Simultaneous estimation of uv spectroscopy and method development and validation for drospirenone and ethinyl estradiol by RP-HPLC

Simultaneous estimation of uv spectroscopy and method development and validation for drospirenone and ethinylestradiol by RP-HPLC

1. SIMULTANEOUS ESTIMATION OF UV SPECTROSCOPY AND METHOD DEVELOPMENT AND VALIDATION FOR DROSPIRENONE AND ETHINYL ESTRADIOL BY RP-HPLC

2. Guide Dr.V.KIRAN KUMAR, M.Pharm,Ph.D Principal & Professor Project Submitted T o Osmania University , Hyderabad Presented By R. Shiva sai kiran H. No : 25641 8 8810 04 B.Pharmacy 4 th year MOTHER TERESA COLLEGE OF PHARMACY

3. INTRODUCTION Analytical chemistry is a branch of chemistry involved in separating, identifying and determining the relative amounts of the components making up a sample of matter. It is mainly involved in the qualitative analysis or detection of compounds and quantitative analysis of the compounds. A qualitative method yields information about the identity of atomic or molecular species or functional groups in the sample. A quantitative method, in contrast provides numerical information as to the relative amount of one or more of these components.

4. CLASSIFICATION OF ANALYTICAL METHODS Analytical methods are classified into two categories; they are classical methods and instrumental methods. Classical methods Analysis of substances was carried out by separating the components of interest in a sample by precipitation, extraction or distillation. For qualitative analysis, the separated components were then treated with reagents that yielded products that could be recognized by their colors, their boiling or melting points, their solubilities in a series of solvents, their optical activities, their odors or their refractive indexes. For quantitative analysis, the amount of analyte was determined by gravimetric or by volumetric measurements.

5. Instrumental methods Measurement of physical properties of analytes such as conductivity, electrode potential, light absorption or emission, fluorescence, mass-to-ratio began to be used for quantitative analysis of various inorganic and biochemical analytes. Highly efficient chromatographic and electrophoretic techniques began to replace distillation, extraction and precipitation for the separation of components of complex mixtures prior to their qualitative and quantitative determination. These newer methods for separating and determining chemical species are collectively known as instrumental methods of analysis. Most of the instrumental methods fit into one of the following three categories viz.., spectroscopy, electrochemistry and chromatography.

6. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) A variety of methods are available for analyzing pharmaceutical compounds. High Performance/Pressure Liquid Chromatography (HPLC) is one of the best methods of choice for analyzing a variety of natural and synthetic compounds. It is because it offers high performance over ambient pressure. The phenomenal growth in chromatography is largely due to the introduction of the technique called high-pressure liquid chromatography, which is frequently called high-performance liquid chromatography (both are abbreviated as HPLC).

7. It allows separations of a large variety of compounds by offering some major improvements over the classical column chromatography, TLC, GC; and it presents some significant advantages over more recent techniques such as supercritical fluid chromatography (SFC), capillary electrophoresis (CE), and electro kinetic chromatography. Chromatographic separations are based on a forced transport of the liquid (mobile phase) carrying the analyte mixture through the porous media and the differences in the interactions at analytes with the surface of this porous media resulting in different migration times for a mixture components.

8. High surface area of the interface between mobile and stationary phases is essential for space discrimination of different components in the mixture. Analyte molecules undergo multiple phase transitions between mobile phase and adsorbent surface. Average residence time of the molecule on the stationary phase surface is dependent on the interaction energy. The nature of the stationary and the mobile phases, together with the mode of the transport through the column, is the basis for the classification of Chromatographic methods .

9. TYPES OF HPLC : The four main types of HPLC techniques are 1. Normal-Phase Chromatography. 2. Reversed-Phase Chromatography. 3. Ion-Exchange Chromatography. 4. Size-Exclusion Chromatography.

10. Reversed-Phase Chromatography Reversed-Phase Chromatography, the most widely used chromatographic mode, is used to separate neutral molecules in solution on the basis of their hydrophobicity. As the name suggests, Reversed-Phase Chromatography is the reverse of Normal-Phase Chromatography in the sense that it involves the use of a non-polar stationary phase and a polar mobile phase. As a result, a decrease in the polarity of the mobile phase results in a decrease in solute retention .

11. Reversed-Phase Chromatography is the most popular mode for the separation of low molecular weight (<3000), neutral species that are soluble in water or other polar solvents. It is widely used in the pharmaceutical industry for separation of species such as steroids, vitamins, and β-blockers. Because of the mobile phase in Reversed-Phase Chromatography is polar, Reversed-Phase Chromatography is suited to the separation of polar molecules that either are insoluble in organic solvents or bind too strongly to the polar, normal-phase materials.

12. REVIEW OF LITERATURE   Andi Suhendi et al (2016) The objective of this research was to develop and validate a simple, specific and accurate reverse phase of high performance of liquid chromatographic method for the determination of levonorgestrel (LVG) and ethinylestradiol (EE) in tablets. The chromatographic system included column SunFire ODS (150 mm × 4.6 mm i.d., particle size at 5 μm), mobile phase consisting of acetonitrile: methanol: aquabidest (60:15:25) with the flow rate of 1 mL/minute and effluents monitored at 230 nm. The validation of RP HPLC method for the simultaneous determination of LVG and EE was determined by accuracy, precision, linearity, and limit of detection (LOD) as well as the limit of quantitation (LOQ) parameters.

13. Tejal Rathod et al (2014 ) A simple, accurate, precise, sensitive, specific and reliable stability indicating RP- HPLC method was developed for simultaneous estimation of Ethinyl estradiol (EE) and Levonorgestrel (LEV) in Pharmaceutical dosage form. The developed method with mobile phase Acetonitrile: Water (75: 25), Analytica brownee C-18 (150×4.6 mm, 3µm particle size) as a stationary phase and flow rate was 0.8 ml/min. Detection was carried out at 230 nm in PDA detector .

14. The calibration curve of Ethinyl estradiol and Levonorgestrel was found to be linear in the range of 4-14 µg/ml and 20-70 µg/ml respectively. The proposed method has been validated for precision, accuracy, robustness. As the proposed method can effectively separate the drugs from all their degradation products, it can be employed as stability indicating method.

15. DRUG PROFILE DROSPIRENONE IUPAC Name: (1R,2R,4R,10R,11S,14S,15S,16S,18S,19S)-10,14 dimethylspiro[ hexacyclo [9.8.0.0², ⁴ .0 ⁵ ,¹ ⁰ .0¹ ⁴ ,¹ ⁹ .0¹ ⁶ ,¹ ⁸ ]nonadecane-15,2'-oxolan]-5-ene-5',7- dione

16. Chemical formula : C24H30O3   Molecular weight : 366.4932 g/mol. Category : Contraceptive Agents, Female   BRAND NAME : Slynd Drospirenone and ethinyl estradiol in combination suppress the release of follicle stimulating hormone (FSH) and luteinizing hormone (LH), preventing ovulation. Other changes induced by this drug which may aid in the prevention of pregnancy include alterations in cervical mucus consistency, hindering sperm movement, and lowering the chance of embryo implantation.

17. ETHINYL ESTRADIOL Iupac name : (1S,10R,11S,14R,15S)-14-ethynyl-15- methyltetracyclo [8.7.0.0², ⁷ .0¹¹,¹ ⁵ ]heptadeca-2(7),3,5-triene-5,14-diol Molecular formula : C20H24O2  Molecular weight : 296.4034 Category : Cytochrome P-450 CYP2C8 Inhibitors,Cytochrome P-450 CYP3A4 Substrates Brand name: Ethinorm E  

18. AIM AND OBJECTIVE Literature review reveals that there is no analytical method reported for the analysis of Drospirenone and Ethinyl Estradiol by simultaneous estimation by RP-HPLC. Spectrophotometer, HPLC and HPTLC are the reported analytical methods for compounds either individually or in combination with other dosage form. Hence, it was felt that, there is a need of new analytical method development for the simultaneous estimation of Drospirenone and Ethinyl Estradiol in pharmaceutical dosage form.

19. Present work is aimed to develop a new, simple, fast, rapid, accurate, efficient and reproducible RP-HPLC method for the simultaneous analysis of Drospirenone and Ethinyl Estradiol. The developed method will be validated according to ICH guidelines. Objective of the work The analytical method for the simultaneous estimation of Drospirenone and Ethinyl Estradiol will be developed by RP-HPLC method by optimizing the chromatographic conditions. The developed method is validated according to ICH guidelines for various parameters specified in ICH guidelines, Q2 (R1).  

20. PLAN OF WORK To develop a new analytical method for the simultaneous estimation of Drospirenone and Ethinyl Estradiol by RP-HPLC. The dissertation work has been carried out in the following steps: Selection Of Drug Literature Review Analytical Method Development:- Selection of λ max Selection of column Selection of mobile phase

21. Selection of flow rate Optimized chromatographic Analytical Method Validation

22. EXPERIMENTAL WORK

23. Materials and Methods List of chemicals and standards used S.No Chemicals Manufacturer Name Grade 1. Water Merck HPLC grade 2. Methanol Merck HPLC grade 3. Acetonitrile Merck HPLC grade 4. Ortho phosphoric acid Merck G.R 5. KH 2 PO 4 Merck G.R 6. K 2 HPO 4 Merck G.R 7. 0. 22µ Nylon filter Advanced lab HPLC grade 8. 0.45µ filter paper Millipore HPLC grade 9. Drospirenone and Ethinyl Estradiol In - House In- House

24. Instruments used S.No Instrument name Model number Soft ware Manufacturers Name   1 HPLC-auto sampler –UV detector Separation module2695, UV.detector2487 Empower- software version-2   Waters 2 U.V double beam spectrometer UV 3000+ U.V win soft ware Lab India   3 Digital weighing balance(sensitivity 5mg)   ER 200A   -   Ascoset 4 pH meter AD 102U - ADWA 5 Sonicator SE60US - Enertech

25. Method development for the simultaneous estimation of Drospirenone and Ethinyl Estradiol by using RP-HPLC. Selection of mobile phase Selection of detection wavelength Selection of column Selection of solvent delivery system Selection of flow rate Selection of column temperature Selection of diluent Selection of test concentration and injection volume

26. Selection of mobile phase Sodium acetate buffer : Methanol (30 : 70% v/v) Buffer pH should be between 2 to 8. Below 2: siloxane linkages are cleaved. Above 8: dissolution of silica. pH selected: 3 ±0.05 pH controls the elution properties by controlling the ionization characteristics. Reasons: To decrease the retention and improve separation. Good Response, Area, Tailing factor, Resolution.

27. Selection of wavelength: 10 mg of Drospirenone and Ethinyl Estradiol was dissolved in mobile phase. The solution was scanned from 200-400 nm the spectrum was obtained. The overlay spectrum was used for selection of wavelength for Drospirenone and Ethinyl Estradiol. The isobestic point was taken as detection wavelength. The overlay spectrums are shown in Fig.

28. Selection of column Heart of HPLC made of 316 grade stainless steel packed with stationary phase. Silica based columns with different cross linking’s in the increasing order of polarity are as follows: ß ------- Non-polar----------moderately polar--------Polar à C18< C8< C6< Phenyl < Amino < Cyano < Silica In reverse phase chromatography, hydrophobic interaction between drug molecule and the alkyl chains on the column packing material. Column is selected based on solubility, polarity and chemical differences among analysts and Column selected: i.e. Thermosil C18 column (4.6 x125 mm) 5 µ

29.   Selection of solvent delivery system Always preferable solvent delivery system. More chance of getting reproducible result on retention time of analytes. More economic than gradient technique. Selection of flow rate Acceptable limit: - Not more than 2.5 ml/min Flow rate selected was 1ml/min Flow rate is selected based on Retention time, Column back pressure, Peak symmetry, Separation of impurities.

30. Selection of diluent Selection of diluent is based on the solubility of the analyte Diluent selected: Sodium acetate buffer : Methanol (30 : 70% v/v) 7 Selection of column temperature: Preferable temperature is ambient or room temperature. Reasons: To elute all impurities along with analyte with in 10.0 min of run time. Less retention time Good peak shape Higher theoretical plates. Good resolution.

31. Selection of test concentration and injection volume Test concentration is finalized after it is proved that API is completely extractable at the selected test concentration. Test concentration is fixed based upon the response of API peak at selected detector wavelength. And the test concentration selected is 10 ppm. Injection volume selected was 10µL. Reason: good peak area, retention time, peak symmetry.

32. Chromatographic trials for simultaneous estimation of Drospirenone and Ethinyl Estradiol by RP- HPLC. Chromatogram showing trial-1 injection

33. Chromatographic conditions   Column : Thermosil C18 4.6x150mm, 5µm Mobile phase ratio : MeOH: H2O (60:40%v/v) Detection wavelength : 252 nm Flow rate : 1ml/min Run time : 10min   OBSERVATION The trial shows no proper separation peaks in the chromatogram, so more trials were required for obtaining peaks

34. Trial – 2( Chromatogram showing trial-2 injection) Retention time : 4.015 min & 4.638 mins Observation In this trial two peaks were separated but don’t have proper resolution. Still more trials were required for proper peaks

35. Procedure Preparation of phosphate buffer :- 6.8 grams of sodium acetete was weighed and taken into a 1000ml beaker, dissolved and diluted to 1000ml with HPLC water and pH was adjusted to 3 with orthophosphoric acid. The resulting solution was sonicated and filtered. Preparation of mobile phase :- Mix a mixture of above buffer 30 ml (30%) and 70 ml of Methanol (HPLC grade- 70%) and degassed in ultrasonic water bath for 5 minutes. Filter through 0.22 µ filter under vacuum filtration. Diluents preparation :- Mobile phase was used as the diluent.

36. Preparation of the individual Ethinyl Estradiol standard preparation :- 10 mg of Ethinyl Estradiol working standard was accurately weighed and transferred into a 10 ml clean dry volumetric flask and add about 2 ml of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution).Further pipette out 1.5 ml from the above stock solution into a10 ml volumetric flask and was diluted up to the mark with diluent. Preparation of the individual Drospirenone standard preparation:- 10 mg of Drospirenone working standard was accurately weighed and transferred into a 10 ml clean dry volumetric flask and add about 2ml of diluent and sonicate to Dissolve it completely and make volume up to the mark with the same solvent (Stock solution).Further pipette out 3 ml from the above stock solution into a 10 ml volumetric flask and was diluted up to the mark with diluent.

37. Assay Assay preparation of the Drospirenone and Ethinyl Estradiol standard and sample solution Sample solution preparation: 1mg of Ethinyl Estradiol and 10 mg Drospirenone tablet powder were accurately weighed and transferred into a 10 ml clean dry volumetric flask, add about 2ml of diluent and sonicate to dissolve it completely and making volume up to the mark with the same solvent(Stock solution). Further pipette 10ml of the above stock solution into a 100ml volumetric flask and was diluted up to the mark with diluent Standard solution preparation :- 1mg Ethinyl Estradiol and 10 mg Drospirenone working standard was accurately weighed and transferred into a 10ml clean dry volumetric flask and add about 2ml of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution).Further pipette out 1ml of the above stock solution into a 10ml volumetric flask and was diluted up to the mark with diluent.

38. Procedure :- 10 m L of the blank, standard and sample were injected into the chromatographic system and areas for the Drospirenone and Ethinyl Estradiol the peaks were used for calculating the % assay by using the formulae. System suitability:- Tailing factor for the peaks due to Drospirenone and Ethinyl Estradiol in standard solution should not be more than 1.5. Theoretical plates for the Drospirenone and Ethinyl Estradiol peaks in standard solution should not be less than 2000 .

39. ANALYTICAL METHOD VALIDATION Validation parameters Specificity Linearity Range Accuracy Precision Repeatability Intermediate Precision Detection Limit Quantitation Limit Robustness

40. RESULTS AND DISCUSSIONS The present investigation reported in the thesis was aimed to develop a new method development and validation for the simultaneous estimation of Drospirenone and Ethinyl Estradiol by RP-HPLC method. Literature reveals that there are no analytical methods reported for the simultaneous estimation Drospirenone and Ethinyl Estradiol by RP-HPLC method. Hence, it was felt that, there is a need of new analytical method development for the simultaneous estimation of Drospirenone and Ethinyl Estradiol in pharmaceutical dosage form.

41. M ethod Development The detection wavelength was selected by dissolving the drug in mobile phase to get a concentration of 10µg/ml for individual and mixed standards. The resulting solution was scanned in U.V range from 200-400nm. The overlay spectrum of Drospirenone and Ethinyl Estradiol was obtained and the isobestic point of Drospirenone and Ethinyl Estradiol showed absorbance’s maxima at 252 nm. The chromatographic method development for the simultaneous estimation of Drospirenone and Ethinyl Estradiol were optimized by several trials for various parameters as different column, flow rate and mobile phase, finally the following chromatographic method was selected for the separation and quantification of Drospirenone and Ethinyl Estradiol in API and pharmaceutical dosage form by RP- HPLC method .

42. The spectrums are shown in Fig. Spectrum showing overlapping spectrum of Drospirenone and Ethinyl Estradiol

43. Optimized chromatographic conditions for simultaneous estimations of Drospirenone and Ethinyl Estradiol by RP-HPLC method . Column : Thermosil C18 (4.0×125 mm) 5.0 µm Column temperature : Ambient Wavelength : 252 nm Mobile phase ratio : 70:30 Methanol: Sodium acetate buffer Flow rate : 0.7 ml/min Auto sampler temperature : Ambient Injection volume : 10µl Run time : 10.0 minutes

44. Assay calculation for Drospirenone and Ethinyl Estradiol The assay study was performed for the Drospirenone and Ethinyl Estradiol . Each three injections of sample and standard were injected into chromatographic system. The chromatograms showing assay of sample injection-1,2.

45.   Name RT Area Height(µV) USP   Tailing USP   plate count 1 Drospirenone 2.582 5224568 546584.3 1.4 4.255.7 2 Drospirenone 2.577 5237671 584237.2 1.4 4.882.2 3 Drospirenone 2.568 5248562 575438.3 1.4 4.253.4 4 Drospirenone 2.566 5347231 553245.2 1.4 4.625.3 Mean     5344012       Std.Dev     3252.4       %RSD     0.37      

46. Chromatogram showing assay of Standared injection -1,2

47.   Name RT Area Height(µV) USP   Tailing USP   plate count 1 Ethinyl   Estradiol 3.407 2744568 246582.3 1.4 5255.7 2 Ethinyl   Estradiol 3.408 2737671 234238.2 1.4 5882.2 3 Ethinyl   Estradiol 3.418 2765584 235726.2 1.4 5772.3 4 Ethinyl   Estradiol 3.417 2722354 234482.7 1.4 5442.5 Mean     5344012       Std.Dev     3252.4       %RSD     0.42      

48. The retention time of Drospirenone and Ethinyl Estradiol was found to be 2.566 mins and 3.417 mins respectively. The system suitability parameters for Drospirenone and Ethinyl Estradiol such as theoretical plates and tailing factor were found to be 4678, 1.3 and 6287, 1.2. the % purity Drospirenone and Ethinyl Estradiol in pharmaceutical dosage form was found to be 99.24 and 101.2% respectively.

49. VALIDATION REPORT Specificity Linearity Accuracy Precision Repeatability Intermediate precision/Ruggedness Repeatability Detection limit Robustness

50. Specificity The specificity test was performed for Drospirenone and Ethinyl Estradiol. Chromatogram showing standard injection

51. Chromatogram showing sample injection It was found that there was no interference of impurities in retention time of analytical peak.

52. Linearity :- Chromatograms showing linearity level-1 to level 5 (5ppm-25 ppm of Drospirenone and 50ppm -250ppm of Ethinyl Estradiol ) injections. Drospirenone r 2 = 0.999

53. S.No Linearity Level Concentration Area 1 I 5 ppm 471543 2 II 10 ppm 656277 3 III 15 ppm 794999 4 IV 20 ppm 946124 5 V 25 ppm 1002139 Correlation Coefficient 0.999

54. Linearity Results for Ethinyl Estradiol S.No Linearity Level Concentration Area 1 I 50ppm 56472 2 II 100 ppm 73841 3 III 150ppm 92655 4 IV 200ppm 111541 5 V 250ppm 130567 Correlation Coefficient 0.999 140000 120000 y = 371.78x + 37248 R² = 0.9997 100000 80000 60000 Series1 Linear (Series1) 40000 20000 50 100 150 200 250 300

55. Accuracy The accuracy study was performed for 50%, 100% and 150 % for Drospirenone and EthinylEstradiol.Each level was injected in triplicate into chromatographic system. Chromatograms showing accuracy-50% and 150% injection-1 ,2,3

56. Chromatogram showing accuracy -100% injection-1,2,3.

57. Showing accuracy results for Drospirenone %Concentratio n (at specification level)     Average area   Amount added (mg)   Amount found (mg)     %   Recovery     Mean recovery 50% 2630409 5 4.96 99.91%     99.56% 100% 5277055 10 9.98 99.18% 150% 7514836 15 15.02 99.60%

58. Showing accuracy results for Ethinyl Estradiol : %Concentration   (at specification level)   Average area Amount added (mg) Amount found (mg)   %   Recovery   Mean recovery 50% 1366666 0.5 0.99 99.53%     99.47% 100% 2777487 1.0 1.05 99.38% 150% 4151234 1.5 1.495 99.52% The accuracy study was performed for % recovery of Drospirenone and Ethinyl Estradiol. The % recovery was found to be 99.56% and 99.47% respectively (NLT 98% and NMT 102%)

59. Precision Repeatability:- The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits. Showing% RSD results for Drospirenone -

60. Showing %RSD results for Ethinyl Estradiol   The Method precision study was performed for the %RSD of Drospirenone and Ethinyl Estradiol was found to be 0.82 and 0.86 (NMT 2).

61. Intermediate precision/Ruggedness Showing results for intermediate precision of Drospirenone

62. The intermediate precision was performed for %RSD of Drospirenone and Ethinyl Estradiol was found to be 0.19 and 0.44 respectively (NMT 2).

63. Detection li m it LOD’s can be calculated based on the standard deviation of the response (SD) and the slope of the calibration curve (S) at levels approximating the LOD according to the formula. The standard deviation of the response can be determined based on the standard deviation of y-intercepts of regression lines. Showing R esults F or Limit O f Detection

64. Showing results for Limit of Detection : Drug name Standard deviation(σ) Slope(s) LOD(µg) Drospirenone 373625.50 581075863 3.17 Ethinyl   Estradiol   5772.40   476579210   0.0172 The LOD was performed for Drospirenone and Ethinyl Estradiol was found to be 3.17and 0.0172 respectively.

65. Quantitation limit LOQ’s can be calculated based on the standard deviation of the response (SD) and the slope of the calibration curve (S) according to the formula. Again, the standard deviation of the response can be determined based on the standard deviation of y- intercepts of regression lines. Showing results for Limit of Quantitation

66. Drug name Standard deviation(σ) Slope(s) LOQ(µg) Drospirenone 372727.80 574265980 5.80 Ethinyl   Estradiol   5761.30   478828490   0.212 The LOQ was performed for Drospirenone and Ethinyl Estradiol was found to be 5.80 and 0.212 respectively.

67. Robustness The robustness was performed for the flow rate variations from 0.4ml/min to 0.6ml/min and mobile phase ratio variation from more organic phase to less organic phase ratio for Drospirenone and Ethinyl Estradio . The method is robust only in less flow condition and the method is robust even by change in the Mobile phas e ±5%. Chromatogram showing less flow rate 0.8ml/min

68. Fig.No.63. Chromatogram showing less flow rate 1.2 ml/min   The results are summarized on evaluation of the above results, it can be concluded that the variation in flow rate affected the method significantly. Hence it indicates that the method is robust even by change in the flow rate ±0.2ml/min. The method is robust only in less flow condition.

69.   S. No   Flow rate (ml/min) System suitability results USP Plate Count SP Tailing 1 0.8 5339 1.4 2 1 4668 1.3 3 1.2 5216 1.4     S.No     Flow rate (ml/min) System suitability results USP Plate Count SP Tailing 1 0.8 7036 1.3 2 1 6089 1.2 3 1.2 6998 1. 3 Table.No.18. Showing system suitability results for Drospirenone Table.No.19. Showing system suitability results for Ethinyl Estradiol

70. CONCLUSION A new method was established for simultaneous estimation of Drospirenone and Ethinyl Estradiol by RP-HPLC method. The chromatographic conditions were success fully developed for the separation of Drospirenone and Ethinyl Estradiol by using Thermosil C18 column (4.0×125mm) 5µ, flow rate was 0.7 ml/min, mobile phase ratio was (70:30 v/v) methanol: Sodium acetate buffer pH 3 (pH was adjusted with orthophosphoricacid ), detection wavelength was 252nm. The instrument used was WATERS HPLC Auto Sampler, Separation module 2690, photo diode array detector 996, Empower-software version-2. The retention times were found to be 2.566 mins and 3.417 mins . The % purity of Drospirenone and Ethinyl Estradiol was found to be 101.27% and 99.97% respectively.

71. The system suitability parameters for Drospirenone and Ethinyl Estradiol such as theoretical plates and tailing factor were found to be 4668, 1.3 and 6089 and 1.2, the resolution was found to be 6.0. The analytical method was validated according to ICH guidelines (ICH, Q2 (R1)). The linearity study n Drospirenone and Ethinyl Estradiol was found in concentration range of 5µg-25µg and 50µg-250µg and correlation coefficient (r 2 ) was found to be 0.999 and 0.999, % recovery was found to be 99.56% and 99.48%, %RSD for repeatability was 0.86 and 0.82, % RSD for intermediate precision was 0.44 and 0.19 respectively. The precision study was precise, robust, and repeatable.LOD value was 3.17 and 5.68, and LOQ value was 0.0172 and 0.2125 respectively. Hence the suggested RP-HPLC method can be used for routine analysis of Drospirenone and Ethinyl Estradiol in API and Pharmaceutical dosage form .

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