INTRODUCTION:

Ethinylestradiol (EE), chemically [19-nor-17α-pregna-1, 3, 5(10)-trien-20-yne-3, 17-diol] is orally bio-active estrogen used for the management of menopausal symptoms and female hypogonadism¹. Norgestimate (NGM), (13-ethyl-17-ethynyl-3-hydroxyimino- 1,2,6,7,8,9,10,11,12,14,15,16- dodecahydrocyclopenta[a] phenanthren-17-yl) acetate is a form of progesterone used for the regulation of ovulation and menstruation².

Literature survey reveals High Performance Liquid Chromatographic (HPLC)^3-7and LC-MS⁸ methods for determination of EE either in single or in combination with other drugs. Spectrophotometric⁹method for quantification of EE is also reported. HPLC¹⁰and LC-MS¹¹methods have been reported for the determination of NGM either in single or in combination with other drugs. High Performance Liquid Chromatographic (HPLC) method has been reported for determination of EE and NGM in combined tablet dosage form¹².

No work has been reported for the determination of the EE and NGM in combined tablet dosage form by HPTLC method. This paper presents HPTLC method for determination of Ethinylestradiol and Norgestimate in combined tablet dosage form.

The proposed method is optimized and validated as per the International Conference on Harmonization (ICH) guidelines¹³.

EXPERIMENTAL:

Reagents and chemicals:

Analytically pure samples of EE and NGM were kindly supplied by Talent Healthcare (Haridwar, Uttaranchal). Benzene, Triethylamine and Ethyl acetate (all AR grade) were used for the method development. The pharmaceutical dosage form used in this study was ORTHO TRI-CYCLEN tablets (Ortho-McNeil Pharmaceutical Corporation) labeled to contain 0.035 mg of Ethinylestradiol and 0.215 mg of Norgestimate/tablet were procured from local market.

Instrumentation and chromatographic conditions:

The samples were spotted in the form of bands of width of 6 mm with space between bands of 5 mm, with a 100 µL sample syringe (Hamilton, Bonaduz, Switzerland) on precoated silica gel aluminium plate 60 F₂₅₄ (10 ×10) with 250 µm thickness (E. MERCK, Darmstadt, Germany) using a CAMAG Linomat 5 sample applicator (Switzerland). The plates were prewashed with methanol and activated at 110 C for 5 minutes, prior to chromatography. The slit dimensions 5 mm × 0.45 mm and scanning speed of 20 mm/sec was employed.

The linear ascending development was carried out in 10 cm × 10 cm twin trough glass chamber (CAMAG, Muttenz, Switzerland) using mobile phase. The optimized chamber saturation time for mobile phase was 20 min. The length of chromatogram run was 9 cm and development time was approximately 20 min. TLC plates were dried in a current of air with the help of a hair drier. Densitometric scanning was performed on CAMAG thin layer chromatography scanner 3 at 280 nm for all developments operated by WINCATS software version 1.4.2. The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 200 to 300 nm.

Preparation of Standard Stock Solutions:

Standard stock solution of EE was prepared by dissolving 10 mg of drug in 10 mL of methanol to get concentration of 1 mg/mL from which 1 mL was further diluted to 10 mL to get the solution having concentration of 100 ng/µL. Standard stock solution of NGM was prepared by dissolving 5 mg of drug in 10 mL of methanol to get concentration of 500 ng/µL.

Selection of Detection Wavelength:

After chromatographic development bands were scanned over the range of 200-300 nm and the spectra were overlain. It was observed that both drugs showed considerable absorbance at 280 nm. So, 280 nm was selected as the wavelength for detection (Figure 1).

Fig. 1: Overlain spectra of Ethinylestradiol and Norgestimate

Preparation of Calibration Curve:

The standard stock solutions of EE (100 ng/µL) and NGM (500 ng/µL) were applied by overspotting on TLC plate in range of 2 – 10 µL with the help of CAMAG 100 µL sample syringe, using Linomat 5 sample applicator. The plate was developed and scanned under above established chromatographic conditions. Each standard in five replicates was analyzed and peak areas were recorded. Calibration curves of EE and NGM were plotted separately of peak area vs respective concentration of EE and NGM.

Analysis of Tablet Formulation:

For NGM, twenty tablets were weighed accurately and finely powdered. A quantity of powder equivalent to 1 mg of drug was weighed and dissolved in 5 mL of methanol. The solution was filtered using Whatman paper No. 41 and the volume was made upto10 ml with same solvent. Twenty µL volume of this solution was applied on TLC plate. For EE, quantity of powder equivalent to 0.2 mg of drug was weighed and dissolved in 5 mL of methanol. The solution was filtered using Whatman paper No. 41 and the volume was made upto10 ml with same solvent. Twenty µL volume of this solution was applied on TLC plate.

After chromatographic development peak areas of the bands were measured at 280 nm and the amount of drug present in sample was estimated from the respective calibration curves. Procedure was repeated six times for the analysis of homogenous sample.

Robustness Studies:

In the robustness study, the influence of small, deliberate variations of the analytical parameters on peak area of the drugs was examined. Factors varied were mobile phase composition (± 0.1%), volume of mobile phase (± 0.5 %), time from application to development (0, 10, 20, and 30 min) and from development to scanning (0, 30, 60, and 90 min). One factor at a time was changed to estimate the effect. Robustness of the method was checked at a concentration level of 400 ng/band and 2000 ng/band for EE and NGM respectively.

Recovery Studies:

To check the accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre-analyzed sample solution at three different levels 50, 100 and 150 %. Chromatogram was developed and the peak areas were noted. At each level of the amount, three determinations were carried out.

RESULTS AND DISCUSSION:

Different mobile phases containing various ratios of Benzene, Ethyl acetate, Triethylamine, Glacial acetic acid, Ammonia, Chloroform were examined (data not shown). Finally the mobile phase containing Benzene: Ethyl acetate: Triethylamine (7: 2: 1 v/v/v) was selected as optimal for obtaining well defined and resolved peaks. The optimum wavelength for detection and quantitation used was 280 nm. The retention factors for EE and NGM were found to be 0.62 ± 0.102 and 0.82 ± 0.123 respectively. Representative densitogram of mixed standard solution of EE and NGM is shown in Figure 2.

Straight-line calibration graphs were obtained for EE and NGM in the concentration range 200-1000 ng/band for EE and 1000-5000 ng/band for NGM, with high correlation coefficient > 0.998. The proposed method was also evaluated by the assay of commercially available tablets containing EE and NGM. The % assay (Mean ± S.D.) was found to be 99.4 % ± 0.157 for EE and 99.8 % ± 0.14 for NGM. Robustness of the method checked after deliberate alterations of the analytical parameters showed that areas of peaks of interest remained unaffected by small changes of the operational parameters (% RSD < 2). The results are given in Table I.

Table I: Robustness Data in Terms of Peak Area (% RSD)

Sr. No.	Parameter Varied	EE	NGM
1	Mobile phase composition (±0.1%)	0.88	0.72
2	Volume of mobile phase (±0.5%)	1.06	1.41
3	Time from application to development (Mins.)	0.73	0.56
4	Time from development to scanning (Mins.)	1.23	0.75

Table II: Recovery Studies of EE and NGM

Drug	Amount taken (ng/band)	Amount added (ng/band)	Total amount found (ng/band)	% Recovery	% RSD^a
EE	400	200	593.33	98.88	0.356
	400	400	790.30	98.79	0.474
	400	600	992.66	99.26	0.796
NGM	2000	1000	2991.12	99.70	0.557
	2000	2000	3997.35	99.93	0.669
	2000	3000	4998.56	99.96	0.564

^aAverage of three determinations

Fig. 2: Representative chromatogram of mixed standard solution of EE (500 ng/band, R_f = 0.62 ± 0.102) and NGM (500 ng/band, R_f= 0.82 ± 123).

For EE, the recovery study results ranged from 98.79 to 99.26 % with % RSD values ranging from 0.346 to 0.796 %. For NGM, the recovery results ranged from 99.70 to 99.96 % with % RSD values ranging from 0.557 to 0.669 %. Results of recovery studies are reported in Table II.

CONCLUSION:

The validated HPTLC method employed here proved to be simple, fast, accurate, precise and robust, thus can be used for routine analysis of EE and NGM in combined tablet dosage form.

ACKNOWLEDGEMENTS:

The authors wish to express their gratitude to Talent Healthcare, Haridwar for the sample of pure EE and NGM. Thanks are also extended to Principal, Dr. A. R. Madgulkar for providing infrastructure facilities and her constant support.

REFERENCES:

1. Block JH, and Beale John M. Wilson and Giswold’s Textbook of Organic Medicinal and Pharmaceutical Chemistry. 11th Edition, Lippincott Williams & Wilkins, New York. 2004.

2. Sitruk-Ware LR and Daniel R Progestins and Antiprogestins in Clinical Practice. Eastern Hemisphere Distributon, New York. 2000.

3. Strusiak SH, Hoogerheide JG and Gardner MS. Determination of ethinylestradiol in solid dosage forms by high performance liquid chromatography. J. Pharm. Sci. 1981; 71: 636.

4. Prasad SD, Reddy GC and Mukkanti K. Simultaneous HPLC estimation of levonorgestrel & ethinylestradiol from tablets. Ind. J. Pharm. Sci. 2004; 66: 231.

5. Prabhakar B and Deshpande SG. Simultaneous estimation of ethinyl estradiol and levonorgestrel from transdermal patches by HPLC. Ind. J. Pharm. Sci. 1999; 61:12.

6. Laban A, Markovic S and Stankov M. Simultaneous determination of gestodene and ethinylestradiol in contraceptive formulations by RP-HPLC. Analytical Letters. 2004; 37: 273.

7. Reif VD, Eickhoff WM and Jackman JK. Automated stability indicating high performance liquid chromatographic assay for ethinylestradiol and norgestrel tablets. Pharmaceutical research. 1987; 4: 54.

8. Wenkul L, Ying L and Austin CL. Simultaneous determination of norethindrone & ethinylestradiol in human plasma by HPLC with tandem mass spectroscopy. J. Chromatogr. B 2005; 825: 223.

9. Mishap PR., Namdeo A and Jain NR. Spectrophotometric methods for simultaneous estimation of ethinylestradiol and levogestrel from tablets. Ind. J. Pharm. Sci. 1999; 61: 122.

10. Wong FA, Juzwin SJ, Tischio NS and Flor SC. Determination of norgestimate in serum by automated high-performance liquid chromatography and subsequent radioimmunoassay. J. Liq. Chromatogr. & Rel. Techno. 1995; 18: 1851.

11. Wong FA, Edom RW, Duda M and Tischio JP. Determination of norgestimate and it’s metabolite in human serum by high-performance liquid chromatography with tandem mass spectrometric detection. J. chromatogr. B 1999; 734: 247.

12. Lane PA, Mayberry DO and Young RW. Determination of norgestimate and ethinylestradiol in tablets by high performance liquid chromatography. J. Pharm. Sci. 2006; 76: 44.

13. ICH Harmonised Tripartite Guideline (Nov 2005) Validation of Analytical Procedures: Text and Methodology Q2 (R1).

Received on 16.11.2010 Modified on 24.11.2010

Research J. Pharm. and Tech. 4(4): April 2011; Page 582-584