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International Journal of Pharmaceutical Sciences and
Clinical Research 2023; 3(1):37-44

RESEARCH ARTICLE

Development and Validation of a New Robust RP-HPLC Method for the

Simultaneous Quantitation of Levonorgestrel and Ethinylestradiol in Bulk and
Pharmaceutical Dosage Form
D. Susmitha1, V. Pavan Kumar1*, K. Sai Rajesh2, Narayanaswamy Harikrishnan3
Department of Pharmaceutical Analysis, Seven Hills College of Pharmacy (Autonomous), Tirupati,
1

Andhra Pradesh, India, 2Department of Pharmaceutical Analysis, A.S.N College of Pharmacy, Tenali,
Andhra Pradesh, India, 3Department of Pharmaceutical Analysis, Faculty of Pharmacy, Dr. M. G. R. Educational
and Research Institute, Chennai, Tamil Nadu, India

Received: 25-01-2023 Revised: 12-02-2023 Accepted: 05-03-2023

ABSTRACT
The present study was mainly focused on developing and validating a new isocratic, simple, rapid,
precise, accurate, and stable reverse phase high-performance liquid chromatography method for a
combination of levonorgestrel and ethinylestradiol. The separation was achieved on a C18 µ-bondopak
column (250 mm*4.6 mm) using a mobile phase consisting of buffer, water, and acetonitrile in the ratio
of 40:20:40 (buffer-0.1% v/v triethylamine pH-3.0 followed by 0.9 mL/min flow rate. The wavelength
detection was at 260 nm. The chromatographic retentions were stable at 3.604 min for levonorgestrel and
5.142 min for ethinylestradiol. Linearity concentrations were established in the range of 20–100 µg/mL
for levonorgestrel and 10–50 µg/mL for ethinylestradiol and the correlation coefficient for the above
drugs was 0.999. The relative standard deviation of inter-and intra-day precision was <2%. The proposed
method provides a useful tool for the quantification of levonorgestrel and ethinylestradiol for their assay.
This method is simple, accurate, and reproducible and can be successfully employed for routine quality
control analysis of drugs in pure as well as in pharmaceutical dosage form. The main advantage of the
developed method was its high specificity for the estimation of levonorgestrel and ethinylestradiol.
Keywords: Accuracy, Ethinylestradiol, Levonorgestrel, Validation

INTRODUCTION with chemical formula C21H28O2 and molecular

mass of 312.47 g/mol is a hormonal medication
Analytical chemistry plays an important role and is used in a number of birth control methods.[3]
for the development of drugs in pure and also The chemical structure of levonorgestrel is shown
in marketed formulations[1] and also ensures in Figure 1. It mainly works by preventing
the amount of particular drugs can be easily ovulation or fertilization from occurring.[3] In an
determined.[2] Levonorgestrel is chemically intrauterine device such as mirena among others,
known as (8R,9S,10R,13S,14S,17R)-13-ethyl-17- it is effective for the long-term prevention of
ethynyl-17-hydroxy-1,2,6,7,8,9,10,11,12,14,15,16- pregnancy. Various intrauterine levonorgestrel
dodecahydrocyclopenta[a]phenanthren-3-one devices are available worldwide, including four
FDA-approved intrauterine systems: mirena
*Corresponding Author: (52 mg); liletta (52 mg); kyleena (19.5 mg); skyla
V. Pavan Kumar, (13.5 mg); and other levosert (52 mg) and jydess
E-mail: pavanvarikuti87@gmail.com (13.5 mg).[4]

© 2023, IJPSCR. All Rights Reserved 37

 Susmitha, et al.: RP-HPLC Method Development and Validation for Quantitation of Levonorgestrel and Ethinylestradiol

Ethinylestradiol is chemically (8R,9S,13S,14S,17R) subsequently validated to assess the performance

-17-ethynyl-13-methyl-7,8,9,11,12,14,15,16- characteristics.[8-13]
octahydro-6H-cyclopenta[a]phenanthrene-3,17-
diol with a chemical formula C20H24O2. Veeran
EXPERIMENTAL
et al.[5] have mentioned its molecular mass was
found to be 296.403 g/mol. AL-Japairai et al.[6] Chemicals and materials
mentioned that it is one of the synthetic estrogens
that decreases luteinizing hormone to decrease Levonorgestrel and ethinylestradiol reference
endometrial vascularization and Bao et al.[7] have standards were obtained as gift samples from
identified that some of the drugs activity mainly Spectrum Laboratories, Hyderabad, Telangana, and
depends on design parameters and also on in vitro the marketed formulation Duoluton L produced by
release from intrauterine systems and also some of Zydus Pharmaceuticals Pvt. Ltd. was obtained from
the drugs such as ethinylestradiol acts by decreasing the local market. Acetonitrile and phosphate buffer
gonadotrophic hormone to prevent ovulation. The were purchased from Merck Ltd. (Mumbai, India).
chemical structure of ethinylestradiol is shown in Milli-Q water was used throughout the study.
Figure 2.
The present study mainly focuses on developing Wavelength detection
and validating a reproducible, less cost, simple,
and rapid liquid chromatography method for Stock solutions of levonorgestrel and
determining levonorgestrel and ethinylestradiol ethinylestradiol of 1 mg/mL were prepared in the
in marketed formulations. Instead of routine mobile phase and subsequent dilutions were done
analysis, the use of a rapid and uncomplicated to get the final concentrations of 10 µg/mL.[14]
method is a matter of highly importance. The Both the solutions were scanned in the range of
present strategy mainly focuses on developing a 200–400 nm by UV spectrophotometer and the
novel method having a shorter run time and also spectra were recorded at 260 nm and overlay UV
symmetrical peaks for both of the drugs. The spectrum of levonorgestrel and ethinylestradiol is
liquid chromatography method was designed and shown in Figure 3.

Preparation of buffer solution (10 mM

Potassium dihydrogen phosphate)
Weigh accurately 1.3609 g of potassium dihydrogen
phosphate and transfer it into a 1000 mL volumetric

Figure 1: Structure of Levonorgestrel

Figure 3: Overlay Spectrum of Levonorgestrel and

Figure 2: Structure of ethinylestradiol Ethinylestradiol at 260nm

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 Susmitha, et al.: RP-HPLC Method Development and Validation for Quantitation of Levonorgestrel and Ethinylestradiol

flask by dissolving in HPLC grade water and the 7 mL of diluent and sonicate the above solution to
final volume was adjusted up to the mark with the dissolve completely the above samples and made
same water up to 1000 mL. up to the mark with the same solvent. From this
pipette, out 0.3 mL of the above stock solution and
transfer into 10 mL volumetric flask and fill the
Preparation of mobile phase
diluent up to the mark.
Mixture of buffer solution, water, and acetonitrile
in the ratio of 40:20:40 (400 mL of phosphate Chromatographic conditions
buffer, 200 mL of water, 400 mL of acetonitrile)
was transferred into 1000 mL volumetric flask and The liquid chromatographic analysis was
kept under sonication for 10 min, degassed and performed using a Shimadzu HPLC system
filtered through 0.45 um membrane filter. equipped with a PDA Detector. Chromatographic
separation of both the drugs was achieved using
a C18 µ-bondopak column having dimensions
Diluent preparation
of 250*4.6 mm, 5 µm particle size, and in an
The mobile phase is used as diluent.
isocratic mode with mobile containing a mixture
of phosphate buffer, water, acetonitrile in the
Preparation of standard solution of ratio of 40:20:40 (v/v). Elution of drugs was
levonorgestrel carried out at room temperature with a flow rate
of 0.9 mL/min, injection volume of 20 µL, and a
A 10mg of levonorgestrel working standard was total run time of 12 min. Before injecting blank
accurately weighed and transferred into a 10 mL and drug solution, the chromatographic system
clean dry volumetric flask and add about 7 mL was equilibrated for 80 min with the degassed
of diluents is added and sonicated to dissolve mobile phase. The blank solution was filtered
completely and volume was made up to the mark through 0.22 µm nylon filter and injected to
with the same solvent. Further, 0.3 mL of the above check the solvent interference.
stock solution was pipetted into a 10 mL volumetric
flask and diluted up to the mark with diluent
Procedure
20 µL of the standard, samples are injected into
Preparation of standard solution of the chromatographic system and the areas for
Ethinylestradiol levonorgestrel and ethinylestradiol peaks are
measured and the % assay is calculated using the
A 10 mg of ethinylestradiol working standard was
formulae.
accurately weighed and transferred into a 10 mL
clean dry volumetric flask and add about 7 mL
of diluents is added and sonicated to dissolve RESULTS
completely and volume was made up to the mark
with the same solvent. Further, 0.3 mL of the above Method development
stock solution was pipetted into a volumetric flask Initially, mobile phase optimization was carried out
and diluted up to the mark with diluent. with shimadzu C18 µ-bondopak column using buffer,
acetonitrile, and water at different concentrations.
Preparation of sample solution During these trials, the peaks are not eluted
properly. Finally, the mobile ratio was changed to
Weigh accurately 10 tablets and crushed in a 40:20:40 v/v at a flow rate of 0.9 mL/min. With
mortar and pestle and weight equivalent to 10 mg this composition of mobile phase, the peaks were
of levonorgestrel and ethinylestradiol samples into eluted satisfactorily. The peaks were well separated
a 10 mL clean dry volumetric flask and add about by following chromatographic parameters. The

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 Susmitha, et al.: RP-HPLC Method Development and Validation for Quantitation of Levonorgestrel and Ethinylestradiol

run time of the method was 12 min. The detection are summarized. The chromatogram of the standard
wavelength was at 260 nm. The prescribed method solution is shown in Figure 4 and the chromatogram
was validated as per the ICH guidelines. of the sample solution is shown in Figure 5

System suitability Linearity

The retention times of levonorgestrel and The peaks were eluted at different concentrations
ethinylestradiol were found to be 3.6 min and of standard solution and the calibration curve was
5.1 min, respectively. No major deviations were plotted using peak area against concentration. The
observed in the retention times during the analysis. regression coefficient was found to be 0.999 for
The percentage of related standard deviation the above two drugs. The linear concentrations of
(%RSD) of the lowest concentration of individual levonorgestrel and ethinylestradiol are in the range
six peaks was <2%. System suitability parameters of 10–50 µg/mL for ethinylestradiol and 20–100 µg/

Figure 4: Chromatogram of Standard Solution

Figure 5: Chromatogram of Sample Solution

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 Susmitha, et al.: RP-HPLC Method Development and Validation for Quantitation of Levonorgestrel and Ethinylestradiol

mL for levonorgestrel, respectively. The linearity developed method can be adopted in industry and
results of levonorgestrel are tabulated in Table 1 as well as academics for the assay of levonorgestrel
and linearity results of ethinylestradiol are tabulated and ethinylestradiol.
in Table 2. Calibration graphs of levonorgestrel and
ethinylestradiol are shown in Figures 6 and 7.
Precision

Accuracy Precision studies indicate that the developed

method has accepted values of validation for the
The accuracy of a method mainly depends on estimation of the above drugs in combination. The
the recovery estimation study. Recovery a study repeatability results of levonorgestrel are tabulated
was performed by spiking the known amount in Table 5 and ethinylestradiol are tabulated
of standard drug corresponding to 50,100,150% in Table 6. The %RSD value was found to be
of label claim had been added to the marketed <2. Deviations were not observed in inter- and
drug sample. Recovery studies were found to be
satisfactory and are in the range of 98–102%. The
results of accuracy for levonorgestrel are tabulated
in Table 3 and for ethinylestradiol accuracy results
are tabulated in Table 4. The result has shown that
the proposed method was accurate. Hence, the

Table 1: Linearity values of levonorgestrel

Injection no. Concentration (µg/mL) Peak area
1 20 756893
2 40 1583786
3 60 2241779
Figure 6: Calibration Graph of Levonorgestrel
4 80 2997571
5 100 3744463

Table 2: Linearity values of ethinylestradiol

Injection no. Concentration (µg/mL) Peak area
1 10 496895
2 20 993771
3 30 1490657
4 40 2017542
5 50 2484427
Figure 7: Calibration Graph of Ethinylestradiol

Table 3: Accuracy results of levonorgestrel

% Con Area Amount added Amount found % Recovery Mean recovery
50 3513866 5 5.10 101.3 100.5%
100 4735089 10 9.94 99.4
150 5911797 15 14.8 99.2

Table 4: Accuracy results of ethinylestradiol

% Con Area Amount added Amount found % Recovery Mean recovery
50 2332745 5 5.10 101.8 100.5%
100 3132694 10 9.99 99.9
150 3918996 15 14.9 99.1

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 Susmitha, et al.: RP-HPLC Method Development and Validation for Quantitation of Levonorgestrel and Ethinylestradiol

intra-day analysis which reveals that the proposed the peak response area with slope, where k =3.3 and
method is more precise. 10 for LOD and LOQ. Sensitivity means lowest
amount of sample detected with signal response,
i.e., 3:1 and 10:1, calculated in terms of %RSD
Specificity
should not be more than 10%. LOD chromatogram
The most important aspect of this method was for levonorgestrel and ethinylestradiol is shown in
its usage in the formulation analysis. Hence, Figure 8 and LOQ chromatogram for levonorgestrel
the marketed formulations were collected and and ethinylestradiol is shown in Figure 9.
analyzed by employing the above method. The test
samples are prepared by weighing a known amount
of sample and diluted with the mobile phase. Later Robustness
the solution was filtered through a membrane filter The robustness of the method was studied by
and the solution was further diluted to get the injecting the standard solution with variation
equivalent concentration like that of the standard in the optimized conditions of the method. It
solution. The two samples are injected separately includes change in flow rate and also column
and the concentration was calculated as per the temperature. The values indicate there are no
standard formula. The results were found to be
significant deviations in results. System suitability
within the acceptable range.
results (flow rate change) for levonorgestrel
and ethinylestradiol were tabulated below in
Limit of detection (LOD) and limit of Tables 7 and 8. System suitability results (change
quantification (LOQ) in mobile phase composition) for levonorgestrel
and ethinylestradiol are shown in Tables 9 and 10.
LOD and LOQ were calculated for sensitivity
measurement by dividing K* standard deviation of
Table 7: System suitability results (flow rate change) for
Table 5: Repeatability results of levonorgestrel levonorgestrel
Injection Area S. No. Flow rate (mL/min) System suitability results

Injection‑1 2235418 USP plate count USP tailing

Injection‑2 2240677 1. 0.8 884.4 1.57

Injection‑3 2249491 2. 1.0 1235.1 1.1

Injection‑4 2245823 3. 1.2 969.3 1.6

Injection‑5 2251693
Average 2244605 Table 8: System suitability results (flow rate change) for
Standard deviation 6657.7 ethinylestradiol
%RSD 0.33 S. No. Flow rate (mL/min) System suitability results
RSD: Related standard deviation USP plate count USP tailing
1. 0.8 1748.5 1.23
Table 6: Repeatability results of ethinylestradiol 2. 1.0 1548.2 1.2
Injection Area 3. 1.2 1948.1 1.2
Injection‑1 1501418
Injection‑2 1486941
Injection‑3 1490657
Table 9: System suitability results (mobile phase) for
levonorgestrel
Injection‑4 1487327
S. No. Change in mobile System suitability results
Injection‑5 1490385
phase composition USP plate count USP tailing
Average 1491347
1. 10% less 2257.1 1.51
Standard deviation 5882.5
2. Actual 2457.2 1.12
%RSD 0.39
3. 10% more 2633.1 1.61
RSD: Related standard deviation

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 Susmitha, et al.: RP-HPLC Method Development and Validation for Quantitation of Levonorgestrel and Ethinylestradiol

Figure 8: LOD Chromatogram of Levonorgestrel and Ethinylestradiol

Figure 9: LOQ Chromatogram of Levonorgestrel and Ethinylestradiol

Table 10: System suitability results (mobile phase) for Table 11: Ruggedness results of levonorgestrel
ethinylestradiol Injection No. Area
S. No. Change in mobile System suitability results 1 2194759
phase composition USP plate count USP tailing 2 2195701
1. 10% less 2357.1 1.22 3 2196192
2. Actual 2501.3 1.2 4 2195327
3. 10% More 2799.2 1.2 5 2200952
Average 2196586
Ruggedness Standard deviation 2496.1
%RSD 0.11
Ruggedness was deliberate by changing the RSD: Related standard deviation

instruments, columns, solvents, and their

proportions. No major changes are observed
DISCUSSION
during the analysis and hence the above proposed
method is a rugged method. The ruggedness results The marketed pharmaceutical dosage forms
for levonorgestrel are shown in Table 11 and for contain the above-mentioned drugs and hence it
ethinylestradiol are tabulated in Table 12. is most important to have a mono method for the

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 Susmitha, et al.: RP-HPLC Method Development and Validation for Quantitation of Levonorgestrel and Ethinylestradiol

Table 12: Ruggedness results of ethinylestradiol IFPMA Switzerland; 1995. p. 11260-2.

2. International Conference on Harmonization (ICH) Q2B.
Injection no. Area
Validation of Analytical Procedures: Methodology,
1 1456297
Federal Register. Vol. 62. 1997. p. 27463-7.
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3 1456514 levonorgestrel [Last accessed on 2023 Jan 10].
4 1454578 4. Available from: https://en.wikipedia.org/wiki/
5 1451484 ethinylestradiol [Last accessed on 2023 Jan 10].
Average 1455258 5. Veeran MG, Karthikeyan C, Bharaniraja B, Painuly D,
Standard deviation 2347.5
Aprem AS. RP-HPLC method validation for fast
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RSD: Related standard deviation
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185-93.
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By keeping in mind, the above criteria a simple, Almurisi SH. A bioanalytical method for quantification
rapid method has been developed for the estimation of telmisartan in rat plasma; development, validation
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Technol 2021;14:2139-43.
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