INTRODUCTION:

Bicalutamide (CAS 90357-06-5) is a non-steroidal androgen receptor antagonist¹. Generally, it is used in combination with other medications for the treatment of metastatic prostate cancer. Bicalutamide was developed from a series of nonsteroidal compounds related to flutamide that showed a range of pharmacologic activity from full androgen agonist to pure antiandrogen, including progestational and anti-progestational properties¹.

Figure 1: Chemical structure of Bicalutamide

Bicalutamide (Figure 1) is chemically N- [4-Cyano-3-(trifluoro methyl) phenyl]-3- [(4-fluoro phenyl) sulfonyl]-2-hydroxy-2-methyl propenamide with molecular formula C₁₈H₁₄F₄N₂O₄S and molecular weight 430.37 gm. Bicalutamide and its impurities were estimated by different analytical techniques using HPLC, UPLC, LC-MS/MS etc in pharmaceutical formulations as well as biological fluids. Palleshwar Rao et al., developed a stability indicating liquid chromatographic method² for the estimation of process related and degradation impurities of Bicalutamide tablet dosage forms using Waters Symmetry C18 column with PDA detector using a mixture of 0.1%v/v Trifluoro acetic acid and 0.05% w/v aq. sodium-1-octane sulphonic acid as mobile phase A and 0.1%v/v trifluoroacetic acid in acetonitrile as mobile phase B (65:35) (Isocratic mode) (Detection wavelength 270 nm). Nageswara Rao et al., developed a HPLC method³ for the Isolation and characterization of process related impurities and degradation products of Bicalutamide using 0.01M KH₂PO₄ (pH 3.0): Acetonitrile (50:50, v/v) with PDA detector (Detection wavelength 215nm) and the degradants as well as impurities were characterized using ESI-MS/MS, H¹ NMR and FT-IR spectral techniques. Lakshmana Rao et al., developed HPLC method⁴ using RP-C18 column for the estimation of Bicalutamide in pure and pharmaceutical dosage forms using a mobile phase consisting of phosphate buffer (pH 2.8) and acetonitrile (60:40) with flow rate 1.0ml/min and the linearity was found to be 25-150µg/ml. Nanduri et al., developed a stability-indicating HPLC and UPLC methods⁵ for the determination of Bicalutamide along with six new process related impurities using Zorbax SB phenyl column and HSS T3 column respectively using buffer solution consisting of 0.01M sodium dihydrogen ortho phosphate (pH adjusted to 6.0 with sodium hydroxide) and 0.001M sodium octane-1-sulphonic acid and 0.0025M tetra butyl ammonium hydrogen sulphate. A Zorbax SB phenyl column and (Gradient mode) a mixture of solution A (buffer and acetonitrile 95:5, v/v) and solution B (acetonitrile and buffer 90:10, v/v) were used (Detection wavelength 220nm). Saravanan et al., developed a stability-indicating LC assay method⁶ for Bicalutamide using Waters Symmetry shield RP18 HPLC column and mobile phase consisting of a mixture of phosphate buffer and acetonitrile with flow rate 1.8ml/min on gradient mode (Detection wavelength 270nm). LC-MS/MS^7-12 and HPLC¹³ methods were also proposed by different authors for the estimation of Bicalutamide in biological fluids. In the present study the authors have proposed a simple and specific stability indicating RP-HPLC method for the estimation of Bicalutamide in tablets.

EXPERIMENTAL:

Preparation of mobile phase:

0.68384 grams of potassium dihydrogen phosphate was accurately weighed and transferred in to a 1000ml volumetric flask and about 500ml of diluent was added to dissolve. The pH was adjusted to 2.90 using ortho phosphoric acid and finally the volume was made up to volume with HPLC grade water (Mobile phase A). A mixture of Acetonitrile: Methanol: Water (80: 10: 10, v/v) was used as mobile phase B. The diluent solution was prepared mixing water and Acetonitrile in 50:50 (v/v) ratio and was sonicated and filtered.

Preparation of stock solution:

25.7mg of Bicalutamide was weighed accurately and transferred in to a 25mL volumetric flask (1000µg/mL) and dissolved in HPLC grade Acetonitrile, sonicated and filtered before use. The stock solution (1000µg/mL) was further diluted with the diluent as per the requirement for the linearity, precision, accuracy, robustness and other studies and all the solutions were filtered through 0.45μm membrane filter before injection.

Instrumentation and optimised chromatographic conditions:

Shimadzu HPLC Model no. iSeries 2050C 3D with Lab solutions Software with PDA detector and Inertsil C₈ column (150 x 4.6mm., 5mm) were used for the present study. Mobile phase mixture consisting of phosphate buffer (pH adjusted to 2.90 using ortho phosphoric acid) was used as mobile phase A and a mixture of Acetonitrile: Methanol: Water (80: 10: 10, v/v) was used as mobile phase B with flow rate 1.3mL/min were the optimised chromatographic conditions (Detection wavelength 270nm).

Method validation¹⁴:

Linearity, Precision, Accuracy and Robustness studies:

0.1-120µg/mL Bicalutamide solutions were prepared from the Bicalutamide stock solution and diluted with the diluent (Water: Acetonitrile (50:50, v/v) and injected (n = 3) into the HPLC system and the peak area was noted from the respective chromatograms. Finally, the mean peak area (n=3) was calculated and a calibration curve was drawn by plotting the Bicalutamide concentration on the x-axis and the corresponding mean peak area on the y-axis. The LOD and LOQ were calculated from the S/N ratio. Intraday and interday precision studies were performed on the same day and on three different days (10, 20 and 40µg/mL) and the peak area of the chromatograms were recorded during the study from which the mean peak area (n = 3) was calculated. The percentage relative standard deviation was also calculated from the mean peak area and the standard deviation.

Accuracy study was performed by standard addition method i. e. spiking of Bicalutamide tablet formulation (20µg/mL) solution with 50%, 100%, 150% of Bicalutamide API and these solutions were injected (n=3) in to HPLC system and the chromatograms were recorded and the peak areas were noted from which the mean peak area and the % RSD were calculated from the linear regression equation. The robustness of the method was proved by incorporating a very small changes in the optimized chromatographic conditions such as flow rate (±0.1mL/min; 1.2 and 1.4mL/min), mobile phase composition (Tetra butyl ammonium hydrogen sulphate: Acetonitrile (±2% v/v; 52: 48 and 48: 52), detection wavelength (±2nm; 268 and 272 nm) and pH (±0.1unit; 2.80 and 3.00).

Stress degradation studies:¹⁵

Stress degradation or stress degradation studies were performed to determine the stability of Bicalutamide (100µg/mL) towards acidic hydrolysis, basic hydrolysis, oxidation and thermal degradation. The specificity of the method was determined from the stability studies and therefore Bicalutamide was exposed to different stress conditions as given below. Acidic degradation was performed by treating Bicalutamide (100µg/mL) solution with 2.0mL of 2N HCl solution for 4hours followed by neutralization with 2.0mL 2N sodium hydroxide solution, made up to volume and then 5µl of the resultant solution was injected in to the HPLC system. Thermal degradation was performed by heating the Bicalutamide (100µg/mL) solution at 80ºC for 4 hours and then cooled, diluted with mobile phase and 5 μl of the resulting solution was injected in to the HPLC system. Oxidative degradation was performed by heating Bicalutamide (100µg/mL) solution with 2.0mL 10% hydrogen peroxide solution in dark room for 4 hours and then the stressed sample was diluted with mobile phase and then 5µl of the resulting solution was injected in to the HPLC system. Alkaline degradation was performed by treating Bicalutamide (100µg/mL) solution with 2.0mL of 2N sodium hydroxide solution for 4 hours followed by neutralization with 2.0mL 2N HCl solution, made up to volume and then 5µl of the resultant solution was injected in to the HPLC system.

Assay of Bicalutamide tablets:

Bicalutamide was obtained as gift sample from Cipla Ltd (India). Bicalutamide is available as tablets with different brand names Calutide-50 Cipla Ltd (India), Bicatero (Hetero Labs) (India) with label claim 50mg and Bicaltab from GLS Pharma with label claim 150 mg. Twenty tablets of two different brands of Bicalutamide tablets were procured, powdered and tablet powder equivalent to 25mg of Bicalutamide was accurately weighed and transferred in to two different volumetric flasks and extracted with HPLC grade Acetonitrile and the mixture was sonicated, and filtered. The resultant solutions were diluted with the diluent as per the requirement and 100µg/mL of each of the marketed formulation solution was injected (n=3) in to the HPLC system and the peak area was noted from the respective chromatograms and then the mean peak area was calculated. The percentage of purity of Bicalutamide was calculated from the linear regression equation for the marketed formulations.

RESULTS AND DISCUSSION:

A new stability indicating RP-HPLC method has been developed and validated for the determination of Bicalutamide in tablets. Some of the important parameters of the previously published analytical methods were discussed in detail in Table 1.

Table 1: Literature survey of Bicalutamide

Mobile phase (v/v)	Detection wavelength (nm)	Linearity (µg/mL)	Comment	Ref
Mobile phase A: 0.1% Trifluoro acetic acid: 0.05% aq. sodium-1-octane sulphonic acid Mobile phase B: 0.1%v/v trifluoroacetic acid in Acetonitrile (65:35)	270	-	HPLC (Process related impurities)	2
0.01 M KH₂PO₄ (pH 3.0) buffer: Acetonitrile (50:50)	215	-	HPLC, ESI-MS/MS, IR and NMR (Related impurities)	3
Phosphate buffer (pH 2.8): Acetonitrile (60:40)	-	25-150	HPLC	4
Sodium dihydrogen ortho phosphate buffer (pH 6.0) and 0.001M sodium octane-1-sulphonic acid and 0.0025M tetra butyl ammonium hydrogen sulphate. Mobile phase A: Buffer: Acetonitrile (95:5) Mobile phase B: Acetonitrile: Buffer (90:10)	220	-	Stability indicating HPLC UPLC and 6 process related impurities	5
Phosphate buffer: Acetonitrile	270	-	Stability indicating RP-HPLC	6
Mobile phase A: Phosphate buffer (pH adjusted to 2.9 using ortho phosphoric acid) Mobile phase B: Acetonitrile: Methanol: Water (80: 10: 10)	270	0.1-120	Stability indicating RP-HPLC	Present work

Shimadzu HPLC Model no. iSeries 2050C 3D with Lab solutions Software with PDA detector and Inertsil C₈ column (150 x 4.6 mm., 5mm) were used for the present study. Phosphate buffer (pH adjusted to 2.90 using ortho phosphoric acid) was used as mobile phase A and a mixture of Acetonitrile: Methanol: Water (80: 10: 10, v/v) was used as mobile phase B with flow rate 1.3 mL/min were the optimised chromatographic conditions (Detection wavelength 270 nm). The method was linear over the concentration range 0.1-120 mg/ml with linear regression equation, y = 20351x + 980.75 (R² = 0.9999). The LOQ and LOD were found to be 0.0919 mg/ml and 0.0301 respectively. The injection volume was 10 µl and the run time was 20 min (Column oven temperature 30ºC and sample cooler 25ºC). A mixture of water: Acetonitrile (50:50, v/v) was used as diluent.

Table 2: Linearity of Bicalutamide

Conc. (µg/mL)	*Mean peak area
0	0
0.1	2078
0.2	4132
1	21057
2	40823
5	101891
10	203645
20	407182
40	809549
50	1021054
80	1652678
100	2039173
120	2425794

*Mean of three replicates

Method validation:

Bicalutamide has shown linearity over the concentration range 0.1-120mg/ml (Table 2) with linear regression equation, y = 20351x+980.75 (R² = 0.9999). The typical chromatogram obtained for the blank and Bicalutamide API were shown in Figure 2. The calibration was shown in Figure 3. The LOQ and LOD were found to be 0.0919 mg/ml and 0.0301 respectively. The %RSD was found to be 0.0039-0.0273 (Intraday) and 0.0133-0.1611 (Inter-day) (Table 3) in precision studies which is less than 2.0 indicating that the method is precise. The % recovery in accuracy studies was found to be 99.30-99.65% (Table 4) and % RSD was (0.0808-0.7159) less than 2% indicating that the method is accurate. The %RSD in robustness study was found to be 0.0031 - 0.0457 which was less than 2% indicating that the method is robust (Table 5).

Figure 3: Calibration curve of Bicalutamide

Table 3: Precision studies of Bicalutamide

Intraday precision study
Conc. (µg/mL)	Mean peak area	Statistical analysis *Mean peak area ± SD (% RSD)
10	203645	203644 ± 55.5068 (0.0273)
10	203588
10	203699
20	407182	407199 ± 16.0935 (0.0039)
20	407214
20	407201
40	809549	809584.6667 ± 32.3162 (0.0039)
40	809612
40	809593
Interday precision study
Conc. (µg/mL)	Day 1	Day 2	Day 3	Statistical analysis *Mean peak area ± SD (% RSD)
10	203645	203127	203734	203502 ± 327.7941 (0.1611)
20	407182	407266	407364	407270.6667 ± 91.0897 (0.0224)
40	809549	809694	809483	809575.3333 ± 107.9367 (0.0133)

*Mean of three replicates

Table 4: Accuracy study

Spiked Conc. (µg/mL)	*Conc. obtained (µg/mL) ± SD (%RSD)	% Recovery
10 (50%)	9.95 ± 0. (0.0808)	99.50
20 (100%)	19.93 ± 0.1252 (0.6282)	99.65
30 (150%)	29.79 ± 0.2133 (0.7159)	99.30

*Mean of three replicates

Table 5: Robustness study (40 µg/mL)

Parameter	Condition	Mean peak area*	Statistical Analysis *Mean±SD (% RSD)
Flow rate (± 0.1mL/min)	1.2 1.3 1.4	809017	809431.3333± 369.8166 (0.0457)
		809549
		809728
Detection wavelength (± 2 nm)	268 270 272	809651	809574 ± 68.0368 (0.0084)
		809549
		809522
Mobile phase composition 0.002% TEA (pH 2.31): Acetonitrile (± 2 %) (v/v)	48:52 50:50 52:48	809587	809577.3333 ± 24.9466 (0.0031)
		809549
		809596
pH (± 0.1 unit)	2.80 2.90 3.00	809253	809538.6667 ± 280.6427 (0.0347)
		809549
		809814

*Mean of three replicates

Assay of Bicalutamide tablets:

Two different brands of Bicalutamide tablets with label claim 50mg were chosen and the proposed optimized RP-HPLC method was applied for the quantification of Bicalutamide and the percentage of purity was calculated using the linear regression equation and the percentage of purity was found to be 99.42-99.74 (Table 6). The representative chromatogram obtained for the tablet formulation was shown in Figure 2C.

Table 6: Assay of Bicalutamide tablets

S. No.	Brand name	Label claim (mg)	*Observed amount (%w/w)	% Recovery*
1	Brand I	50	49.71	99.42
2	Brand II	50	49.87	99.74

*Mean of three replicates

Stress degradation studies:

Bicalutamide was eluted at 7.275 min with theoretical plates 7707 (> 2000) and tailing factor 1.085 (< 1.5).

During the acidic degradation Bicalutamide has shown 3.68% degradation and Bicalutamide was eluted at 7.275 min with theoretical plates 7738 and tailing factor 1.130 with extra peaks at 3.744min and 4.341min with resolution greater than 2%. During the thermal degradation Bicalutamide has shown 0.06% degradation and Bicalutamide was eluted at 7.285min with theoretical plates 7690 and tailing factor 1.133 with extra peaks at 4.352min with resolution greater than 2%. During the oxidative degradation Bicalutamide has shown 0.98% degradation and Bicalutamide was eluted at 7.285min with theoretical plates 7705 and tailing factor 1.132 with extra peak at 4.363min with resolution greater than 2%. During the alkaline degradation Bicalutamide has shown 7.18% degradation and Bicalutamide was eluted at 7.285min with theoretical plates 7748 and tailing factor 1.105 with extra peaks at 3.744min and 4.352min with resolution greater than 2%. The method is specific as Bicalutamide peak was clearly eluted even in presence of the degradants during all the stress degradation studies. The system suitability parameters were within the acceptable criteria (Table 7) and the resultant chromatograms obtained during the stability studies were shown in Figure 4.

Table 7: Stress degradation studies of Bicalutamide

Stress condition	R_t (min)	Mean peak area	% Recovery	% Drug degradation	Theoretical Plates (>2000)	Tailing factor (<1.5)	Resolution (>2)
Standard drug	7.275	2039654		-	7707	1.085	-
Acidic degradation 2N HCl / 4 hrs	3.744 4.341 7.275	1964575	96.32	3.68	7738	1.130	9.288 3.100
Alkaline degradation 2N NaOH/4 hrs	3.744 4.352 7.285	1893268	92.82	7.18	7748	1.105	9.147 2.957
Oxidation degradation H₂O₂/ 4 hrs	4.363 7.285	2019587	99.02	0.98	7705	1.132	7.870
Thermal degradation 80ºC / 4 hrs	4.352 7.285	2038442	99.94	0.06	7690	1.133	7.675

*Mean of three replicates

Acidic degradation

Thermal degradation

Oxidative degradation

Alkaline degradation

Figure 4: Representative chromatograms of Bicalutamide (100µg/mL) during stress degradation studies

CONCLUSION:

Bicalutamide is an anti-cancer drug and a a new stability indicating RP-HPLC method has been developed and validated as per ICH guidelines. The method is simple and specific and there is no interference of the degradants and the system suitability parameters were within the acceptable criteria. The proposed method can be applied for the quantification of Bicalutamide in pharmaceutical formulations.

ACKNOWLEDGEMENT:

The authors are grateful to Cipla Ltd (India) for providing the gift samples of Bicalutamide. The authors declare no conflict of interest.

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Received on 06.01.2025 Revised on 11.04.2025

Accepted on 19.06.2025 Published on 02.08.2025

Available online from August 08, 2025

Research J. Pharmacy and Technology. 2025;18(8):3817-3823.

DOI: 10.52711/0974-360X.2025.00549

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

GITAM School of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, India.