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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access  Full Text Article                                                                                           Research Article 

Method Development and Validation for Quantitative Estimation of Bortezomib in its Bulk and Pharmaceutical Formulation

Banothu Srikanth¹*, Badi Pavani², Ollepu Keerthana ², Iqbal Hussain², H. Indira², Adike Vamshi², Faruk Mondal²

¹ Assistant Professor, Department of Pharmaceutical Analysis, St. Mary's group of institution, Deshmukhi (Village), Pochampally (Mandal), Yadadri Bhuvanagiri (Dist), Telangana-508284, India.

² St. Mary's group of institution, Deshmukhi (Village), Pochampally (Mandal), Yadadri Bhuvanagiri (Dist), Telangana-508284, India.

INTRODUCTION

Chromatography is a powerful separation tool that is used in all branches of science, and is often the only means of separating components from complex mixtures. It is mostly implemented in science subjects such as chemistry and life sciences, especially in biochemistry. Chromatography comes from the Greek words, chroma meaning 'color' and graphein meaning 'to write'. Chromatography can be analytical or preparative, based on the main objective for which it has been followed. Analytical chromatography makes use of only a small amount of a mixture and determines the components of that particular mixture. Depending on the techniques used in chromatography, the process is broadly classified as, Adsorption chromatography, Partition chromatography ¹. New types of chromatography developed during the 1930s and 1940s made the technique useful for many separation processes. Chromatography technique was developed substantially as a result of the work of Archer John Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s. They established the principles and basic techniques of partition chromatography, and their work encouraged the rapid development of several chromatographic methods like, Paper Chromatography, Gas Chromatography, High Performance Liquid Chromatography. Advances are continually improving the technical performance of chromatography, allowing the separation of increasingly similar molecules. Chromatography is used as a technique to separate the additives, vitamins, preservatives, proteins and amino acids. Some other uses are in the detection of drugs or medications in the urine. It is used by pharmaceutical companies to prepare large amounts of pure materials that are further required in making medicines. Also, it is used to check the presence of any contamination in the manufactured compounds. It is also very popular in forensic science for investigative purposes ².

MATERIALS AND METHODOLOGY:

Materials:

For the determination of Bortezomib in tablet dosage form by HPLC method the following Chemicals were used such as Methanol, Distilled water, Acetonitrile, Formic acid.

Method Development 

Chromatographic trails for estimation of Bortezomib by RP-HPLC.

Table 1: Trial 1: Chromatographic trails for estimation of Bortezomib by RP-HPLC

Figure 1: CMG showing trial-1 chromatogram

Table 2: Trail 2: CMG showing trial-1 chromatogram 

Figure 2: CMG showing trial-2 chromatogram

Figure 3: CMG showing Optimized chromatogram

Preparation of the optimized mobile phase:

Preparation of 0.1% formic acid 

Weigh 0.1ml of formic acid and transferred to 100 ml volumetric flask, dissolved in sufficient quantity of water and then diluted to the mark with water ³.

Preparation of mobile phase

Preparation of mobile phase by using Methanol and water in the ratio of Acetonitrile : 0.1% formic acid (50: 50 v/v). The mobile phase was filtered through 0.45 um membrane filter paper. After filtration it was ultra sonicated for 10 minute on ultra sonicator ⁴.

Preparation of stock solution of Bortezomib 

API of Bortezomib (10mg) accurately weighed and transferred to 10 ml volumetric flask, dissolved in mobile phase. The solution contains 1000ug/ml of Bortezomib .The solution was filtered through 0.45 um membrane filter paper and firs few drops of filtrate were discarded ⁵.

Preparation of sample solution:

Take 25 mg equivalent tablet powder of GSF and dilute up to 25 ml with Acetonitrile : 0.1% formic acid (50: 50 v/v). Sonicate it for 10 minutes. Filter the solution through Whatmann filter paper no. 41. This solution was used as sample solution ⁶.

Procedure: 20 µL of the blank, standard and sample was injected in to the chromatographic system and areas for the Bortezomib peaks were used for calculating the % assay by using the formulae ⁷.

System suitability 

• Tailing factor for the peaks due to Bortezomib in standard solution should not be more than 1.5.
• Theoritical plates for the Bortezomib peaks in standard solution should not be less than 1.5.

Analytical Method Validation

Validation parameters

1. Specificity
2. Linearity
3. Range
4. Accuracy
5. Precision

1. Repeatability
2. Intermediate precision

1. Limit of Detection
2. Limit of Quantitation
3. Robustness

1. Specificity ⁸: In the case of assay, demonstration of assay specificity is required to show that the procedure is unaffected by the impurities or exipients. Specificity of an analytical method indicates that the analytical method is its able to measure accurately and specifically the analyte of interest without any interference from blank. So here, the specificity was determined by the comparision of the chromatograms of 

1. Blank (mobile phase)
2. Standard  sample solutions of Bortezomib 
3. Sample solution of Bortezomib 

Acceptance criteria:

Chromatogram of standard and sample should be identical with near Retention time.

2. Linearity ⁹

Preparation of solution: 

API of Bortezomib (10mg) accurately weighed and transferred to 10 ml volumetric flask, dissolved in sufficient quantity of methanol and then diluted to the mark with mobile phase.

Preparation of level – 1 (20µg/ml of Bortezomib )

From the standard solution (SS) 0.2 ml was taken in to 10 ml volumetric flask and diluted up to mark with methanol.

Preparation of level – 2 (40µg/ml of Bortezomib )

From the standard solution (SS) 0.4 ml was taken in to 10 ml volumetric flask and diluted up to mark with diluents.

Procedure:

Each level was injected in to the chromatographic system and peak area was measured. Plot a graph of peak area versus concentration (on x –axis concentration and on y axis peak area) and the correlation was calculated. 

Acceptance criteria

Correlation coefficient should be not less than 0.9994.

3. Range ¹⁰

Based on precision, linearity and accuracy data it can be concluded that the assay method is precise, linear and accurate in the range of 20-120 µg/ml of Bortezomib respectively.

4. Accuracy ¹¹

The accuracy of the test method is demonstrated by % of recovery. Accuracy was performed in three different three concentration levels and injected three times (Like 50%, 100%, and 150%). The observations are mentioned below.

Preparation of sample solutions

Preparation of 50% solution (with respect to target assay concentration)

From the stock solution 0.4ml was taken in to 10ml volumetric flask and diluted up to the mark with diluents.

Preparation of 100% solution (with respect to target assay concentration)

From the stock solution 0.8 ml was taken in to 10ml volumetric flask and diluted up to the mark with diluents.

Procedure:

The standard solutions of accuracy 50%, 100% and 150% were injected in to chromatographic system. Calculate the amount found and amount added for Bortezomib and calculate the individual % recovery and mean % recovery values. 

Acceptance criteria

% Recovery at each spike level shall be not less than 98.0 and not more than 102.0.

5. Precision ¹²

Preparation of solution

From the stock solution 0.8 ml was taken in to 10ml volumetric flask and diluted up to the mark with diluents

Procedure

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

Acceptance criteria

The % RSD for the area of five standard injections results should not be more than 2.

To evaluate the intermediate precision (also known as ruggedness) of the method, precision was performed on different day by using different make column of same dimensions.

Preparation of solution

From the stock solution 0.8 ml was taken in to 10ml volumetric flask and diluted up to the mark with methanol.

Procedure

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

Acceptance criteria

The % RSD for the area of five sample injections results should not be more than 2%.

6. Limit of detection (LOD) 

LOD’s can be calculated based on the standard deviation of the response (SD) and the slope of the calibration curve (s) at levels approximating the LOD according to the formula. The standard deviation of the response can be determined based on the standard deviation of y- intercepts of regression lines ¹³.

Formula:    LOD = 3.3 X                

Where

SD – Standard deviation (SD)                 S – Slope

Acceptance criteria

The values should not more than 3 for LOD solution.

7. Limit of quantification (LOQ) 

LOQ’s can be calculated based on the standard deviation of the response (SD) and the slope of the calibration curve (s) according to the formula. Again, the standard deviation of the response can be determined based on the standard deviation of y- intercepts of regression lines ¹⁴.

Formula:      LOD = 10 X                 

Where

SD – Standard deviation (SD)                      S – Slope

Acceptance criteria

The values should not more than 10 for LOQ solution.

8. Robustness ¹⁵

As Part of the robustness, deliberate change in the flow rate, mobile phase composition was made to evaluate the impact on the method

1. The flow rate was varied at ± 10%. Standard solution 40 µg/ml of Bortezomib was prepared and analysed using the varied flow rates along with method flow rate. 
2. The Temperature was varied (± 5^oC) Standard solution 40 µg/ml of Bortezomib was prepared and analysed using the varied flow rates along with method flow rate. 

RESULTS AND DISCUSSION

Validation Report:

Specificity:

Specificity by Direct comparison method

There is no interference of mobile phase, and placebo with the analyte peak and also the peak purity of analyte peak which indicate that the method is specific for the analysis of analytes in their dosage form [Table 3].

Table 3: Specificity Data

System suitability: System suitability test was an integral part of method development and has been used to ensure adequate performance of the chromatographic system [Table 4].

Table 4: Results of System Suitability

Results for intraday and inter day precision

Table 5: Intraday and Inter day precision

Table 6: Linearity

Linearity and range

Figure 4: Chromatogram showing Linearity 20 µg/mL

Figure 5: Chromatogram showing Linearity 40 µg/mL

Accuracy

Accuracy of the method was determined by Recovery studies [Table 7]. To the formulation (pre analyzed sample), the reference standards of the drugs were added at the level of 50%, 100% [Figure 6,7].

Figure 6: Chromatogram showing Accuracy 50% injection-1

Figure 7: Chromatogram showing Accuracy 100% injection-2

Table 7: Results of accuracy

LOD & LOQ:

Table 8: Results of LOD & LOQ:

Robustness:                     

Figure 8: Chromatogram showing flow rate 0.9mL/min

Figure 9: Chromatogram showing flow rate 1 mL/min

Figure 10: Chromatogram showing flow rate 1.1 mL/min

Change of Flow rate (± 0.1mL)

Table 9: Robustness

Change in Temperature (± 5^oC)

Table 10: Robustness

CONCLUSION:

The analytical method was validated according to ICH guidelines (ICH, Q2 (R1). The linearity study for Bortezomib was found in concentration range of 20µg-120µg/ml and correlation coefficient (r²) was found to be 0.9994, % recovery was found to be 101.37%, % RSD for repeatability was 1.778, % RSD for intermediate precision was 1.537 respectively. The precision study was precise, robust, and repeatable. LOD value was 2.6402, and LOQ value was 7.281 respectively. Hence the suggested RP-HPLC method can be used for routine analysis of Bortezomib in API and pharmaceutical dosage form.

Acknowledgement: I would like to thank Principal sir (Dr. Kamal Hassan) St. Mary’s Group of Institutions, Deshmukhi (Village), Pochampally (Mandal), Yadadri Bhuvanagiri(Dist), Telangana-508284, India.

Conflict of Interest: The authors attest that they have no conflict of interest in this study.

Funding: No Funding

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