Development and Validation of UV Spectrophotometric Area under Curve
Method for Estimation of Ezetimibe in Bulk and Tablet Dosage Form
K. V. TALWADEKAR1 AND H. K. JAIN1*
1
Department of Quality Assurance Techniques, STES’s Sinhgad College of Pharmacy,
Vadgaon (Bk), Off. Sinhgad Road, Pune -411041, Maharashtra, India.
Abstract
The aim of present work was to develop an accurate, precise, reproducible and economical
UV spectrophotometric method for estimation of Ezetimibe. This method was based on area
under curve of UV spectrum between 232 to 242 nm and validated as per ICH guideline Q2
(R1). The method has followed linearity in the range of 2-12µg/ml. The value of correlation
coefficient was 0.999. Values for the intra-day and inter-day precision were satisfactory
which indicated that method is precise. Results of the recovery studies (99.98% to 100.29 %)
showed accuracy of the method. LOD and LOQ were calculated as 0.480 µg/ml and
1.851 µg/ml, respectively. The developed method can be used for routine estimation of
Ezetimibe in bulk and tablet dosage forms.
INTRODUCTION
Ezetimibe is lipid lowering agent and it acts by inhibiting intestinal absorption of cholesterol
and phytosterols. It interferes with a specific CH transport protein NPC1C1 in the intestinal
mucosa
and
reduces
absorption
of
both
dietary
and
biliary
cholesterol[2].
Chemically it is [(3R,4S) -1- (4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)3hydroxypropyl]-4(4-hydroxyphenyl)-2-azetidinone] (Fig. 1)[1]. It is a white, crystalline powder and it is freely
to very soluble in ethanol, methanol, acetone and practically insoluble in water[3]. Literature
survey revealed that some HPLC methods have been reported for estimation of Ezetimibe as
single drug or in combination. [4,5,6,10,11]. Ezetimibe was also estimated by TLC [10].
Only few paper have been available in the literature using spectrophotometry for analysis of
Ezetimibe as single drug & combination[7,8,9]. These techniques were simultaneous equation
method, derivative spectroscopy, but none of these methods have utilized integration
technique so far. In this context UV spectrophotometry further explore by using area under
curve method for estimation of ezetimibe in bulk and tablet dosage form.
1
Figure 1 - Chemical structure of Ezetimibe.
MATERIALS AND METHODS
Apparatus and Instrumentation
Shimadzu UV spectrophotometer 1800 with matched quartz cells was used and UV Prob
Software, was employed for this work. Electronic balance (Shimadzu, AX 200, Japan) was
used for weighing purpose. An Ultrasonic Cleaning Bath sonicator (Biosystem) was used for
sonication. Galsswares (Borosil) used for this study were calibrated .
Materials
Active pharmaceutical ingredient (API) of Ezetimibe was procured as a gift sample from
Genpharma International Pvt. Ltd, (Pune, India).Commercially available tablets (Zetia
containing 10 mg of Ezetimibe ) were obtained from local pharmacy. Methanol (as a solvent)
used was of AR-grade and was purchased from Merck India Ltd., Mumbai.
Method Development
Preparation of standard solution
For the prepartion standard stock solution of Ezetimibe, 10 mg of API was accurately
weighed and transferred to 10 ml of volumetric flask. The drug was dissolved with sonication
in methanol for 10 min and volume was made up to the mark by using methanol. The
standard stock solution (1000 μg/ml) of API was further diluted with methanol to get the
concentration of 10 μg/ml.
Selection of wavelength range
Baseline correction was done by using methanol as blank. The standard solution of 10μg/ml
of Ezetimibe was scanned between 400 nm to 200 nm in UV spectrophotometer. Wavelength
range was selected around wavelength maxima (232 nm). Working standards were prepared
between 2-12 μg/ml. Various wavelength range were tried around wavelength maxima
(232nm). On the basis of linear relationship between area and corresponding concentration
final range between 222-242 nm was selected.
2
Area under curve (Area calculation)
This method involves calculation of integrated value of absorbance with respect to
wavelength in indicated range. The area bounded by the curve and horizontal axis was
calculated by are calculation processing item. The baseline was represented by horizontal
axis.
Preparation of calibration curve
Working solutions of the concentration of 2, 4, 6, 8, 10 and 12 μg/ml were prepared from
standard stock solution by further dilution with methanol. These solutions were scanned
between 400 to 200 nm and area under curve (AUC) was integrated in the range of 222-242
nm. The calibration curve was plotted between area under curve against concentration (Fig.
3).
Assay of Tablet Formulation
Twenty tablets were weighed individually and average weight of tablet was calculated. These
tablets were crushed and powdered by using glass mortar. The powder of tablet equivalent to
10 mg of Ezetimibe was accurately weighed, and transferred to a 100 ml of volumetric flask
and diluted up to mark with methanol. This solution was filtered with Whatmann filter paper
No.41 and the first few ml of filtrate was discarded. Further dilution of standard stock
solution was done with same solvent to obtained 10 µg/ml of solution and subjected for UV
analysis. This procedure was repeated in triplicate (Table 1).
Method validation[12]
The objective of validation of an analytical procedure is to demonstrate whether the
procedure is suitable for its intended purpose. The proposed method was validated for various
parameters such as Linearity, Accuracy, Precision, Limit of detection (LOD) and Limit of
Quantitation (LOQ) as per the ICH Q2 (R1) guidelines.
Linearity and Range
The linearity was determined by using working standard solutions of Ezetimibe between 2-12
μg/ml. Area under curve was calculated in wavelength range of 222-242 nm. Calibration
curve of Area under curve vs. Concentration was plotted and simple linear regression was
performed (Figure 3). Regression equation and correlation coefficient were obtained. The
range of solution has been decided according to statistical parameters of generated equation.
3
Method Precision Repeatability
The precision of the method was checked by repeatedly injecting (n = 6) standard solutions of
Ezetimibe (10 μg/ml). In the range of 222-242 nm area under curve of each of these solutions
was measure Percentage relative standard deviation (RSD) was calculated (Table 2).
Intermediate Precision (Reproducibility)
The intra-day precision of the proposed method was determined by analyzing the
corresponding responses 3 times on the same day and inter-day precision of the proposed
method was determined by analyzing response on 3 different days over a period of 1 week for
3 different concentrations of standard solutions of Ezetimibe (5, 10 and 15 μg/ml). The
results were reported in terms of relative standard deviation (RSD). The results are tabulated
in Table 3.
Accuracy
The accuracy for the analytical procedure was determined at 80 %, 100 % and 120 % levels
of standard solution. Area under curve was measured in the range of 222-242 nm. Results of
above procedure were expressed in terms of % recoveries. Three determinations at each level
were performed and % RSD was calculated. The results were tabulated in Table 4.
Limit of Detection (LOD) and Limit of Quantitation (LOQ)
Six sets of known concentrations (2-12 μg/ml) were prepared. Calibration curves were
plotted for each set. LOD and LOQ were calculated using the formulae .
𝑆𝐷
𝑆
𝑆𝐷
𝐿𝑂𝑄 = 10
𝑆
𝐿𝑂𝐷 = 3.3
Where, SD is standard deviation of y-intercept of the calibration curves, S is mean slope of
six calibration curves
Result and Discussion
For the determination of Ezetimibe in tablet dosage form An attempt was made to develop a
simple and specific AUC spectrophotometric method. The generated regression equation was
222
222
∫242 𝐴𝑑𝜆 = 0.136𝐶 + 0.057 (R2=0.999), Where ∫242 𝐴𝑑𝜆 is area under curve between 222
to 242 nm, C is concentration and R is correlation coefficient. R2 value is 0.999 which
indicate that developed method was linear. As % R.S.D values for intraday as well interday
precision were satisfactory therefore proposed method was found to be precise. It can be said
that this method was accurate as drug at each of the 80 %, 100 % and 120 % levels showed
4
good recoveries (99.98 % to 100.29 %). The LOD and LOQ value were found to be 0.480
μg/ml and 1.851 μg/ml, respectively which shows that method is sensitive. The result of the
analysis of formulation by the developed method was consistent with the label claim, highly
reproducible and reliable. The method can be used for the routine analysis of the Ezetimibe
in tablet dosage form. The validation parameters are summarised in Table 5.
Area under curve
Figure 2: UV spectrum of Ezetimibe (10 μg/ml) in methanol
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
y = 0.1362x + 0.0579
R² = 0.999
0
5
10
15
Concentration
Figure 3: Calibration Curve of Ezetimibe (2-12 μg/ml)
5
CONCLUSION
From these result it can be concluded that the proposed method was accurate, precise and
consistent for determination of Ezetimibe in tablet dosage form. Validation of this method
was carried out as per the ICH guidelines. It can be concluded that this this method can be
used for routine estimation of Ezetimibe in pharmaceutical dosage forms
ACKNOWLEDGEMENT
The authors are grateful to Genpharma international Pvt. Ltd (Pune India) for providing API
of Ezetimibe as gift sample and Dr. K.N. Gujar, Principal, Sinhgad College of Pharmacy,
Vadgaon (Bk.), Pune for providing necessary facilities for this project.
REFERENCES.
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ezetimibe, International Journal of Research in Pharmaceutical and Biomedical Sciences
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formulations, Research Journal of Pharmaceutical, Biological and Chemical Sciences
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Estimation of Simvastatin and Ezetimibe In Bulk Drug and its combined dosage form ,
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and Ezetimibe in their combined dosage forms, International Journal of Pharmacy and
Pharmaceutical Sciences2010;2:131-138.
6
9.Shravya A, Chandan RS, Gurupadyya BM, Sireesha M. Spectrophotometric determination
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AM, Marwa FM, and Nadia FY. Development and
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11.Rajamanickam V, Rajasekaran A, Stephen B,
Rathinaraj and Anandarajagopal
K .
Development and Validation of Analytical Methods for Simultaneous Estimation
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7
Table 1. Assay of Tablet Dosage Form.
%RSD*
Sr No
Sample solution conc.
Amount Found
1
10
101.23
2
10
100.93
3
10
100.89
niss *n=3 , % RSD – Relative standard deviation.
Mean Amount Found (%)
101.01
0.183
8
Table 2. Repeatability Results for Ezetimibe.
Drug
Concentration of drug (μg/ml)
%RSD*
Ezetimibe
*n=6
10
0.721
9
Table 3. Precision Results for Ezetimibe
Drug
Concentration of drug (μg/ml)
% RSD intraday
% RSD Interday
Ezetimibe
5
10
15
0.376
0.373
0.380
0.390
0.401
0.391
10
Table 4. Accuracy Results for Ezetimibe
Accuracy level
Amount added
(μg/ml
% Recovery
I (80%)
II (100%)
III (120%)
8
10
12
99.98
100.20
100.29
Mean %
Recovery
% RSD
100.15
0.159
11
Table 5. Summary of Validation Parameters.
Parameter
λ max
Linearity range
Regression Equation(y=mx+c)
Slope (m) ± SD*
Intercept (c) ± SD*
Correlation Coefficient (R2)
Precision (% R.S.D)
Repeatability
Intraday
Interday
Accuracy (Mean % Recovery
LOD
LOQ
Result
232
2-12µg/ml
y=0.136x + 0.057
0.136
0.057
0.999
0.721
0.376
0.394
100.15%
0.480 μg/ml
1.851 μg/ml
12