Estimation of Ezetemibe by Visible Spectrophotometry

 

R.B. Desi Reddy, G. Naga Sowjanya, V. Shanthi, P. Rizpah, G. Geetha, B.R. Maneesha

Nalanda Institute of Pharmaceutical Sciences, Kantepudi, Sattenapalli.

*Corresponding Author E-mail: naga.sowji@gmail.com

 

ABSTRACT:

Development of new analytical methods for the determination of drugs in pharmaceutical dosage forms is more important in pharmacokinetic, toxicological and biological studies. Ezetemibe is a hypo-lipidaemic agent. Two simple and sensitive spectrophotometric methods (Method A and Method B) were developed for the estimation of Ezetemibe in bulk and pharmaceutical preparations. The two methods are based on the formation of yellow chromophores with PDAB and Picric acid exhibiting maximum absorption at 420nm and 400nm respectively. These two methods obey Beer’s law in the concentration range of 10-50μg/ml and 10-30μg/ml respectively. The methods were validated for use in routine quality control studies of Ezetemibe. Interference studies were conducted and it was found that the common excipients usually present in dosage forms do not interfere in the proposed methods. The optical characteristics, regression analysis data and precision of the methods were calculated. The accuracy of the methods was evaluated by estimating the amount of Ezetemibe in previously analyzed samples to which known amounts of Ezetemibe were spiked. The accuracy of the methods was also confirmed by comparison of the results obtained by proposed and reference methods. The methods were validated for use in routine quality control of Ezetemibe in bulk and pharmaceutical preparations.

 

 

INTRODUCTION:

Ezetimibe is chemically (3R, 4S)-1-(P-fluorophenyl)-3-[(3)-3(P-fluorophenyl)-3-hydroxy phenyl] – 4- (p-hydroxyl phenyl)-2-azetidinone.^(1,10) It is a hypolipaedemic agent. It is freely soluble in ethanol, methanol and acetone and practically insoluble in water. The presence of phenolic group and tertiary nitrogen enables the use of condensation reaction with PDAB (method A) and salt formation picric acid (method B) respectively.

 

REAGENTS AND PREPARATION:

a)     PDAB:  (0.1% w/v)

a.     125 mg of PDAB was weighed accurately and dissolved in a mixture of 65ml

b.     concentrated Sulphuric acid, 35ml methanol and 0.05ml of ferric chloride

b)    PICRIC ACID:  (0.4% w/v)

a.     400 mg of picric acid was dissolved in 100ml of chloroform.

c)     Concentrated Sulphuric acid

d)    Methanol

e)     Chloroform

 

STANDARD SOLUTION:

Method A: 100 mg of pure drug was dissolved in few ml of methanol and made up to volume with methanol in a 100ml volumetric flask. From this stock solution, working standard solution (100µg/ml) was prepared. ^{( 2,4-8,10)}

 

Method B: 100mg of pure drug was dissolved in few ml chloroform and made up to volume with chloroform in 100ml volumetric flask. From this stock solution, working standard solution (100µg/ml) was prepared.^{( 2,4-8,10)}

 

METHODOLOGY:

Method A: Aliquots of standard solution containing 1ml, 1.5ml, 2ml, 2.5ml, 3ml (10-30µg/ml) of 100µg/ml were added into 5 different volumetric flasks. To these flasks, 2ml of PDAB, followed by 2ml conc.H₂SO₄ were added and kept aside for sometime. Then they are made up to the mark with methanol and absorbance of the developed yellow chromogenic species was measured at 420 nm respectively against blank. A calibration curve was plotted with concentration on X-axis and absorbance on Y-axis. ^(3,6,7,9,10)

 

Method B:-aliquots of standard solution containing 1ml, 1.5ml, 2ml, 2.5ml, 3ml (10-30µg/ml) of 100µg/ml were added into 5 different volumetric flasks. To these flasks, 2ml of picric acid was added and kept aside for some time. Then they are made up to the mark with chloroform and the absorbance of the developed yellow chromogenic species was measured at 400 nm respectively against the blank. A calibration curve was plotted with concentration on X-axis and absorbance on Y-axis. ^(3,6,7,9,10)

 

DISCUSSION:

From the data concerning to the proposed method, the recommended procedure is accurate, sensitive and precise and is adapted to micro determination of ezetimibe in bulk form and its pharmaceutical preparations. It may be used in the routine determination of ezetimibe. The chemistry involved in the above proposed method to give various colored chromogens can be explained in the following scheme –

Method A: PDAB is an aromatic aldehyde which reacts with amines, indoles, pyrroles and forms Schiff’s base through condensation reaction. PDAB reacts with phenolic group of ezetimibe undergoes condensation reaction and forms a Schiff’s base which is an yellow colored compound with λmax of 420 nm.

 

Method B : Addition of picric acid to drugs containing different functional group bearing compounds in alkaline medium lead to the colorimetric determination of primary and secondary amines and other compounds. Therefore picric acid reacts with ezetimibe and forms a molecular salt, a yellow colored compound with λmax of 400nm.

 

 

OPTIMUM CONDITIONS:

TABLE I:

PARAMETER	METHOD A		METHOD  B
	Optimum range	Conditions in procedure	Optimum conditions	Conditions in procedure
λmax (nm)	400-430	420	390-410	400
Effect of volume of PDAB/picric acid (ml)	0.8-2	2	0.5-1.5	1.0
Volume of conc.H2SO4(ml)/ chloroform	0.8-2	2	made up to volume	made up to volume
Effect of temp. on colored species(oc)	Lab temp (28+5)	Lab temp (30)	Lab temp (28+5)	Lab temp (30)
Stability of colored product	4.0 hrs	5 min	2.0 hrs	5 min

 

RESULTS:

TABLE II:

S. no	Parameter	Method A	Method B
1.	Λmax	420	400
2.	Beer’s law limits(µg/ml)	10-50	10-30
3.	Molar absorptivity	4134.94	4175.88
4.	Detection limits(µg/ml)	1.357	0.983
5.	Sandell’s sensitivity (µg/cm2 /0.01 absorbance unit)	0.09901	0.098039
6.	Correlation co-efficient	0.999	0.999
7.	% relative standard deviation	4.047	1.461
8.	% error in bulk samples**	0.09	0.05

 ** Average of three determinations

 

CONCLUSION:

The molar absorptivity and Sandell’s sensitivity values show the sensitivity of both the methods while the precision is confirmed by %RSD (% relative standard deviation) ,which are mentioned earlier in table II. The results are in good agreement with the limits of standards. The reproducibility, repeatability and accuracy of these methods were found to be good. The proposed methods were simple, sensitive, accurate, precise and reproducible and can be successfully applied for the routine estimation of ezetimibe in bulk and pharmaceutical preparations.

 

ACKNOWLEDGEMENT:

Authors are thankful to management of Nalanda Institute of Pharmaceutical Sciences, Kantepudi for providing us with required provisions.

 

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Received on 26.06.2012       Modified on 14.07.2012

Accepted on 12.08.2012      © RJPT All right reserved