Simultaneous Spectrophotometric Estimation of Ezetimibe and ...

Asian J. Research Chem.. 2(1): Jan..-Mar. 2009 ,

www.ajrconline.org

ISSN 0974-4169 RESEARCH ARTICLE

Simultaneous Spectrophotometric Estimation of Ezetimibe and Atorvastatin in Pharmaceutical Dosage Form Godse VP.* Deodhar MN, Bhosale AV, Sonawane RA, Sakpal PS, Borkar DD and Bafana YS

Dept. of Pharmaceutical Analysis, Seth Govind Raghunath Sable College of Pharmacy,Saswad, Pune- 412 301, India * Corresponding Author E-mail: [email protected]

ABSTRACT

Two simple, sensitive accurate, precise, rapid and economical methods were developed for the estimation of Ezetimibe and Atorvastatin in two components solid dosage form. First method is based on simultaneous equations and second method is based on absorbance ratio method. Ezetimibe has absorbance maxima at 232.5 nm and Atorvastatin has Absorbance maxima at 246.5 nm in methanol. The linearity was obtained in the concentration range 5-30 mcg/ml for both Ezetimibe and Atorvastatin.In the first method the concentration of the drugs were determined by using simultaneous equations and in second method, concentration of the drugs were determined by using ratio of absorbance at isoabsorptive point and at max of one of the drug. The results of analysis have been validated statistically and by recovery studies.

KEY WORDS

Ezetimibe, Atorvastatin, Simultaneous Equations, Absorbance Ratio, Iso-absorptive Point.

INTRODUCTION:

Atorvastatin (ATV) is chemically [ R, R]-2-(4fluorophenyl)-beta, delta-dihydroxy 5- (1-methylethyl)-3phenyl4[(phenyl amino)carbonyl]-1H- pyrrole-1- heptanoic acid and Ezetimibe(EZE) is (3R,4S)-1-(4-fluorophenyl)-3((3S)-3-(4-fluorophenyl)-3-hydroxypropyl)-4-(4hydroxyphenyl)-2-azetidinone. Ezetimibe and Atorvastatin both used as antilipidemic agents. Literature survey reveals that HPLC1-4, Electrochemical methods5, and tendem MS6-8 method for estimation of atorvastatin in pharmaceutical dosage form and from human plasma. Similarly, survey of literature for ezetimibe revealed several methods based on HPLC9-10 for estimation of ezetimibe in pharmaceutical dosage form. However no method has been developed for estimation of these drugs in combined dosage form. This paper presents two simple rapid, reproducible and economical methods for simultaneous estimation in tablet dosage form.

MATERIALS AND METHODS:

Instruments and Reagents A Jasco model V-530 double beam UV-Vis Spectrophotometer with spectral width of 2 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched quartz cells was used to measure the absorbance of resulting solutions.

Received on 04.03.2009 Accepted on 25.03.2009

Modified on 13.03.2009 © AJRC All right reserved

Asian J. Research Chem. 2(1): Jan.-March, 2009;Page 86-89

A Shimadzu analytical balance, an ultra sonic cleaner, Ezetimibe (Glenamark pharmaceuticals Ltd., Mumbai) and Atorvastatin (Bal Pharma, Bangalore) and methanol were used in the study. Preparation of standard drug solutions The standard stock solutions of EZE and ATV were prepared by dissolving 10 mg of each drug in 10 ml volumetric flask separately using methanol. Final working standard solutions of 10 mcg/ml of each EZE and ATV were prepared by diluting 0.1 ml of the above solution to 10 ml with methanol. PROCEDURE Method 1: Simultaneous Equation Method Working standard solutions were scanned in the entire range of 200-400 nm to determine the max of both the drugs. The max of EZE and ATV were found to be 232.5 nm and 246.5 nm respectively. Six standard solutions having concentrations 5, 10, 15, 20, 25, 30 mcg/ml for both EZE and ATV were prepared in methanol using the final working standard solution (10 mcg/ml). The absorbances of resulting solutions were measured at 232.5 nm and 246.5 nm and calibration curves were plotted at these wavelengths. The absorptivity coefficients of these two drugs were determined by using calibration curve equation. Two simultaneous equations were formed using these absorptivity coefficient values. A1 = 42.04 C x + 35.99.Cy A2 = 36.4 C x + 41.76 Cy

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Where, Cx and Cy are concentrations of EZE and ATV, respectively in gm/100ml in sample solution. A1 and A2 are absorbance of the sample solution containing EZE and ATV at 232.5 nm and 246.5 nm, respectively.

Where, Q x and Q y are Q values, a x, a y are absorptivities at isobestic point for EZE and

The concentration of Cx and Cy can be obtained as

Qx Qy

A2 aY1 – A1 aY2 C x = ________________ ax2 aY1 – ax1 aY2 And,

(01)

A1ax2 – A2 ax1 C y = ________________ ax2 aY1– ax1 aY2

Qy -Qx

×

A ay

(04)

The Q-values and absorptivities for both drugs were calculated as follows, Absorbance of ATV at 232.5 nm Q y = __________________________________________ Absorbance of ATV at 238.5 nm Qx

=

1.0773 0.920985

a x = 39.52 a y = 39.02

(02)

The concentrations of two drugs in the mixture can be calculated using equations Q0 - Qy A Cx = × (03) Q x- Q y ax Similarly, Q 0- Q x

= =

Q0= Absorbance of sample at 232.5nm/ Absorbance of sample at 238.5nm; A= Absorbance of sample at 238.5nm

Method 2: Absorbance Ratio Method (Q -Analysis) Absorbance ratio method uses the ratio of absorbance at two selected wavelengths one at iso-absorptive point and other being the max of one of the two components. From the overlay spectra of two drugs, it is evident that EZE and ATV show an iso-absorptive point at 238.5nm and the second which is the max of EZE. Six standard solutions having concentration 5,10,15,20,25, and 30 mcg/ml for both EZE and ATV were prepared in methanol using final working standard solution ( 10 mcg/ml) and the absorbance at 238.5 nm (iso-absorptive point) and 232.5 nm( max of EZE) were measured and absorptivity coefficients were calculated using calibration curve.

Cy =

ATV respectively. These values were found to be

Absorbance of EZE at 232.5 nm _________________________________________ Absorbance of EZE at 238.5 nm

Absorbance of ATV at 238.5 nm a y = _________________________________________ Concentration of ATV gm/lit Absorbance of EZE at 238.5nm a x =__________________________________________ Concentration of EZE in gm/lit

Fig.1:-Overlain Spectra of Atorvastatin (246.5 nm) and nm).

Ezetimibe (232.5 nm), Iso-absorptive point (238.5

Estimation of Ezetimibe and Atorvastatin in Tablet An Accurately measured equivalent quantity of tablet powder equivalent to 10 mg 0f Ezetimibe and 10 mg of Atorvastatin was transferred to a 100ml volumetric flask and dissolved in 50 ml methanol and sonicated for 10 min. The solution was filtered through whatman filter paper No.41 and residue was washed thoroughly with methanol. The filtrate and washings were combined in a 100 ml volumetric flask and diluted to the mark with methanol. The solution was further diluted with the same solvent to get a final concentration of 10 mcg/ml for both the drugs in the sample were determined using equations 1 and 2. For method 2 the absorbance of the sample solution i. e. A1 and A2 were recorded at 238.5 nm (iso-absorptive point) and 232.5 nm ( - max of EZE), respectively and ratio of absorbance were calculated i. e. Qo. Relative concentrations of two drugs were calculated using equations 3 and 4. The analysis procedure was repeated five times with tablet formulations. The result of analysis of tablet formulation is shown in Table 1. To study the accuracy and precision of above proposed methods recovery studies were carried out by addition of known amount of standard drug solution of EZE and ATV to preanalysed formulation. The resulting solution was then analyzed by proposed methods. Results of recovery studies were found to be satisfactory and are

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Table 1: Estimation of Ezetimibe and Atorvastatin in tablet. Method Name

Label Claim* mg/tablet

Amount found* mg/tablet

Percentage labeled claim

of

Standard deviation

Coefficient Variance

EZE ATV EZE ATV EZE ATV EZE ATV EZE 10 10 9.86 10.25 98.62 102.5 0.13 0.18 0.13 SEM 10 10 10.1 9.95 101.4 99.5 0.20 0.25 0.20 ARM *Average of five determination; SEM-Simultaneous Equation Method; ARM-Absorption Ratio Method

ATV 0.177 0.25

of

Standard Error EZE 0.05 0.09

ATV 0.08 0.11

Table 2: Recovery Study Data Method Name SEM

Level of % Recovery*

% Recovery Found

Standard Deviation

Coefficient of Variance

EZE ATV EZE ATV EZE ATV 50 102.32 99.08 0.094 0.0837 0.1675 0.0845 100 102.0 99.73 0.122 0.1545 0.2435 0.1549 150 100.9 98.97 0.049 0.4410 0.5432 0.4455 50 102.19 100.42 0.193 0.2255 0.189 0.0245 ARM 100 102.25 100.9 0.204 0.2857 0.199 0.2832 150 100.98 100.06 0.049 0.1557 0.049 0.1556 *Average of three determination; SEM-Simultaneous Equation Method; ARM-Absorption Ratio Method

reported in Table 2. Placebo study was also carried out without drug substance

RESULTS AND DISCUSSION:

The proposed methods were found to be simple, sensitive, accurate, precise, economical and rapid for routine simultaneous estimation of these two drugs. The value of Standard deviation and Coefficient of variation were satisfactory and recovery studies were indicative of the accuracy of the proposed methods. In Simultaneous Equation Methods (Method 1), two wavelengths of respective absorbance maxima i. e. 232.5 nm for EZE and 246.5 nm for ATV were used for the analysis of drugs. The criteria for obtaining maximum precision, by this method were calculated and found to be outside the range of 0.1-2. In Absorbance Ratio Method (Method 2), the primary requirement for developing a method for analysis is that the entire spectra should follow the Beer’s Law at all wavelengths, which was fulfilled in case of both these drugs. The two wavelengths used for the analysis of both drugs were 238.5 nm (iso-absorptive point) and 232.5 nm ( max of EZE). The validation parameters were studied at all the three wavelengths for both the methods. Accuracy was determined by calculating the % recovery and precision was calculated as repeatability (Standard deviation and relative standard deviation) and inter and intraday variation (%CV) for both the drugs. Both the methods were successfully used to determine the amount of EZE and ATV present in tablet. The results obtained are in well agreement with the corresponding labeled amount (Table I). Placebo study shows that the additives usually present in the pharmaceutical formulations did not interfere with determination of EZE and ATV.

Standard Error EZE 0.054 0.122 0.028 0.111 0.117 0.028

ATV 0.0484 0.0549 0.2549 0.1303 0.1089 0.0900

CONCLUSION:

The methods described in this paper for the simultaneous estimation of Ezetimibe and Atorvastatin are found to be simple, sensitive, accurate, precise, rapid and economical, hence both the methods could be successfully employed for the routine analysis of EZE and ATV in their combined dosage form.

ACKNOWLEDGEMENT:

The authors are grateful to Glen mark Pharmaceuticals Ltd. Mumbai, for providing gift samples of Atorvastatin and Bal pharma, Bangalore for providing gift samples Ezetimibe.

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