Simultaneous Quantification of Drospirenone, Ethinyl Estradiol and Levomefolate by Stability Indicating RP-HPLC Method

Drospirenone is a synthetic progestogen, progestin, with antimineralocorticoid and progestational activity [1]. Drospirenone is used in menopausal hormone therapy, treatment of premenstrual dysphoric disorder and acne [2-4]. Drospirenone is also an important ingredient in most of the oral contraceptive pills. Chemically, drospirenone is described as (6R,7R,8R,9S,10R,13S,14S,15S,16S,17S)-1,3',4',6,6a,7,8,9 ,10,11,12,13,14,15,15a,16-Hexadecahydro-10,13-dimetylspiro-[17Hdicyclopropa[6,7:15,16]cyclopenta[a]phenantrene-17,2'(5'H)-furan]3,5'(2H)-dione (Figure 1). Drospirenone exerts its activity through binding strongly and specifically to progesterone receptor [5,6]. Drospirenone-progesterone receptor complex produces an activated complex which binds to specific sites in DNA. As the result, luteinizing hormone activity is suppressed and ovulation is inhibited. This activated complex also changes the cervical membrane and endometrium.

Drospirenone, ethinyl estradiol and levomefolate combination is available in oral contraception tablet dosage form with brand names Safyral, Beyaz and Rajani [16][17][18][19]. In this combination, drospirenone and ethinyl estradiol prevent pregnancy by repressing ovulation. These two drugs also make changes in cervical mucus and endometrial which inhibits penetration of sperm and lessen the implantation, respectively. Levomefolate in the tablet increases the levels of folate levels in women who opt oral contraceptive [20].
To the best of our knowledge, there is no report for the simultaneous determination of drospirenone, ethinyl estradiol and levomefolate in bulk and combined tablet dosage form by stability indicating reverse phase high performance liquid chromatographic (RP-HPLC) method. Therefore, the main aim of this investigation was to develop and validate a stability indicating RP-HPLC method to determine drospirenone, ethinyl estradiol and levomefolate simultaneously in the presence of their stress degradation products [21][22][23].

Experimental Materials
Drospirenone, ethinyl estradiol and levomefolate reference drug standards were provided kindly by Rainbow Pharma Training Labs (Hyderabad, India). Safyral (Bayer Health Care Pharmaceuticals Inc. Whippany, NJ) tablets labeled to contain 3 mg drospirenone, 0.03 mg ethinyl estradiol and 0.451 mg levomefolate were obtained from a local pharmacy market. HPLC grade acetonitrile and methanol were purchased from Merck India Ltd (Mumbai, India). Analytical reagent orthophosphoric acid, hydrogen peroxide, hydrochloric acid and sodium hydroxide were supplied by Sd. Fine Chemicals Ltd (Mumbai, India). HPLC grade water was prepared using Milli-Q system (Millipore, USA).

Instrumentation
Waters Alliance HPLC system 2695 Module with a 2998 PDA detector, degasser, auto sample injector and column oven were used in the present analysis. Data acquisition and processing was done with Empower 2 software. Method development and validation was done using Waters, C18, 5 µm, 250 mm × 4.6 mm analytical column.

Optimized HPLC conditions
Isocratic elution was performed with a mobile phase comprised of filtered (using a 0.45 μm membrane filter) and degassed 0.1% orthophosphoric acid: methanol: acetonitrile (60:20:20 v/v/v) adjusted to pH 4.8 and pumped at a flow rate of 1.0 mL/min. The column temperature was set at 27°C. The samples were injected at 10 µL injection volume and eluted samples were analyzed at a wavelength of 245 nm. The total runtime was 8 min.

Standard stock and working solutions
The standard stock solution (Drospirenone-1200 µg/mL, Ethinyl estradiol-12 µg/mL and Levomefolate-180.4 µg/mL) was prepared by dissolving an accurately weighed 30 mg, 0.30 mg and 4.51 mg of drospirenone, ethinyl estradiol and levomefolate, reference standard respectively in 25 mL of mobile phase in a volumetric flask (25 mL). The working standard solutions in the range 30-240 µg/mL of drospirenone, 0.3-2.4 µg/mL of ethinyl estradiol and 4.51-36.08 µg/mL of levomefolate were obtained by appropriately diluting the standard stock solution with mobile phase.

Construction of calibration curve
Aliquots (10 μL) of working standard solutions were injected into the HPLC system and eluted by the mobile phase under the optimum HPLC conditions. The peak area response of drug versus the final concentration of drug (μg/mL) was plotted. On the other hand, the corresponding regression equations were derived.

Analysis of drospirenone, ethinyl estradiol and levomefolate in tablet sample solution
Ten tablets were crushed into powder. The tablet powder weight equivalent to 30 mg, 0.30 mg and 4.51 mg of drospirenone, ethinyl estradiol and levomefolate, respectively was transferred to 25 mL volumetric flask and sonicated with 10 mL of mobile phase for 20 min. The volume was diluted to 25 mL with mobile phase and filtered through 0.45 μm membrane filter. The stock tablet sample solution was then diluted aptly with mobile phase to get the final concentration 120 µg/mL, 1.2 µg/mL and 18.04 µg/mL of drospirenone, ethinyl estradiol and levomefolate, respectively. 10 μL of working tablet sample solution prepared was injected into the HPLC system and analyzed by the developed method. The nominal content of drospirenone, ethinyl estradiol and levomefolate in the tablet was calculated either using the corresponding calibration graph or corresponding regression equation.

Stress degradation studies
The stress degradation studies were performed through the analysis of tablet sample solution (drospirenone-120 µg/mL, ethinyl estradiol-1.2 µg/mL and levomefolate -18.04 µg/mL), which was exposed to accelerated degradation conditions as per the ICH guidelines [21]. The results are compared to a reference standard solution prepared in the same day.

Acid and alkaline hydrolysis
Volumetric flasks (100 mL) containing 10 mL of tablet sample solution (drospirenone-1200 µg/mL, ethinyl estradiol-12 µg/mL and levomefolate-180.4 µg/mL) were mixed with 10 mL of 0.1 N proportions of 0.1% orthophosphoric acid, acetonitrile and methanol with different pH units in isocratic elution mode were investigated to obtain optimum resolution, symmetric peak shape and optimal sensitivity in reasonable time. Best results were obtained with a mixture of 0.1% orthophosphoric acid, acetonitrile and methanol in the ratio of 60:20:20 (v/v/v) with pH 4.8 units was employed as the mobile phase. The flow rate of mobile phase, for improved resolution and quick separation, was adjusted to 1.0 mL/min. Room temperature was adequate for the separation and analysis of selected drug combination and so the same was used in the whole separation and analysis. Detection at 245 nm was used as it was observed as the optimum detection wavelength for the three analytes (drospirenone, ethinyl estradiol and levomefolate). At this detection wavelength (245 nm), the peak area response for the three analytes was high. Representative chromatogram of the finalized chromatographic conditions, showing drospirenone, ethinyl estradiol and levomefolate, is illustrated in Figure 2.

Method validation
The method was validated following ICH and FDA guidelines for system suitability, selectivity, specificity, linearity, sensitivity, accuracy, precision and robustness [22,23].

System suitability
System suitability parameters like peak tailing, plate count, resolution, and percent relative standard deviation for retention time and peak area response were calculated to demonstrate that the HPLC system performed well. For this study, standard solution (drospirenone-120 µg/mL, ethinyl estradiol-1.2 µg/mL and levomefolate-18.04 µg/mL) was injected into the HPLC system in five replicates. The obtained values were in the acceptable limits as given in Table 1.

Selectivity
The selectivity of the method was evaluated by comparison of chromatograms of blank mobile phase, placebo blank (mixture of excipients), tablet sample solution with standard solution. The representative chromatograms of the four samples are shown in Figure  3a HCl solution for acidic degradation acid or 10 mL of 0.1 N NaOH solution for alkaline degradation. The solutions were sonicated at room temperature for 30 min. After this period, the acid and alkali degraded solutions were neutralized with apt volume of 0.1 N NaOH and 0.1N HCl, respectively. The resulting solutions were diluted with mobile phase to get a concentration of 120 µg/mL, 1.2 µg/mL and 18.04 µg/ mL drospirenone, ethinyl estradiol and levomefolate, respectively. The solutions were filtered and injected.

Thermal and photo degradation
10 mL of tablet sample solution (drospirenone-1200 µg/mL, ethinyl estradiol-12 µg/mL and levomefolate-180.4 µg/mL) was transferred to volumetric flask (100 mL) and exposed to 105°C for 30 min in oven (for thermal degradation) or exposed to sun light for 24 hr (for photo degradation). After the specified period of degradation, the resulting solution was diluted with mobile phase for a concentration of 120 µg/mL, 1.2 µg/mL and 18.04 µg/mL drospirenone, ethinyl estradiol and levomefolate, respectively. The solutions were filtered and injected.

Oxidative and neutral degradation
10 mL of 30% hydrogen peroxide solution (for oxidative degradation) or 10 mL of deionised water (for neutral degradation) was added into a 100 mL volumetric flask containing 10 mL tablet sample solution (drospirenone-1200 µg/mL, ethinyl estradiol-12 µg/ mL and levomefolate-180.4 µg/mL). After sonication for 30 min at room temperature, the solutions were diluted to 100 mL with mobile phase until a concentration 120 µg/mL, 1.2 µg/mL and 18.04 µg/mL of drospirenone, ethinyl estradiol and levomefolate, respectively was obtained. These solutions were filtered and injected.

Optimization of HPLC conditions
Preliminary studies involved testing different mobile phase compositions, pH, flow rates, temperatures and detection wavelength for the effective separation and simultaneous analysis of drospirenone, ethinyl estradiol and levomefolate. Trial experiments were conducted using a Waters C18 column with a length of 250 mm, internal diameter of 4.6 mm and particle size of 5 μm as the stationary phase. Different in the tablets did not interfere with the peaks of drospirenone, ethinyl estradiol and levomefolate. This proved the method selectivity.

Specificity
Stress degradation was done to demonstrate the method specificity, stability of the drugs, detect the possible degradation products and stability indicating properties of the developed method. Stress degradation was carried out by exposing tablet sample solution to stress conditions of hydrolysis (acid, alkali and neutral), oxidation, photo and thermal. Stressed samples were analyzed by the proposed method. The corresponding peaks were checked for the peaks interference, retention times, and peak purity. The percentage of degradation in all the stressed samples was also determined.
The chromatograms of acid, base, hydrogen peroxide, heat, sun light and water degraded tablet sample solution are shown in Figure   4a-4e. The chromatograms showed no interference between the peaks of studied drugs (levomefolate, ethinyl estradiol, drospirenone) and the degradation product produced in the applied stress conditions. The percentage of recovery and degradation results of the forced degradation studies are summarized in Table 2. The applied stress conditions were enough to degrade the three drugs. The percent degradation value comparison of the three drugs showed that the order of stability is: levomefolate>ethinyl estradiol>drospirenone. The degradation product was observed at retention times of 2.882 min (acid hydrolysis, Figure 4a Figure 4f). The peak of degradation product is well resolved from the analytes peaks using the proposed method. The homogeneity of the peaks of studied drugs was checked using photodiode array detector. The results were shown in Table 2. The increased peak threshold value than peak purity angle value (Table  2) confirmed the purity and homogeneity of levomefolate, ethinyl estradiol, drospirenone peaks in all the stress conditions applied. The results of stress degradation studies proved the specificity and stability indicating properties of the developed HPLC method.

Sensitivity
The method sensitivity was determined with respect to limit of detection (LOD) and limit of quantitation (LOQ). The LOD and LOQ were assessed at a signal-to-noise ratio of 3:1 and 10:1, respectively using the developed method by analyzing different dilute solutions of drospirenone, ethinyl estradiol and levomefolate. The determined LOD values are 0.010 μg/mL, 0.109 μg/mL and 0.126 μg/mL for ethinyl estradiol, levomefolate and drospirenone, respectively. The determined LOQ values are 0.032 μg/mL, 0.363 μg/mL and 0.420 μg/mL for ethinyl estradiol, levomefolate and drospirenone, respectively. The values showed that the sensitivity of the method was good.

Precision
The precision of the HPLC method for drospirenone, ethinyl estradiol and levomefolate was evaluated by analyzing standard solution (drospirenone-120 µg/mL, ethinyl estradiol-1.2 µg/mL and levomefolate-18.04 µg/mL) six times. Percentage relative standard deviation (%RSD) of peak area response of the studied drugs was used to assess the precision. The results of precision exhibited %RSD below 0.5% (Table 3), indicating the excellent precision of the method.

Accuracy
The method accuracy for drospirenone, ethinyl estradiol and levomefolate was determined by analyzing standard solution (drospirenone-120 µg/mL, ethinyl estradiol-1.2 µg/mL and levomefolate-18.04 µg/mL) six times. The accuracy of the results was demonstrated by calculating the percent recovery. The results showed good accuracy performance for the determination of the three analytes (Table 4).

Recovery
The newly developed HPLC method was further evaluated for its accuracy by the analysis of the placebo spiked with pure drospirenone, ethinyl estradiol and levomefolate at three different concentration levels. Recovery of the spiked drospirenone, ethinyl estradiol and levomefolate was determined by the proposed method three times. The recovery values (Table 5), indicating that the developed method ensure the acquisition of reliable accurate data for drospirenone, ethinyl estradiol and levomefolate at different concentrations.
The study was carried out on the same day with standard solution      of concentration 120 µg/mL of drospirenone, 1.2 µg/mL of ethinyl estradiol and 18.04 µg/mL of levomefolate. In each case, resolution, plate count and peak tailing were calculated. The calculated values were within the acceptance limits (Table 6). Therefore, the method is considered as robust.

Conclusion
For the first time, a stability indicating HPLC with photodiode array detector method has been developed and validated for the simultaneous assay of drospirenone, ethinyl estradiol and levomefolate in bulk and tablet dosage form. All validation parameters satisfied the acceptance criteria of the ICH guideline. The developed method is good enough to separate the peaks of drospirenone, ethinyl estradiol and levomefolate from the degradation products produced during stress degradation. Therefore, it was concluded the developed and validated stability indicating method can be employed for the routine estimation of drospirenone, ethinyl estradiol and levomefolate in quality control laboratories and for stability studies.