alexa Method Development and Validation for Simultaneous Estimation of Ethinyl Estradiol and Drospirenone and Forced Degradation Behavior by HPLC in Combined Dosage Form | Open Access Journals
ISSN : 2153-2435
Pharmaceutica Analytica Acta
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Method Development and Validation for Simultaneous Estimation of Ethinyl Estradiol and Drospirenone and Forced Degradation Behavior by HPLC in Combined Dosage Form

Praveen C*, Ranganath MK and Divakar P

Department of Pharmaceutical Analysis, Krupanidhi College of Pharmacy, Bangalore, India

*Corresponding Author:
Praveen C
Department of Pharmaceutical Analysis
Krupanidhi College of Pharmacy
Bangalore, India
Tel: +91-959-103-1033
E-mail: [email protected]

Received date: April 01, 2013; Accepted date: April 23, 2013; Published date: April 25, 2013

Citation: Praveen C, Ranganath MK, Divakar P (2013) Method Development and Validation for Simultaneous Estimation of Ethinyl Estradiol and Drospirenone and Forced Degradation Behavior by HPLC in Combined Dosage Form. Pharmaceut Anal Acta 4:231. doi: 10.4172/2153-2435.1000231

Copyright: © 2013 Praveen C, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

A simple, accurate, rapid and precise isocratic High performance liquid chromatographic (HPLC) method was developed and validated for the determination of ethinyl estradiol and drospirenone in tablet formulation. The method employs Waters HPLC system on Thermo Hypersil BDS C18 Column (4.6×250 mm and 5 μm) and flow rate of 1.0 ml/min with a load of 15 μl. Acetonitrile and ammonium acetate buffer was used as mobile phase in the composition of 30:70. The detection was carried out at 258 nm. Linearity ranges for ethinyl estradiol and drospirenone were 0.06- 0.18 μg/ml, 6-18 μg/ml respectively. Retention Time of ethinyl estradiol and drospirenone were found to be 1.4 min, 5.3 min respectively. Percent Recovery study values of ethinyl estradiol and drospirenone were found to be within 97-103%. The combination product is exposed to acid/base, hydrolytic, photolytic and peroxide stress conditions and the stressed samples were analyzed. This developed method was successfully utilized for the quantitative estimation of ethinyl estradiol and drospirenone in pharmaceutical dosage forms. This method was validated for accuracy, precision, linearity and Robustness as per ICH guidelines.

Keywords

Ethinyl estradiol; Drospirenone; RP HPLC; Forced degradation

Introduction

Ethinyl estradiol is also known as ethynyl estradiol (EE) which is a derivative of 17β – estradiol. It is the first orally active semi synthetic steroidal estrogen that is used for the management of menopausal symptoms and female hypogonadism. Ethinyl estradiol is an orally bioactive estrogen used in almost all modern formulations of combined oral contraceptive pills. Chemically it is 19-Nor-17α-pregna-1, 3, 5(10)-trien-20-yne-3, 17-diol.

Drospirenone is an analogue of the antimineralocorticoid spironolactone that is synthesized from androstenone. Unlike other progestogens, drospirenone, an analogue of spironolactone, has biochemical and pharmacologic profiles similar to endogenous progesterone, especially regarding antimineralocorticoid and antiandrogenic activities. As a combination, oral contraceptive, drospirenone with ethinyl estradiol, is effective and has positive effects on weight and lipid levels (Figures 1 and 2) [1,2].

pharmaceutica-analytica-acta-Ethinyl-estradiol

Figure 1: Ethinyl estradiol.

pharmaceutica-analytica-acta-Drospirenone

Figure 2: Drospirenone.

Earlier publications have described spectroscopic and chromatographic methods for the quantification of ethinyl estradiol and drospirenone individually. A high-performance liquid chromatography (HPLC) methodology useful for the quantification of drospirenone in tablet dosage form was reported [3].

So far to our present knowledge, HPLC methods were available in the literature for analyzing ethinyl estradiol and drospirenone with other drug combinations in pharmaceutical dosage forms [4,5]. It felt necessary to develop a simple, precise and rapid spectrophotometric method for the quantitative determination of ethinyl estradiol and drospirenone in combined dosage form.

Forced degradation studies were used in the development of this methodology as a stability indicating parameter. The devised method was found to be selective, reliable, faster and straight forward than other reported methods. Though no attempt was made to identify the degradation products, described method can be used as stability indicating method for the assay of ETH and DRO in their combined dosage form.

Materials and Methods

Apparatus / Instruments used

S.No Name Model Manufacturer
1 HPLC 2695 Waters
2 Detector (PDA) 2998 Waters
3 UV-VIS Double Beam pectrophotometer 3200 Labindia
4 Sonicator - Labindia
5 Weighing balance Bsa224s-cw Sartorius

Chemicals / Reagents used

S.No Name Grade Manufacturer
1 Ammonium acetate - Fisher Scientific
2 Acetonitrile HPLC Merk
3 Methanol HPLC Merk
    HPLC Double Milli-QRO
4 Double distilled Water distilled purification system

Marketed formulation

Brand Name Content Marketed Company
Drostol™ Ethinyl estradiol & Drospirenone RANBAXY
  (0.03 mg & 3 mg respectively) Laboratories Ltd

Working standards

Pharmaceutical grade ethinyl estradiol and drospirenone were kindly supplied as a gift sample by Lara Drugs Private Limited, Hyderabad, Andhra Pradesh, India.

Method development

Preparation of mobile phase: The mobile phase was prepared by mixing ammonium acetate: acetonitrile in the ratio of (70:30) and was filtered and degassed.

Preparation of standard drug solutions: Accurately weigh and transfer 24 mg drospirenone and 0.24 mg ethinyl estradiol into a 50 ml volumetric flask. Add about 30 ml of methanol and sonicate to dissolve it completely and make volume up to the mark with the same solvent (stock solution). Further pipette out 5 ml of stock solution into a 10 ml volumetric flask and dilute up to the mark with methanol to get a solution of drospirenone (0.12 mcg/ml) and ethinyl estradiol (0.0012 mcg/ml).

Accurately weigh about 24 mg of the drospirenone and transfer into a 50 ml clean, dry standard volumetric flask, add 25 ml of methanol, sonicate for 30 minutes and make up with methanol (stock solution). Further pipette out 5 ml of stock solution into a 10 ml volumetric flask and dilute up to the mark with methanol to get a solution of drospirenone (0.12 mcg/ml).

Accurately weigh about 0.24 mg of the ethinyl estradiol and transfer into a 50 ml clean, dry standard volumetric flask, add 25 ml of methanol, Sonicate for 30 minutes and make up with methanol (stock solution). Further pipette out 5 ml of stock solution into a 10 ml volumetric flask and dilute up to the mark with methanol to get a solution of ethinyl estradiol (0.0012 mcg/ml).

Preparation of sample drug solution: Transfer 1250.8 mg (10 tablets) of marketed sample into a 50 ml of volumetric flask, add 25 ml of methanol, sonicate it for 10 minutes and make up with methanol (stock solution). Transfer 10 ml of stock solution into 50 ml of volumetric flask dilute to volume with methanol.

  Ethinyl estradiol Drospirenone
Parameters RT USP Tailing USP Plate Count RT USP USP Plate
FLOW1 1.805 1.706 3598 6.392 1.202 8468
FLOW2 1.209 1.75 3540 4.367 1.162 7074
TEMP1 1.45 1.704 3607 5.236 1.186 7752
TEMP2 1.447 1.753 3590 5.16 1.173 7891

Table 1: Robustness results for ethinyl estradiol and drospirenone.

Ethinyl estradiol Drospirenone
Sample Name RT Area RT Area
ACID 1.452 2125132 5.188 1507952
BASE 1.450 2266435 5.183 1591152
PEROXIDE 1.454 2263747 5.192 1578856
WATER 1.455 2227253 5.194 1545941
LIGHT 1.456 2281245 5.195 1583567
Mean   2232762   1561493
SD   63362   34558
%RSD   2.8   2.2

Table 2: Results of forced degradation studies.

Drug Conc.(µg/ml) Equation of regression line R2
ETH 0.06 – 0.18 Y= 29020x + 42584 0.998
DRO 6 – 18 Y= 20634x + 13023 0.999

Table 3: Linearity results for ethinyl estradiol and drospirenone.

Drug %RSD (intra-day) %RSD (inter-day)
ETH 0.717 0.91
DRO 1.414 0.57

Table 4: Precision results for ethinyl estradiol and drospirenone.

ETHINYL ESTRADIOL
Spiked Level Sample Weight Sample Area mcg/ml added mcg/ml found % Recovery % Mean
50% 625.40 1484085 0.059 0.06 101 101
50% 625.40 1476424 0.059 0.06 100
50% 625.40 1462429 0.059 0.06 99
50% 625.40 1489768 0.059 0.06 101
50% 625.40 1490425 0.059 0.06 101
50% 625.40 1483474 0.059 0.06 101
100% 1250.80 2943921 0.119 0.12 100 100
100% 1250.80 2915651 0.119 0.12 99
100% 1250.80 2957407 0.119 0.12 100
150% 1876.20 4423964 0.178 0.18 100 99
150% 1876.20 4409785 0.178 0.18 100
150% 1876.20 4420727 0.178 0.18 100
150% 1876.20 4302456 0.178 0.17 97
150% 1876.20 4427862 0.178 0.18 100
150% 1876.20 4338740 0.178 0.18 98

Table 5: Accuracy results for ethinyl estradiol.

Chromatographic Run: Standard solution containing a mixture of drospirenone and ethinyl estradiol was loaded into the injector and HPLC parameters were entered as per table 7. This method was saved and then sample was injected and run for 7 min.

Chromatographic conditions for the optimized method

Parameters Description
Column C18 Thermo Hypersil BDS (250×4.6×5 mm)
Mobile phase Ammonium acetate : Acetonitrile (70:30)
Injection volume 15 µl
Flow rate 1 ml/min
Detector Wavelength PDA at 258 nm
Column Temperature 40°C
Auto Sampler Temperature 25°C
Run Time 7 min

Results and Discussion

Validation

The method was validated for accuracy, precision, linearity, limit of detection, limit of quantitation and robustness as per ICH guidelines (Figures 3 and 4).

pharmaceutica-analytica-acta-standard-solution

Figure 3: Chromatogram of standard solution of ethinyl estradiol and Drospirenone.

pharmaceutica-analytica-acta-Chromatogram

Figure 4: Chromatogram of DROSTAL™ (Tablet Formulation).

Specificity and selectivity

It is the extent to which the procedure applies to analyte of interest and is checked by examining the formulation samples for any interfering peaks. The specificity of the method was evaluated with regard to interference due to presence of blank and any other excipients. The figure shows that drug was clearly separated from blank and its excipients. Figure 2 shows chromatogram for the formulation show that the selected drugs were clearly separated. Thus the proposed HPLC method is selective.

Forced Degradation studies of the drug product were carried out under stress conditions. The drug product in solution state was conducted with 0.1N HCl for 30 min. Base hydrolysis of drug product was conducted by 0.1N NaOH for 30 min. For oxidative stress, sample solutions of drug product in 3% hydrogen peroxide were kept (Tables 1 and 2).

Linearity

The linearity of the method was determined at five concentration levels ranging from 0.06 - 0.18 μg/ml for ethinyl estradiol and 6 - 18 μg/ml for drospirenone. The regression equation of calibration curves (Figures 3 and 6) were Y= 29020x + 42584 for ethinyl estradiol and Y= 20634x + 13023 for drospirenone and are summarized in table 3, Figures 5 and 6.

pharmaceutica-analytica-acta-Linearity-Ethinyl

Figure 5: Linearity of Ethinyl estradiol.

pharmaceutica-analytica-acta-Linearity-Drospirenone

Figure 6: Linearity of Drospirenone.

DROSPIRENONE
SpikedLevel SampleWeight SampleArea mcg/mladded mcg/mlfound %Recovery %Mean
50% 625.40 1000737 6.056 5.96 98 98
50% 625.40 997394 6.056 5.94 98
50% 625.40 987911 6.056 5.89 97
50% 625.40 1001888 6.056 5.97 99
50% 625.40 1007424 6.056 6.00 99
50% 625.40 1006174 6.056 6.00 99
100% 1250.80 2045007 12.120 12.19 101 101
100% 1250.80 2034841 12.120 12.13 100
100% 1250.80 2064497 12.120 12.30 102
150% 1876.20 3087552 18.178 18.40 101 102
150% 1876.20 3086164 18.178 18.39 101
150% 1876.20 3100722 18.178 18.48 102
150% 1876.20 3112915 18.178 18.55 102
150% 1876.20 3122673 18.178 18.61 102
150% 1876.20 3130111 0.178 18.65 103

Table 6: Accuracy results for Drospirenone.

Validation Parameters Ethinyl estradiol Drospirenone
Mobile Phase 70:30 (Ammonium Acetate: ACN) 70:30 (Ammonium Acetate: ACN)
Flow Rate 1 ml/min 1 ml/min
Detection wave Length PDA at 258 nm PDA at 258 nm
Rt 1.438 5.321
Run Time 7 min 7 min
Asymmetry 1.693 1.188
Theoretical Plates 3636 7728
LOD 0.00026 ppm 0.0925 ppm
LOQ 0.00087 ppm 0.308 ppm
Linearity R2=0.998 R2=0.999
Precision % RSD < 2 % RSD < 2
Recovery 99-101% 98-102%

Table 7: System suitability parameters for HPLC.

Precision

Precision of the method was studied as repeatability, intra-day and inter day variations. The intra-day precision was determined by analyzing ETH and DRO six times each on same day (intra-day study). This was repeated on the second day (inter-day study) and the results were shown in table 4, figures 7-10.

pharmaceutica-analytica-acta-ETH-DRO

Figure 7: Chromatogram of ETH and DRO [Flow variation 1].

pharmaceutica-analytica-acta-Chromatogram

Figure 8: Chromatogram of ETH and DRO [Flow variation 2].

pharmaceutica-analytica-acta-ETH

Figure 9: Chromatogram of ETH and DRO [Temperature variation 1].

pharmaceutica-analytica-acta-DRO

Figure 10: Chromatogram of ETH and DRO [Temperature variation 2].

Accuracy

The accuracy of the method was determined by recovery studies. The recovery studies were performed by standard addition method at 50% for six times, 100% for three times, 150% for six times and summarized in tables 5 and 6.

Limit of detection (LOD) and Limit of quantitation (LOQ)

LOD is the ability of analytical method to detect the lowest concentration of the analyte. LOQ is the lowest concentration of the analyte with acceptable precision and accuracy. It can be calculated based on the signal to noise ratio. The LOD of ETH and DRO were 0.00026 ppm and 0.0925 ppm. The LOQ of ETH and DRO were 0.00087 ppm and 0.308 ppm respectively.

Robustness

Robustness of the method was determined by making slight changes in the flow rate and column temperature. It was observed that there were no marked changes in the retention time and area of the chromatograms which demonstrated that the RP HPLC method developed was robust and data are summarized in table 1.

Conclusion

The Proposed RP-HPLC and UV-Spectrophotometric method were suitable techniques for simultaneous determination of Ethinyl estradiol and Drospirenone in combined dosage combinations without any interferences form each other and excipients. All the parameters for both the drugs had met the criteria of ICH guidelines for method validation. The low values of % RSD indicate the method is precise and accurate.

From the forced degradation studies it can be concluded that there is no other co-eluting peak with the main peaks and the method is specific for the estimation of Ethinyl estradiol and Drospirenone in presence of its degradation products and impurities. Result of validation parameter demonstrates that the analytical procedure is suitable for its intended purpose and meets the criteria defined in ICH Q2A/B.

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