AV. Bukkapatnam*, Mukthinuthalapati Mathrusri Annapurna and K. C. Routhu

Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy, GITAM University, Visakhapatnam 530045, India

*Corresponding Author:
AV. Bukkapatnam
Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy, GITAM University, Visakhapatnam 530045, India
E-mail: venki.b11@gmail.com
Date of Submission 02 April 2020
Date of Revision 12 October 2021
Date of Acceptance 17 August 2022
Indian J Pharm Sci 2022;84(4):1071-1082  

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DOI: 10.36468/pharmaceutical-sciences.1001

Abstract

Abiraterone acetate is an androgen biosynthesis inhibitor used in the treatment of prostate cancer. This study focuses on a simple, economical and systematic liquid chromatographic and mass spectroscopic method to study the stress degradation behavior of abiraterone acetate under various stress conditions along with its validation. The chromatographic separation of abiraterone acetate and its major degradation product is achieved on Capcell PAK C18 MG-III (100×4.6 mm, 3 μm) column using combination of 0.1 % acetic acid and acetonitrile as mobile phase with a flow rate of 1.2 ml/min and the wavelength of detection is selected as 251 nm. The drug was degraded extensively in acidic and basic hydrolysis. A tentative degradation pathway of drug in acidic and alkaline condition was predicted. The developed analytical method was found selective, accurate, precise and robust in accordance with International Council for Harmonization guidelines. The method will also suffice the suitability for the assay of abiraterone acetate in bulk and finished products.

Keywords

Abiraterone acetate, anti-prostate cancer agent, liquid chromatographic and mass spectrophotometry, stability indicating, International Council for Harmonization guidelines

Metastatic Castration Resistant Prostate Cancer (MCRPC) is a major category of malignance, which is one of the most common cancers in the United States and the 2nd most common cause of cancer death among men. It causes 258 400 mortality rates yearly[1,2]. The therapy for metastatic prostate cancer involves suppressing testosterone production through androgen deprivation therapy. Abiraterone Acetate (ABT), the first compound that increases the overall survival in MCRPC patients, through the inhibition of adrenal gland production of testosterone. Abiraterone inhibits the enzymatic activity of steroid 17a-monooxygenase, a member of the cytochrome P450 family that catalyzes the 17a-hydroxylation of steroid intermediates involved in testosterone synthesis from the adrenal glands. ABT is an orally active acetate salt of the steroidal compound abiraterone with anti-androgen activity[3]. According to the available literature, abiraterone was determined using different techniques like spectrofluorimetry[4], High-Performance Liquid Chromatography (HPLC) fluorescence detection[5], Liquid Chromatography coupled to Electrospray Ionization Mass Spectrometry (LC-ESI-MS)[6], Reverse Phase-HPLC-Ultraviolet (RPHPLC/ UV)[7-10], Liquid Chromatography/ESI Tandem Mass Spectrometric (LC/ESI-MS/MS) LC-MS/MSESI[ 11,12] and Ultra Performance Liquid Chromatography (UPLC)-MS/MS in biological fluids[13,14]. As most of the methods available from literature are bioanalytical methods for biological fluids. Moreover, according to the International Council for Harmonization (ICH), Stress degradation studies for the compounds are mandatory not only to establish the inherent stability characteristics, but also to identify the degradation products. Therefore, there is a large scope for the development of a simple, short and better analytical method for the study of degradation behavior ABT and also identification of degradant production is very essential.

The main objective of this research work is to develop a fast and simple enough robust isocratic liquid chromatographic method that is compatible with LCMS, not only to quantify ABT in the presence of the degradation product under various stress degradation conditions but also, to identify the degradation product. This further facilitates to the prediction of the degradation pathway.

Materials and Methods

Chemicals and reagents:

ABT standard (3S,8R,9S,10R,13S,14S)- 2,3,4,7,8,9,10,11,12,13,14,15-dodecahydro-10,13- dimethyl-17-(pyridin-3-yl)-1H-cyclopenta[a] phenanthren-3-yl acetate) (C26H33NO2; molecular weight-391.55 g/mol) (purity 99.8 %) was obtained as a gift sample from Dr. Reddy’s Ltd., India. The structure of the compound is shown in fig. 1. ABT is marketed as film coated tablets (label claim: 250 mg), with the brand names Zytiga® (Janssen-Cilag Ltd, India); Xibra (Cipla labs, India) and Abirapro (Glenmark Pharmaceuticals, India).

IJPS-chemical-structure

Fig. 1: Chemical structure of ABT

Acetonitrile (ACN), glacial acetic acid, Hydrochloric acid (HCl), Sodium hydroxide (NaOH), Hydrogen peroxide (H2O2) and HPLC water were all procured from Merck, India. All the reagents and chemicals used were all of HPLC grade.

Instrumentation and chromatographic conditions:

The analysis was carried out on UFLC Shimadzu Prominence System (CBM-20Alite) model equipped with SPD-M20A prominence Photodiode Array (PDA) detector. For the identification of major degrading product, the method is transferred to Agilent 1260 Series Infinity instrument equipped with 1260 Quad pump; 1260 auto-sampler device; 1260 thermostatted column compartment oven; 1260 variable wavelength detector; 6120 quadrupole mass spectrophotometer. The separation is achieved on Capcell PAK C18 MG-III (100×4.6 mm, 3 μm) column using combination of 0.1 % acetic acid and ACN (11:89, % v/v) in isocratic mode with a flow rate of 1.2 ml/min. The injection volume is 20 μl and wavelength of detection is set at 251 nm. The column is maintained at ambient temperature (25°). For identification of degradation product mass conditions are mass to charge (m/z) range 50-1000 m/z; nebulizer and curtain gas nitrogen; nebulizer gas pressure 35 psi; dry gas flow 12 l/min; dry gas temperature 250° and capillary voltage 4000 V.

Validation:

The method so optimized was validated as per ICH guidelines Q2(R1) guidelines, 2005[15]. The linearity (0.1-1000 μg/ml) and precision (50, 100 and 150 μg/ ml) studies were conducted. The accuracy studies were accomplished with three pre-delineates spiked concentration levels (80, 100 and 120 %). In robustness study the chromatographic conditions were marginally modified for flow rate (1.1 and 1.3 ml/min); percentage organic phase (87 and 91 % v/v) and detection wavelength (249 and 253 nm). The robustness study was carried at a concentration level of 10 μg/ml of the standard. All the validation data obtained was taken in triplicate.

Forced degradation studies:

To confirm that the analytical method is stability indicating, ABT was exposed to stress under various conditions to accomplish forced degradation studies[16]. As the present ICH guidelines did not make any statement regarding the detailed degradation conditions in the stress testing, the presently used forced degradation conditions were all based on trial and error. Acidic degradation was performed by refluxing stock solution (1 ml) with 1 ml of 0.1 N HCl for a time of 30 min at 80° in a thermostat, the solution was cooled, neutralised and then diluted as per requirement. Alkaline degradation was conducted by heating stock solution (1 ml) along with 1 ml of 0.1 N NaOH for a period of 30 min at 50°, later it was cooled, neutralised and then diluted as per requirement. During oxidative degradation, stock solution (1 ml) was left in a 10 ml volumetric flask with 1.0 ml of 30 % H2O2 for a period of 30 min at 40°, later cooled and then diluted. Thermal degradation was conducted by exposing a definite quantity of ABT standard in solution state to heat in a hot air oven maintained at 80° continuously for 7 d and the resultant was cooled and diluted before the study. Photolytic degradation was governed by exposing the solid drug sample to light source radiation with a wavelength range of 290-700 nm in the Ultra-Violet (UV) chamber, continuously for about 7 d. The sample was spread as a thin layer and sealed with a transparent cover to minimize the effects of the changes in the physical state. The solution was prepared with ACN and further dilution with diluent.

Assay of marketed formulations:

Twenty tablets of each available brands of ABT were procured from the local pharmacy store, weighed and crushed into fine powder. Powder equivalent to 25 mg of ABT was accurately weighed and extracted with mobile phase. The solution was filtered through 0.45 μm membrane prior analysing and the peak area was recorded from the respective chromatogram.

Results and Discussion

A comparative study of the previously published methods with the present method was summarized detail in Table 1. In the initial trials the experiments were conducted with different columns such as Sunfire C18 column (150×4.6 mm, 5 μm), Phenomenex Luna C18 column (250×4.6 mm, 5 μm) and Capcell PAK C18 MGIII (10×4.6 mm, 3 μm). After reviewing the results of column trials, it was found that the peak shape, retention time, tailing factor and column efficiency were found better with Capcell PAK C18 MG-III (100×4.6 mm, 3 μm), hence this column is selected for this method of analysis. In overall reviewing the results of mobile phases, their ratios and the flow rates, mobile phase comprising of 0.1 % acetic acid:ACN (11:89 v/v) and with a flow rate of 1.2 ml/min was selected as the optimum mobile phase for better peak shape, tailing factor and theoretical plates low retention time, etc. The overall analysis was performed with 251 nm as wavelength of detection ( fig.2).

Method Reagent/Mobile phase (% v/v) Flow rate (ml/min) Column Remarks Reference
LC-MS/MS 2 % Propan-2-ol:ACN:Ammonium acetate (gradient mode) - Luna C5 (50 mm×2.1 mm, 5 μm) Human plasma [10]
LC-MS Ammonium acetate:CAN (10:90) 0.7 Atlantis d C18 Rat and human plasma [8]
UPLC-MS/MS ACN:Water:Formic acid (90:10:0.1) 0.3 UPLC BEH™ C18 Human plasma [9]
LC-UV-ESI-MS 0.1 % Formic acid:ACN (70:30) 0.5 Agilent Zorbax Extend-C18 (150 mm×4.6 mm, 5 µm) Stability indicating [6]
HPLC ACN:Water:Potassium dihydrogen phosphate (pH 3.0), (55:5:40) 1 Betasil C18 Rat plasma [8]
HPLC ACN:Glycine buffer (pH 9.0) (60:40) 0.9 C8 Xterra® MS Fluorescent detection [5]
LC-MS Ammonium acetate (pH 3.5):ACN (10:90) 0.6 Kromasil C18 (250 mm×4.6 mm, 4.0 µm) Stability indicating [15]
HPLC Potassium phosphate buffer: ACN (40:60) 1 Hypersil ODS C18 (150 mm×4.6 mm, 5 µm) Stability indicating [15]
LC-MS-NMR Water:ACN (gradient) 1 Waters X-terra MC C18 (250 mm×4.6 mm, 5 µm) Related substance [16]
HPLC ACN:0.1 % Orthophosphoric acid (15:85) 1 Hypersil BDS C18 (250 mm×4 mm, 5 μm) Stability indicating [14]
HPLC Potassium phosphate buffer:ACN (40:60) 1 Hypersil ODS C18 (150 mm×4.6 mm, 5 μm) Stability indicating [13]
LC-UV-ESI-MS 0.1 % Acetic acid:ACN (11:89) 1.2 Capcell PAK C18 MG-III (100×4.6 mm, 3 µm) Stability indicating Present method

Table 1: Comparison of the Previously Reported Methods with the Present Method

IJPS-Absorption

Fig. 2: UV Absorption spectra of ABT (at 10 μg/ml)

The method was validated as per ICH guidelines. The method shows a linearity range of 0.1-1000 μg/ml, with their percentage Relative Standard Deviation (% RSD) in limits. The linearity data with their linear regression equations and their correlation coefficients, limit of detection and limit of quantitation were clearly presented in Table 2. The method accuracy was demonstrated by the recovery test at three dissimilar concentrations in which known amount of standard was supplemented to aliquots of sample solutions and then diluted to yield the total concentrations of 80, 100 and 120 μg/ml (80, 100 and 120 %) for which percentage recovery was found to be 97.96 %-99.48 % (Table 3).

Parameter Result
Calibration curve y=28388.67x+110388.00
Slope 28388.67
Intercept 110388
R2 0.9994
Limit of detection (μg/ml) 0.03275
Limit of quantitation (μg/ml) 0.0992
Range (μg/ml) 0.1-1000

Table 2: Linearity of Abt

Concentration (µg/ml) Intraday precision Interday precision Accuracy Overall % recovery
*Measured concentration (µg/ml)±SD % RSD SEM *Measured concentration (µg/ml)±SD % RSD SEM Spiked concentration (µg/ml) Total concentration (µg/ml) *Concentration found (µg/ml)±SD % RSD SEM % Recovery
50 49.76±0.19 0.39 0.11 49.48±0.50 1.01 0.29 80 % 80 79.58±0.42 0.47 0.3 99.48 Mean 98.9
100 98.08±0.66 0.67 0.38 100.05±0.46 0.46 0.27 100 % 100 97.96±0.40 0.41 0.2 97.96 SD 0.8
150 149.44±0.25 0.16 0.14 149.36±0.69 0.46 0.4 120 % 120 118.98±0.27 0.25 0.1 99.15 % RSD 0.81

Table 3: Precision and accuracy studies of Abt

For precision studies conducted at three concentrations (i.e., 50, 100 and 150 μg/ml) for both intraday and interday precision for which the % RSD was found to be was found to be 0.16 %-0.67 % (intraday precision) and 0.46 %-1.01 % (interday precision) (Table 3). The robustness of the method was estimated by assaying the sample in diverse analytical conditions by deliberately making slight fluctuations the original condition. From the results (Table 4), it was shown that the system suitability parameters, retention times and the assays for the test solution were not much affected there by signifying that the method is robust.

Parameter (condition) *% Assay±SD % RSD SEM *Retention time±SD % RSD SEM
Mobile phase flow rate (±0.1 ml/min)
1.1 ml/min 100.22±0.21 0.21 0.12 5.94±0.01 0.09 0
1.3 ml/min 99.34±0.31 0.31 0.18 4.94±0.02 0.42 0.01
Detection wavelength (±2 nm)
249 nm 98.74±0.59 0.6 0.34 5.39±0.00 0.04 0
253 nm 98.54±0.16 0.16 0.09 5.39±0.00 0.03 0
Mobile phase composition (% ACN) (±2 % v/v)
09:91 v/v 98.53±0.15 0.16 0.09 5.294±0.01 0.09 0
13:87 v/v 99.76±0.28 0.28 0.16 5.434±0.01 0.23 0.01

Table 4: Robustness Study of Abt

The stability indicating capability of the method was established from the separation of ABT peak from the degradation peaks of degraded samples. Typical chromatograms obtained following the assay of stressed samples are shown in fig. 3. The drug shows degradation in acidic (62.72 %) and alkaline (16.99 %) conditions. Whereas, it shows almost no decomposition when exposed to thermal (1.09 %), photolytic (0.34 %) and oxidative degradation (0.26 %) (Table 5). The Three- Dimensional (3D) chromatographs for the degradation studies were obtained from the PDA data which shows the selectivity of the wavelength and the degradation peaks at the wide range of wavelength (fig. 4). The system suitability parameters for all the degradation studies supporting the selectivity of the method were shown in Table 5.

Stress Condition/media/duration % Recovery* % Drug degradation* Theoretical plates (N) Tailing factor Resolution (R) Peaks (min) m/z values Peak purity
Standard 100 - 11398.5 1.25 - 5.43 392.3 1
Acidic/0.1 N HCl/80°/1 h 37.28 62.72 11591.64 1.12 16.15 5.43 392.3 1
            2.77 350.3  
Alkaline/0.1 N NaOH/80°/1 h 83.01 16.99 11251.61 1.23 15.718 5.47 392.3 0.9999
            2.75 350.3  
Oxidative/30 % H2O2/40°/1 h 99.74 0.26 9338.09 1.27 - 5.47 392.3 0.9999
Thermal/80°/7 d 98.91 1.09 9348.4 1.24 - 5.42 392.3 1
Photolytic/7 d 99.66 0.34 10861.28 1.31 - 5.45 392.3 0.9999

Table 5: Forced degradation studies of Abt

IJPS-Chromatograms

Fig. 3: Typical Chromatograms of ABT (a): Standard; (b): Acidic; (c): Alkaline; (d): Oxidative; (e): Thermal and (f): Photolytic degradations (each at 10 μg/ml)

IJPS-degradations

Fig. 4: Typical 3D Chromatograms of ABT (a): Standard; (b): Acidic; (c): Alkaline; (d): Oxidative; (e): Thermal and (f): Photolytic degradations (each at 10 μg/ml)

Stress degradation studies for ABT were conducted with different conditions such as acidic, alkaline, oxidative, thermal and photolytic degradations. After the degradations the separation was achieved using HPLC and further the samples were processed for LC-MS study for the characterization of ABT and its degradants (fig. 5-fig. 10). The positive ESI-MS of ABT shows an abundant [M+H]+ ion at m/z 392.3 (fig. 5).

IJPS-mass-spectrum

Fig. 5: LC-MS data of ABT standard (a): Chromatogram and (b) Mass spectrum of the ABT

IJPS-acidic

Fig. 6: LC-MS data of ABT under acidic degradation (a): Chromatogram; (b): Mass spectrum of the DP1 and (c): Mass spectrum of the ABT

IJPS-alkaline

Fig. 7: LC-MS data of ABT under alkaline degradation (a): Chromatogram; (b): Mass spectrum of the DP1 and (c): Mass spectrum of the ABT

IJPS-oxidation

Fig. 8: LC-MS data of ABT under oxidation (a): Chromatogram and (b): Mass spectrum of the ABT

IJPS-thermal-degradation

Fig. 9: LC-MS data of ABT under thermal degradation (a): Chromatogram and (b): Mass spectrum of ABT

IJPS-photolytic

Fig. 10: LC-MS data of ABT under photolytic degradation (a): Chromatogram and (b): Mass spectruvm of ABT

During acidic and alkaline degradations ABT shows sufficient degradation forming a single degradation peak (DP)1 with the retention time at 3.5 min as depicted in fig. 6 and fig. 7. The degradation product DP1 shows an abundant [M+H]+ ion at m/z 350.3 [M+H]+; C24H31NO, from the structure of ABT, DP1 can be assigned with the structure (3S,8R,9S,10R,13S,14S)- 2,3,4,7,8,9,10,11,12,13,14,15-dodecahydro-10,13-dimethyl-17-(pyridin-3-yl)-1H-cyclopenta[a] phenanthren-3-ol. The degradation pathway for the acidic/alkaline hydrolysis of ABT was predicted as shown in fig. 11. From this it was predicted that, the degradation product might be formed by preferably cleaved ABT, which lose one acetyl group.The proposed method was applied to the available marketed formulations (Zytiga®, Xibra® and Abirapro®) for the determination of ABT. The percentage recovery was found to be 97.54 %-98.87 % (Table 6). The resultant chromatograms obtained from the extraction of marketed formulations were shown in fig. 12.

Formulation Manufacturer Labelled claim (mg) Amount found* (mg) % Recovery*±SD (% RSD)
Zytiga Janssen-Cilag Ltd, India 250 243.85 97.54±0.44 (0.45)
Xibra Cipla labs, India 250 247.18 98.87±0.13 (0.13)
Abirapro Glenmark Pharmaceuticals, India 250 244.63 97.85±0.28 (0.29)

Table 6: Analysis of abiraterone acetate in commercial formulations

IJPS-pathway

Fig. 11: Predicted degradation pathway of ABT in the acidic and alkaline hydrolysis

IJPS-abirapro

Fig. 12: Representative chromatograms of ABT (a): Blank; (b): Standard; (c): Zytiga®; (d): Xibra® and (e): Abirapro® (each at 10 μg/ml)

In this work, an attempt made to develop a fast simple and robust stability-indicating RP-HPLC compatible with LC-ESI/MS for the determination of ABT in the presence of degradation product which was validated as per ICH guidelines and applied for the determination of ABT in pharmaceutical dosage form (film coated tablets). In the forced degradation studies, it was observed that ABT is more sensitive towards the acidic and slightly towards alkaline environment.

From the LC-ESI/MS study both the acidic and alkaline degradation show the same degradant and were identified thereby providing a prospect for the prediction of degradation pathway. This method can be successfully applied to perform long-term and accelerated stability studies of ABT formulations. This study might also be helpful in designing of the formulations.

Acknowledgments:

The authors are grateful to University Grants Commission (UGC), New Delhi, India for the financial support (UGC Ref No: 42-674/2013(SR), 2013) and also GITAM University, Visakhapatnam for providing research facilities.

Conflict of interest:

The authors have no conflict of interest.

References