Simultaneous Determination of Ethinylestradiol and Levonorgestrel in Oral Contraceptives by Derivative Spectrophotometry Juan J. Berzas*, Juana Rodr�ýguez and Gregorio Casta�neda Departamento de Qu�ýmica Anal�ýtica y Tecnolog�ýa de Alimentos, Facultad de Ciencias, Universidad de Castilla-La Mancha, 13071 Ciudad Real, Spain A method for determining ethinylestradiol (ETE) and levonorgestrel (LEV) in mixtures by first-derivative spectrophotometry is described. The procedure does not require any separation step.Measurements are made at the zero-crossing wavelengths and the calibration graphs are linear up to 26 and 33 mg ml21 of ETE and LEV, respectively. The method was applied to the determination of both compounds in five different Spanish commercial low-dose oral contraceptives. Similar results were obtained by an HPLC method. Keywords: Ethinylestradiol; levonorgestrel; derivative spectrophotometry; oral contraceptives At present there are three types of oral contraception available.In the sequential type, estrogen is administered alone for the first week, followed by a lower dosage of the estrogen in conjunction with a progestogen for the remainder of the course. In the second, commonly used, type both an estrogen and a progestogen are present in the tablets (as either a single dose or in three different doses). In the progestogen type, a progestogen alone is administered. Ethinylestradiol (ETE) is a semisynthetic estrogen female sex hormone and levonorgestrel (LEV) is a synthetic steroid with an extremely potent progestational action. The formulation of these steroids in tablets of low dosage, i.e., 30–250 mg per tablet, presented a challenging analytical problem.A sensitive, accurate and rapid procedure is desirable for content uniformity testing of the dosage form. The structure of LEV has a characteristic D4-3-keto group in the A-ring with a different chromophoric power to ETE. The most commonly encountered estrogen is ETE, which is present at a very low dosage level (30–100 mg per tablet) in combination with an orally active synthetic progestin (one of the most commonly used is LEV), which is present at a level of from 5 to 30 times that of the estrogen.Oral contraceptives have had an enormous positive impact on public health for the past three decades and, in the main, there has been a remarkably low incidence of troublesome side-effects. Although estrogens are implicated in an increased incidence of breast and endometrial cancer, epidemiological studies have not provided convincing evidence to support a direct correlation between the use of oral contraceptives and an increase in breast cancer. The modern low-dose oral contraceptives (estrogen–progestogen) require a sensitive analysis method which is unaffected by the small amount of the estrogen and the large excess of progestogen.There have been several reports1–11 of the determination of levonorgestrel or ethinylestradiol, including the use of radioactively labelled derivatives,1,2 dansyl or other fluorescent derivatives,3–5 spectrophotometry or photometry6–9 and gel or column chromatography, but the methods are complicated.10,11 No references were found to the simultaneous determination of ETE and LEV using spectrophotometric methods.The determination of ETE in the presence of noresthisterone by derivative spectrophotometry in methanol or ethanol has been reported,12,13 the recoveries in different tablets being 120–80% for both compounds.The simultaneous determination of ETE and mestranol by derivative spectrophotometry has been proposed, either in a solution of methanol and chloroform14 or in NaOH in methanol.15 In derivative UV/VIS spectrophotometry, the information contained in the spectrum is presented in a potentially more useful form, greatly increasing the versatility of the technique16 –18 and offering a convenient solution to a number of well defined analytical problems, such as the resolution of multi-component systems, removal of sample turbidity, matrix background and enhancement of spectral details.19 Although the use of derivative spectra is not new, it has only become practical in recent years with the development of microcomputer technology, which allows the almost instantaneous generation of derivative spectra.In this paper we demonstrate the ease with which the derivative methods circumvent the problem of overlapping spectral bands and sample turbidity, allowing the simultaneous determination of ETE and LEV without prior separation.The method yielded accurate, rapid and reproducible results for five different commercial products, two of them with three different dosages. The results obtained by the proposed method were compared with those obtained by HPLC with spectrophometric detection, very similar to the method proposed in the US Pharmacopeia. 20 Experimental Apparatus A Beckman (Fullerton, CA, USA) DU-70 spectrophotometer equipped with 1.0 cm quartz cells and connected to an IBM-PS 2 Model 30 computer, fitted with Beckman Data Leader software,21 and an Epson FX-850 printer was used for all absorbance measurements. A Shimadzu (Kyoto, Japan) high-performance liquid chromatograph equipped with a Nova-Pak C18 column (15 3 0.39 cm id, 4 mm), a diode-array detector, a Rheodyne injection valve and connected to a computer fitted with CLASS LC-10 software was used.A Crison (Barcelona, Spain) MicropH 2002 pH meter was used for the pH measurements. Standard Solutions All chemicals and solvents were of analytical-reagent grade. ETE and LEV were obtained from Sigma (St. Louis, MO, USA) and stock standard solutions were prepared in absolute ethanol Analyst, January 1997, Vol. 122 (41–44) 41(100 mg ml21). The purities of the ETE and LEV reported by Sigma were 98.6% and 99.6%, respectively, determined using an HPLC method with spectrophotometric detection at 280 and 242 nm.Procedure Calibration Stock standard solutions of ETE and LEV were placed in 25 ml calibrated flasks to give final concentrations of up to 26 and 33 mg ml21, respectively, adding absolute ethanol to dilute the contents to 25 ml (the resulting final solution was 100% in ethanol). This high percentage of ethanol was necessary to obtain total dissolution of the drugs from the oral contraceptive tablets.The absorption spectra of the samples were recorded against an ethanol blank between 315 and 210 nm at a scan speed of 120 nm min21 and stored in the computer. Firstderivative spectra were obtained with Dl = 8 nm and ETE was determined by measuring the signal of the first derivative spectrum at 293.0 nm (1D293) (zero-crossing point for LEV), and by using an appropriate calibration graph, their concentrations could be determined. These calibrations were performed by varying the concentration of the estrogen, in the absence of the other hormone.The LEV content was also determined by measuring the signal at 249.0 nm (1D249) (zero-crossing point for ETE). These spectra were not treated with a smoothing function because the noise level was low. Assay of pharmaceutical preparations Twenty tablets were finely powdered and an appropriate portion (equivalent to the median mass of two tablets) was dissolved in 8 ml of absolute ethanol by sonication for 15 min, followed by shaking by mechanical means for 20 min.The mixture was filtered, using a Swinnex polypropylene disc filter holder of 13 mm diameter (Millipore, Bedford, MA, USA) with an FH 0.5 mm Fluoropore (PFTE) membrane, into a 10 ml calibrated flask. The residue was washed twice with the same solvent and diluted to volume. The absorption spectra were recorded against an absolute ethanol blank and stored in the IBM-PS computer. For determining ETE and LEV, the absorption spectra were handled as in first-derivative spectrophotometry.Comparison with HPLC reference method Twenty tablets were finely powdered and an appropriate portion (equivalent to the median mass of two tablets) was dissolved in 8 ml of absolute ethanol by sonication for 15 min, followed by shaking by mechanical means for 20 min. The mixture was filtered using a Swinnex polypropylene disc filter holder of 1ter with an FH 0.5 mm Fluoropore (PFTE) membrane into a 10 ml calibrated flask.The residue washed twice with the same solvent and diluted to volume. A 2.5 ml portion of this solution was diluted with water in a 10 ml calibrated flask; the reason for this dilution is to obtain a higher polarity and lower concentration in the sample. HPLC determination was performed on a Nova-Pak C18 60 A column (15 3 0.39 cm id) containing 4 mm packing. The mobile phase was deaerated acetonitrile–methanol–water (3.5 + 1.5 + 4.5) and spectrophotometric detection was performed at 215 nm.The flow rate was about 1 ml min21.20 The differences between the two methods are the dissolution of the tablets (using ethanol–water or acetonitrile–methanol–water) and the column used [C18 (15 3 0.39 cm id) or C8 (15 3 0.46 cm id)]. Results and Discussion Method Development The influence of pH on the absorption spectra of ETE (e280 nm = 2253 l mol21 cm21 in absolute ethanol) and LEV (e240 nm = 17 155 l mol21 cm21 in absolute ethanol) was studied, with a total content of ethanol of 25%.The LEV spectrum showed only a maximum at 246 nm, which remained unchanging in the pH range 1.0–12.4. The ETE spectrum showed a maximum at 280 nm in the pH range 1.0–9.5 and for more alkaline solutions two different bands at 296 and 240 nm. The stability of ETE in very acidic solutions was not very good. For this reason, the best results for analytical purposes were obtained in the pH range 4.0–9.5. The preparation of the samples with 100% of ethanol resulted in spectra very close to those obtained at the optimum pH.Samples were prepared in absolute ethanol solution and the addition of a buffer solution was not necessary. Under these conditions, dilute solutions of ETE and LEV were stable for at least 12 h. The use of absolute ethanol permits the best recovery of the hormones in the oral contraceptive tablets. Derivative Spectrophotometry In Fig. 1 the zero-order spectra of ETE and LEV in the wavelength range 210–315 nm are shown.It can be seen that the absorption spectrum of LEV is very overlapped with the ETE spectrum. The determination of ETE directly could be easy at the start, but the small content of this steroid and the high content of LEV in commercial tablets (the ETE : LEV ratio is normally 1 : 4 or 1 : 5) presumes a large contribution of the spectrum of LEV to the maxima in the spectrum of ETE. The spectra of real samples after dissolution of the hormones and filtration show a very small overlap, not perceptible at the beginning, but the spectrum showed a small y-axis displacement of the absorbance owing to the overlap.This behaviour is revealed when the spectrum of an artificial binary mixture is compared with that of a real contraceptive tablet of similar concentration (Fig. 2). Derivative spectrophotometry is a suitable technique to overcome this problem. The zero-crossing method is the most common procedure for the preparation of analytical calibration graphs.In practice, the measurement selected is that which exhibits the best linear response, gives a zero or near zero intercept on the ordinate of the calibration graphs and is less affected by the concentration of any other component. The shape of the first derivative spectrum is adequate for determining ETE in the presence of LEV and vice versa. Fig. 3 shows the firstderivative absorption spectra of a solution of ETE and a solution of LEV, both solutions in 100% ethanol.It can be seen that owing to the overlapping spectra of these compounds, the zero-crossing method is the most appropriate approach for resolving mixtures of these compounds and it was used in this work with satisfactory results. Fig. 1 Absorption spectra of ethinylestradiol (26.01 mg ml21, broken line), levonorgestrel (7.17 mg ml21, dotted line) and their mixture (continuous line). 42 Analyst, January 1997, Vol. 122Preliminary experiments showed that the signals of the first derivative at 293.0 nm (working zero-crossing wavelength of LEV) are proportional to the ETE concentration and the signals of the first derivative at 249.0 nm (working zero-crossing wavelengths of ETE) are proportional to the LEV concentration.Selection of Optimum Instrumental Conditions The main instrumental parameters that affect the shape of the derivative spectra are the wavelength scanning speed, the wavelength increment over which the derivative is obtained (Dl) and the smoothing. These parameters need to be optimized to give a well resolved large peak, i.e., to give good selectivity and higher sensitivity in the determination. Generally, the noise level decreases with increase in Dl, thus decreasing the fluctuations in the derivative spectrum.However, if the value of Dl is too large, the spectral resolution is very poor. Therefore, the optimum value of Dl should be determined by taking into account the noise level, the resolution of the spectrum and the sample concentration.Some values of Dl were tested and 8.0 nm was selected as the optimum in order to obtain a satisfactory signal-to-noise ratio. In this way, a smoothing function was not necessary. Having established the experimental conditions, the calibration graphs were tested between 2.0 and 26.0 mg ml21 of the ETE in the absence of LEV at 293.0 nm for the first-derivative spectra. The calibration graphs were also tested between 4.0 and 30.0 mg ml21 of LEV in the absence of ETE at 243.0 nm for the first-derivative spectra (Fig. 4). Good linearity was observed in all cases. Statistical Study Tables 1 and 2 summarize the most characteristic statistical data obtained from the different calibration graphs and the reproducibility of the reagent blank and a standard. The reproducibility of particular concentrations of ETE (10.40 mg ml21) and LEV (10.60 mg ml21) were evaluated over 2 d by performing 10 absorption spectrophotometric measurements each day on 10 different samples. The results (Table 1) show that the repeatability for both hormones on each day was satisfactory.The comparison of the average concentrations with the Snedecor test did not show any significant difference at a confidence level of 5%. Determination of ETE and LEV in Synthetic Mixtures Some binary mixtures of ETE and LEV were prepared from the stock standard solutions in the proportions from 1 + 1 to 1 + 4 and were analysed by the proposed derivative spectrophotometric method.Some of these proportions between the two hormones were the same as in commercial contraceptives with the object of checking the relationships of more commercial interest. Table 3 shows the results of the analyses of different mixtures. The recoveries were between 94 and 104% for ETE and between 99 and 100% for LEV for the wavelengths studied. These results show that the method is effective for the simultaneous determination of ETE and LEV by first-derivative spectrophotometry.Fig. 2 (a) Absorption spectra of (A) synthetic mixture of ETE (5.00 mg ml21) and LEV (25.00 mg ml21) and (B) commercial solution of he Ovoplex pills, containing the same concentrations of ETE and LEV. (b) Zoom of the spectra between 270–315 nm. Fig. 3 First-derivative spectra (Dl = 8 nm) of ethinylestradiol (26.01 mg ml21, broken line), levonorgestrel (7.17 mg ml21, dotted line) and their mixture (continuous line).Fig. 4 First-derivative spectra (Dl = 8 nm) for different concentrations of levonorgestrel: a, 2.59; b, 5.18; c, 7.77; d, 10.36; e, 12.95; f, 19.42; g, 25.90; and h, 32.63 mg ml21. Table 1 Precision of the determination of the concentration of ETE (10.40 mg ml21) and LEV (10.60 mg ml21) on different days (n = 10 determinations on each day) Ethinylestradiol Levonorgestrel Average/ s/ RSD Average/ s/ RSD mg ml21 mg ml21 (%) mg ml21 mg ml21 (%) Day A 10.26 0.16 1.55 10.53 0.07 0.65 Day B 10.38 0.08 0.75 10.60 0.08 0.73 Analyst, January 1997, Vol. 122 43Determination of ETE and LEV in Commercial Contraceptives The Spanish pharmacological industry has at present five different low-dose commercial oral contraceptives (Neogynona, Microgynon, Ovoplex, Triciclor and Triagynon) containing ETE and LEV. Two of these contraceptives (Triciclor and Triagynon) have in the formulation three different doses (different proportions of ETE and LEV); in these contraceptives the amount of LEV starts at 0.050 mg per tablet at the beginning of the treatment, later it is 0.075 mg per tablet and at the end is 0.125 mg per tablet.Each oral contraceptive packet contains 21 tablets, six of them corresponding to the lowest dosage of LEV, five to the intermediate dosage and ten to the highest dosage. The amount of ETE starts at 0.03 mg per tablet, becomes 0.04 mg per tablet and at the end is once more 0.03 mg per tablet. The results obtained for the determination of ETE and LEV mixtures in commercial pharmaceuticals are given in Table 4.The relative differences between HPLC and derivative spectrophotometric results were between ±6% for the LEV determination in all oral contraceptives except Triagynon B and C. The relative differences for ETE were ±4% in all the commercial formulations except Triciclor C. The anomalous results found for Triagynon and Triciclor could be due to the presence of some interferences due to the excipients or of dyes that coated these tablets.In all cases the recoveries were calculated with respect to the results obtained by the HPLC method. Good correlations between the two methods were found. The indicated value is the mean of two different analyses of the same commercial batch. Conclusions The proposed derivative method is very suitable for the simultaneous determination of ETE and LEV and can be employed to analyse commercial formulations of low-dose oral contraceptives.The proposed method gave good results when compared with the HPLC method. The authors are grateful to Dr. V. Trigo (Wyeth-Orfi Laboratories) and Dr. C. Barona (Shering Laboratories). Financial support from the DGICYT of the Ministerio de Educaci�on y Ciencia of Spain (Project PB-94-0743) is acknowledged. References 1 Pollow, K., Sinnecker, R., and Pollow, B., J. Chromatogr., 1974, 90, 402. 2 Verma, P., Curry, C., Crocker, C., Titus Dillon, P., and Ahluwalia, B., Clin.Chim. Acta, 1975, 63, 363. 3 Skrivanek, J. A., Ruhlig, M., and Schraer, R., Abstr. Pap. Am. Chem. Soc. 166th Meet. Biol., 1973, 58. 4 Penzes, L. P., and Oertel, G. W., J. Chromatogr., 1972, 74, 359. 5 Short M. P., and Rhodes, C. T., Can. J. Pharm. Sci., 1973, 8, 26. 6 Moreti, G., Cavina, G., Pacioti, P., and Siniscalchi, P., Farmaco, Ed. Prat., 1972, 27, 537. 7 Eldawy, M. A., Tawfik, A. S., and Elshabouri, S. R., J. Pharm. Sci., 1975, 64, 1221. 8 Wu, J.Y. P., J. Assoc. Off. Anal. Chem., 1974, 57, 747. 9 Szepesi, G., and G�or�og, S., Analyst, 1974, 99, 218. 10 Lisboa, B. P., and Strassner, M., J. Chromatogr., 1975, 111, 159. 11 Graham, R. E., and Kenner, C. T., J. Pharm. Sci., 1973, 62, 1845. 12 Korany, M. A., El-Yazbi, F. A., Abdel-Razak, O., and Elsayed, M. A., Pharm. Weekbl., Sci. Ed., 1985, 7(4), 163. 13 Cao, Y., and Zhang, J., Yaowu Fenxi Zazhi, 1984, 4(1), 31. 14 Corti, P., Lencioni, E., and Sciarra, G. F., Boll. Chim.Farm., 1983, 122(6), 281. 15 Corti, P., and Sciarra, G. F., Boll. Chim. Farm., 1981, 120(12), 701. 16 Talsky, G., Mayring, L., and Kreuzer, H., Angew. Chem., 1978, 17, 785. 17 O’ � Haver, T. C., Anal. Chem., 1979, 51, 91A. 18 Fell, A. F., and Smith, G., Anal. Proc., 1982, 19, 28. 19 Cottrell, C. T., Anal. Proc., 1982, 19, 43. 20 United States Pharmacopeia, XXIII Revision, US Pharmacopeial Convention, Rockville, MD, 1995, pp. 881–883. 21 Data Leader Software Package, Beckman, Fullerton, CA, 1987.Paper 6/04558H Received July 1, 1996 Accepted October 15, 1996 Table 2 Calibration data for the determination of ETE and LEV Standard deviation Inter- Range/ cept Slope LOD/ LOQ/ Equation r mg ml21 3105 3105 mg ml21 mg ml21 Ethinylestradiol— 1D293 = 5.8 3 1025 + 72.5 3 1025 C* 0.9999 26 4.2 0.3 0.3 0.9 Levonorgestrel— 1D249 = 1.4 3 1023 + 2.3 3 1023 C* 0.9996 33 40.7 2.3 0.8 2.5 * C = concentration in mg ml21. Table 3 Compositions and recoveries for artificial mixtures Ethinylestradiol Levonorgestrel Sample Actual/ Found*/ Recovery* Actual/ Found*/ Recovery* No.mg ml21 mg ml21 (%) mg ml21 mg ml21 (%) 1 2.06 2.02 98 3.88 3.86 99 2 4.13 4.23 103 7.77 7.77 100 3 6.19 6.12 99 7.77 7.77 100 4 2.06 2.15 104 10.36 10.30 99 5 4.13 3.96 96 10.36 10.35 100 6 2.06 2.01 97 10.36 10.33 100 7 3.10 2.92 94 15.54 15.48 100 8 8.26 8.28 100 20.72 20.58 99 * Average of two determinations. Table 4 Relative differences between HPLC and derivative spectrophotometric methods in the assays of commercial formulations Ethinylestradiol Levonorgestrel Found/mg Found/mg per tablet Relative per tablet Relative Commercial difference difference formulation HPLC 1D293 (%) HPLC 1D249 (%) Ovoplex 0.0460 0.0439 24 0.2300 0.2320 +1 Microginon 0.0295 0.0300 +2 0.1444 0.1505 +4 Neogynona 0.0461 0.0452 22 0.2383 0.2369 21 Triagynon A* 0.0304 0.0314 +3 0.1223 0.1152 26 Triagynon B* 0.0369 0.0361 22 0.0708 0.0775 +9 Triagynon C* 0.0285 0.0292 +2 0.0467 0.0526 +13 Triciclor A* 0.0249 0.0246 21 0.1271 0.1272 0 Triciclor B* 0.0315 0.0325 +3 0.0681 0.0699 +3 Triciclor C* 0.0249 0.0305 +22 0.0488 0.0522 +6 * A = yellow, B = white, C = brown. 44 Analyst, January 1997, Vol. 122 Simultaneous Determination of Ethinylestradiol and Levonorgestrel in Oral Contraceptives by Derivative Spectrophotometry Juan J. Berzas*, Juana Rodr�ýguez and Gregorio Casta�neda Departamento de Qu�ýmica Anal�ýtica y Tecnolog�ýa de Alimentos, Facultad de Ciencias, Universidad de Castilla-La Mancha, 13071 Ciudad Real, Spain A method for determining ethinylestradiol (ETE) and levonorgestrel (LEV) in mixtures by first-derivative spectrophotometry is described.The procedure does not require any separation step. Measurements are made at the zero-crossing wavelengths and the calibration graphs are linear up to 26 and 33 mg ml21 of ETE and LEV, respectively. The method was applied to the determination of both compounds in five different Spanish commercial low-dose oral contraceptives.Similar results were obtained by an HPLC method. Keywords: Ethinylestradiol; levonorgestrel; derivative spectrophotometry; oral contraceptives At present there are three types of oral contraception available. In the sequential type, estrogen is administered alone for the first week, followed by a lower dosage of the estrogen in conjunction with a progestogen for the remainder of the course. In the second, commonly used, type both an estrogen and a progestogen are present in the tablets (as either a single dose or in three different doses).In the progestogen type, a progestogen alone is administered. Ethinylestradiol (ETE) is a semisynthetic estrogen female sex hormone and levonorgestrel (LEV) is a synthetic steroid with an extremely potent progestational action. The formulation of these steroids in tablets of low dosage, i.e., 30–250 mg per tablet, presented a challenging analytical problem.A sensitive, accurate and rapid procedure is desirable for content uniformity testing of the dosage form. The structure of LEV has a characteristic D4-3-keto group in the A-ring with a different chromophoric power to ETE. The most commonly encountered estrogen is ETE, which is present at a very low dosage level (30–100 mg per tablet) in combination with an orally active synthetic progestin (one of the most commonly used is LEV), which is present at a level of from 5 to 30 times that of the estrogen.Oral contraceptives have had an enormous positive impact on public health for the past three decades and, in the main, there has been a remarkably low incidence of troublesome side-effects. Although estrogens are implicated in an increased incidence of breast and endometrial cancer, epidemiological studies have not provided convincing evidence to support a direct correlation between the use of oral contraceptives and an increase in breast cancer.The modern low-dose oral contraceptives (estrogen–progestogen) require a sensitive analysis method which is unaffected by the small amount of the estrogen and the large excess of progestogen. There have been several reports1–11 of the determination of levonorgestrel or ethinylestradiol, including the use of radioactivelyves,1,2 dansyl or other fluorescent derivatives,3–5 spectrophotometry or photometry6–9 and gel or column chromatography, but the methods are complicated.10,11 No references were found to the simultaneous determination of ETE and LEV using spectrophotometric methods.The determination of ETE in the presence of noresthisterone by derivative spectrophotometry in methanol or ethanol has been reported,12,13 the recoveries in different tablets being 120–80% for both compounds. The simultaneous determination of ETE and mestranol by derivative spectrophotometry has been proposed, either in a solution of methanol and chloroform14 or in NaOH in methanol.15 In derivative UV/VIS spectrophotometry, the information contained in the spectrum is presented in a potentially more useful form, greatly increasing the versatility of the technique16 –18 and offering a convenient solution to a number of well defined analytical problems, such as the resolution of multi-component systems, removal of sample turbidity, matrix background and enhancement of spectral details.19 Although the use of derivative spectra is not new, it has only become practical in recent years with the development of microcomputer technology, which allows the almost instantaneous generation of derivative spectra.In this paper we demonstrate the ease with which the derivative methods circumvent the problem of overlapping spectral bands and sample turbidity, allowing the simultaneous determination of ETE and LEV without prior separation. The method yielded accurate, rapid and reproducible results for five different commercial products, two of them with three different dosages.The results obtained by the proposed method were compared with those obtained by HPLC with spectrophometric detection, very similar to the method proposed in the US Pharmacopeia. 20 Experimental Apparatus A Beckman (Fullerton, CA, USA) DU-70 spectrophotometer equipped with 1.0 cm quartz cells and connected to an IBM-PS 2 Model 30 computer, fitted with Beckman Data Leader software,21 and an Epson FX-850 printer was used for all absorbance measurements. A Shimadzu (Kyoto, Japan) high-performance liquid chromatograph equipped with a Nova-Pak C18 column (15 3 0.39 cm id, 4 mm), a diode-array detector, a Rheodyne injection valve and connected to a computer fitted with CLASS LC-10 software was used.A Crison (Barcelona, Spain) MicropH 2002 pH meter was used for the pH measurements. Standard Solutions All chemicals and solvents were of analytical-reagent grade. ETE and LEV were obtained from Sigma (St.Louis, MO, USA) and stock standard solutions were prepared in absolute ethanol Analyst, January 1997, Vol. 122 (41–44) 41(100 mg ml21). The purities of the ETE and LEV reported by Sigma were 98.6% and 99.6%, respectively, determined using an HPLC method with spectrophotometric detection at 280 and 242 nm. Procedure Calibration Stock standard solutions of ETE and LEV were placed in 25 ml calibrated flasks to give final concentrations of up to 26 and 33 mg ml21, respectively, adding absolute ethanol to dilute the contents to 25 ml (the resulting final solution was 100% in ethanol).This high percentage of ethanol was necessary to obtain total dissolution of the drugs from the oral contraceptive tablets. The absorption spectra of the samples were recorded against an ethanol blank between 315 and 210 nm at a scan speed of 120 nm min21 and stored in the computer. Firstderivative spectra were obtained with Dl = 8 nm and ETE was determined by measuring the signal of the first derivative spectrum at 293.0 nm (1D293) (zero-crossing point for LEV), and by using an appropriate calibration graph, their concentrations could be determined.These calibrations were performed by varying the concentration of the estrogen, in the absence of the other hormone. The LEV content was also determined by measuring the signal at 249.0 nm (1D249) (zero-crossing point for ETE). These spectra were not treated with a smoothing function because the noise level was low.Assay of pharmaceutical preparations Twenty tablets were finely powdered and an appropriate portion (equivalent to the median mass of two tablets) was dissolved in 8 ml of absolute ethanol by sonication for 15 min, followed by shaking by mechanical means for 20 min. The mixture was filtered, using a Swinnex polypropylene disc filter holder of 13 mm diameter (Millipore, Bedford, MA, USA) with an FH 0.5 mm Fluoropore (PFTE) membrane, into a 10 ml calibrated flask.The residue was washed twice with the same solvent and diluted to volume. The absorption spectra were recorded against an absolute ethanol blank and stored in the IBM-PS computer. For determining ETE and LEV, the absorption spectra were handled as in first-derivative spectrophotometry. Comparison with HPLC reference method Twenty tablets were finely powdered and an appropriate portion (equivalent to the median mass of two tablets) was dissolved in 8 ml of absolute ethanol by sonication for 15 min, followed by shaking by mechanical means for 20 min.The mixture was filtered using a Swinnex polypropylene disc filter holder of 13 mm diameter with an FH 0.5 mm Fluoropore (PFTE) membrane into a 10 ml calibrated flask. The residue washed twice with the same solvent and diluted to volume. A 2.5 ml portion of this solution was diluted with water in a 10 ml calibrated flask; the reason for this dilution is to obtain a higher polarity and lower concentration in the sample.HPLC determination was performed on a Nova-Pak C18 60 A column (15 3 0.39 cm id) containing 4 mm packing. The mobile phase was deaerated acetonitrile–methanol–water (3.5 + 1.5 + 4.5) and spectrophotometric detection was performed at 215 nm. The flow rate was about 1 ml min21.20 The differences between the two methods are the dissolution of the tablets (using ethanol–water or acetonitrile–methanol–water) and the column used [C18 (15 3 0.39 cm id) or C8 (15 3 0.46 cm id)].Results and Discussion Method Development The influence of pH on the absorption spectra of ETE (e280 nm = 2253 l mol21 cm21 in absolute ethanol) and LEV (e240 nm = 17 155 l mol21 cm21 in absolute ethanol) was studied, with a total content of ethanol of 25%. The LEV spectrum showed only a maximum at 246 nm, which remained unchanging in the pH range 1.0–12.4. The ETE spectrum showed a maximum at 280 nm in the pH range 1.0–9.5 and for more alkaline solutions two different bands at 296 and 240 nm.The stability of ETE in very acidic solutions was not very good. For this reason, the best results for analytical purposes were obtained in the pH range 4.0–9.5. The preparation of the samples with 100% of ethanol resulted in spectra very close to those obtained at the optimum pH. Samples were prepared in absolute ethanol solution and the addition of a buffer solution was not necessary. Under these conditions, dilute solutions of ETE and LEV were stable for at least 12 h.The use of absolute ethanol permits the best recovery of the hormones in the oral contraceptive tablets. Derivative Spectrophotometry In Fig. 1 the zero-order spectra of ETE and LEV in the wavelength range 210–315 nm are shown. It can be seen that the absorption spectrum of LEV is very overlapped with the ETE spectrum. The determination of ETE directly could be easy at the start, but the small content of this steroid and the high content of LEV in commercial tablets (the ETE : LEV ratio is normally 1 : 4 or 1 : 5) presumes a large contribution of the spectrum of LEV to the maxima in the spectrum of ETE.The spectra of real samples after dissolution of the hormones and filtration show a very small overlap, not perceptible at the beginning, but the spectrum showed a small y-axis displacement of the absorbance owing to the overlap. This behaviour is revealed when the spectrum of an artificial binary mixture is compared with that of a real contraceptive tablet of similar concentration (Fig. 2). Derivative spectrophotometry is a suitable technique to overcome this problem. The zero-crossing method is the most common procedure for the preparation of analytical calibration graphs. In practice, the measurement selected is that which exhibits the best linear response, gives a zero or near zero intercept on the ordinate of the calibration graphs and is less affected by the concentration of any other component. The shape of the first derivative spectrum is adequate for determining ETE in the presence of LEV and vice versa.Fig. 3 shows the firstderivative absorption spectra of a solution of ETE and a solution of LEV, both solutions in 100% ethanol. It can be seen that owing to the overlapping spectra of these compounds, the zero-crossing method is the most appropriate approach for resolving mixtures of these compounds and it was used in this work with satisfactory results.Fig. 1 Absorption spectra of ethinylestradiol (26.01 mg ml21, broken line), levonorgestrel (7.17 mg ml21, dotted line) and their mixture (continuous line). 42 Analyst, January 1997, Vol. 122Preliminary experiments showed that the signals of the first derivative at 293.0 nm (working zero-crossing wavelength of LEV) are proportional to the ETE concentration and the signals of the first derivative at 249.0 nm (working zero-crossing wavelengths of ETE) are proportional to the LEV concentration.Selection of Optimum Instrumental Conditions The main instrumental parameters that affect the shape of the derivative spectra are the wavelength scanning speed, the wavelength increment over which the derivative is obtained (Dl) and the smoothing. These parameters need to be optimized to give a well resolved large peak, i.e., to give good selectivity and higher sensitivity in the determination. Generally, the noise level decreases with increase in Dl, thus decreasing the fluctuations in the derivative spectrum.However, if the value of Dl is too large, the spectral resolution is very poor. Therefore, the optimum value of Dl should be determined by taking into account the noise level, the resolution of the spectrum and the sample concentration. Some values of Dl were tested and 8.0 nm was selected as the optimum in order to obtain a satisfactory signal-to-noise ratio. In this way, a smoothing function was not necessary.Having established the experimental conditions, the calibration graphs were tested between 2.0 and 26.0 mg ml21 of the ETE in the absence of LEV at 293.0 nm for the first-derivative spectra. The calibration graphs were also tested between 4.0 and 30.0 mg ml21 of LEV in the absence of ETE at 243.0 nm for the first-derivative spectra (Fig. 4). Good linearity was observed in all cases. Statistical Study Tables 1 and 2 summarize the most characteristic statistical data obtained from the different calibration graphs and the reproducibility of the reagent blank and a standard.The reproducibility of particular concentrations of ETE (10.40 mg ml21) and LEV (10.60 mg ml21) were evaluated over 2 d by performing 10 absorption spectrophotometric measurements each day on 10 different samples. The results (Table 1) show that the repeatability for both hormones on each day was satisfactory. The comparison of the average concentrations with the Snedecor test did not show any significant difference at a confidence level of 5%.Determination of ETE and LEV in Synthetic Mixtures Some binary mixtures of ETE and LEV were prepared from the stock standard solutions in the proportions from 1 + 1 to 1 + 4 and were analysed by the proposed derivative spectrophotometric method. Some of these proportions between the two hormones were the same as in commercial contraceptives with the object of checking the relationships of more commercial interest. Table 3 shows the results of the analyses of different mixtures.The recoveries were between 94 and 104% for ETE and between 99 and 100% for LEV for the wavelengths studied. These results show that the method is effective for the simultaneous determination of ETE and LEV by first-derivative spectrophotometry. Fig. 2 (a) Absorption spectra of (A) synthetic mixture of ETE (5.00 mg ml21) and LEV (25.00 mg ml21) and (B) commercial solution of he Ovoplex pills, containing the same concentrations of ETE and LEV.(b) Zoom of the spectra between 270–315 nm. Fig. 3 First-derivative spectra (Dl = 8 nm) of ethinylestradiol (26.01 mg ml21, broken line), levonorgestrel (7.17 mg ml21, dotted line) and their mixture (continuous line). Fig. 4 First-derivative spectra (Dl = 8 nm) for different concentrations of levonorgestrel: a, 2.59; b, 5.18; c, 7.77; d, 10.36; e, 12.95; f, 19.42; g, 25.90; and h, 32.63 mg ml21. Table 1 Precision of the determination of the concentration of ETE (10.40 mg ml21) and LEV (10.60 mg ml21) on different days (n = 10 determinations on each day) Ethinylestradiol Levonorgestrel Average/ s/ RSD Average/ s/ RSD mg ml21 mg ml21 (%) mg ml21 mg ml21 (%) Day A 10.26 0.16 1.55 10.53 0.07 0.65 Day B 10.38 0.08 0.75 10.60 0.08 0.73 Analyst, January 1997, Vol. 122 43Determination of ETE and LEV in Commercial Contraceptives The Spanish pharmacological industry has at present five different low-dose commercial oral contraceptives (Neogynona, Microgynon, Ovoplex, Triciclor and Triagynon) containing ETE and LEV.Two of these contraceptives (Triciclor and Triagynon) have in the formulation three different doses (different proportions of ETE and LEV); in these contraceptives the amount of LEV starts at 0.050 mg per tablet at the beginning of the treatment, later it is 0.075 mg per tablet and at the end is 0.125 mg per tablet. Each oral contraceptive packet contains 21 tablets, six of them corresponding to the lowest dosage of LEV, five to the intermediate dosage and ten to the highest dosage. The amount of ETE starts at 0.03 mg per tablet, becomes 0.04 mg per tablet and at the end is once more 0.03 mg per tablet.The results obtained for the determination of ETE and LEV mixtures in commercial pharmaceuticals are given in Table 4. The relative differences between HPLC and derivative spectrophotometric results were between ±6% for the LEV determination in all oral contraceptives except Triagynon B and C.The relative differences for ETE were ±4% in all the commercial formulations except Triciclor C. The anomalous results found for Triagynon and Triciclor could be due to the presence of some interferences due to the excipients or of dyes that coated these tablets. In all cases the recoveries were calculated with respect to the results obtained by the HPLC method. Good correlations between the two methods were found. The indicated value is the mean of two different analyses of the same commercial batch.Conclusions The proposed derivative method is very suitable for the simultaneous determination of ETE and LEV and can be employed to analyse commercial formulations of low-dose oral contraceptives. The proposed method gave good results when compared with the HPLC method. The authors are grateful to Dr. V. Trigo (Wyeth-Orfi Laboratories) and Dr. C. Barona (Shering Laboratories). Financial support from the DGICYT of the Ministerio de Educaci�on y Ciencia of Spain (Project PB-94-0743) is acknowledged.References 1 Pollow, K., Sinnecker, R., and Pollow, B., J. Chromatogr., 1974, 90, 402. 2 Verma, P., Curry, C., Crocker, C., Titus Dillon, P., and Ahluwalia, B., Clin. Chim. Acta, 1975, 63, 363. 3 Skrivanek, J. A., Ruhlig, M., and Schraer, R., Abstr. Pap. Am. Chem. Soc. 166th Meet. Biol., 1973, 58. 4 Penzes, L. P., and Oertel, G. W., J. Chromatogr., 1972, 74, 359. 5 Short M.P., and Rhodes, C. T., Can. J. Pharm. Sci., 1973, 8, 26. 6 Moreti, G., Cavina, G., Pacioti, P., and Siniscalchi, P., Farmaco, Ed. Prat., 1972, 27, 537. 7 Eldawy, M. A., Tawfik, A. S., and Elshabouri, S. R., J. Pharm. Sci., 1975, 64, 1221. 8 Wu, J. Y. P., J. Assoc. Off. Anal. Chem., 1974, 57, 747. 9 Szepesi, G., and G�or�og, S., Analyst, 1974, 99, 218. 10 Lisboa, B. P., and Strassner, M., J. Chromatogr., 1975, 111, 159. 11 Graham, R. E., and Kenner, C. T., J. Pharm. Sci., 1973, 62, 1845. 12 Korany, M. A., El-Yazbi, F. A., Abdel-Razak, O., and Elsayed, M. A., Pharm. Weekbl., Sci. Ed., 1985, 7(4), 163. 13 Cao, Y., and Zhang, J., Yaowu Fenxi Zazhi, 1984, 4(1), 31. 14 Corti, P., Lencioni, E., and Sciarra, G. F., Boll. Chim. Farm., 1983, 122(6), 281. 15 Corti, P., and Sciarra, G. F., Chim. Farm., 1981, 120(12), 701. 16 Talsky, G., Mayring, L., and Kreuzer, H., Angew. Chem., 1978, 17, 785. 17 O’ � Haver, T. C., Anal. Chem., 1979, 51, 91A. 18 Fell, A. F., and Smith, G., Anal. Proc., 1982, 19, 28. 19 Cottrell, C. T., Anal. Proc., 1982, 19, 43. 20 United States Pharmacopeia, XXIII Revision, US Pharmacopeial Convention, Rockville, MD, 1995, pp. 881–883. 21 Data Leader Software Package, Beckman, Fullerton, CA, 1987. Paper 6/04558H Received July 1, 1996 Accepted October 15, 1996 Table 2 Calibration data for the determination of ETE and LEV Standard deviation Inter- Range/ cept Slope LOD/ LOQ/ Equation r mg ml21 3105 3105 mg ml21 mg ml21 Ethinylestradiol— 1D293 = 5.8 3 1025 + 72.5 3 1025 C* 0.9999 26 4.2 0.3 0.3 0.9 Levonorgestrel— 1D249 = 1.4 3 1023 + 2.3 3 1023 C* 0.9996 33 40.7 2.3 0.8 2.5 * C = concentration in mg ml21. Table 3 Compositions and recoveries for artificial mixtures Ethinylestradiol Levonorgestrel Sample Actual/ Found*/ Recovery* Actual/ Found*/ Recovery* No. mg ml21 mg ml21 (%) mg ml21 mg ml21 (%) 1 2.06 2.02 98 3.88 3.86 99 2 4.13 4.23 103 7.77 7.77 100 3 6.19 6.12 99 7.77 7.77 100 4 2.06 2.15 104 10.36 10.30 99 5 4.13 3.96 96 10.36 10.35 100 6 2.06 2.01 97 10.36 10.33 100 7 3.10 2.92 94 15.54 15.48 100 8 8.26 8.28 100 20.72 20.58 99 * Average of two determinations. Table 4 Relative differences between HPLC and derivative spectrophotometric methods in the assays of commercial formulations Ethinylestradiol Levonorgestrel Found/mg Found/mg per tablet Relative per tablet Relative Commercial difference difference formulation HPLC 1D293 (%) HPLC 1D249 (%) Ovoplex 0.0460 0.0439 24 0.2300 0.2320 +1 Microginon 0.0295 0.0300 +2 0.1444 0.1505 +4 Neogynona 0.0461 0.0452 22 0.2383 0.2369 21 Triagynon A* 0.0304 0.0314 +3 0.1223 0.1152 26 Triagynon B* 0.0369 0.0361 22 0.0708 0.0775 +9 Triagynon C* 0.0285 0.0292 +2 0.0467 0.0526 +13 Triciclor A* 0.0249 0.0246 21 0.1271 0.1272 0 Triciclor B* 0.0315 0.0325 +3 0.0681 0.0699 +3 Triciclor C* 0.0249 0.0305 +22 0.0488 0.0522 +6 * A = yellow, B = white, C = brown. 44 Analyst, January 1997, Vol. 1