1. INTRODUCTION:

Endometriosis, fibrocystic breast disease, hereditary angioedema, and other disorders are treated with the drug danazol, which is also marketed under the brand names Danocrine and other names. In 1971, danazol—which had been first discovered in 1963—became a recognised medicinal compound. Danazol, also known as 2,3-isoxazol-17-ethynyltestosterone, is a synthetic androstane steroid and a derivative of testosterone and ethisterone (17-ethynyltestosterone), has largely been replaced by gonadotropin-releasing hormone analogues (GnRH analogues) due to their improved side-effect profiles, particularly their lack of masculinizing side effects. This particular derivative of ethisterone has a 2,3-isoxazole moiety in place of the C3 ketone (i.e., an isoxazole ring is bonded to the A ring at the C2 and C3 locations). Ethisterone has little androgenic action and is a weak progestin. The drug has the properties of a weak progestogen, weak antigonadotropin, weak steroidogenesis inhibitor, and a functional antiestrogen. It also exhibits weak androgen and anabolic activity. Due to its lipophilicity and ability to partition across cell membranes, danazol is likely to penetrate extensively into tissue compartments. Albumin, SHBG, and CBG are known to bind danazol to plasma proteins.

Figure 1: Chemical Structure of Danazol

In the current work, a stability-indicating reverse phase HPLC (RP-HPLC) method was developed and evaluated for identifying Danazol in pharmaceutical dosage form. As far as we are aware, there aren't many reports regarding the Danazol stability test in the literature. Furthermore, we are uncertain whether there is a technique with the sensitivity, specificity, simplicity, accuracy, selectivity, and robust liquid stability needed to determine the Danazol. While studying various literature, we also found that there are no methods related to the Pharmacological, toxicological and therapeutic determination of drugs major degradant.

2. EXPERIMENTAL:

2.1. MATERIALS AND METHODOLOGY

Chemicals and reagents: Pharmaceutical grade Danazol was obtained as a gift sample from Cipla Ltd. L.B.S Marg, Vikhroli and all chemicals used were of HPLC grade Acetonitrile, Methanol, HCl, NaOH, H2O2 and was purchased from S D fine-Chem limited (India).

2.2 Instrumentation, condition, and optimization for HPLC

Waters Alliance e2695 separation module, with the Detector is a Photodiode array (model 2998) with wavelength range of 190-800nm. The data acquisition was done using Waters Empower 3 Software was used to carry out chromatographic separation.

With a flow rate of 1 ml/min, isocratic elution was carried out using acetonitrile and Water (90:10, v/v). Preparation of standard stock solution: 10mg of Danazol was dissolved in 10mL methanol. This gave 1000µg/mL standard stock solution for Danazol. From this 1mL solution was pipette out and dissolved in 10mL of diluents to make 100µg/mL solution of Danazol. From this solution 1mL solution was withdrawn and diluted to 10mL with diluents to make 10µg/mL solution of Danazol

Table 1: Chromatographic Conditions

Parameters	Conditions
Column	Sapphirus c18 HPLC classic 5µm length: 250 x4.6mm
Flow rate	1.0ml/min
Column temperature	25°c
Wavelength	285nm
Injection volume	10µl
Mobile phase	Acetonitrile: Water (90:10)

2.3. Method development and Validation:

Following ICH Q2(R1) requirements, the analytical technique was created and verified Using optimized HPLC data, several analytical variable factors including specificity, sensitivity, precision, accuracy, linearity, ruggedness, robustness, and system appropriateness were evaluated.

2.3.1 Specificity:

Specificity was carried out as blank, standard and test solution was injected and interference was examined. Blank preparation- Diluent i.e., mobile phase was used as blank. Standard preparation- 10mg of Danazol was accurately weighed and transferred to 10mL volumetric flask and volume was made up to mark by Diluent i.e., Mobile phase (1000µg/mL) from this 0.1mL solution is withdraw and make volume up to 10mL with diluents to get concentration of 10µg/mL. Test preparation- 50mg of drug was added in 50mL of diluent i.e., Mobile phase, out of which 1ml of solution was pipette out and again diluted by mobile phase to make up 100µg/mL stock solution. From 100µg/mL 1ml of solution was pipette out and 10µg/mL was prepared. Then each solution blank, standard and test were injected separately into HPLC system and observed that Danazol peak is not affected by excipients and diluents.

2.3.2 Predicted linearity:

From the above standard stock solution of 100µg/mL of Danazol was diluted with diluent to get concentration of 1, 2, 3, 4, 5µg/mL. Then subjected to chromatographic analysis, Calibration curve of concentration vs area was plotted and quantitation and detection limit was calculated.

Preparation of calibration curve standard: From the standard stock solution of 100µg/mL of Danazol was diluted with diluent to get calibration curve standards with concentration of 2, 4, 6,8,10, 12µg/mL.

Calibration experiment and regression analysis By using the chromatographic condition the calibration standard curve was analyzed. After the completion of calibration experiment on Y axis peak area were plotted against respective concentration on X-axis and linear regression were performed to get least square line and linear regression type Y= a+bx where b is slope and a is Y-intercept.

2.3.3. Linearity: Linearity study was conducted by analyzing six standard solution covering range of 2µg/mL to 12µg/mL. From primary stock solution of 1000µg/mL, 0.2mL, 0.4mL, o.6mL, 0.8mL, 1mL, and 1.2mL are pipette out into 10mL volumetric flask and make up volume up to 10mL by diluent to make 10µg/mL solution. A calibration curve with concentration vs. Peak area was plotted by injecting above prepared solutions.

2.3.4. Accuracy and Precision: The accuracy and precision were evaluated by preparing solution with 9µg/mL, 10µg/mL, 11µg/mL of Danazol (80%, 100%, 120%). Solution of 5ppm of test sample was prepared and was added in the standard solution of 4ppm, 5ppm, 6ppm, to make up 9µg/mL, 10µg/mL, 11µg/mL respectively. Resulting mixture is analyzed in triplicated over three days. The % recovery of added drug and %RSD were taken to measure accuracy and precision.

2.3.5. Detection limit: Based on standard deviation of response and slop of calibration curve, the detection limit (DL) was calculated.

2.3.6. Quantitation limit: Based on standard deviation of the response and slop of calibration curve, the quantitation limit (QL) was calculated.

2.3.7. Robustness:

Robustness was performed by using Stat-Ease Design of Experiments (DOE) Software. By applying central composite design for 2 factors i.e., Composition of Organic Phase and Flow rate.

The robustness of the method was checked by analyzing 10µg/mL solution of Danazol in deliberately changing parameters like composition of mobile phase and flow rate. Then results were compared with results obtained in optimized condition. Change in response of Danazol and system suitability parameters was recorded.

2.4. Forced Degradation Studies:

In compliance with the ICH Q1A (R2) specifications, study on forced degradation that included acid hydrolysis, alkali hydrolysis, thermal degradation, and oxidative degradation were carried out.

2.4.1. Alkali degradation:

Alkali degradation study was performed by dissolving 10mg of Danazol in 10ml of (0.1N) concentrated sodium hydroxide and was condensed on water bath at 80°C for 1Hr. Sample are withdrawn after 05, 10, 15minutes interval, 10µg/mL conc. Solution is prepared and subjected to chromatographic analysis.

2.4.2 Acid degradation:

Acid degradation study was performed by dissolving 10mg of Danazol in 10ml of (0.1N) concentrated hydrochloric acid and was condensed on water bath at 80°C for 1Hr. Sample are withdrawn after 30,45,60 min interval, 10µg/mL conc. Solution is prepared and subjected to chromatographic analysis.

2.4.3. Dry heat degradation:

Drug sample is placed in oven at 100°C for 1 hour and then 10µg/mL concentration solution of drug is prepared and subjected to chromatographic analysis.

2.4.4. Wet heat degradation:

Hydrolytic degradation study was performed by dissolving 10 mg of Danazol in 10 mL of water and heated on water bath at 80°C for 1 hour. Sample are withdrawn after 5 min interval, 10µg/mL concentration solution is prepared and subjected to chromatographic analysis.

2.4.5. Oxidative degradation:

Oxidative degradation study was performed by dissolving 10 mg of Danazol in 10 mL of 3 % and 30% hydrogen peroxide. Then kept in dark condition for 24 hours, 10µg/mL concentration solution is Prepared and subjected to chromatographic analysis.

2.4.6. Photolytic degradation:

Drug sample is placed in sunlight for 1hour and then 10µg/mL concentration solution of drug is prepared and subjected to chromatographic analysis.

3. RESULTS AND DISCUSSION:

Melting point of danazol was found to be 220°C.

Danazol was found to be soluble in Methanol and Acetonitrile.

The maximum absorption of wave length (λmax) of Danazol was found be 285nm.

In a C18 column, the chromatographic conditions were optimized. From standard solution, the danazol peak area and retention duration were computed.

Figure 2: Representative chromatogram of Danazol in optimized chromatographic condition

Table 2: System Suitability Parameters.

Sr. no	Parameters	Danazol
1.	Retention time	5.644
2.	Theoretical plates	17625
3.	Tailing factor	1.1
4.	Area	293812

3.1. Method Validation:

3.1.1. Specificity:

Chromatogram was recorded for the blank solution and it was subjected to the analysis which shows that there is no peak at the retention time of Danazol. The HPLC chromatogram recorded for blank, standard and formulation showed that Danazol peak not affected.

3.1.2. Assay of Pharmaceutical Formulation (Capsule):

Assay of Danazol of label claim 50 mg was performed in triplicate and the percentage recovery was found to be 99.9%.

3.1.3. Linearity:

After calibration experiment it was observed that Danazol was linear in the range of 2- 12µg/mL. The plot of peak area vs. concentration was subjected to least square regression. The equation was y = 21346x + 3977.8 Where X is concentration (µg/mL) and y is the peak area. The regression coefficient was found to be 0.9995.

After performing predicted linearity,

3.1.4. The Detection limit was found to 0.6903µg/mL.

3.1.5. The Quantitation limit was found to be 2.0919µg/mL.

3.1.6. Accuracy and precision:

The accuracy was evaluated by fortifying drug solution with the drug according to 80%, 100% and 120% label claim. Percent recovery of added drug was taken as a measure of accuracy.

The result obtained for accuracy and precision experiment as shown in Table. From the data obtained for accuracy and precision studies, it has been found that mean value of amount of drug found very close to amount of drug added; To determine intra-day and inter-day variability, one-way ANOVA was conducted and the F-value for each level was determined by taking ratio of between mean square to within mean square.

Figure 3: Calibration Curve for linearity

Table 3: Linearity data for Danazol

Concentration. (µg/mL)	Retention time (min)	Area				± SD	%RSD
Concentration. (µg/mL)	Retention time (min)	1	2	3	Mean	± SD	%RSD
2	5.650	46094	46095	46094	46094	0.57735	0.001253
4	5.651	87564	87566	87562	87564	2	0.002284
6	5.650	133594	133588	133591	133591	2	0.001497
8	5.651	177150	177146	177142	177146	4	0.002258
10	5.658	218094	218082	218088	218088	6	0.002751
12	5.658	257911	257907	257915	257911	5	0.001939
Equation		y=21346x+ 3977.8
Regression		0.9995

Table 4: Accuracy and Precision Data for Danazol

Amount Added (mg)	Amount found (mg)			Within mean square	Between mean square	F-value
Amount Added (mg)	Day 1	Day 2	Day 3	Within mean square	Between mean square	F-value
80% (9 µg/mL)	8.95	8.88	8.99	0.011	0.0014	0.38182
	9.00	9.00	8.99
	9.94	8.95	8.94
Mean	8.96	8.94	8.97
% Recovery	99.55	99.33	99.66
S.D	0.32146	0.060277	0.028868
R.S.D.	0.358633	0.67399	0.322422
100% (10 µg/mL)	9.95	9.84	9.98	0.0044	0.00275	1.87879
	9.92	9.89	9.93
	9.94	9.95	9.94
Mean	9.93	9.95	9.94
% Recovery	99.36	99	99.4
S.D.	0.015275	0.041633	0.015275
R.S.D.	0.153726	0.420397	0.153623
120% (11 µg/mL)	11.05	10.89	10.87	0.02047	0.03742	5.04853
	10.92	11.01	10.83
	10.98	11.02	10.84
Mean	10.98	10.94	10.84
% Recovery	99.83	99.75	98.53
S.D.	0.065574	0.072342	0.026456
R.S.D.	0.597217	0.659251	0.244073

Table 5: Robustness data

Sr.no	Factor 1 (ACN: Water)	Factor 2 Flow Rate (mL/Min)	Retention time (min)	Theoretical Plate	Asymmetric Factor	Capacity Factor
1.	85:15	0.8	7.509	18527	1.2	6.51
2.	95:05	0.8	5.821	18909.3	1.2	4.82
3.	85:15	1.2	4.938	16716.8	1.2	3.93
4.	95:05	1.2	3.819	16408.1	1.2	2.84
5.	82.92:17.08	1	6.457	18498.7	1.2	5.55
6.	97.07:02.93	1	4.515	17746.6	1.2	3.56
7	90:10	0.717	7.507	19311.2	1.2	6.57
8.	90:10	1.28	4.445	16650.0	1.2	3.48
9.	90:10	1	5.504	18101.8	1.2	4.5
10.	90:10	1	5.504	18101.8	1.2	4.5
11.	90:10	1	5.504	18101.8	1.2	4.5
12.	90:10	1	5.504	18101.8	1.2	4.5
13.	90:10	1	5.504	18101.8	1.2	4.5

3.1.8. Forced Degradation Studies:

Danazol was subjected to variety of stressed condition such as acid, alkali, wet heat, dry heat, oxidation, and light. Acid degradation of Danazol was carried out at 80°C in 0.1 N HCl for 1 Hr. on water bath. Degradation is observed at 30,45,60 min i.e., reduction in peak area and degradant peak is observed. Base degradation was conducted at 80°C in 0.1N NaOH solution for 15 min. Degradation is observed at 10 and 15 minutes i.e., reduction in peak area and degradant peak is observed. For oxidative stress drug dissolved in 30% H2O2 and 3% H2O2 at room temperature for 24 hours. No Degradant peak was observed. For wet heat degradation, drug was dissolved in water and subjected to 80°C on water bath for 45min. Degradation is observed at 45 min as reduction in peak area. Drug was placed in oven at 100°C for dry heat degradation for 1 hour, Degradation was found at 45 mins. Photodegradation was performed by spreading drug on petri dish and direct exposure to sunlight. Peak area was not reduced and hence no degradation was found.

Table 6: Forced Degradation studies

Sr.No	Stressed conditions	Drug decomposed (%)	Drug recovered (%)
1.	Alkali hydrolysis	44.34	55.66
2.	Acid hydrolysis	43.32	56.68
3.	Thermal degradation	23.93	76.07
4.	Oxidative degradation	No deg	-
5.	Photolytic degradation	No deg	-

3.1.9. Enrichment of alkali degradation product:

Molecular Formula: C21H29NaO2

Molecular weight: 336.44g/mol

Melting point: 225°C which was more than pure drug sample.

Color: Beige

Solubility:

Compound was found to be soluble in Methanol and Acetonitrile.

Figure 4: Predicted structure of major degradant product

3.1.10. ADMET and PKPD Modeling:

ADMET and PKPD analysis:

Compound is categorized under small molecule, with molecule weight ranging below 500D i.e. 336.44 with molecular formula C₂₁H₂₉NaO₂. There is total 23 heavy atoms present in the molecule, with zero (0). Aromatic heavy atoms. Fraction Csp³ is 0.75 defines that the compound exhibit high degree of saturation. Molecule has Moderate solubility with respect to water. GI absorption of the ideal drug and impurity is high, with high BBB permeability. The model predicts to be the given compound is likely to be a substrate of P-glycoprotein for the ideal drug compound, whereas the impurity which is synthesized is not. No. of Violations (n violations) = 1 or < 0 that means both the compound that can easily binds to the receptor. Bioavailability score was found to be 0.55 which is above 50% chance of bioavailability in human system.

3.1.11. Predicted Pharmacological Activities by PASS online Server:

· The pharmacological activities were predicted by using PASS (Predicted Activity Spectra for Substances) online server

· Results of Pa > 0.7 were considered

3.1.12. Determination of drug- likeliness properties using Molsoft:

The drug likeliness properties of danazol and its impurities were evaluated using Molsoft (www.molsoft.com)

Danazol: Drug likeliness Score was found to be 0.66.

Impurity: Drug Likeliness Score was found to be 0.15

From the Molsoft it was observed that the degradation product has low drug likeliness score as compared to the Standard drug.

4. CONCLUSION:

For the Danazol, a simple, precise, accurate, robust, HPLC technique was created and verified. The drug was found to be more sensitive to alkaline and acidic hydrolysis and all the degradation products were found to be well separated from the principal peak, indicating that the Danazol peaks in all obtained chromatograms were extremely pure. Danazol was subjected to stress conditions including acidic, alkaline, oxidation, and thermal degradation. According to the developed method, the Danazol is stable under thermal and oxidative stress but unstable under acidic and alkaline environments. The created technique was approved in accordance with ICH Q2 (R1) Guidelines. Linearity, accuracy, precision, specificity, and robustness were the validation parameters that were examined. Drug-likeness properties of these degradation products were predicted along with Danazol using MolSoft software. From the Molsoft it was observed that the degradation product has low drug likeliness score as compared to the Standard drug. Impurity showed very low drug likeliness score and were not be useful as drug with respect to the pharmacokinetic properties. From ADMET SAR 2.0 toolbox, test degradation product found non-toxic with respect to carcinogenicity, eye corrosion, eye irritation, Ames mutagenicity and hepatoxicity. From the PASS server data, it can be concluded that the Impurity has good Testosterone 17beta-dehydrogenase (NADP+) inhibitor activity.

5. ACKNOWLEDGEMENT:

The Authors are grateful to Cipla Ltd. L.B.S Marg, Vikhroli., India for providing gift sample of Danazol. The Authors are thankful to MET’s Institute of Pharmacy Bhujbal Knowledge City, Nashik, for providing necessary chemicals and analytical facilities.

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Received on 13.11.2024 Revised on 21.02.2025

Accepted on 26.04.2025 Published on 10.07.2025

Available online from July 17, 2025

Asian J. Pharm. Res. 2025; 15(3):263-268.

DOI: 10.52711/2231-5691.2025.00042

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