1. INTRODUCTION:

Danazol was first used in 1969 in clinical trials for the treatment of a variety of gynaecological and endocrinological disorders in the USA. It has been available for human used in the UK since 1974 and the danocrine brand of Danazol was released for marketing in Australia in May 1978. In other countries the drug is being used clinically in the management of endometriosis [1], benign breast disorders [2], angioneurotic oedema and has been suggested as an oral contraceptive in both male and females [3].

Danazol is an aromatic heteropolycyclic compound with IUPAC name 17α – Pregna – 2, 4-dien-20-yno [2, 3-d] – isoxazol -17-ol (Figure 1). Danazol is an endocrine metabolic agent or anti gonadotropin drug approved by the US Food and Drug Administration as an anterior pituitary suppressant in the treatment of endometriosis and fibrocystic breast disease. The mechanism of action of Danazol involves in the anterior pituitary suppression by inhibiting the pituitary output of gonadotropins [4]. To date, analytical methods like UPLC and micelluar chromatographic techniques described in literature for determination of Danazol [5,6,7]. In the present study was designed to develop and validate a stability indicating reverse phase HPLC (RP-HPLC) method in order to determine the Danazol in pharmaceutical dosage form. To the best of our knowledge there is no report in the literature related to Danazol stability test, as well as a method with sensibility, specificity, simple, precise, accurate, selective and robust liquid stability enough to be determining the Danazol and it could be valid as an alternative method.

Figure 1 Chemical structure of Danazol

2. MATERAILS AND METHODOLOGY:

Chemicals and reagents

Danazol standard drug was obtained from Micron pharmaceuticals, Mumbai, India and all chemicals used were of HPLC grade: acetonitrile, methanol, and potassium dihydroxyl ortho phosphate purchased from Merck (India).

HPLC instrumentation and conditions and optimization

Chromatographic separation was achieved by using a Shimadzu Model CBM-20 A/20 HPLC system, equipped with an SPD M20A prominence photodiode array detector (150X4.6 mm, 5μm particle size) at 25⁰C. Isocratic elution was performed using acetonitrile and potassium dihydroxyl ortho phosphate (70:30, v/v) with flow rate of 1ml/min. The retention of the drug was found to be 4.9 min.

Danazol solution was prepared by about 10mg of drug was taken in 10ml standard flask. To that 5−8ml of acetonitrile was added and kept in a sonicator for 5−10min and the final volume was adjusted to 10ml to produce 1000μg/ml solution and kept in refrigerator for further use.

Method development and validation

The analytical method was developed and validated according to ICH Q2 (R1) guidelines [8,9,10]. Analytical variable parameters such as specificity, sensitivity, precision, accuracy, linearity, ruggedness, robustness and system suitability were tested using optimized HPLC data.

Specificity:

The specificity and peak purity were carried out to determine whether there are any interference due to presence of impurities and including degradation products and in order to prove the method is specific and selective, the standard peak of the drug and sample peak were compared to the R_Tagainst the blank and placebo chromatogram.

Sensitivity:

Sensitivity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc. The developed method was carried out for sensitivity studies based upon limit of detection (LOD) and quantification (LOQ). The LOD and LOQ were carried out by injecting 3 injections of each and the peak was determined. LOD and LOQ were calculated based upon their signal to noise ratio by injecting the 3 replicate injections of solutions respectively.

Precision:

The precision of the method was determined at 2 levels of 6 injections of 3 different concentrations such as 20, 80, and 120μg/ml. The precision is expressed as % RSD and mean, standard deviation were calculated. The formula for % RSD was calculated.

Intra run precision:

Intra run precision was calculated by injecting 2 levels of 6 injections of 3 different concentrations such as 20, 80, and 120μg/ml. The precision is expressed as % RSD. Mean and standard deviation were calculated.

Intraday precision:

Intraday precision was calculated by injecting 2 levels of 6 injections of 3 different concentrations such as 20, 80, and 120μg/ml. The precision is expressed as % RSD. Mean and standard deviation were calculated. The results were obtained on the same day.

Inter day precision:

Inter day precision was calculated by injecting 2 levels of 6 injections of 3 different concentrations such as 20, 80, and 120μg/ml. The precision is expressed as % RSD. Mean and standard deviation were calculated. The results obtained over at least 2 days.

Accuracy:

Accuracy of the developed method was determined based on the recovery studies. Recovery studies were carried out by adding known concentration of standard solution of 20mcg/ml to the sample solution of 20, 80 and 120mcg/ml. The result of accuracy was noted for 6 replicates at 3 different concentrations and mean; standard deviation and % nominal were calculated.

Linearity:

From the stock solution, suitable dilutions were prepared using acetonitrile as solvent at the range of 20, 40, 60, 80, 100, and 120μg/ml by measuring against the blank solution. The standard curve was plotted against between the concentration and peak area and the intercept, slope values were noted.

Ruggedness:

Ruggedness of the method was studied by changing the experimental conditions such as apparatus, instruments, reagents, solvents and column. The chromatographic parameters such as RT, asymmetrical factor were evaluated respectively and the results are noted.

Robustness:

Robustness of the method was studied by injecting the standard solution with slight variation in ± 1 % of mobile phase, ± 0.1 of the pH value and ± 0.1% of flow rate, and the results are noted.

Forced Degradation Studies:

Forced degradation studies were carried out as per ICH Q1A (R₂) guidelines and the parameters such as acid hydrolysis, alkali hydrolysis, thermal degradation and oxidative degradation were carried out.

Hydrolytic studies:

Acid hydrolysis: About 10 mg equivalent weight of Danazol was taken in 50 ml standard flask. To that 10 –20 ml of acetonitrile was added and kept in the sonicator for 10–15 minutes and 1 ml of 0.1N HCl was added and the final volume was adjusted to 50ml with the same solvent. From this solution, 1ml was taken in 10ml standard flask and the final volume was adjusted to 10ml by using acetonitrile as solvent and injected into HPLC for 0 hr. This same procedure was carried out at regular time intervals such as 1hr, 2hr, 4hr, 6hr and 24hr respectively and the readings were recorded.

Alkali hydrolysis:

About 10 mg equivalent weight of Danazol was taken in 50 ml standard flask. To that 10 –20 ml of acetonitrile was added and kept in the sonicator for 10–15 minutes and 1 ml of 0.1N NaOH was added and the final volume was adjusted to 50ml with the same solvent. From this solution, 1 ml was taken in 10 ml standard flask and the final volume was adjusted to 10 ml by using acetonitrile as solvent and injected into HPLC for 0hr. This same procedure was carried out at regular time intervals such as 1hr, 2hr, 4hr, 6hr and 24hr respectively and the readings were recorded.

Thermal degradation:

About 10 mg equivalent weight of Danazol was taken in 50 ml standard flask. To that 10 –20 ml of acetonitrile was added and kept in the sonicator for 10–15 minutes and the final volume was adjusted to 50ml with the same solvent and then it was heated at 60⁰C constantly. From this solution, 1 ml was taken in 10 ml standard flask and the final volume was adjusted to 10 ml by using acetonitrile as solvent and injected into HPLC for 0 hr. This same procedure was carried out at regular time intervals such as 1hr, 2hr, 4hr, 6hr and 24hr respectively and the readings were recorded.

Oxidative degradation:

About 10 mg equivalent weight of Danazol was taken in 50 ml standard flask. To that 10 –20 ml of acetonitrile was added and kept in the sonicator for 10–15 minutes and 1 ml of 30% of HClO₄ (Perchloric acid)was added and the final volume was adjusted to 50ml with the same solvent. From this solution, 1 ml was taken in 10 ml standard flask and the final volume was adjusted to 10 ml by using acetonitrile as solvent and injected into HPLC for 0hr. This same procedure was carried out at regular time intervals such as 1hr, 2hr, 4hr, 6hr and 24hr respectively and the readings were recorded.

3. RESULTS AND DISCUSSION:

Melting point of Danazol drug was found to be 225⁰C. The maximum absorption of wave length for drug was observed at 285nm.The solubility of Danazol was determined in various solvents such as methanol, acetonitrile, water, phosphate buffer solution of pH 1.2, 4.5, 6.8, and 7.2 respectively. The concentration of Danazol was determined by using UV visible spectrometer at 285nm. The results showed that maximum solubility was observed in acetonitrile (7.5mg/ml) followed by methanol (6.8mg/ml) and showed less soluble in water (5mg/ml).

A chromatographic condition was optimized in a C18 column. The parameters for the optimized conditions given in Table 1 and chromatogram showed in Figure 2. It was evaluated for the % RSD. The % RSD of Danazol peak area and retention time were calculated from 6 replicate injections of standard solution and the result showed no much difference and it was not more than 2.0. The tailing factor for Danazol peak was not more than 2.0. The theoretical plates of Danazol peak was not less than 2000.

Figure 2. Optimized HPLC chromatogram

Table 1. Optimized chromatographic conditions

Parameters	Conditions
Column	Water symmetry C18, 150X4.6mm, 5μ
Flow rate	1.0ml/min.
Column temperature	25⁰C
Wave length	285nm
Injection volume	20 μl
Mobile phase	25mM potassium dihydrogen phosphate buffer: acetonitrile (70:30)
pH	4.5

System suitability was done by spiking 6 replicate injections of standard solution and calculated (Table 2) for % RSD, it showed below 2.0% which revealed that system was in suitable condition.

Table 2. System suitability

S.No	Concentration	RT	Peak area
1	20	4.947	1654.519
2	20	4.943	1659.521
3	20	4.987	1640.041
4	20	4.843	1650.021
5	20	4.913	1638.442
6	20	4.909	1617.059
	Mean	4.92	1643.267
	Standard deviation	0.04	15.2116
	% RSD	0.81	0.9256

The specificity and peak purity were carried out to determine whether there was any interference due to presence of impurities, degradation products or other components that may be present at the retention time of analytical peak and affect the peak purity and specificity of the analytical method. The analysis of was performed over a wavelength range of 200-400nm. The retention time (RT-4.9) of standard solution of 100mcg/ml sample and blank injected and results were recorded show in Figure 3 and it describes there were no peak observed in blank with respective to drug peak, which revealed that the selected method is specific. The developed method also no interference with other matrix substances.

Figure 3. HPLC Chromatogram of specificity

The LOD and LOQ was calculated (Table 3) based on the signal to noise ratio. The lowest detectable concentration was set as 1ng/ml (LOD) and the lowest quantified concentration was set as 3ng/ml (LOQ) and it was found to be sensitive and the chromatogram shown in Figure 4 and 5.

Table 3. LOD and LOQ studies for Danazol

S.No	LOD(ng)	Peak area	LOQ(ng)	Peak area
1	1ng	0.592	3	0.961

Figure 4. HPLC Chromatogram for LOD

Figure 5. HPLC Chromatogram for LOQ

Linearity and range of the developed method was determined by plotting calibration curve using different concentrations range of standard Danazol (10-120μg/ml) solution. Standard solution was used for plotting calibration curve (Figure 6) was plotted using peak area v/s concentration. It was estimated that perfect linear graph was observed between peak area and concentration with the range of 10-120μg/ml. The linear regression equation for Danazol was found to be y=84.452 + 35.538 with correlation coefficient (R²) value 0.9986 and given in Table 4. The 3D view overlay of linearity chromatogram shown in Figure in 7.

Table 4. Linearity studies for Danazol

S.No	Concentration	Peak area
1	10	860.987
2	20	1616.714
3	40	3426.886
4	60	4916.951
5	80	6561.729
6	100	8632.231
7	120	10050.231

Figure 6. Calibration curve for Danazol

Figure 7. 3D view of linearity chromatogram

Precision studies were determined in intraday and inter day runs. Six replicate at three different concentration levels of lower, middle and high quality control sample of Danazol 20, 60, 120μg/ml was spiked and the mean, standard deviation and % RSD was calculated and found to be within the limit as shown in Table 5,6 and 7. The results showed that percentage of coefficient of variation (% RSD) of the analytes was determined and was found to be <5%.

Table 5. Intraday Precision studies for the Danazol

S. No	Concentration (μg/ml)	Measured concentration (μg/ml)	Average	SD	% RSD
1	20	19.17	19.03	0.18	0.94
		19.22
		18.99
		19.11
		18.98
		18.72
2	80	75.90	77.88	0.18	0.23
		78.16
		77.76
		77.62
		77.89
		77.95
3	120	118.56	119.65	1.80	1.50
		121.80
		118.48
		118.56
		122.15
		118.35

Table 6. Inter day Precision studies for the Danazol (Day 1)

S. No	Concentration (μg/ml)	Measured concentration (μg/ml)	Average	SD	% RSD
1	20	19.17	19.03	0.29	1.50
		19.23
		19.33
		18.58
		19.13
		18.75
2	80	74.89	75.01	0.81	1.07
		73.99
		74.72
		74.56
		75.74
		76.21
3	120	118.35	120.96	1.45	1.19
		120.96
		121.80
		122.63
		121.43
		120.61

Table 7. Inter day Precision studies for the Danazol (Day 2)

S.No	Concentration (μg/ml)	Measured concentration(μg/ml)	Average	SD	% RSD
1	20	19.23	19.06	0.26	1.36
		18.75
		18.72
		19.22
		19.11
		19.33
2	80	77.62	76.59	1.54	2.0
		74.72
		76.21
		78.16
		77.95
		74.89
3	120	118.56	119.37	1.47	1.23
		121.80
		118.35
		118.56
		118.35
		120.61

Accuracy of the developed analytical method was estimated by performing recovering studies and recovery of the Danazol was consistent at all levels (Table 8) and the percentage nominal of the Danazol are in between 98.87% to 100.69% .The recovery level 1,2 and 3 chromatograms shown in Figure 8, 9 and 10. The Danazol solution was found to be stable at 24hrs and described in Table 9.

Table 8. Accuracy studies for the Danazol

S.No	Level	Concentration (μg/ml)		Measured Concentration (μg/ml)	Mean	SD (n=3)	% C.V.	% Nominal
1	1	20	20	40.13	40.07	0.55	1.3	98.87
				41.23
				40.76
2	2	80	20	102.01	101.7	0.36	0.3	100.69
				101.3
				101.79
3	3	120	20	139.78	140.8	1.01	0.7	99.34
				141.00
				141.80

Figure 8. HPLC chromatogram of recovery at level 1

Figure 9. HPLC chromatogram of recovery at level 2

Figure 10. HPLC Chromatogram of recovery at level 3

Table 9. Solution stability

S. No	Time in Hr.	Added concentration (μg/ml)	Measured concentration (μg/ml)	% Nominal
1	0	10	10	100
2	2	10	10	100
3	12	10	9.8	98
4	24	10	9.5	95

Ruggedness of the method was analysed by changing the experimental conditions like solvents, operators, instruments and column of similar type and the method was found to be rugged, since there was no change in the method.

Robustness of the method was studies by spiking the standard solutions with slight variations in the optimized conditions like ± 1% in the ratio of acetonitrile in the mobile phase, ± 0.1ml of the flow rate and ±0.5% in pH conditions. The method was found to be robust, since there was no change in the chromatogram (Figure 11, 12).

Figure 11. HPLC Chromatogram of Flow Rate at 1.2ml/min

Figure 12. HPLC Chromatogram of Flow Rate at 0.8ml/min

Forced degradation studies were carried out for Danazol as per ICH Q1A (R2) guidelines, using 0.1N HCl (at 90⁰C for 20 min), 0.1N NaOH (at 90⁰C for 60 min), thermal degradation by heating 200μg/ml (at 60⁰C for 30min) and 30% HClO₄ (at 90⁰C for 30 min). During degradation studies Danazol exhibited different percentage of degradation at various conditions. Among all the stress studies major degradation occurred much in alkali, followed by acid, thermal, and oxidative stress 55.92%w/w, 11.21% w/w, 3.12% w/w and 1% w/w respectively (Table 10). The chromatogram results are specified in the Figure 13, 14, 15 and 16.

Table 10. Forced degradation studies of Danazol

S.No	Stress conditions	Drug recovered (%)	Drug decomposed (%)
1	Standard drug	100	100
2	Alkali hydrolysis	44.08	55.92
3	Acidic hydrolysis	88.79	11.21
4	Thermal degradation	96.88	3.12
5	Oxidative hydrolysis	100	1.21

Figure 13. HPLC Overlay Chromatograms for alkali Hydrolysis

Figure 14. HPLC Overlay Chromatograms for acid Hydrolysis

Figure 15. HPLC Overlay Chromatograms for thermal degradation

Figure 16. HPLC Overlay Chromatograms for oxidative degradation

4. CONCLUSION:

The developed HPLC method for Danazol was found to be simple, precise, accurate, and reproducible and costs effective. Statistical analysis of the developed method confirms that the proposed method is an appropriate method for their quantification in the formulation. Therefore, they can be useful for routine analyses for the qualitative and quantitative analysis.. This developed method can also be used regular for the in-process quality control of the sample. This method gives a sound knowledge regarding stability studies. The results of forced degradation studies shows that the major route of degradation is in alkali hydrolysis followed by thermal, oxidation and acidic respectively. The developed method concludes that the Danazol was found to be stable in acidic, thermal and oxidative stress conditions and unstable in alkaline conditions. The information presented here gives an idea for the researcher who is working in area like product development and finish product testing.

5. CONFLICT OF INTREST:

The authors confirm that this article content has no conflict of interest.

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Received on 10.09.2016 Accepted on 25.10.2016

Asian J. Pharm. Ana. 2016; 6(4): 227-234.

DOI: 10.5958/2231-5675.2016.00034.X