INTRODUCTION:

Etonogestrel is a long-acting synthetic derived progestin contraceptive used in various devices such as contraceptive rings and intradermal implants, Etonogestrel molecule is a 3-ketodesogestrel or 19-nortestosterone which is a synthetic biologically active metabolite of progestin desogestrel.²

Ethinylestradiol was first synthesized in 1938 by Hans Herloff Inhoffen and Walter Hohlweg at Schering.¹ It was developed in an effort to create an estrogen with greater oral bioavailability.¹

These properties were achieved by the substitution of an ethinyl group at carbon 17 of estradiol.¹ Ethinylestradiol soon replaced mestranol in contraceptive pills.

Structure of Etonogestrel

Chemical formula : C₂₂H₂₈O₂

Average : 324.4565

physical state : solid

Melting Point : 196-200ºC

pK Values : 17.99

IUPAC name:

(1R,3aS,3bS,9aR,9bS,11aS)-11a-ethyl-1-ethynyl-1-hydroxy-10-methylidene-1H,2H,3H,3aH,3bH,4H,5H,7H,8H,9H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-7-one

Structure of ethinylestradiol

Molecularweight : 296.4 g/mol

Monoisotopic : 296.17763 g/mol

Chemical formula : C20H24O2

Appearance : powder

physical state : powder

Solubility : 17% in EtOH, 25% in Et2O, 20% in Me2CO, 25% in dioxane

Melting Point : 182 to 184 °C (360 to 363 °F)

pK Values : 10.33

IUPACname: (1R, 3aS, 3bR, 9bS, 11aS)-1-ethynyl-11a-methyl-1H, 2H, 3H, 3aH, 3bH, 4H, 5H, 9bH, 10H, 11H, 11aH-cyclopenta[a]phenanthrene-1,7-diol)

MATERIALS AND METHODS:

Instruments used:

· Waters HPLC System series with Binary pumps, Photo Diode array detector and manual sampler integrated with empower software

· Lab india UV double beam spectrophotometer with UV win5 software was used for measuring absorbances of Ethinylestradiol and Etonogestrel solutions.

Materials:

· Ethinylestradiol and Etonogestrel pure drugs (API), Combination Ethinylestradiol and Etonogestrel tablets (DESOGEN) were obtained from Ramkem. Distilled water, Acetonitrile, Phosphate Buffer, Methanol, Potassium dihydrogen ortho phosphate Buffer, Ortho-phosphoric acid.

Chromatographic conditions:

Mobile phase: Na₂HPO₄Acetonitrile: (70:30 v/v)

Flow rate: 1ml/min

Column: BDS C18 (4.6 x 150mm, 5µm)

Detector wave length: 230nm

Column temperature: 30°C

Injection volume : 20mL

Run time: 5min

Diluent: Acetonitrile and Water in the ratio of 50:50 % V/V

Methods:

Preparation of Standard stock solutions: Accurately weighed 6mg of Etonogestrel, 0.75mg of Ethinylestradiol and transferred to 50ml and 50ml individual volumetric flasks and 3/4^th of diluents was added to these flask and sonicated for 10 minutes. Flask were made up with diluents and labeled as Standard stock solution. (120µg/ml of Etonogestrel and 15µg/ml Ethinylestradiol).

Preparation of Standard working solutions (100% solution): 1ml from each stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (12µg/ml of Etonogestrel and 1.5µg/ml of Ethinylestradiol).

Preparation of Sample stock solutions: 10 number of was transferred into a 10ml volumetric flask, 5ml of diluents was added and sonicated for 25min, further the volume was made up with diluent and filtered by HPLC filters. (120µg/ml of Etonogestrel and 15µg/ml Ethinylestradiol).

Preparation of Sample working solutions (100% solution): 1ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (12µg/ml of Etonogestrel and 1.5µg/ml Ethinylestradiol).

Preparation of Buffer:

0.1% OPA Buffer: 1ml of Ortho phosphoric acid was diluted to 1000ml with HPLC grade water to get 0.1% OPA Solution.

RESULTS AND DISCUSSION:

System suitability parameters:

The system suitability parameters were determined by preparing standard solutions of Etonogestrel (12ppm) and Ethinylestradiol (1.5ppm) 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%.

Specificity:

Checking of the interference in the optimized method. We should not find interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific.

Precision:

From a single volumetric flask of working standard solution six injections were given and the obtained areas were mentioned above. Average area, standard deviation and % RSD were calculated for two drugs. % RSD obtained as 0.9% 0.6% respectively for Ethinylestradiol and Etonogestrel. As the limit of Precision was less than “2” the system precision was passed in this method.

System precision table of Ethinylestradiol and Etonogestrel

S. No	Area of Etonogestrel	Area of Ethinylestradiol
1.	234714	41522
2.	234589	41562
3.	238625	41463
4.	239564	41154
5.	236738	41869
6.	238694	41498
Mean	237154	41511
S.D	2147.1	228.4
%RSD	0.9	0.6

Linearity:

Six linear concentrations of Etonogestrel (3-18µg/ml) and Ethinylestradiol (0.375-2.25µg/ml) were injected in a duplicate manner. Average areas were mentioned above and linearity equations obtained for Etonogestrel was y = 19522x + 504.5 and of Ethinylestradiol was y = 27207x + 358.2 Correlation coefficient obtained was 0.999 for the two drugs.

Linearity table for Ethinylestradiol and Etonogestrel.

Etonogestrel		Ethinylestradiol
Conc (μg/mL)	Peak area	Conc (μg/mL)	Peak area
0	0	0	0
3	59810	0.375	10741
6	115535	0.75	20661
9	181057	1.125	31762
12	232455	1.5	41369
15	291156	1.875	50249
18	353418	2.25	61984

Calibration curve of Etonogestrel

Calibration curve of Ethinylestradiol

Accuracy:

Three levels of Accuracy samples were prepared by standard addition method. Triplicate injections were given for each level of accuracy and mean %Recovery was obtained as 100.55% and 100.20% for Ethinylestradiol and Etonogestrel respectively.

Accuracy table of Etonogestrel:

% Level	Amount Spiked (μg/mL)	Amount recovered (μg/mL)	% Recovery	Mean % Recovery
50%	6	6.0	99.5	100.55%
	6	6.0	99.7
	6	6.0	100.2
100%	12	11.9	99.4
	12	12.2	101.5
	12	12.0	99.7
150%	18	18.3	101.8
	18	18.3	101.7
	18	18.3	101.4

Accuracy table of Ethinylestradiol:

% Level	Amount Spiked (μg/mL)	Amount recovered (μg/mL)	% Recovery	Mean % Recovery
50%	0.75	0.74	98.55	100.20%
	0.75	0.74	98.70
	0.75	0.75	99.73
100%	1.5	1.51	100.47
	1.5	1.49	99.60
	1.5	1.52	101.14
150%	2.25	2.28	101.26
	2.25	2.26	100.41
	2.25	2.29	101.96

Sensitivity:

Sensitivity table of Ethinylestradiol and Etonogestrel

Molecule	LOD	LOQ
Etonogestrel	0.03	0.10
Ethinylestradiol	0.02	0.06

Robustness:

Robustness conditions like Flow minus (0.9ml/min), Flow plus (1.1ml/min), mobile phase minus (75B:25A), mobile phase plus (65B:35A), temperature minus (25°C) and temperature plus (35°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit.

Robustness data for Ethinylestradiol and Etonogestrel

S. No	Condition	% RSD of Ethinylestradiol	% RSD of Etonogestrel
1	Flow rate (-) 1.1ml/min	0.3	1.3
2	Flow rate (+) 0.9ml/min	1.4	0.1
3	Mobile phase (-) 75B:25A	1.0	0.7
4	Mobile phase (+) 65B:35A	0.9	0.5
5	Temperature (-) 25°C	0.5	0.5
6	Temperature (+) 35°C	1.1	0.9

Assay:

Femilon, bearing the label claims Etonogestrel 0.12mg, Ethinylestradiol 0.015mg. Assay was performed with the above formulation. Average %Assay for Ethinylestradiol and Etonogestrel obtained was 99.61 and 99.66% respectively.

Assay Data of Etonogestrel

S. No	Standard Area	Sample area	% Assay
1	234714	236483	99.62
2	234589	234888	98.95
3	238625	238013	100.26
4	239564	236442	99.60
5	236738	237193	99.92
6	238694	235807	99.33
Avg	237154	236471	99.61
Stdev	2147.1	1081.4	0.46
%RSD	0.9	0.5	0.5

Assay Data of Ethinylestradiol

S. No	Standard Area	Sample area	% Assay
1	41522	41719	100.40
2	41562	41238	99.24
3	41463	41173	99.09
4	41154	41787	100.56
5	41869	41029	98.74
6	41498	41535	99.96
Avg	41511	41414	99.66
Stdev	228.4	311.2	0.75
%RSD	0.6	0.8	0.75

DEGRADATION:

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.

Regarding the pH adjustment in mobile phase for the acid and base degradation studies have movement in retention time of drugs. But due to neutralized acid sample with 2N Base solution and base sample with 2N Acid solution there will be no change in retention time

Degradation Data of Etonogestrel

S. No	Degradation Condition	% Drug UnDegraded	% Drug Degreaded
1	Acid	92.14	7.86
2	Alkali	95.89	4.11
3	Oxidation	94.93	5.07
4	Thermal	95.70	4.30
5	UV	97.83	2.17
6	Water	99.80	0.20

Degradation Data of Ethinylestradiol

S. No	Degradation Condition	% Drug UnDegraded	% Drug Degreaded
1	Acid	93.07	6.93
2	Alkali	92.63	7.37
3	Oxidation	91.93	8.07
4	Thermal	98.42	1.58
5	UV	98.63	1.37
6	Water	99.20	0.80

CONCLUSION:

A simple, Accurate, precise method was developed for the simultaneous estimation of the Ethinylestradiol and Etonogestrel in Tablet dosage form. Retention time of Ethinylestradiol and Etonogestrel were found to be 2.163 min and 2.824. % RSD of the Ethinylestradiol and Etonogestrel were and found to be 0.8 and 0.5 respectively. %Recovery was obtained as 100.20% and 100.55% for Ethinylestradiol and Etonogestrel respectively. LOD, LOQ values obtained from regression equations of Ethinylestradiol and Etonogestrel were 0.03, 0.10 and 0.01, 0.02 respectively. Regression equation of Etonogestrel is y = 19522x+504.57, and y = 27207x+358.27 of Ethinylestradiol. Retention times were decreased and that run time was decreased, so the method developed was simple and economical that can be adopted in regular Quality control test in Industries.

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Received on 04.12.2023 Modified on 20.01.2024

Asian J. Pharm. Ana. 2024; 14(2):76-80.

DOI: 10.52711/2231-5675.2024.00014

S No	Ethinylestradiol			Etonogestrel
Inj	RT (min)	USP Plate Count	Tailing	RT (min)	USP Plate Count	Tailing	USP Resolution
1	2.111	3945	1.13	2.813	8132	1.34	4.4
2	2.126	4322	1.11	2.822	8203	1.36	4.3
3	2.127	3997	1.11	2.824	8296	1.38	4.4
4	2.129	4198	1.18	2.824	8320	1.40	4.3
5	2.129	4238	1.17	2.825	8369	1.40	4.2
6	2.129	4115	1.11	2.830	8178	1.33	4.4

Pavithra Bodagala, Y Divya, C Parthiban, M Sudhakar