin of HPLC: High performance liquid chromatography, HPLC, is an analytical technique in general use. It evolved from preparative column chromatography and its performance (efficiency, resolution) has been greatly enhanced by the use of elaborate stationary phases composed of spherical particles with diameters of between 2 and 5 um. However, because the particles are small, the head pressure needed to force the mobile phase through the column packing must be greatly increased compared to that used in preparative liquid chromatography. Because of this, the letter P in HPLC has occasionally corresponded to the word pressure. The forced migration of a liquid phase through a stationary phase is encountered in many chromatographic techniques. One of the aspects particular to HPLC is that of the partition mechanisms between analyte, mobile phase and stationary phase. These are based on coefficients of adsorption or partition.

MATERIAL AND EQUIPMENTS

Experimental Work

Preparation of Buffer solution: Transfer 2.46 g of sodium dihydrogen phosphate monohydrate into a 1000 mL water and mix. Filter through 0.45µm nylon membrane filter.

Preparation of mobile phase: Mix Acetonitrile and above buffer solution in the ratio of 200:800(v/v) respectively. Degas by sonication for 10 minutes.

Chromatographic Parameters: Use suitable High-Performance Liquid Chromatography equipped with following:

Preparation of diluent: Mix Acetonitrile and water in the ratio of 100:900 (v/v) respectively.

Preparation of blank: Filter the diluents through 0.45µm nylon syringe filter and use as a blank.

Preparation of standard stock solution: Weigh and transfer accurately about 50mg of Carbimazole working standard into a 100mL volumetric flask. Add 70 mL of diluent, sonicate to dissolve and dilute up to the volume with diluent.

Preparation of standard solution: Transfer 5.0 mL of standard stock solution into a 50 mL volumetric flask and dilute up to the volume with diluent. Filter the solution through 0.45 µm nylon syringe filter and discard first 5 mL of filtrate.

Preparation of sample solution: Determine the average weight of tablets. Crush tablets to fine powder. Weigh and transfer the sample equivalent to 50mg of Carbimazole into a 100 mL volumetric flask, add 70mL of diluent, sonicate for 15minutes with intermediated shaking and make up the volume with diluent. Filter the solution through 0.45µm nylon syringe filter. Transfer 5.0mL of sample solution into a 50 mL volumetric flask and make up the volume with diluent.

Procedure: Separately inject 10µL of blank, standard solution and sample solution into the chromatographic system. Record the chromatograms and measure the peak areas count for Carbimazole peak. The retention time of Carbimazole peak is about 6.2 minutes.

General Sequence

Evaluation of system suitability:

From standard solution, The Theoretical plate for Carbimazole peak from first injection of standard solution should not be less than 3000. The Tailing factor for Carbimazole peak from first injection of standard solution should not be more than 2.0 The % RSD for the peak areas of Carbimazole peak from five replicate injections of standard solution should not be more than 2.0 The % RSD for the peak areas counts of Carbimazole peak from five replicate injections and bracketing standard should not be more than 2.0 and should meet system suitability criteria with point No.1 and 2.

Calculations

Where,

AS= Average peak area counts of Carbimazole peak from five replicate injections of standard solution.

AT= Average peak area counts of Carbimazole peak from two replicate injections of sample solution.

WS= Weight of Carbimazole working standard takenin mg

AW= Average of tablets

WT= Weight of sample

P= Percent Potency of Carbimazole working standard on as Is basis

LC= Label claim of Carbimazole in mg per tablet.

RESULT AND DISCUSSION

The work was carried out for development and validation of analytical method for assay of carbimazole tablet.

HPLC method development:

Mobile phase preparation: Acetonitrile: buffer solution (200:800v/v).

Preparation of diluents: Acetonitrile and water in the ratio of 100:900 (v/v).

Chromatographic parameters: Use suitable High-Performance Liquid Chromatography equipped with following:

Specificity:

Specificity by interference:

Analyzed blank, worst case placebo (duplicate preparation), standard solution, as such test solution and test solution spiked with impurities at 1% level.The % assay for as such test solution and spiked test solution are compiled in table-1 and the retention time, peak purity data of spiked sample with impurities are compiled in table- 2

Acceptance Criteria:

There should be no interference from blank and placebo at the retention time of Carbimazole peak.

1. The % difference of test solution and spiked test solution should be within ± 2.0

2. Peak purity for Carbimazole peak should pass in spiked test solution.

Linearity:

The linearity of Carbimazole was performed using standard solution in the range of 10mcg/mL to 90 mcg/mL (about 20% to 180% of the test concentration). A graph was plotted with concentration mcg/mL on X axis and peak area of Carbimazole on Y axis. Slope, y-intercept and correlation coefficient is determined. The results are tabulated in table – 4 and graphically represented as below.

Acceptance criteria:

The correlation coefficient ‘r’ should not be less than 0.999 The Bias at 100% response should be within ±2.0

Fig 1: Linearity plot

Precision:

System precision

Prepared standard solution as per test method and injected for six times into HPLC system. The mean and % RSD for peak areas of Carbimazole was calculated. The results are tabulated in table – 5.

Acceptance criteria: The %RSD for the peak area of Carbimazole peak from six replicate injections of standard solution should not be more than 2.0

Method precision: Prepared six samples of Carbimazole tablets 5mg and 20 mg and analysed as per the test method. Calculated the % RSD for assay of six samples preparation and results are tabulated in table – 6.

Acceptance criteria:1. % RSD of the assay results of six sample solutions should not be more than 2. The individual % assay value should be with in specification limit.

Intermediate precision (Ruggedness):

Performed ruggedness by preparing six sample solutions of same batch of Carbimazole tablets 100mg, by different analyst, different column serial no and different system on different day. Evaluated the system suitability criteria. Calculated % RSD of the assay result of six sample solutions of intermediate precision and overall % RSD of assay results of twelve sample solutions (six sample solutions from method precision and six sample solutions from intermediate precision). The results are tabulated in table-7.

Acceptance criteria:

1. % RSD of the assay results of six sample solutions should not bemorethan2.0

2. Overall % RSD of the assay results of twelve sample solutions (six sample solutions of method precision and six sample solutions of intermediate precision) should not be more than 2.0

3. The individual % assay value should be within specification limit.

Accuracy: Known amount of Carbimazole was spiked in placebo at about 20% and 180% of test concentration. The working level concentration for Carbimazole is 50µg/mL. The amount of Carbimazole was quantified as per the test method. The % recovery was calculated from the amount found and amount added. The results are tabulated in table 9.8.

Acceptance criteria:

Individual % Recovery of Carbimazole at each level should be in between 98.0 and 102.0.

Range:

Conclusion:

The method was found linear in the range 20 % to 180 % of test concentration i.e.10 mcg/mL to 90 mcg/mL.The method was found accurate from 20 % to 180 % of test concentration. The method was found precise as % RSD of the assay results is found within the acceptance range. Also % assay results are within the specification limit. The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity. So the range is concluded from linearity, precision and accuracy.

Solution Stability: The stability of Carbimazole Tablets BP 20 mg was verified by analyzing sample and standard solution initially and at different time interval. Calculated the similarity factor for standard solution and for test solution calculated % difference in assay results with respect to initial results. The results are tabulated in table–9 and table-10.

Acceptance criteria: The standard similarity factor should be in the range of 0.98 to1.0 The % difference in assay results from initial to different time intervals should be with in ±2.0.

Table 14 Standard solution

Table 15 Sample solution

Robustness:

• Robustness of the method was verified by deliberately varying the following conditions.
• By changing the flow rate by ?10%.
• By changing the column oven temperature by?5°C.
• By changing the organic content in mobile phase by ??5% absolute.
• By changing method wavelength ?2nm.
• The system suitability and % assay was evaluated at each condition. The robustness parameters results are tabulated in table-11.

Acceptance criteria:

The system suitability criteria should comply.

The % difference between the mean assay results of varied conditions and mean assay of as such condition (as per methodology) using the same batch should be within ± 2.0%.                                      

Filter Validation:

For sample: Selection of filter was evaluated by performing assay on sample solution of 5mg strength. Centrifuged sample, 0.45 µm nylon filter and 0.45 µm PVDF filter were analyzed against the unfiltered standard solution. The % assay was calculated for each filter and compared the results with centrifuged sample solution results. The results are tabulated in table–12 and table-13.

Acceptance criteria:

For sample: The % difference of centrifuged sample results and filtered sample results should be within ± 2.0

For standard: Selection of filter was evaluated by standard solution filter through 0.45µm. Nylon filter and 0.45µmPVDFand calculated %difference for peak area against peak area of unfiltered standard solution. The results are tabulated in table – 14 and table 15.

Acceptance criteria:

For standard: The % difference between mean area of unfiltered standard and mean area of filtered standard should be within ± 2.0

Typical Chromatogram of Blank

A. Typical Chromatogram of Placebo

B. Typical Chromatogram of Placebo

C. Typical Chromatogram of Standard

CONCLUSION

Based upon above results it is concluded that the assay method is specific. The detector response of Carbimazole is linear in the concentration range from10.071mcg/mL to90.641 mcg/mL i.e. 20% to 180% level. The % RSD of six replicate injections of standard solution indicates an acceptable level of precision. The% RSD value indicates an acceptable level of precision for the assay results. Hence it is concluded that the assay method is precise. The% RSD value indicates an acceptable level of precision for the assay results. Hence it is concluded that the assay method is precise and reproducible The analytical method meets the pre-established acceptance criteria for accuracy study as per protocol. Hence it is concluded that the assay method is accurate.Based upon above results it is concluded that the standard and test solution are stable up to 35hrs and 36 hrs respectively at 10ºC.Based upon above results it is concluded that the assay method is robust for change in flow rate, change in column oven temperature, change in wavelength, change inorganic content in mobile phase for Carbimazole tablets5mg and 20mg.Based on above study it is concluded that 0.45µmPVDF filter and 0.45µmNylon filter is found to be suitable for both standard and sample solution for filtration.

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