INTRODUCTION:

Abiraterone is a derivative of steroidal progesterone and is an innovative drug that offers clinical benefit to patients with hormone refractory prostate cancer. Abiraterone is administered as an acetate salt prodrug because it has a higher bioavailability and less susceptible to hydrolysis than abiraterone itself [1-3]. FDA approved on April 28, 2011. Abiraterone acetate, sold under the brand name Zytiga among others, is an antiandrogen medication which is used in the treatment of prostate cancer. It is specifically indicated for use in conjunction with castration and prednisone for the treatment of metastaticcastration-resistaprostate cancer (mCRPC). Abiraterone-acetate chemical name is {[(3S,8R,9S,10R,13S,14S)-10,13-dimethyl-17pyridin-3-yl-2,3,4,7,8,9, 11,12,14,15 decahydro -1H-cyclopenta[a]phenanthren-3yl] acetate} - is an acetyl ester of Abiraterone.

A literature review revealed that different bioanalytical methods including spectrofluorimetric method [4-8] for the estimation of Abiraterone acetate and Abiraterone in their formulations. Hence our plan to develop a new economic UPLC method for routine analysis.

Fig 1. Abiraterone structure

MATERIALS AND METHODS:

Equipments and Apparatus:

UPLC WATERS make Acquity with Auto Injector and PDA Detector. Software used is Empower 2. UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2mm and 10mm and matched quartz was be used for measuring absorbance for Abiraterone solutions, Sonicator (Ultrasonic sonicator), PH meter (Thermo scientific), Micro balance (Sartorius) and Vacuum filter pump.

Reagents used:

Abiraterone, Acetonitrile, Glacial Acetic acid, HPLC Grade.

ANALYTICAL METHODOLOGY:

Diluent:

Based up on the solubility of the drugs, diluent was selected, Acetonitrile and Water taken in the ratio of 50:50

Preparation of Standard stock solutions:

Accurately weighed 6.25mg of Abiraterone transferred 25ml of volumetric flask, and 3/4^th of diluents was added and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution (250µg/ml of Abiraterone)

Preparation of Standard working solutions (100% solution):

1ml of Abiraterone from each stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (25µg/ml of Abiraterone)

Preparation of Sample stock solutions:

5 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 100 ml volumetric flask, 50ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters. (2500 µg/ml of Abiraterone)

Preparation of Sample working solutions (100% solution):

0.1ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (25µg/ml of Abiraterone)

Preparation of buffer:

0.1%OPA Buffer:

1ml of Perchloric acid was diluted to 1000ml with HPLC grade water. 0.01% KH₂PO₄Buffer

METHOD DEVELOPMENT:

Based on drug solubility and pkaValue following conditions has been used to develop the method estimation of Abiraterone.

Table 1: Optimized Chromatographic Conditions

Column	SB C8 100 x 3.0 mm, 1.8m.
Mobile phase	0.01% KH₂PO₄: Acetonitrile (60:40)
Flow rate	0.3 ml/min
Detector	PDA 235nm
Temperature	30⁰C
Injection Volume	0.5µL
Pump mode	Isocratic

Fig. 2: Optimized chromatogram

METHOD VALIDATION:

System suitability parameters:

The system suitability parameters were determined by preparing standard solutions of Abiraterone (25ppm) and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined.

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

Table 2: System suitability

	PEAK NAME	RT	AREA	USP Plate Court	USP Tailing
1	Abiraterone	1.026	883921	2553	1.48
2	Abiraterone	1.030	902081	2586	1.38
3	Abiraterone	1.031	887571	2660	1.36
4	Abiraterone	1.031	902557	2606	1.39
5	Abiraterone	1.030	893641	2636	1.41
6	Abiraterone	1.030	884060	2697	1.38
Mean	Abiraterone		892305
Std. dev			8522.2
% RSD			1.0

Precision:

Precision of the developed method was assessed by measuring the response on the same day (intraday precision) and next two consecutive days (inter day precision). The precision of the method was assessed by six replicates.

Repeatability:

Six working sample solutions of 25ppm are injected and the % Amount found was calculated and %RSD was found to be 0.2.

Table 3: Repeatability data

S. No	Peak Area	Peak Area
1	886373	889627
2	889423	872821
3	900449	888776
4	890875	876971
5	890144	870300
6	896505	893790
AVG	892295	882048
ST. DEV.	5180.2	9894.6
%RSD	0.6	1.1

Linearity:

To demonstrate the linearity of assay method, inject 5 standard solutions with concentrations of about 6.25ppm to 37.5ppm of Abiraterone. Plot a graph to concentration versus peak area. Slope obtained was y = 35175x + 15266 and Correlation Co-efficient was found to be 0.999 and Linearity plot was shown in fig.3.

Table4: Linearity Data:

Linearity Level (%)	Concentration (ppm)	Area
0	0	0
25	6.25	243919
50	12.5	453675
75	18.75	686254
100	25	899068
125	31.25	1123876
150	37.5	1316761

Fig. 3. Linearity plot

Accuracy:

Three Concentrations of 50%, 100%, 150% are Injected in a triplicate manner and %Recovery was calculated as 99.99%. And chromatograms were shown in table 5.

Table 5: Accuracy data:

% Level	Amount Spiked (μg/mL)	Amount recovered (μg/mL)	% Recovery	Mean %Recovery
50%	25	12.306326	98.45	99.99%
	25	12.502715	100.02
	25	12.453305	99.63
100%	25	25.193945	100.78
	25	25.143539	100.57
	25	25.045828	100.18
150%	25	37.149851	99.07
	25	37.471613	99.92
	25	37.973902	101.26

Robustness:

Small Deliberate change in the method is made like Flow minus, flow plus, Mobile phase minus, Mobile phase plus, Temperature minus, Temperature Plus. %RSD of the above conditions is calculated. Robustness conditions like Flow minus (0.2ml/min), Flow plus (0.4ml/min) , mobile phase minus, mobile phase plus, temperature minus (25°C) and temperature plus (35°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much effected and all the parameters were passed. %RSD was within the limit.

Table 6: Robustness Data:

PARAMETER		%RSD Abiraterone Acetate
Flow rate (±0.2ml/min)	0.2 mL/min	0.2
Flow rate (±0.2ml/min)	0.4 mL/min	0.3
Mobile phase (±5)	70:30	0.8
Mobile phase (±5)	60:40	1.0
Temperature (±2°C)	28°C	0.8
Temperature (±2°C)	32°C	0.3

Degradation studies:

Degradation studies were performed with the formulation and the degraded samples were injected. Assay of the injected samples was calculated and all the samples passed the limits of degradation.

Table 7: Degradation Data of Abiraterone

S. No	Degradation Condition	% Drug UN Degraded	% Drug Degraded
1	Acid	94.09	5.91
2	Alkali	95.58	4.42
3	Oxidation	96.28	3.72
4	Thermal	97.06	2.94
5	UV	98.31	1.69
6	Water	98.31	1.69

CONCLUSION:

Chromatographic conditions used are stationary phase SB C8 (100mm*3.0mm 1.8m.) Mobile phase 0.01N KH₂PO₄: Acetonitrile in the ratio of 60:40v/v and flow rate was maintained at 0.3ml/min, detection wave length was 235 nm, column temperature was set to 30^oC and diluent was mobile phase Conditions were finalized as optimized method. System suitability parameters were studied by injecting the standard six times and results were well under the acceptance criteria. Linearity study was carried out between 25% to150 % levels, R² value was found to be as 0.999. Precision was found to be 0.6 for repeatability and 1.1 for intermediate precision. LOD and LOQ are 0.09µg/ml and 0.27µg/ml respectively. By using above method assay of marketed formulation was carried out 99.60% was present. Degradation studies of Abiraterone were done, in all condition’s purity threshold was more than purity angle and within the acceptable range and can be used for routine analysis of Abiraterone.

REFERENCES:

1. Food and Drug Administration Approves Abiraterone for Treatment of Men with Advanced Prostate Cancer.

2. Salem M, Garcia JA. Abiraterone acetate, a novel adrenal inhibitor in metastatic castration-resistant prostate cancer. Curr Oncol Rep 2011(13):92-96.

3. National Institute for Health and Care Excellence (NICE). Guide to the processes of technology appraisal. London: NICE; 2014.https://www.nice.org.uk/article/pmg19/chapter/foreword. Accessed 21 Mar 2016.

4. Martins V, Asad Y, Wilsher N, Raynaud F, A validated liquid chromatographic– tandem mass spectroscopy method for the quantification of abiraterone acetate and abiraterone in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2006; 843(2):262-267.

5. Gaurav S, Punde R, Farooqui J, Zainuddin M, Rajagopal S, Mullangi R, Development and validation of a highly sensitive method for the determination of abiraterone in rat and human plasma by LC-MS/MS-ESI: application to a pharmacokinetic study. Biomed Chromatogr. 2012;26(2):761-8.

6. Kumar SV, Rudresha G, Gaurav S, Zainuddin, Dewang P, Kethiri RR, Rajagopal S, Mullangi R, Validated RPHPLC/UV method for the quantitation of abiraterone in rat plasma and its application to a pharmacokinetic study in rats. Biomed Chromatogr. 2013; 27(2): 203-7.

7. Khdera A, Darwish I, Bamanea F, Analysis of abiraterone stress degradation behavior using liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry. J Pharm Biomed Anal. 2013; 23:74-77.

8. Gong A, Zhu X, β-cyclodextrin Sensitized Spectrofluorimetry for the Determination of Abiraterone Acetate and Abiraterone. J. Fluoresc. 2013; 23(6):1279-86. 2014. https://www.nice.org.uk/article/pmg19/chapter/foreword. Acces- sed 21 Mar 2016

Received on 23.01.2019 Modified on 18.02.2019

Research J. Pharm. and Tech. 2019; 12(6):3029-3032.

DOI: 10.5958/0974-360X.2019.00512.2