Development and Standardization of Chromatographic Technique for Quantification of Bicalutamide in Pure and Pharmaceutical Dosage Forms

Introduction

Analytical chemistry deals with separation, identification, characterization and determination of the amount of components of a sample.^1,2 In Analytical chemistry, ‘Quality’ is associated with accuracy and reproducibility; other criteria can be cost, speed and information.^3,4 Quantitative analysis deals with the determination of how much of one or more constituents are present.^5,6 HPLC or High Performance Liquid Chromatography is the most common analytical technique used for separation, identification, purification and quantification of compounds. Even though HPLC technique is helpful to the greatest extent for biotechnological, biomedical and biochemical investigations along with the pharmaceutical industry, the above mentioned areas presently include only about 50% of HPLC users.^7,8 Validation is the action of providing evidence that the method, development activity, material and equipment is giving the required results. The primary purpose of validation is to write procedures for manufacture and control over the process which ensures that the drug output possesses the character, stability, quality and purity as described in the process. Standard of product, safety and efficacy are a must for the products. Steps involved in the manufacturing process should be ensured that the end-product meets every standard parameter.^9,10,11

Bicalutamide (BTM)¹ is a non-steroidal antiandrogen, devoid of other endocrine activity. It binds to androgen receptors without activating gene expression, and thus inhibits the androgen stimulus and also inhibits the prostatic tumors. It is a white to off-white crystalline powder with molecular formula C₁₈H₁₄F₄N₂O₄S and molecular weight of about 430.37. It is soluble in acetonitrile, acetone, moderately soluble in water and insoluble in n-hexane. It is mainly used as an anti-cancer drug.¹²

Material and Methods

Reagents and chemicals

Bicalutamide was obtained as a gift sample from Micro Labs. Acetonitrile HPLC grade was procured from Merck Ltd., methanol HPLC grade was procured from Qualigen and water HPLC grade was obtained from Merk Ltd. 

Instrumentation and chromatographic conditions

The chromatographic column used was a reverse phase 4.6×250 mm Phenomenex C18 HPLC column with 5 µm particles. The column and the HPLC system were kept at ambient conditions. Mobile phase used was Acetonitrile:Methanol (60:40) supplied at a rate of 1.0 mL/min. Injection volume of 20 mL was used. The analyte was analyzed by a UV detector set at 275 nm.

Method Development

Selection of solvent system

Mobile phase was selected in connection with its components and proportions. The solvent system with Methanol:Acetonitrile:Water (40:40:20) was used and then the other ratio of the same components 60:30:10, 30:50:20 and 40:20:40 was also tried. But with this mobile phase, proper elution of peaks was not obtained. The peak was broad. The other composition Acetonitrile:Methanol was also tried by using different ratios. Finally, a mobile phase constituting of acetonitrile and methanol in the ratio of 60:40 was employed. It produced good resolution, reasonable retention times and acceptable peak symmetry for both the drugs.

Selection of wavelength

Accurately weighed 50 mg of BTM was transferred into 100 mL volumetric flask and the volume was made up to the mark using methanol. From this, 1 mL of the stock solution was taken and was further diluted with methanol up to 10 mL to maintain the concentration of 10 µg/mL. It was scanned on UV-Visible spectrophotometer in the range of 200 nm to 400 nm wavelength.

Preparation of mobile phase

The selected mobile phase containing acetonitrile and methanol in a ratio of 60:40 v/v was prepared and ultrasonicated for ten minutes. The mobile phase was then filtered through a 0.45 µ membrane filter.

Preparation of standard stock solutions

Accurately weighed 50 mg of standard BTM was transferred into 100 mL volumetric flask and volume was made up to the mark using mobile phase. Occasional shaking and sonication was done for 10 minutes to dissolve the drug.

Preparation of working standards

50 mg of the drug sample was weighed accurately, transferred to 100 mL volumetric flask, was dissolved in methanol and the volume was made up to 100 mL with methanol. From the above 1 mL, 2 mL, 4 mL, 6 mL, 8 mL solutions were taken and diluted to 10 mL with methanol to prepare concentrations of 10, 20, 40, 60, 80 µg/mL.

Method validation

The proposed method was standardized in terms of various validation parameters such as specificity, selectivity, linearity, range, Limit of Detection (LOD) and Limit of Quantification (LOQ), precision, and repeatability as per ICH guidelines.

System suitability

In order to check the suitability of instrument for the planned analysis system, suitability was performed on a daily basis by injecting 100 μg/mL of drug solution.

Specificity and selectivity

20 μL of mobile phase solvent and 100 μg/mL drug solution were injected to the HPLC system to observe the interference of analyte at retention time.

Linearity and range

Serial dilution of the drugs was prepared ranging from 10-100 μg/mL, then injected in the HPLC in triplicate with injection volume of 20 μL. Chromatograms were integrated and data was collected. By using mean peak area of analyte vs concentration, calibration curve was plotted and correlation coefficient and linear regression were calculated.

LOD and LOQ

Limit of Detection (LOD) and Limit of Quantification (LOQ) values were procured from the statistical methods and reported. LOD was determined by putting the Formula STEYX/SLOPE x 3.3 into the excel sheet of linearity. STEYX/ SLOPE x 3.3. LOQ was determined by putting the Formula STEYX/SLOPE x 10 into the excel sheet of linearity. STEYX/ SLOPE x 10.

Precision and Repeatability

It was performed by injecting three replicates of different concentrations of drug solution into HPLC and the area was calculated from chromatograms obtained and % RSD (Relative Standard Deviation) was calculated.

Assay

Solid dose formulation containing 50 mg of BTM dose was obtained from the local market of Belgaum. Twenty tablets of BTM were weighed and triturated to make fine powder. Powder equivalent to 50 mg of BTM was weighed and transferred into 100 mL of volumetric flask, dissolved in sufficient amount of mobile phase, was sonicated for 10 minutes and volume was made up to the mark using the same to obtain 500 μg/mL of drug solution. The above solution was filtered and 10 mL of filtrate was taken and transferred into 100 mL of graduated flask to obtain 50 μg per ml of BTM. This was injected into HPLC and drug content was calculated.

Results

Method development

The method development was started with solubility analysis of BTM in different solvents. Several trials were made by using different solvent compositions utilizing acetonitrile and methanol. Mixture of acetonitrile and water gives poor elution of analyte and hence methanol in combination with acetonitrile was used to obtain the intense and sharp peak with minimum tailing.

Selection of wavelength of detection of analyte

The solution containing BTM was scanned in the UV Spectrophotometer between the range of 400-200 nm and spectrum was obtained. The drug showed maximum absorbance at 275 nm and detection was executed at 290 nm. The UV spectrum is shown in Figure 2.

Determination of Retention Time

20 μL of 100 μg/ml drug solution was injected into HPLC and chromatogram was obtained. The retention time of drug was found to be at 4.7 minutes as shown in Figure 3. The developed method parameters are displayed in Table 1.

Specificity and selectivity

Developed method was determined to be specific and selective as there was no interference of some peak at the retention time of BTM. Chromatogram of BTM is shown in Figure 4.

Linearity and Range

Method used was established to be linear between concentration range of 10-100 μg/mL with correlation coefficient 0.999 and % curve fitting at 99.99. The data obtained was displayed in Table 2. Calibration curve was constructed and showed in Figure 5.

LOD and LOQ

LOD and LOQ values of Bicalutamide were found to be 3.41 μg/mL and 10.34 μg/mL respectively by statistical calculations.

Precision

The % RSD of area obtained in each replicate of standard solution and sample solution were calculated by injecting three replicates solution of analyte in different concentrations. Table 3 and 4 presents the data of precision and repeatability study.

Assay

Three replicates of sample solution were injected into the HPCL and data obtained from chromatograms % drug content in Bicalutamide tablet was calculated. Quantity of drug present in the marketed tablet formulation was established to be 47.6 mg and % assay was established to be 98.60%. Assay Chromatogram is presented in Figure 6 and results are presented in Table 5.

Discussion

The chromatographic analytical development was initiated with solubility testing of BTM in various solvents. Few trials were made by using different solvent compositions using the acetonitrile and methanol. Mixture of acetonitrile and water gives poor elution of analyte and hence methanol in combination with acetonitrile was used to obtain the intense and sharp peak with minimum tailing. The drug showed maximum absorbance at 275 nm and detection was executed at 290 nm. In order to determine the retention time of analyte, about 20 μL of 100 μg/ml drug solution was injected into HPLC and chromatogram was obtained. The retention time of drug was found to be at 4.7 minutes. Developed method was specific and selective as there was no interference of some peak at the retention time of BTM. Method used was established to be linear between concentration range of 10-100 μg/mL with correlation coefficient of 0.999 and % curve fitting at 99.99. The method was found to be precise and repeatable with % RSD of area obtained in each replicate of standard solution and sample solution were less than 2%. Quantity of drug present in the marketed tablet formulation was established to be 47.6 mg and % assay was established to be 98.60% which indicates the applicability of proposed method.

Conclusion

For the estimation of BTM in bulk, the new HPLC analytical technique was developed and validated as per the ICH guidelines. All the results obtained were found to be within the acceptance limit of the guidelines. The developed method was found to be simple, selective, specific, precise, and rugged and can be adapted for the routine quality control analysis of BTM in bulk and dosage forms.