Stability Indicating RP-HPLC method for the determination of Abiraterone (An Anti-Cancer Drug)

 

Mukthinuthalapati Mathrusri Annapurna*, Debi Prasad Pradhan, Krishna Chaitanya Routhu

Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy,

GITAM (Deemed to be University), Visakhapatnam, Andhra pradesh-530045, India.

 

ABSTRACT:

Abiraterone is used for the treatment of prostate cancer for men. Abiraterone is available as Abiraterone acetate. Food and drug administration approves Abiraterone for treatment of men with advanced prostate cancer. A new stability indicating RP-HPLC method has been proposed for the quantification of Abiraterone. Capcell PAK C18 column (100 mm × 4.6 mm i.d., 3 µm particle size) was used for the chromatographic study of Abiraterone acetate with mobile phase mixture 0.1 % acetic acid and acetonitrile (11:89 v/v) (Flow rate: 1.2 mL/min) on Shimadzu Model CBM-20A/20 Alite HPLC system (PDA detector). Forced degradation studies were performed and the method was validated.

 

 

 

INTRODUCTION:

Abiraterone (C₂₄H₃₁NO) is an irreversible inhibitor of 17α-hydroxylase/C17, 20-lyase (CYP17) which is play a part in the production of androgens in the testes and adrenal glands^1-2. Abiraterone (Fig 1) is practically insoluble over a wide pH range of reagents and slightly soluble in HCl and organic solvents. Abiraterone was determined by LC-MS^3-4, LC-MS/MS⁵, spectrofluorimetry⁶, liquid chromatography with fluorescence detection⁷ in cancer patients and RP-HPLC methods for the quantification of Abiraterone in rat plasma ^8-9 and for dosage forms^10-11 have been so far in the literature. In the present study the authors have proposed a stability indicating liquid chromatographic method for the determination of Abiraterone in tablet formulations and the method was validated.

Figure 1: Chemical Structure of Abiraterone

 

MATERIALS AND METHODS:

Abiraterone is available as tablets with brand names ZYTIGA (Janssen-Cilag Ltd, India), XIBRA (Cipla labs, India) and ABIRAPRO (Glenmark Pharmaceuticals, India) with label claim: 250 mg. HPLC grade acetic acid and acetonitrile were procured from Merck. Abiraterone gift sample was supplied from Glenmark Pharmaceuticals, India

 

Method validation

HPLC instrument and chromatographic conditions

Shimadzu Model CBM-20A/20 Alite HPLC system with PDA detector and Capcell PAK C18 (100 mm × 4.6 mm i.d., 3 µm particle size) column was chosen or the chromatographic study. A mixture of 0.1 % acetic acid and acetonitrile (11:89, %v/v) was used as the mobile phase with flow rate 1.2 mL/min.

 

Procedure:

Abiraterone stock solution (1000 μg/mL) was prepared by dissolving 25 mg of Abiraterone in mobile phase in a 25 mL volumetric flask and a series of (1-1000 μg/mL) diluted solutions were prepared with the diluent (0.1% acetic acid: acetonitrile 20:80). The peak area of chromatograms was noted down and a calibration curve was plotted (Concentration vs peak area). The limit of quantification (LOQ) and limit of detection (LOD) were calculate based on the calibration curve response.

 

The method was validated by performing different studies – linearity, precision, accuracy and robustness. Precision study was performed on the same day as well as on three different days. Robustness study was performed by modifying the chromatographic conditions slightly and there by measuring the percentage relative standard deviation of the method developed.

 

Forced degradation studies:

Abiraterone standard drug solutions were exposed to different stress conditions such as acidic (0.1N HCl, 80°C, 30 min), alkaline (0.1N NaOH, 80°C, 30 min), oxidation (3% H₂O₂), thermal (105°C, 30 min) and photolytic (UV radiation; 365 nm, 3 hours) degradations. The resulting acidic and alkaline stressed sample solutions were neutralised and all the solutions were cooled and diluted to proceed for the chromatographic study.

 

Assay of Abiraterone marketed formulations:

Twenty tablets of three different brands of Abiraterone were purchased form the local pharmacy store and extracted for their active pharmaceutical ingredients and processed for the chromatographic study.

 

RESULTS AND DISCUSSION:

A stability indicating liquid chromatographic method has been developed for the determination of Abiraterone in pharmaceutical formulations (Tablets). A mixture of acetic acid (0.1 %) and acetonitrile (11:89, %v/v) was used as the mobile phase with flow rate 1.2 mL/min.

 

Method optimization:

Method optimization was done using Shimadzu Model CBM-20A/20 Alite HPLC system, equipped with SPD M20A prominence PDA detector. During the method optimization process different columns (Table 1) (Figure 2), mobile phase composition (Table 2), flow rate (Table 3) and diluent composition (Table 4) were used and the detailed reports were tabulated. Capcell C18 Capcell PAK C18 MG-III (100 × 4.6 mm, 3 µm) column was chosen finally for the chromatographic study. Finally a mobile phase consisting of a mixture of 0.1 % acetic acid and acetonitrile (11:89, %v/v) with flow rate 1.2 mL/min. was selected.in which Abiraterone was eluted at 5.327 ± 0.29 mins as a sharp peak (UV detection 252 nm). 

 

 

 

Table.1. Optimization (Variation of columns)

 

 

Figure 2: Representative chromatograms of Abiraterone (Column variation)

 

 

Table. 2. Optimization (Variation in mobile phase composition)

 

 

 

Table. 3. Optimization (Variation of flow rate)

 

Table.4. Optimization (Variation in diluent composition)

 

Method validation¹²

Abiraterone was eluted at 5.30 ± 0.08 min. Abiraterone has shown linearity 1-1000 μg/mL (R² = 0.9999) (Table 5) with linear regression equation y = 25841.1139x – 3883.8764 and the LOD and LOQ were found to be 0.2283 μg/mL and 0.69191 μg/mL respectively (Figure 3). The complete separation of the analysis was finished in less than 10 min. The theoretical plates were > 2000, capacity factor > 2 and the tailing factor was < 2.

 

Table. 5. Linearity of Abiraterone

*Mean of three replicates

 

Figure 3: Calibration curve of Abiraterone

 

Precision, Accuracy and Robustness

The method precision was performed by assaying the drug samples (50, 100 and 250 μg/mL) on the same day (intra-day precision) and on three different days (inter-day precision) and the % RSD was 0.16-0.58 (intra-day) and 0.37-0.97 (inter-day) (Table 6) i.e. < 2.0 indicating that the method is precise. Accuracy studies were performed by spiking pure drug solutions with Abiraterone tablets extracted solution (50 μg/mL) (80%, 100% and 120%). The percentage recovery was 99.13-99.79 (Table 7) with % RSD 0.11-0.45 (<2.0) indicating that the method is accurate. The robustness study results were discussed in Table 8 and here also the % RSD was found to be less than 2.0 (0.79-1.55) indicating that the optimised method is robust.

 

Table.6. Precision studies of Abiraterone

*Mean of three replicates

 

Table. 7. Accuracy studies of Abiraterone

*Mean of three replicates

 

Table. 8. Robustness studies of Abiraterone

*Mean of three replicates

 

 

Assay of Abiraterone marketed formulations

Abiraterone tablet formulations were evaluated by using the above optimised procedure and found that the recovery was 99.34% - 99.67% (Table 9). There is no interference of excipients.

 

Table. 9. Assay of marketed formulations of Abiraterone

Brand Name	Label Claim (mg)	Drug obtained	% Recovery
Brand I	250	248.96	99.58
Brand II	250	248.34	99.34
Brand III	250	249.18	99.67

 

 

 

Forced degradation studies¹³

Abiraterone standard drug solutions were exposed to different stress conditions and during acidic degradation 65.25 % and during alkaline degradation 14.78 % of the drug was decomposed (Figure 4). In oxidation, thermal and photolytic degradations less than 2% degradation was reported indicating that the drug is resistant towards these conditions (Table 10). The system suitability parameters were well in acceptance criteria. The overlay chromatogram was shown in Figure 5 and that of the peak purity results and 3D chromatograms in Figure 6 and Figure 7 respectively.

 

Figure 4: Chromatograms of Abiraterone: Acidic (5.370 min); Alkaline (5.344 min) degradations

 

Figure 5: Overlay chromatogram of Abiraterone: A) Blank [B] Diluent [C] Acidic [D] Alkaline [E] Oxidative [F] Thermal and [G] Photolytic degradations

 

Table. 9. Stress degradation studies of Abiraterone

Stress conditions	*Drug recovered (%)	*Drug decomposed (%)	Peaks observed	Theoretical plates	Tailing factor	Peak Purity Index	Single point threshold
Abiraterone standard (Control)	100.00	-	5.388	11500.55	1.25	1.000000	0.999841
Acidic degradation 1 ml 0.1N HCl, 80°C, 30 mins	34.75	65.25	2.726 5.370	11563.26	1.13	1.000000	0.999790
Alkaline degradation 0.1 ml 0.1N NaOH, 80°C, 30 mins	85.22	14.78	2.721 5.344	11232.81	1.24	0.999998	0.994050
Oxidative degradation 1 ml 3% H2O2, 80°C, 30 mins	98.89	1.11	5.278	9333.82	1.28	1.000000	0.999959
Thermal degradation 105°C, 30 mins	99.73	0.27	5.279	11026.43	1.29	1.000000	0.999971
Photolytic degradation 365 nm, 3 hours	99.84	0.16	5.351	10740.18	1.31	0.999995	0.999942

*Mean of three replicates

Figure 6: Peak purity plots of [A] Abiraterone [B] Acidic [C] Alkaline [D] Oxidative [E] Thermal and [F] Photolytic degradations

 

Figure 7: 3D chromatograms of [A] Abiraterone during [B] Acidic [C] Alkaline [D] Oxidative [E] Thermal [F] Photolytic degradations

 

CONCLUSIONS:

The proposed method was applied for the determination of Abiraterone in laboratory prepared tablets with different excipients and there is no interference of excipients. Abiraterone is sensitive towards acidic and alkaline environments and highly resistant towards temperature, UV radiation and oxidation conditions. The proposed method is economical and simple for the assay of Abiraterone.

 

ACKNOWLEDGEMENT:

The authors are grateful to M/s GITAM (Deemed to be University), Visakhapatnam, India for providing the research facilities as well as Glenmark Pharmaceuticals, India for providing the gift samples of Abiraterone. The authors have no conflict of interest.

 

REFERENCES:

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9.     Ramesh Mullangi, Vijay Kumar S, Rudresha G, Sandip Gurav, Mohd Zainuddin, Purushottam Dewang, Raghava Reddy Kethiri and Sriram Rajagopal. Validated RP-HPLC/UV method for the quantitation of Abiraterone in rat plasma and its application to a pharmacokinetic study in rats. Biomedical Chromatography. 27; 2013: 203-207.

10.   Jala Chandra Reddy B and Sarada NC. Development and validation of a novel RP-HPLC method for stability-indicating assay of Abiraterone acetate, Journal of Liquid Chromatography & Related Technologies. 39(7); 2016: 354-363.

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Accepted on 20.07.2018           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(7): 3007-3012.