A Spectrophotometric Assay for the Simultaneous Analysis of Mifepristone and Misoprostol in Tablets Using Vierodt’s and Absorbance Ratio Methods

 

Devadasu C.H.*, Harika S., Mallikarjuna T., Adilakshmi G., Sreenath A. and Swetha Ratna G.

Department of Pharmaceutical Analysis, Vignan Pharmacy College, Vadlamudi, Guntur-522 213, AP, India.

Corresponding author: devdaspharma@gmail.com

 

 

ABSTRACT:

Two simple, accurate and rapid methods have been developed for the quantitative determination of mifepristone and misoprostol in their dosage form. Method-I is based on constructing and solving two simultaneous equations at 304nm and 257.6nm for mifepristone and misoprostol respectively. In the quantitative assay of two components in admixture by the absorbance ratio method, absorbances are measured at 264.6nm (iso-absorptive point) and 304nm (Method-II). These two methods were validated in terms of linearity, accuracy, precision, limit of detection, limit of quantitation and robustness. The linearity was in the concentration range of 8-24µg/ml for both the methods.

 

KEYWORDS: Mifepristone, Misoprostol, Simultaneous equations, Absorbance ratio, Spectrophotometry

 

INTRODUCTION:

Mefipristone1-2 is a progesterone receptor antagonist with partial agonistic activity. Mefipristone is indicated in combination with misoprostol for the medical termination of pregnancy of first trimester. Chemically it is (11β-17 β)-11-[4-(dimethylamino) phenyl]-17-hydroxy-(propynyl) estra-4, 9-dien-3-one. Misoprostol3-4 is a cytoprotective prostaglandin PGE1 analogue, exhibits uterotonic and ervical-ripening actions and therapeutically it is an antiulcerative agent. Chemically misoprostol is (11α, 13E)-11, 16-dihydroxy-16-methyl-9-ooxoprost-13-en-1-oic acid methyl ester. Medical termination of an early pregnancy using a combination of mifepristone followed by synthetic prostaglandin is effective and safe 5

 

Many pharmaceutical industries are manufacturing these two drugs as a combinational dosage form or a kit of both the drugs for the effectiveness of medical termination of pregnancy.

 

A thorough literature survey reveals that there were reported some analytical methods which include a radio chromatographic assay for misoprostol with iloprost6, a HPLC-UV method with solid-phase microextraction7, A HPLC, HPLC-RIA and HPLC using norethisterone internal standard methods   for mifepristone in human plasma8-10, a HPLC method for mifepristone in wild canid serum11 ,  a supercritical fluid extraction –liquid chromatography method12, a LC-MS method for misoprostol in human plasma13, a GC-MS14 method to determine misoprostol human breast milk       and a HPLC method to determine stability and recovery of carboprost and misoprostol  in infuision preparations15

 

EXPERIMENTAL DETAILS:

Materials and methods:

A Systronics Double beam UV- visible spectrophotometer 2203 with 1 cm matched quartz cells was used for all spectral and absorbance measurements. All the chemicals and reagents used were of analytical grade and solutions were prepared in double distilled water. Mifepristone pure material is a gift sample of Hetero Pharmaceutical industries ltd Hyderabad and misoprostol is a gift sample of Sun Pharma ltd., the commercial formulations (tablets) of these drugs in their combination were procured from the local market.

 

RECOMMENDED PROCEDURE:

Method-I, Simultaneous Equation Method16-17 (Vierodt’s Method):

Standard stock solutions containing 100µg/ml of mifepristone and 100µg/ml of misoprostol were prepared in ethanol in separate volumetric flasks. From both the stock solutions, different concentrations of 4, 8,12,16,20 and 24 µg/ml were prepared in ethanol and the absorbances were recorded at 304nm and 257.6nm for mifepristone and misoprostol respectively. The absorptivities(A1%,1cm) of both the drugs at their respective  wavelengths were calculated. Similarly recorded the absorbances of the sample solution (formulation) containing both the drugs  at the two above specified wavelengths. The absorbances of mixture and absorptivities of the standard solutions at their wavelengths were substituted in the following equations to obtain the concentration of individual drugs present in the mixture.

            A2. ay1 – A1. ay2

CX=    -------------------------

             ax2.ay1– ax1.ay2

 

            A1. ax2 – A2. ax1

CY=    -------------------------

          ax2.ay1 – ax1.ay2

Where

A1, A2 are the absorbance of the mixture at λ1(304nm) and λ2(257.6nm)

ax1 and  ax2 are the absorptivities of mifepristone at λ1(304nm) and λ2(257.6nm)

ay1 and ay2 are the absorptivities of misoprostol  at λ1(304nm) and λ2(257.6nm)

CX and CY are the concentrations (g/100ml) of mifepristone and misoprostol

 

Method-II, Absorbance Ratio Method18,19:

Standard stock solutions containing 100µg/ml of mifepristone and 100µg/ml of misoprostol were prepared in ethanol in separate volumetric flasks. from the overlain spectra of the two drugs, two wavelengths were selected i.e. 264nm (isoabsorptive point for two drugs) and 304nm (absorption maximum of mifepristone). From both the stock solutions, different concentrations of 4, 8,12,16,20 and 24 µg/ml were prepared in ethanol and the absorbances were recorded at selected wavelengths. The absorptivities(A1%,1cm) of both the drugs at their respective  wavelengths were calculated. Similarly recorded the absorbance of the sample solution (formulation) containing both the drugs at the two above specified wavelengths. The absorbance of mixture and absorptivities of the standard solutions at their wavelengths were substituted in the following equations to obtain the concentration of individual drugs present in the mixture.

 

              QM – QY

CX =    -------------- (A1/ax1)

              QX– QY

 

             QM – QX

CY =    -------------- (A1/ay1)

             QX– QY

Where,

CX and CY are the concentrations (g/100ml) of mifepristone and misoprostol

A1= Absorbance of sample at isoabsorption point (264nm)

ax1anday1are the absorptivities of mifepristone and misoprostol at isoabsorption   point

QX= Ratio of absorptivities of mifepristone at two wavelengths

QY= Ratio of absorptivities of misoprostol at two wavelengths

QM= Ratio of absorbance of mixture at two wavelengths

 

RESULTS AND DISCUSSION:

The optical characteristics concerning to the proposed methods and the results of the assay of the marketed formulation are presented in table no.1, 2 and 3.

 

Combined Absorption Spectrum Of Mifepristone And Misoprostol

 

Absorption Spectrum Of Mifepristone In Ethanol 40mcg

 

Absorption spectrum of misoprostol in ethanol 40mcg

 

Table no. 1: Assay and Precision results of Mifepristone and Misoprostol in Combined Dosage Form

Drug

Label Claim

%Drug Found by the Proposed Methods

% RSD

M1±S.D*

M2±S.D*

M1

M2

Mifepristone

200mg/tab

98.31±0.276

100.49±0.541

0.2807

0.538

misoprostol

200µg/tab

97.48±0.365

100.38±0.521

0.374

0.519

*=Average of six determinations

 

Table no. 2: Accuracy and Recovery Studies of Mifepristone and Misoprostol in Commercial Formulations

Method

Amount present

Level Added

(%)

Amount of drug added

Amount Recovered(N=6)

%Recovery

MFP

(mg/tab)

MSP

(µg/tab)

MFP

(mg)

MSP

(µg)

MFP±SD*

MSP±SD*

MFP±SD*

MSP±SD*

M1

200

200

80

160

160

359.96 ± 0.98

358.96±0.83

99.73±0.56

99.71±0.25

M2

200

200

80

160

160

358.21±0.82

358.44±0.57

99.50±0.21

99.56±0.62

M1

200

200

100

200

200

396.62±0.59

394.96±0.64

99.15±0.16

98.74±0.45

M2

200

200

100

200

200

400.98±0.48

400.76±0.47

100.24±0.34

100.19±0.22

M1

200

200

120

240

240

438.84±0.29

437.62±0.29

99.73±0.44

99.45±0.59

M2

200

200

120

240

240

439.12±0.36

438.18±0.27

99.80±0.25

99.58±0.48

*= Average of six determinations

MFP=Mifepristone and MSP=Misoprostol

 

Table -3: Optical characteristics and Regression Data for Method I and II

PARAMETER

Method-I

Method-II

 

MFP

MSP

MFP

MSP

λmax(nm)

304

257.6

264. 3

304

Beer’s law limits(µg/ml)

4-20µg/ml

4-20µg/ml

4-20µg/ml

4-20µg/ml

A1%, 1cm (gm/100ml)

At λ304nm=290.5

at λ257.6nm=236.5

At λ304nm =104

at λ257.6nm =259.7

At λ304nm=290.5,

at λ257.6nm=236.5

At λ304nm =104,

at λ257.6nm =259.7

Detection limits(µg/ml)

0.5334181

0.44126

0.5334181

0.44126

Sandell’s sensitivity

(µg/cm2/0.001 absorbance unit)

0.033784

0.036101

0.033784

0.036101

Optimum photometric range(µg/ml)

2-24

2-24

2-24

2-24

Regression equation(y=a+bc) : slope(b)

0.029993

0.027707

0.029993

0.027707

S.D. of slope(Sb)

0.0004

0.000306

0.0004

0.000306

Intercept(a)

-0.00343

-0.003238

-0.00343

-0.003238

S.D. of intercept(Sa)

0.004848

0.0037049

0.004848

0.0037049

Standard error of estimation(Se)

0.006699

0.0051191

0.006699

0.0051191

Correlation coefficient

0.999288

0.999513

0.999288

0.999513

% Relative S.D. *

0.3163

0.1895

0.3163

0.1895

% Range of error (confidence levels)*

95%

99%

0.3319753

0.520624

0.1988864

0.311905

0.3319753

0.520624

0.1988864

0.311905

 

 

Overlain spectra of mifepristone and misoprostol

(Both are prepared in ethanol; 40mcgs each)

Linearity for the proposed methods was established by least square regression analysis of the calibration curves of both the drugs. Linearity was in the range of 4-24 µg/ml for both the drugs. LOD and LOQ were determined based on the standard deviation of response and slope of calibration curve. For Intra day precision studies six concentrations were analyzed by the proposed methods on the same day and for inter-day studies, six concentrations were analyzed by the proposed methods on the three days. Recovery studies were performed at three levels, in which the pre-analyzed sample solutions were spiked with mifepristone and misoprostol at 80%, 100%and120% of the label claim. Three replicate samples of each concentration levels were prepared and the percentage recovery at each level was determined. The results are given in table no. 2.

 

CONCLUSION:

There were few methods reported in the literature for the determination of mifepristone and misoprostol in plasma samples. No spectrophotometric method was reported for the estimation of mifepristone and misoprostol in their combined formulations, thereby the author has developed two rapid, sensitive, accurate and precise methods to determine both the drugs without their prior separation. These methods can be adopted for routine quality control of mifepristone and misoprostol pharmaceutical preparations.

 

ACKNOWLEDGEMENTS:

Dr. P. Srinivasa Babu and Dr. S. Gananathamu for their support offered during the period of the study.

 

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Received on 28.04.2011          Modified on 02.06.2011

Accepted on 10.06.2011         © RJPT All right reserved

Research J. Pharm. and Tech. 5(1): Jan. 2012; Page 46-49