New Simple and Economical Spectrophotometric Methods for Estimation of Ezetimibe in Bulk Drug and Pharmaceutical Dosage Forms
Ramakrishna Kommana*
Dept. of Pharmaceutical Analysis, Gokaraju Rangaraju College of Pharmacy, Osmania University, Hyderabad-90
*Corresponding Author E-mail: rkkommana@yahoo.com
ABSTRACT:
Two accurate, precise, rapid and cost effective methods were developed for the estimation of Ezetimibe in bulk drug and tablet dosage form. Wavelengths selected for quantitation were 233 nm in Methanol and 243 nm in 0.5 M NaOH respectively. In method-I (methanol) linearity for detector response was observed in the concentration range of 2-40 μg/ml and in method-II (0.5 M sodium hydroxide) linearity for detector response was observed in the concentration range of 2-30 μg/ml. The results of the analysis have been validated statistically. The method has been successfully applied in the analysis of marketed formulations.
KEYWORDS: Spectrophotometric analysis, Ezetimibe
Ezetimibe chemically, it is 1-(4-fluorophenyl)-(3R)-[3-(4-fluorophenyl)-(3s)-hydroxy propyl]-4s-(4-hydroxyphenyl)-2-azetidinone5 (Fig. 1). It is mainly used as anti-lipemic drug 1. It inhibits cholesterol absorption in the intestine for the reduction of cholesterol levels in patients with hypercholesterolemia1-3, 7, 9,-13. The drug is marketed as tablet formulation and the product strength is 10 mg. Literature survey revealed HPLC methods in biological fluids16, bulk drug and pharmaceutical dosage forms, but no UV spectrophotometric method was revealed. So, an attempt is made to develop simple, cost effective and accurate methods for estimation of Ezetimibe in bulk drug and in tablet formulation.
Fig.1. Chemical structure of Ezetimibe
MATERIALS AND METHODS:
Instrument: A double-beam Shimadzu UV-Visible Spectrophotometer 1800, with spectral bandwidth of 1.0 nm, wavelength accuracy ± 0.1 nm and a pair of 1-cm matched quartz cells was used to measure absorbance of the resulting solution.
Materials: Standard gift sample of Ezetimibe was provided by M/s. Micro Labs, Hosur. The pharmaceutical dosage forms (tablets) - (Ezedoc – Lupin (10 mg), Ezee – Genix (10 mg), were purchased from local market.
Solvent used: Methanol (Qualigens), Double distilled water, 0.5 M Sodium hydroxide (Qualigens)
Preparation of standard solution for Method I:
10 mg of Ezetimibe bulk drug accurately weighed was dissolved in minimum quantity of methanol and diluting with 100 ml with same solvent. 1.6 ml of this solution was again diluted to 25 ml with the same solvent.
METHOD-I:
Suitable aliquots of the standard solution of Ezetimibe (0.5-11 ml) were taken in 25 ml volumetric flasks. The volume was then made upto the mark with methanol to prepare a series of standard solutions containing 2 – 44 µg/ml. Absorbance was measured at 233 nm against blank (Fig. 3). Linearity was checked, the concentrations ranging from 2-40 mg/ml obeys Beer’s law4, 6.
Estimation of Ezetimibe in Tablets (Method – I):
Twenty tablets (of same respective batch number) of two pharmaceutical companies Ezedoc T1 (Lupin), Ezee T2 (Genix) were accurately weighed and powdered. Weight of powdered tablets equivalent to 10 mg of drug was taken in 25 ml of methanol and vigorously shaken for 10 minutes, filtered through Whatmann filter paper No.41 and made up to 100 ml. Aliquots of 1.0 to 2.0 ml were transferred into a series of 25 ml volumetric flasks and the final volume was brought to 25 ml with methanol. The absorbance was measured at 233 nm against methanol as blank and the amount of Ezetimibe present in the sample solution was calculated. The experiment was repeated six times for each brand of tablets (Table.3 and Table.4). Drug content in each brand of tablet was calculated by using the following formula and was recorded in the tables.
Content of Ezetimibe in average weight of each tablet =
Preparation of standard solution for Method-II:
50 mg of Ezetimibe from bulk drug is dissolved in 0.5 M Sodium hydroxide and make to 500 ml in a calibrated flask with 0.5 M Sodium hydroxide. 4.0 ml of the above solution was pipetted out into a 10 ml standard flask and volume was made up to the mark with 0.5 M Sodium hydroxide. Absorbance of resulting solution was noted at 243 nm against 0.5 M Sodium hydroxide as blank (Fig. 2).
Figure 2: Ultra Violet Absorption Spectrum of Ezetimibe in 0.5M Sodium Hydroxide
METHOD-II:
In UV spectroscopic method using 0.5 M sodium hydroxide as solvent the dilutions ranging from 2 -10 ml from 20 mg/ml and 2.5-5ml from 100 mg/ml were taken and made upto 10 ml. Thus the concentration ranges from 4-50 mg/ml. Linearity was checked, the concentration ranging from 2-30 mcg/ml obeys Beer’s law.
Estimation of Ezetimibe in Tablets (Method –II):
Twenty tablets (of same respective batch number) of two pharmaceutical companies Ezedoc T1 (Lupin), Ezee T2 (Genix) were accurately weighed and powdered. Weight of each tablet powder equivalent to 50 mg of drug is taken in 500 ml of 0.5 M sodium hydroxide and vigorously shaken for 20 minutes, filtered through Whatmann filter paper No.41 and made up to 500 ml. Aliquots of 1.0 to 3.0 ml were transferred into a series of 10 ml volumetric flasks and the final volume was brought to 10 ml with 0.5 M NaOH. The absorbance was measured at 243 nm against 0.5 M NaOH as blank and the amount of Ezetimibe present in the sample solution was computed from it calibration curve. The experiment was repeated six times for each brand of tablets (Table. 5 and Table. 6). The absorbance of resulting solution was noted at 243 nm. Drug content in each brand of tablet was calculated by using the formula mentioned before.
Figure 3: Ultra Violet Absorption Spectrum of Ezetimibe in Methanol
METHOD VALIDATION:
Accuracy of the method was determined by the recovery studies in the tablet dosage form of Ezetimibe. Recovery studies were carried out by addition of known quantities of standard drug solution to pre-analyzed sample at three different concentrations. The experiment was repeated for six times. The percent relative standard deviation
(% RSD)8 was calculated. In UV-Spectroscopic method [I] quantification of raw material was performed 6 times and the mean percentage obtained was 100.26%. To validate statistically standard deviation, percentage relative standard deviation and range of error were performed. The values obtained for standard deviation (0.6705), %RSD (0.669) and range of error 90% (±1.1036), 95% (±0.703) were computed (Table.1 and Table.7). The statistical values were found to be satisfactory. In UV- Spectroscopic method [II] quantification of raw material was performed 6 times and the mean percentage obtained was 101.08. To validate statistically all the parameters mentioned in method [I] of UV-Spectroscopy were followed. The values obtained for standard deviation (0.2316), %RSD (0.2291) and range of error 90% (±0.3812), 95% (±0.2430) were computed (Table.2and Table.8). Comparative results for raw material and formulations in method [I] and method [II] were computed (Table.9 and Table.10).The statistical values were found to be satisfactory.
table 1: Quantification of Raw Material - Method I
|
S. No. |
Quantity weighed (mg) |
Amount found (mg) |
Percentage obtained |
S.D. |
% RSD |
Range of Error |
|
|
90% |
95% |
||||||
|
1 |
10 |
10.08 |
100.8 |
0.6705 |
0.669 |
± 1.1036 |
± 0.703 |
|
2 |
10 |
9.94 |
99.4 |
||||
|
3 |
10 |
9.96 |
99.6 |
||||
|
4 |
10 |
10.10 |
101.0 |
||||
|
5 |
10 |
10.05 |
100.5 |
||||
|
6 |
10 |
9.98 |
99.8 |
||||
table 2: Quantification of Raw Material - Method II
|
S. No. |
Quantity weighed (mg) |
Amount found (mg) |
Percentage obtained |
S.D. |
% RSD |
Range of Error |
|
|
90% |
95% |
||||||
|
1 |
10 |
10.10 |
101.0 |
0.2316 |
0.2291 |
± 0.3812 |
± 0.2430 |
|
2 |
10 |
10.13 |
101.3 |
||||
|
3 |
10 |
10.07 |
100.7 |
||||
|
4 |
10 |
10.12 |
101.2 |
||||
|
5 |
10 |
10.10 |
101.0 |
||||
|
6 |
10 |
10.13 |
101.3 |
||||
table 3: Quantification of Formulation - Method I
Ezedoc (Formulation – 1)
|
S. No. |
Label claim (mg/tablet) |
Amount found (mg) |
Percentage obtained |
S.D. |
% RSD |
Range of Error |
|
|
90% |
95% |
||||||
|
1 |
10 |
9.99 |
99.91 |
0.2087 |
0.2088 |
±0.3435 |
±0.2190 |
|
2 |
10 |
9.95 |
99.58 |
||||
|
3 |
10 |
9.99 |
99.58 |
||||
|
4 |
10 |
9.99 |
99.91 |
||||
|
5 |
10 |
9.99 |
99.91 |
||||
|
6 |
10 |
10.02 |
100.24 |
||||
table 4: Quantification of Formulation - Method I
Ezee (Formulation – 2)
|
S. No. |
Label claim (mg/tablet) |
Amount found (mg) |
Percentage obtained |
S.D. |
% RSD |
Range of Error |
|
|
90% |
95% |
||||||
|
1 |
10 |
9.90 |
99.02 |
0.2996 |
0.3025 |
± 0.4931 |
± 0.3144 |
|
2 |
10 |
9.93 |
99.36 |
||||
|
3 |
10 |
9.93 |
99.36 |
||||
|
4 |
10 |
9.86 |
98.69 |
||||
|
5 |
10 |
9.86 |
98.69 |
||||
|
6 |
10 |
9.90 |
99.02 |
||||
table 5: Quantification of Formulation - Method II
Ezedoc (Formulation 1)
|
S .No. |
Label claim (mg/tablet) |
Amount found (mg) |
Percentage obtained |
S.D. |
% RSD |
Range of Error |
|
|
90% |
95% |
||||||
|
1 |
10 |
10.01 |
100.1 |
0.8359 |
0.8390 |
±1.3759 |
±0.8773 |
|
2 |
10 |
10.01 |
100.1 |
||||
|
3 |
10 |
10.05 |
100.5 |
||||
|
4 |
10 |
10.05 |
100.5 |
||||
|
5 |
10 |
9.98 |
99.88 |
||||
|
6 |
10 |
9.98 |
99.88 |
||||
table 6: Quantification of Formulation - Method II
Ezee (Formulation 2)
|
S. No. |
Label claim (mg/tablet) |
Amount found (mg) |
Percentage obtained |
S.D. |
% RSD |
Range of Error |
|
|
90% |
95% |
||||||
|
1 |
10 |
10.06 |
100.6 |
0.2875 |
0.2867 |
±0.4732 |
±0.3017 |
|
2 |
10 |
10.06 |
100.6 |
||||
|
3 |
10 |
10.03 |
100.3 |
||||
|
4 |
10 |
10.01 |
100.1 |
||||
|
5 |
10 |
9.99 |
99.9 |
||||
|
6 |
10 |
10.01 |
100.1 |
||||
Table 7: Method I - Optical Characteristics and Precision
|
Parameters |
Method UV |
|
lmax(nm) |
233nm |
|
Beers law limit (mg/ml) |
2 - 40mg/ml |
|
Sandell’s sensitivity14 (mg/cm2/0.001 A.U) |
0.021358 |
|
Molar extinction coefficient (L mol–1 cm–1) |
1.9169×104 |
|
Correlation coefficient (r) |
0.9998 |
|
Regression equation (y = mx + c) |
Y = 0.045838(x) +0.0071656 |
|
Slope(m) |
0.045838 |
|
Intercept(c) |
0.0071656 |
|
% Range of error |
|
|
Confidence limit with 0.05 level (95%) |
± 0.703 |
|
Confidence limit with 0.01 level (90%) |
± 1.1036 |
Table 8: Method II - Optical Characteristics and Precision
|
Parameters |
Method UV |
|
Absorption maxima (nm) |
243 |
|
Beers law limit (mg/ml) |
2-30 |
|
Correlation coefficient (r) |
0.9998 |
|
Molar extinction coefficient (L mol–1 cm–1) |
2.3279×104 |
|
Sandell’s sensitivity (mg/cm2/0.001 A.U) |
0.017587 |
|
Regression equation (y = mx + c) |
Y = 0.05648 (x) + 0.0034509 |
|
Slope(m) |
0.05648 |
|
Intercept(c) |
0.0034509 |
|
% Range of error |
|
|
Confidence limit with 0.05 level (95%) |
0.5206 |
|
Confidence limit with 0.01 level (90%) |
0.8164 |
table 9: Comparative Results for Raw Material
|
Method |
Quantity weighed (mg) |
Amount found (mg) |
% obtained |
S.D. |
% RSD |
Range of Error |
|
|
90% |
95% |
||||||
|
UV – I |
10 |
10.02 |
100.26 |
0.6705 |
0.669 |
± 1.1036 |
±0.703 |
|
UV- II |
10 |
10.10 |
101.08 |
0.2316 |
0.2291 |
± 0.3812 |
±0.2430 |
table 10: Comparitive Results for Formulations
|
Method |
Formulation |
Label claim (mg/tablet) |
Amount found (mg) |
% obtained |
S.D. |
%RSD |
Range of Error |
|
|
90% |
95% |
|||||||
|
I |
Ezedoc |
10 |
9.98 |
99.855 |
0.2087 |
0.2088 |
± 0.3435 |
± 0.2190 |
|
Ezee |
10 |
9.90 |
99.0233 |
0.2996 |
0.3025 |
± 0.4931 |
± 0.3144 |
|
|
II |
Ezedoc |
10 |
10.01 |
100.16 |
0.8359 |
0.8390 |
± 1.3759 |
± 0.8773 |
|
Ezee |
10 |
10.02 |
100.2667 |
0.2875 |
0.2867 |
± 0.4732 |
± 0.3017 |
|
RESULTS AND DISSCUSION:
The solubility of Ezetimibe was determined in a variety of solvents using schefter and Higuchi method15. The proposed methods are simple and precise and do not suffer from any interference due to common excipients of tablets. Methods were validated in terms of accuracy and precision. The accuracy of the method was proved by performing recovery studies in the commercially available formulations. Values greater than 99 % indicate that proposed method is accurate for the analysis of drug. The comparative results for raw material and formulations in both the methods were found to be satisfactory. From author point of view comparative results for formulations show UV method [I] is more satisfactory than UV method [II]. In an over view the results indicate that the method is precise enough for the analysis of the drug.
ACKNOWLEDGEMENTS:
The author is thankful to the management, Gokaraju Rangaraju College of Pharmacy for providing facilities for the study.
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Received on 02.10.2010 Modified on 03.11.2010
Accepted on 28.11.2010 © AJRC All right reserved
Asian J. Research Chem. 4(2): February 2011; Page 250-253