INTRODUCTION:

Adenocarcinoma is one of the most common cancers in adults, accounting for 20% of all cancer-related deaths in the Western world.¹ Localized prostate cancer was traditionally treated with radiation therapy, surgical castration, or chemotherapy with first-generation anti-androgens (viz., bicalutamide, nilutamide, flutamide etc)². A significant percentage of patients developed metastatic castration-resistant prostate cancer (mCRPC), an advanced variant of the disease synonymous with tumour growth and a short recovery period of 18-24 months.³ As a result, several new therapies targeting androgen receptor signalling, such as CYP17A1 inhibitors (abiraterone acetate)⁴ and second-generation androgen receptor antagonists (enzalutamide, apalutamide, and darolutamide)^5-7, have emerged in recent years.

Treatment with these medications produced promising outcomes, including increased overall survival and quality of life in mCRPC patients.

Darolutamide (DLT) is a second generation non-steroidal androgen receptor inhibitor. The chemical name is N-{(2S)-1- [3-(3-chloro-4-cyanophenyl)- 1H- pyrazol-1yl] propan-2-yl} -5-(1-hydroxy ethyl) -1H-pyrazole-3-carboxamide. The molecular weight is 398.85 and the molecular formula is C₁₉H₁₉Cl N₆O₂.^8,
9 Figure 1 illustrates the chemical structure of DLT. DLT prevents androgen binding, AR nuclear translocation, and AR-mediated transcription in a competitive manner. Keto-darolutamide, a major metabolite, had comparable in vitro activity to darolutamide. DLT also worked as a progesterone receptor (PR) antagonist in the lab (approximately 1 percent activity compared to AR). In mouse xenograft models of prostate cancer, DLT reduced prostate cancer cell proliferation in vitro and tumour volume.^10-11On July 30, 2019, the Food and Drug Administration approved DLT (NUBEQA, Bayer Health Care Pharmaceuticals Inc.) for non-metastatic castration-resistant prostate cancer. ¹²

Upon reviewing the literature thoroughly three LC-MS/MS methods were found to be reported for quantification of DLT alone¹³ and along with its metabolite (ORM-15341)^{14, 15}. A RP-HPLC-UV method was also reported to quantify DLT in mice plasma along with other second generation non-steroidal anti androgens and their active metabolites.¹⁶ To the best of the authors' knowledge, there is no stability indicating assay procedure for determining DLT in bulk or tablets in the literature. The current project aims to establish and validate a simple and rapid UPLC-PDA system for DLT quantification for this purpose.

MATERIALS AND METHODS:

Chemicals And Reagents:

The standard drug, darolutamide (purity: >98%) was provided by Bayer Health Care Pharmaceuticals in the United States. Sigma-Aldrich included methanol, water, and acetonitrile (LC grade). Sigma-Aldrich issued analytical grade sodium hydroxide (NaOH), hydrogen peroxide (H₂O₂), hydrochloric acid (HCl), and a 0.22 mm membrane filter. Nubeqa tablets containing Darolutamide with the label claim of 300 mg was purchased from the native pharmaceutical market. All chemicals were analytical or LC grade.

UPLC instrumental condition:

An Acquity UPLC system (Waters, Milford, MA, USA) equipped with a model 2996 PDA detector and Empower software was used to develop the method. The UPLC separation of the DLT was obtained with Zorbax SB-C₁₈ (100X2.1 mmX1.8µ) analytical using 8 mM ammonium acetate, pH: 5.4: acetonitrile (66:34) in isocratic mode at a flow rate of 0.4 ml/min and column at room temperature. The PDA detector was used to monitor the drug at 272 nm. The solvents were filtered on a 0.22 mm membrane filter and degassed in an ultrasonic bath before use. The analytical method was optimized using a pure analytical standard. Mobile phase was used as diluent.^17-19

Preparation of standard and sample solutions:

By dissolving the required amount of DLT in the diluent, a DLT stock solution (10g/mL) was prepared. To determine the assay and stability studies, a working solution of 140g/mL was prepared from the above stock solution.

In a clean, dry mortar, 20 tablets is weighted and ground with a pestle. A 10 ml volumetric flask was filled with 10 mg of powder and 7 ml of diluent. To fully scatter the powder, the flask was connected to a rotary shaker and shook for ten minutes. The mixture was sonicated for 10 minutes before being diluted to the required amount with diluent to produce a 1.0 mg/ml solution. This solution was further diluted to obtain final concentration of 140µg/mL. the resulted solution was filtered through a 0.45 µ nylon 66 membrane filter.

Method Validation:

Method validation^20-26 was performed as per ICH guidelines²⁷. The following validation parameter was addressed system suitability, precision, accuracy, specificity, limit of detection and quantitation, robustness and stability of DLT in various stress conditions²⁸.

System suitability:

The system suitability test is an integral part of the validation of the liquid chromatography method performed to verify and assure the continuous performance of a chromatographic system. The repeatability of the system was estimated by 6 repeated injections of standard working solution at 100% of the test concentration (140 µg/mL of DLT). The suitability parameters of the system have been calculated according to the recommendation of ICH.

Specificity:

Specificity is the ability to unequivocally evaluate the analyte in the presence of components that can be assumed to be present (impurities, degrading, matrix, etc.). Specificity has been demonstrated by determining DLT in the presence of excipients of the dosage form.

Linearity:

For the evaluation of linearity, the calibration curve was obtained at 5 concentration levels of standard DLT solutions (70–210 µg/mL). The solutions (3 μl) were injected in triplicate in a chromatographic system with the chromatographic conditions previously provided. For linearity assessment, the peak area and concentrations were subjected to a least squares regression analysis to calculate the calibration equation and the coefficient of determination.

Precision:

The precision of the analytical procedure (intra-assay precision) was investigated by analyzing six sample solutions obtained by multiple sampling of the same homogeneous sample under the prescribed conditions (at 100% of the test concentration of DLT (140 µg/mL)) on the same day, by the same analyst and using the same equipment. The Interday precision of the analytical procedure was investigated by analyzing sample solutions on three consecutive days. The precision of the analytical procedure was expressed as the relative standard deviation of a series of measurements.

Limit of detection and limit of quantification:

The detection limit (LOD) and the limit of quantification (LOQ) of the proposed method were determined by consecutively injecting low concentrations of the standard solutions using the proposed RP-UPLC method. LOD and LOQ were calculated according to ICHguidelines as follows:

LOD = 3.3 SD/S and LOQ = 10 SD/S,

where SD is the standard deviation of the response (y intercept) and S is the slope of the calibration curve obtained.

Accuracy:

To study the accuracy of the proposed analytical method, recovery tests were conducted. To discover whether excipients interfered with the analyte, equivalent amounts at 50, 100 and 150% of DLT were evaluated from tablet formulation and the resulting mixtures were analyzed by the proposed methods. The percent of recovery was calculated as follows:

Amount Added:.

Amount Found:

% Recovery:

Robustness:

The ability of the proposed method to remain unaffected by small (deliberate) variations in parameters was evaluated in order to determine method robustness. Changes were made to the following method parameters: flow rate (±0.1 ml min–1), temperature (± 5 °C) and wavelength of detection (± 2 nm).

Forced degradation studies:

Forced degradation studies were performed on DLT. Intentional degradation was attempted to stress conditions of Acid (0.5 N HCl), Base (0.1 N NaOH), Peroxide (3% H₂O₂), thermal (75°C) and UV light (256 nm) to determine the ability of the proposed method to separate DLT from its degradation products generated during forced degradation studies. For thermal and UV light, study period was 20 and 24 H respectively, whereas for acid, base and oxidation it was7 H, 6H and 5 H respectively. Peak purity test was carried out on the stressed samples by PDA. Assay studies were carried out for stress samples against qualified reference standard and mass balance (% assay + % degradation products) was calculated.

RESULTS AND DISCUSSION:

Method Development:

Various mobile phases have been studied in the development of an UPLC method for DLT analysis. These include: methanol-water, 50:50 (v/v), acetonitrile-water, 30:70 (v/v), methanol-orthophosphoric acid buffer (pH 4.5–6.5), 50:50 (v/v), Methanol buffer- phosphate (pH 3.0–6.5), 25:75 (v/v) and acetonitrile- phosphoric acid buffer (pH 3.2-4.5) 60:40 (v/v). The suitability of the mobile phase was decided based on the sensitivity of the assay, the suitability for stability studies, the ease of preparation and the use of readily available solvents. Therefore, the mobile phase consisting of 8 mM ammonium acetate, pH: 5.4: acetonitrile (66:34), has been found to be optimal for isocratic determination of DLT in pharmaceutical products. The wavelength was selected by scanning the standard DLT solution at more than 200-400 nm and the wavelength of 272 nm was chosen for the detection.

Darolutamide has been identified as a function of retention time compared to the DLT standard. Furthermore, DLT was identified by adding the standard to the sample prior to analysis, which resulted in an increase in the sample peak area that was proportional to the amount added. The mean DLT retention time was approximately 0.83 minutes at a flow rate of 0.4 ml min-1. DLT was rapidly determined as a single sharp peak. No interference was observed from other degradation products. Figure 2 and 3 shows the standard and sample chromatograms respectively.

Figure 2: Standard chromatogram of Darolutamide

Figure 3: Sample chromatogram of Darolutamide

Method Validation:

System suitability:

The results of system suitability test were found within the acceptable range indicating that the system was suitable for the intended analysis (Table 1).

Table 1: Result of System suitability study

S. No	Parameter*	Result
1	Theoretical Plate Count	6782.50
2	Average Peak Area	7347500.5
3	Peak Height	1152600.333
4	RT	0.8335
5	Tailing	1.0
7	S/N	1045

* Average of 6 replicates

Specificity:

In the specificity study, standard DLT solutions and the sample solution were injected and a single peak was obtained, indicating that there was no interference from the excipients used or from the mobile phase. Representative chromatograms of standard and sample were shown in figure 2 and 3 respectively.

Linearity:

In the present study, linearity was studied in the concentration range 70-210 µg/mL DLT and the following regression equation was found by plotting the peak area (y) expressed in mAU versus the DLT concentration (x) expressed in µg/mL: y = 53080x - 15901 (r² = 0.999). The determination coefficient (r²) demonstrates the excellent relationship between the peak area and concentration of DLT. The excipients had no influence and there was no matrix effect observed. Figure 4 shows the linearity curve of DLT.

Figure 4: Linearity curve of Darolutamide

Precision and accuracy

Precision was demonstrated by Interday and intraday variation studies. In the intraday and Interday studies the solutions were injected 6 times and %RSD was calculated which was found to be less than 2%. Accuracy of the proposed methods was demonstrated by analyzing different concentrations covering the points in the calibration range. The mean percentage recovery was found to be 100.87, 99.74 and 100.95 at 50, 100 and 150 % accuracy levels. The precision and accuracy was shown in Table 2 and 3 respectively.

Table 2: Results of Precision

S. No	Intraday precision		Inter day precision
S. No	Peak Area*	% Assay*	Peak Area*	% Assay*
1	7432699	101.03	7348699	99.89
2	7357606	100.01	7346606	99.86
3	7398974	100.57	7345974	99.85
4	7349367	99.90	7376367	100.26
5	7390881	100.46	7405881	100.66
6	7412006	100.75	7122006	96.80
Average	7390255.50	100.45	7324255.50	99.55
STDEV	31902.34	0.43	101835.08	1.38
% RSD	0.43	0.43	1.39	1.39
*mean of three replicates

Table 3: Results of accuracy

Accuracy level	Wt. of Sample	Peak Area	Amount Added	Amount Found	% Reco-very	Mean % Reco-very
50*	10.506	3702716	69.69	70.29	100.87	100.52
100^†	21.011	7253479	139.38	139.01	99.74
150*	31.517	11088563	209.06	211.06	100.95

*Mean of 6 replicates; ^†Mean of 3 replicates

Limit of detection and limit of quantification:

The limit of detection (LOD) and limit of quantification (LOQ) were 1.80 µg/mL and 6.01 µg/mL respectively. The proposed UPLC method for DLT determination was demonstrated to be sensitive for performing the stability indicating assay.

Robustness:

Based on the obtained results the proposed UPLC analytical method was demonstrated to be robust (Table 4).

Table 4: Results of robustness

Parameter	Condition	RT	Peak Area	% Assay
Flow	0.3 ml/min	1.172	7347652	99.87
	0.4 ml/min	0.834	7347501	99.87
	0.5 ml/min	0.747	7398459	100.56
Temp	25 °C	0.826	7465339	101.47
	30 °C	0.834	7347501	99.87
	35 °C	0.83	7342750	99.81
Wave length	270 nm	0.831	7320179	99.50
	272 nm	0.834	7347501	99.87
	274 nm	0.83	7335922	99.71

Method Application:

The validated method was applied for the determination of DLT in commercially available tablets. Figure 2 and 3 shows two typical UPLC chromatograms obtained from the test of the standard DLT reference solution and of the tablet sampling solution, respectively. The results of the trial (n = 6) produced 100.00% (RSD = 0.14%) of the label claim for DLT in Nubeqa 300 mg tablets. The test results indicate that the method is specific for the analysis of DLT without interference from the excipients used to formulate and produce these tablets. Assay results were shown in table 3.

Forced degradation studies:

To ensure the homogeneity and purity of the DLT peak, all forced degradation samples were analysed at an initial concentration of 140 g/mL of DLT using the UPLC conditions described earlier and a PDA detector. In acid, UV, and thermal conditions, substantial degradation of DLT was observed, resulting in the formation of degradants. Stress samples (at 140 g/mL) were compared to a DLT reference level in assay tests. For all strained samples, the mass balance (percent assay + percent sum of all compounds + percent sum of all degradants) was estimated and found to be greater than 99%. The purity and assay of DLT was unaffected by the presence of its degradation products and thus confirms the stability-indicating power of the developed method. Table 5 shows the results of forced degradation studies. Figure 5 shows the chromatograms of forced degradation studies.

Table 5: Forced degradation studies

Condition	Peak Area	% Assay	% Degradation
Acid	6549579	89.02	10.98
Base	6689022	90.92	9.08
H2O2	6789022	92.28	7.72
UV	6614583	89.91	10.09
Heat	6616189	89.93	10.07

CONCLUSION:

In conclusion, a sensitive and selective stability indicating RP-UPLC method has been developed and validated for DLT in API and tablets. Based on the peak purity results obtained from the analysis of force degraded samples using the described method, it can be concluded that the absence of a co-eluent peak together with the main DLT peak indicated that the developed method it is specific for the estimation of DLT in the presence of degradation products. Furthermore, the proposed RP-UPLC method has excellent sensitivity, precision and reproducibility. Although no attempt has been made to identify degraded products, the proposed method can be used as a stability indicator method for Darolutamid dosage.

ACKNOWLEDGMENTS:

Authors are thankful Bayer Health care Pharmaceuticals, USA for providing gift sample of API and for Lotus Laboratories, Bangalore for providing the necessary equipment for the research.

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Received on 16.08.2020 Modified on 21.12.2020

Research J. Pharm. and Tech 2022; 15(1):165-170.

DOI: 10.52711/0974-360X.2022.00027