Determination of ethinylestradiol and drospirenone in oral

contraceptives with HPLC method with UV and fluorescence detection

Zorica Arsova-Sarafinovska1,

*, Azis Pollozhani1, Dimitrovska Aneta

2

1Institute for Health Protection, 50 Divizija 6, Skopje, Republic of Macedonia

2Faculty of Pharmacy, University “Ss. Cyril and Methodius”, Vodnjanska 17, Skopje,

Republic of Macedonia

* Corresponding author

Tel:+389 (0)2 314 7070; Fax: +389 (0)2 322 3354

E-mail address: zarsova2002@yahoo.co.uk

2

Извадок

Целта на ова истражување беше да се развие сензитивен, точен и брз метод за истовремено

определување на стероидни хормони, како што се: етинилестрадиол (ЕЕД) и дроспиренон

(ДРОСП), во ниско-дозирани перорални контрацептивни таблети. Испитуваниот препарат

содржи естроген хормон (нискодозиран) и синтетски прогестоген (присутен во 100 пати

поголема количина). Раздвојувањето на активните компоненти беше изведено на реверзно-

фазна колона Purospher®

STAR RP-18e (150 x 4.0 mm I.D.; 5 µm), со мобилна фаза составена

од 47% ацетонитрил и 53% вода (V/V), изократно и брзина на проток - 1,50 ml /min. Сите

анализи беа изведени на собна температура (24 ± 2°C). УВ апсорпцијата на ДРОСП беше

мерена на бранова должина од 265 nm, а флуоресценцијата на ЕЕД на 310 nm (ексцитација

на 285 nm) со систем на ДАД и флуоресцентен детектор.

Предложениот метод беше валидиран преку опредеување на линеарност, прецизност,

точност и сензитивност.Баждарните дијаграми за ЕЕД и ДРОСП беа конструирани со

стандардни раствори на ЕЕД во концентрации од 0,6 g/ml до 3,0 g/ml и стандардни

раствори на ДРОСП во концентрации од 60,0 g/ml до 300,0 g/ml. Добиените коефициенти

на корелација беа 1,0 и 0,9998 за ЕЕД и ДРОСП, соодветно. Прецизноста на методот беше

потврдена преку испитување на повтроливост и репродуцибилност. Релативните стандардни

девијации добиени при добиени при испитување на повторливоста беа 0,52% и 0,05% за

ЕЕД и ДРОСП, соодветно. Релативните стандардни девијации добиени при испитување на

репродуцибилност беа 1,22% и 0,74% за ЕЕД и ДРОСП, соодветно. Просечниот аналитички

принос беше 98,99% за ЕЕД и 99,85% за ДРОСП. Лимитот на детекција за ЕЕД и ДРОСП

беше 0, 65 ng/ml и 0,0774 g/ml, соодветно, што ја потврдува високата сензитивност на

предложениот метод. Методот беше успешно применет за испитување на параметарот

Воедначеност на дозирани единици.

Предложениот HPLC метод со УВ и флуоресцентна детекција се препорачува како метод на

избор за определување на етинилестрадиол, посебно во препарати каде е присутен во многу

ниски дози, а кои истовремено содржат и многу поголеми количини на синтетски

прогестоген, како дроспиренон.

Клучни зборови: Етинилестрадиол; Дроспиренон; HPLC; Перорални контрацептиви

3

Abstract

The aim of this research was to develop a sensitive, accurate and rapid method for

simultaneous determination of steroid hormones, ethinylestradiol (EED) and drospirenone

(DROSP) in commercially available oral contraceptive tablets. Tested pharmaceutical formulation

contains an estrogen in a small amount with 100-times bigger amount of a synthetic progestin. The

combination of EED and DROSP was analyzed using a Purospher® STAR RP-18e reversed-phase

column (150 X 4.0 mm I.D.; particle size 5 µm) with a mobile phase constituted of 47%

acetonitrile: 53% water (V/V). The elution was carried out at a flow rate of 1.50 ml /min. All

analyses were performed at room temperature (24 ± 2°C). A diode array detector connected in

series with fluorescence detector measured the UV absorbance of DROSP at 265 nm and

fluorescence of EED at 310 nm (excitation at 285 nm).

The proposed method was validated by determination of linearity, precision, accuracy and

sensitivity. Calibration curves for ETE and DROSP were obtained using standard solutions of EED

with concentrations ranged from 0.6 to 3.0 g/ml and standard solutions of DROSP with

concentrations ranged from 60.0 to 300.0 g/ml. Correlation coefficients were 1.0 and 0.9998 for

EED and DROSP, respectively. The precision of the method was confirmed by assessment of

repeatability and reproducibility. Relative standard deviations obtained in the investigation of

repeatability were: 0.52 % and 0.05 % for EED and DROSP, respectively. Relative standard

deviations obtained in the investigation of reproducibility were: 1.22 % and 0.74 % for EED and

DROSP, respectively. The average recovery for samples containing EED and DROSP were 98.99

% and 99.85 % for EED and DROSP, respectively. The limits of detection for EED and DROSP

were 0.65 ng/ml and 0.0774 g/ml, respectively, which indicates an excellent sensitivity of the

proposed method. The method was applied for Uniformity of dosage units testing in commercially

available oral contraceptive tablets.

In conclusion, we recommended HPLC method with UV and fluorescence detection as a

method of choice for determination of ethinylestradiol, present at a very low dosage level in low-

dose oral contraceptives that contain bigger amount of synthetic progestin.

Key words: Ethinylestradiol; Drospirenone; High performance liquid chromatography; Oral

contraceptives

4

Introduction

Oral contraceptives are pharmaceutical formulations containing steroid hormones in a relatively

small amount. The most commonly encountered estrogen is ethinylestradiol (Fig. 1), present at a

very low dosage level (30-100 μg per tablet), in combination with an orally active progestin (such

as drospirenone (Fig. 2)), present at a level of up to 100 times that of the estrogen. Ethinylestradiol

(EED) is a semi synthetic estrogen female sex hormone, while drospirenone (DROSP) is a novel

synthetic progestin available in combined oral contraceptives and menopausal hormonal therapy

(1). Similar to its parent compound spirolactone, an analog of spironolactone, drospirenone has

antimineralocorticoid and antiandrogenic activity. Combined with ethinylestradiol in oral

contraceptive formulations, drospirenone-containing contraceptives have similar efficacy and

safety profiles to other low-dose oral contraceptives, but seem to offer improved tolerability with

regard to weight gain, mood changes, acne and treatment of a severe form of premenstrual

syndrome (2, 3).

Consequently, the modern low-dose oral contraceptives require a sensitive, accurate and

rapid methods of quantitative determination which is unaffected by the small amount of the

estrogen and the large excess of progestogen.

There have been several reports (4-13) on the determination of EED in combination with

an orally active synthetic progestin, including the use of derivative spectrometry (4), high

performance liquid chromatography with fluorescence detection (5), gas chromatography-mass

spectrometry on the pentafluorobenzoyl derivatives (6) and pentafluorobenzyl-trimethylsilyl

derivatives (7), solid phase extraction followed by gas chromatography/MS/MS after derivatization

with mixture of N-methyl-N- (trimethylsilyl)-trifluoroacetamide, trimethylsilylimidazole, and

dithioerytrol (8), solid phase extraction followed by liquid chromatography-diode array detection-

mass spectrometry (9, 10), affinity chromatography with tripeptide column (11) etc. Although the

listed methods produce high sensitivity, they still have drawbacks (e.g. time consuming in

extraction process when solid phase extraction is performed). Furthermore, the target analytes have

to be derivatized if GS-MS is used which makes these procedures inappropriate for routine

analysis. Additionally, spectrometry is susceptible to much interference like those of excipients,

degradation products, and impurities.

On the contrary, liquid chromatography (LC) has only been employed in a few occasions

(12, 13) regardless of its advantages with respect to the already mentioned techniques. Thus, unlike

GC-MS, LC enables the determination of steroid without derivatization and it is not limited by such

factors as properties of the substances (non volatile) and high molecular weight.

5

The aim of this research was to develop a sensitive, accurate and rapid method for

simultaneous determination of steroid hormones, like ethinylestradiol (EED) and drospirenone

(DROSP) in commercially available oral contraceptive tablets.

Experimental

HPLC instrumentation and conditions

HPLC analyses were performed using a Schimadzu LC-2010 chromatographic system

(Schimadzu, Kyoto, Japan) consisting of a LC-20AT Prominence liquid chromatograph pump with

DGU-20A5 Prominence degasser, a SPD-M20A Prominence Diode Array Detector, RF 10AXI

fluorescence detector and a SIL-20 AC Prominence auto sampler. Data analyses were done using

Class VP 7.3 Software.

Chromatographic separation was performed on a Purospher® STAR RP-18e reversed-phase

column (150 X 4.0 mm I.D.; particle size 5 µm), in an isocratic mode with a mobile phase

constituted of 47% acetonitrile: 53% water (V/V).

The elution was carried out at a flow rate of 1.50 ml minˉ1. The injection volume was 10

μl. All analyses were performed at room temperature (24 +/- 2 degrees C). A diode array detector

connected in series with fluorescence detector measured the UV absorbance of DROSP at 265 nm

and fluorescence of EED at 310 nm (excitation at 285 nm).

Preparation of solutions

Commercially available samples, coated tablets, containing an estrogen in a small amount

(30 μg of EED) and 100-times bigger amount of a synthetic progestin (3 mg of DROSP), were used

in this research.

Ethinylestradiol and Drospirenone working standards were supplied by Schering

Deutschland GmbH (Berlin, Germany). HPLC-grade acetonitrile was from Merck (Darmstadt,

Germany). Double-distilled water was used to prepare mobile phase solutions.

A solvent was prepared by mixing 60 volumes of acetonitrile and 40 volumes of water.

All solvents and solutions for HPLC analysis were filtered through a membrane filter (0.45

μm pore size) and vacuum degassed before use.

Calibration curves

6

Stock solution of EED was prepared by dissolving Ethinylestradiol standard substance (25

mg) with 100 ml solvent in a 100-ml volumetric flask. Standard solutions were prepared by dilution

of EED stock solution with solvent to obtain final concentrations ranging from 0.6 μg/ml – 3.0

μg/ml.

Stock solution of DROSP was prepared by dissolving Drospirenone standard substance (30

mg) with 25 ml solvent in a 25-ml volumetric flask. Standard solutions were prepared by dilution

of DROSP stock solution with solvent to obtain final concentrations ranging from 60 μg/ml – 300

μg/ml.

Sample preparation

In HPLC method with UV/ fluorescence detection, each of 10 tablets was transferred in 25-

ml volumetric flask and 20 ml solvent was added. The solution was heated at 60°C in an ultrasonic

bath for 25 minutes, cooled, filled up with the solvent and filtered through 0.045 μm-nylon syringe

filter. 10 μl of the clear solution was injected into chromatograph.

Recovery tests

To study the accuracy of the proposed analytical methods, recovery tests were conducted

using the standard addition method. To discover whether excipients interfered with the analysis,

known amounts of standard were added to tablet formulation samples and the resulting mixtures

were analyzed by the proposed methods. The percent of recovery was calculated using the

calibration equation.

Results and discussion

During the development of the analytical method for quantification of the active

compounds in Drospirenone and Ethinylestradiol tables we have been faced two inherent problems.

They were: a) the low ultraviolet molar absorptivity of EED; and b) the low proportion of EED

compared to DROSP in the pharmaceutical formulation. Fortunately, the molecule of EED has

ability to fluorescence (emit higher wavelength radiation) after being exited by shorter wavelength

energy, which allowed us to measure the concentration of EED with fluorescence detector. On the

contrary, drospirenone has no a natural fluorescence.

In the preliminary research, the absorption spectra of EED and DROSP in solvent (c = 10

μg/ml) were studied. The UV spectrum characteristic of the EED presents two maxima, one at 215

7

nm and another at 280 nm, due to π→π* transitions in the aromatic ring (14). DROSP shows a

characteristic absorption maximum at 265 nm. Because of that, we have chosen to measure the UV

absorbance of DROSP at 265 nm and the fluorescence of EED at 310 nm (excitation at 285 nm)

using a DAD detector connected in series with a fluorescence detector.

Chromatographic separation was performed under the conditions similar to that cited in the

British Pharmacopoeia (BP) (15) separation method for Ethinylestradiol and Levonorgestrel

Tablets, which uses a reversed-phase column (15 cm x 4.6 mm) packed with octadecyl silica gel

(particle size 5 m) and a mobile phase consisted of 49 % acetonitrile and 51 % water, with a flow

rate of 1.5 ml per minute.

As the fluorescence detector is much more sensitive than UV detector (10-1000 times more

sensitive, depending on the compound being measured), we had to dilute the sample solution to

obtain the optimal test concentration. Preliminary tests were performed in order to define the

concentration interval in which the intensity of the detector response is proportional to the

concentration of the analyzed substance. Standard solutions of EED with concentration ranging

from 0.12 μg/ml-12.0 μg/ml were measured and the linearity was proven through the range from

0.12 μg/ml–4.8 μg/ml. Therefore, the concentration of 1.2 μg/ml was chosen as optimal test

concentration.

A chromatogram that represents the separation of ETE and DROSP in mixed standard

solution and sample with UV/ fluorescence detection is shown in Fig. 3. and Fig. 4, respectively.

System suitability test is an integral part of liquid chromatographic method. System

repeatability was estimated by 10 repeated injections of mixed standard solution at 100% of test

concentration (1.2 g/ml EED, 120 g/ml DROSP). The variation in retention times among 10

replicate injections was very low with RSD values: 0,05 % for EED and 0 % for DROSP. The

variation in peak areas among 10 replicate injections was also low with RSD values: 0,52 % for

EED and 0,05 % for DROSP. These results are in accordance with European Pharmacopoeia (16)

and ICH guidelines (17).

An excellent linearity was observed for both analyzed compounds (r2

= 1 for EED and

0.9998 for DROSP). The limit of detection (LOD) for EED was 0.00065 g/ml and the limit of

quantification (LOQ) for EED was 0.00197 g/ml. LOD for DROSP was 0,0774 g/ml and LOQ

for DROSP was 0,2347 g/ml (Table 1.).

The precision of the method was validated performing 6 determinations at 100% of test

concentration that have been done in the same day, by the same analyst and using the same

equipment. Intermediate precision was established by repeating the procedure in two different days

by the same analyst. The variation in results obtained among 6 determinations was very low (≤

2%), which confirmed precision of the method. The statistical data are shown in Table 2.

8

Satisfactory recoveries were observed for both EED and DROSP (98.99 % ± 1.66 % and

99.85 % ± 0.94 %, for EED and DROSP, respectively). The results are shown in Table 3.

The HPLC method with UV/ fluorescence detection, external standard method, was applied

to analyze the parameter Uniformity of Dosage Units in the commercially available samples

(coated tablets containing 30 µg of ETE and 3 mg of DROSP). The results were in good agreement

with the declared values and in accordance with European Pharmacopoeia requirements.

Conclusion

The proposed HPLC method allows simple, accurate, precise and rapid determination of EED and

DROSP in pharmaceutical dosage forms and could, therefore, be easily adopted in routine quality

control analysis. The method performance was fully validated by determination of linearity,

reproducibility, accuracy and sensitivity. The method was applied for determination of Uniformity

of Dosage units. The high sensitivity of HPLC method with UV/ fluorescence detection is its main

advantage. Therefore, it could be recommended as the method of choice for determination of

ethinylestradiol, present at a very low dosage level in low-dose oral contraceptives that contain

bigger amount of synthetic progestin, such as drospirenone.

Fig. 1. Structure of ethinylestradiol (EED)

9

Fig. 2. Structure of drospirenone (DROSP)

Fig. 3. A typical chromatogram of a mixed standard solution (1,2 μg/ml ethinyl estradiol, 120,0 μg/ml

drospirenone) detected by:

a) UV detector at a wavelength of 265 nm (for drospirenone)

b) fluorescence detector at a wavelength of 310 nm (excitation at 285 nm) (for ethinyl estradiol)

10

Fig. 4. A typical chromatogram of a sample solution detected by:

a) UV detector at a wavelength of 265 nm (for drospirenone)

b) fluorescence detector at a wavelength of 310 nm (excitation at 285 nm) (for ethinyl estradiol)

Table 1. Linearity, limit of detection, limit of quantification of the analytical method

EED1 DROSP

2

Concentration range (μg/ml) 0.6 – 3.0 60.0 – 300.0

Slope 2213460 20731

Intercept 9795 10822

Correlation coefficient (r2) 1 0.9998

Limit of detection (µg/ml) 6.5 x 10-4

7.74 x 10-2

Limit of quantification (µg/ml) 1.97 x 10-3

0.2347

1 Ethinylestradiol

2 Drospirenone

11

Table 2. Precision (Repeatability) of the analytical method

EED1 DROSP

2

μg/tabl. % of label claim mg/tabl. % of label claim

X 29.2134 97.38 2.8725 95.75

SD 0.3556 1.1854 0.0214 0.7133

RSD (%) 1.22 0.74

Confidence interval (95%) 28.847 – 29.570 μg/tabl.

96.16 % – 98.57 %

2.850 – 2.895 mg/tabl.

95.00 % – 96.50 %

1 Ethinylestradiol

2 Drospirenone

Table 3. Accuracy of the analytical method

EED1 DROSP

2

Added

amount

(g/ml)

Found

amount3

(g/ml)

Recovery

(%)

Added

amount

(g/ml)

Found

amount3

(g/ml)

Recovery

(%)

0.312 0.306 98.15 30.20 29.62 98.08

0.624 0.631 101.00 60.40 60.88 100.79

0.937 0.901 96.20 90.60 91.01 100.45

1.249 1.242 99.47 120.80 120.81 100.01

1.561 1.548 99.14 151.00 150.75 99.84

1.873 1.973 100.01 181.20 181.13 99.96

X ± SD 98.99 ± 1.66 99.85 ± 0.94

RSD (%) 1.68 0.94

1 Ethinylestradiol

2 Drospirenone

3Average of three determinations

12

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