Simultaneous Analysis of Ezetimibe and Rosuvastatin by Q absorption Ratio Method
Nagvenkar Pratiksha, Nazareth Celina*
*Corresponding Author E-mail: celinanaz@yahoo.com
ABSTRACT:
A Novel UV spectrophotometric method has been developed for the simultaneous estimation of ezetimibe and rosuvastatin based on Q absorption ratio method. The wavelengths of 234.8nm (lsosbestic point) and 242nm (λmax of rosuvastatin) were chosen as analytical wavelengths. The linearity was established in concentration range 2-40µg/mL for ezetimibe and rosuvastatin with correlation coefficient greater than 0.990. Analytical method validation was performed by following ICH guidelines. The % assay for ezetimibe and rosuvastatin in tablet formulation complied with acceptance criteria. The developed method thus can be used as a powerful quality control tool for simultaneous analysis of the drugs in bulk and in formulation by UV spectroscopy.
KEYWORDS: Ezetimibe, Rosuvastatin, UV spectroscopy, Q absorbance ratio method, Simultaneous estimation.
INTRODUCTION:
Ezetimibe (EZE) (Fig. 1) inhibits intestinal absorption of cholesterol and phytosterols and acts as an anti-hyperlipidemic agent. It preferentially localizes at the brush border of the small intestine where it appears to act. It interferes with a specific cholesterol transport protein NPC1C1 in the intestinal mucosa and reduces absorption of both dietary and biliary cholesterol1.
Rosuvastatin (RSV) (Fig. 2) is a hypolipidaemic drug belonging to the class of HMG CoA reductase inhibitor. A synthetic lipid lowering agent, it inhibits the conversion of HMG-CoA to mevalonate by the enzyme HMG-CoA reductase1, primary site of action being the liver. It also inhibits hepatic synthesis of very low density lipoprotein (VLDL) resulting in an overall decrease in plasma LDL and VLDL.
Literature survey reveals that various chromatographic methods like HPTLC2, RP-HPLC3-14, UV spectroscopic methods15-20, have been reported for the analysis of EZE and RSV calcium either alone or in combination with other drugs.
It was observed that the reported UV simultaneous equations method employed methanol as diluent. Spectroscopic grade methanol is an expensive organic solvent. It would be worthwhile to reduce its consumption so as to bring down the cost of analysis. Also, no method has been reported for estimation of EZE and RSV in combined dosage form by Q absorption ratio method. Hence an attempt was made to develop a novel and economical UV spectrophotometric method for the simultaneous estimation of EZE and RSV. The developed method was then applied for the simultaneous estimation of EZE and RSV in their marketed formulation
Fig. 1: Chemical structure of Ezetimibe
Fig. 2: Chemical structure of Rosuvastatin
MATERIALS AND METHODS:
Chemicals and reagents:
Active pharmaceutical ingredients, EZE and RSV calcium were obtained as gift samples from Teva Pharmaceuticals Industries Ltd. and Glenmark Pharmaceuticals Ltd., Goa, India respectively. Tablets ‘Razel Ez 10’ containing ezitimibe 10mg and rosuvastatin calcium 10mg were procured from local pharmacy.
Instrumentation and equipment:
UV spectrophotometer, Shimadzu model UV 1800 was used. A Citizon CY 204 electronic weighing balance was used for weighing the samples. Ultrasonics sonicator was used for sonication.
Choice of diluent:
The choice of diluent was arrived at by using various proportions of methanol and water i.e. (60:40 V/V), (50:50, V/V), (40:60, V/V). The absorbance values of solutions prepared by taking 0.1mL of stock and diluted to 25mL with diluent were recorded. The proportion of methanol: water allowing the lowest amount of methanol and giving a stable absorbance reading was selected.
Preparation of solutions:
Preparation of diluent:
Diluent was prepared by mixing proportionate quantity of methanol and distilled water (V/V) in a volumetric flask. The mixture was sonicated for 5 minutes.
Preparation of stock solution (1000µg/mL):
About 100mg of each EZE and RSV calcium were separately weighed and transferred into two 100mL volumetric flask and dissolved with sufficient volume of methanol. Volume was made up to 100mL with methanol to get concentration of EZE and RSV 1000 µg/mL.
Preparation of working standard solution (200 µg/mL):
Working standard solution of EZE and RSV calcium were separately prepared by taking 5mL of stock solution of each EZE and RSV and diluting it to 25mL using diluent to get concentration of 200µg/mL.
Choice of wavelengths:
The working standard solution of the drugs were scanned in the UV range 220nm to 400nm against the blank. Using the peak pick function, the λ max for the drugs were obtained. The spectra were then overlain to obtain the Isosbestic point. The wavelengths for analysis for the UV method were then finalized as follows: λ1 (Isosbestic point) and λ2 (λmax of drug having longer λmax wavelength).
Determination of Absorptivity (a):
For determining absorptivity of the drugs, aliquots of stock solution of EZE and RSV were delivered into a series of 25mL volumetric flasks and volume was made up with diluent to attain concentrations within the Beer Lambert’s range. Absorbance of the prepared solutions were measured at selected wavelengths. The absorptivity values for EZE and RSV calcium were calculated using the formula as below:
Method Validation:
Linearity:
For determining linearity, aliquots of stock solutions of EZE and RSV were delivered into a series of 25mL volumetric flasks and volume was made up with diluent to attain concentrations ranging from 2-100µg/mL.
Absorbance of the solutions was recorded at predetermined wavelengths and calibration curves were plotted of Absorbance v/s Concentration. The linear regression equations and correlation coefficients (r2) were determined for the drugs at the predetermined wavelengths and the Beer- Lambert’s range was established.
Precision:
Repeatability:
From the working standard solutions of each drug, 2.5 mL was withdrawn and transferred to six 25mL volumetric flasks and volume was made up to the mark with the diluent. Absorbance of these solutions was measured against the blank at predetermined wavelengths. The amount of drugs and % RSD was calculated.
Intra-day Precision:
Intra-day precision was carried out by performing assay procedure on marketed tablet formulation three times on the same day in triplicate. The % assay and % RSD was calculated.
Inter-day Precision:
Inter-day precision was carried out by repeating assay procedure on the marketed tablet formulation on three different days in triplicate. The % assay and % RSD was calculated.
Accuracy:
Accuracy was established by performing % recovery studies by using placebo powder. The accuracy of the method was performed at three concentration levels using stock solution (50%, 100% and 150%) in triplicate and the % recovery was determined.
Robustness:
Robustness of the developed method was determined by performing assay procedure on marketed tablet formulation by employing the following deliberate variations:
· Change of UV spectrophotometer instrument.
· Change of analyst.
· Change of proportion of solvents in diluent i.e. methanol and water (45:55, V/V).
Assay of marketed formulation:
For preparation of sample solution for assay, ten tablets of Razel Ez 10 were accurately weighed and finely powered in a glass mortar and pestle. Tablet powder equivalent to 10mg of RSV calcium was weighed and transferred to 50mL volumetric flask and about 35mL of methanol was added and sonicated for 10minutes. The volume was adjusted with methanol. The resulting solution was filtered through Whatman filter paper no. 1. From above filtrate, 2.5mL was pipetted out into a 25 mL volumetric flask and absorbance was recorded at predetermined wavelengths. Percent purity of the marketed formulation was calculated.
Formula used for calculation of concentration of drug by Q Absorption Ratio Method:
Cx = (QM-Qy/Qx-Qy) X A1/ ax1
Cy = (QM-Qy/Qx-Qy) X A1/ay1
where, Cx = Concentration of drug X; Cy = Concentration of drug Y
QM = Absorbance of tablet solution at λ1/Absorbance of tablet solution at λ2,
Qx = ax2/ax1; Qy = ay2/ay1,
A1 = Absorbance at λ1 (Isosbestic point); A2 = Absorbance at λ2,
ax1= Absorptivity at λ1 of drug X; ay1 =Absorptivity at λ1 of drug Y.
RESULTS AND DISCUSSION:
Method Development:
Choice of diluent
EZE and RSV are both soluble in methanol. Most of the analytical methods reported use of methanol as solvent. In the current developed method, optimum use of methanol so as to bring down the cost of analysis was attempted. Stock solutions were prepared in methanol and choice of diluent was arrived by using various proportions of methanol and water (60:40 V/V), (50:50, V/V), (40:60, V/V) and (30:70, V/V). Mixture of methanol and water in proportion of 40:60, V/V was chosen as diluent with satisfactory results.
Choice of wavelength:
Working standard solutions of EZE and RSV when scanned in the UV range 220-400 nm against blank, showed λmax of the drugs at 231.2 nm and 242 nm respectively. The overlain spectra as in fig. 3 of EZE and RSV showed Isosbestic points at 234.8 nm and 262.3 nm. The Isosbestic point at 234.8 nm was selected for further analysis.
Fig. 3: Overlain spectra of EZE and RSV showing Isosbestic points.
The wavelengths for analysis were arrived as follows: 234.8 nm (Isosbestic point) was selected as λ1 and 242 nm (λmax of RSV) was selected as λ2.
Determination of Absorptivity:
Absorbance of solutions of the drugs within linearity range were recorded at selected wavelengths. The mean absorptivity values obtained are displayed in the table I:
Table I: Mean Absorptivity for EZE and RSV by Q Absorption Ratio Method
|
Sr. No |
Drug |
ax1 (Mean Absorptivity at 234.8 nm) gm -1 cm -1 |
ax2 (Mean Absorptivity at 242 nm) gm -1 cm -1 |
|
1 |
EZE |
40.072 |
40.00 |
|
2 |
RSV |
41.559 |
41.65 |
Method Validation:
Linearity:
The calibration curves are displayed in Fig. 4 and Fig. 5 below.
Fig. 4: Calibration curves of EZE at 234.8 nm and 242 nm.
Fig. 5: Calibration curves of RSV at 234.8 nm and 242 nm.
As seen above, Beer Lambert’s range was established for EZE and RSV calcium from 2-40 (µg/mL).
Precision:
Solutions for precision studies were prepared as per methodology and absorbance values were recorded at selected wavelengths. The results for % assay and % RSD by Q absorption ratio method are depicted in table II.
Table II: Precision data of EZE and RSV by Q Absorption Ratio Method
|
Drug |
Conc. (µg/mL) |
Mean % assay ± SD |
%RSD |
|
Repeatability (n=6) |
|||
|
EZE |
20 |
100.333± 0.516 |
0.514 |
|
RSV |
20 |
105.533 ± 1.865 |
1.760 |
|
Intra-day precision (n=3) |
|||
|
EZE |
20 |
104.066 ± 0.378 |
0.363 |
|
RSV |
20 |
106.266 ± 1.322 |
1.244 |
|
Inter-day precision (n=3) |
|||
|
EZE |
20 |
104.212 ± 0.520 |
0.499 |
|
RSV |
20 |
107.046 ± 0.881 |
0.823 |
The % RSD for repeatability study, intraday and inter-day precision for EZE and RSV calcium by Q Absorption Ratio Method was found to be within the accepted limits; hence the developed method was precise.
Accuracy:
Accuracy of the method was confirmed by carrying out % recovery of standard solution of drug added at three levels (50%, 100%, and 150%) in preanalysed formulation. The results obtained are depicted in table III.
Table III: % Recovery of EZE and RSV by Q Absorption Ratio Method
|
Volume of stock taken (mL) |
Spike Level (%) |
Amt. of pure drug added (µg/mL) |
Abs. at 234.8 nm |
Abs. at 242 nm |
Amt. of std recovered (µg/mL) |
% recovery |
Mean Recovery (%) |
||||
|
EZE |
RSV |
EZE |
RSV |
EZE |
RSV |
EZE |
RSV |
||||
|
0.25 |
50 |
10 |
10 |
0.745 |
0.760 |
9.71 |
9.52 |
97.10 |
95.20 |
97.92 |
95.06 |
|
0.25 |
50 |
10 |
10 |
0.746 |
0.761 |
9.72 |
9.50 |
97.20 |
95.00 |
||
|
0.25 |
50 |
10 |
10 |
0.747 |
0.763 |
9.95 |
9.50 |
99.50 |
95.00 |
||
|
0.5 |
100 |
20 |
20 |
1.68 |
1.705 |
19.70 |
19.98 |
98.90 |
104.4 |
98.60 |
104.40 |
|
0.5 |
100 |
20 |
20 |
1.69 |
1.705 |
18.00 |
19.99 |
98.00 |
104.3 |
||
|
0.5 |
100 |
20 |
20 |
1.68 |
1.704 |
19.70 |
19.98 |
98.90 |
104.4 |
||
|
0.75 |
150 |
30 |
30 |
2.422 |
2.466 |
29.00 |
28.70 |
101.4 |
95.70 |
100.63 |
95.46 |
|
0.75 |
150 |
30 |
30 |
2.422 |
2.466 |
29.99 |
28.70 |
101.4 |
95.70 |
||
|
0.75 |
150 |
30 |
30 |
2.424 |
2.465 |
29.70 |
29.40 |
99.10 |
95.00 |
||
The results of % recovery at 50%, 100%, and 150% for EZE and RSV lie within acceptable criteria of 95-105% establishing the accuracy of the method.
Robustness:
The results for robustness study are depicted in table IV.
Table IV: Results for robustness study by Q Absorption Ratio Method
|
Parameters |
Absorbance |
% Assay (n=6) |
||
|
231.2 nm |
242 nm |
EZE |
RSV |
|
|
Change in UV instrument |
||||
|
Instrument 1 |
0.179 |
1.167 |
95.8 |
98.90 |
|
Instrument 2 |
0.182 |
1.66 |
95.00 |
99.00 |
|
Change of analyst |
||||
|
Analyst 1 |
0.182 |
0.168 |
96.10 |
98.00 |
|
Analyst 2 |
0.184 |
0.166 |
96.00 |
98.20 |
|
Change of diluent proportion |
||||
|
(40:60, V/V ) |
1.181 |
0.166 |
96.00 |
98.40 |
|
(45:55, V/V ) |
1.177 |
0.166 |
95.00 |
97.00 |
As seen, deliberate changes introduced did not significantly affect the results of analysis. Hence, the developed Q absorption ratio method for EZE and RSV calcium was found to be robust.
Assay of Marketed formulation:
The results of marketed formulation are depicted in table V.
Table V: Results of assay of marketed formulation by Q Absorption Ratio Method
|
Drug |
Label claim (mg) |
Amount found (mg) |
% Assay |
|
EZE |
10 |
9.89 |
102.06 |
|
RSV |
10 |
9.90 |
109.00 |
As seen, % assay in marketed formulation for EZE and RSV calcium was within the acceptance criteria (90-110%).
CONCLUSION:
A novel, simple and economical UV spectrophotometric method for simultaneous analysis of EZE and RSV calcium in bulk and in tablet formulation has been developed and validated. The developed UV spectroscopic method based on Q absorption principle employed 40% methanol as diluent for analysis. The method was validated as per ICH guidelines. The % assay of EZE and RSV in tablet dosage form was found to be 102.06 % and 109.00 % respectively, which complied with the acceptance criteria. Hence, the developed method can be used as cost effective alternative to the earlier reported methods for the simultaneous estimation of the drugs both in bulk and in their combined formulation.
ACKNOWLEDGEMENT:
The authors are thankful to Teva Pharmaceuticals Industries Ltd. and Glenmark Pharmaceuticals Ltd., Goa, India for the gift samples of EZE and RSV.
CONFLICTS OF INTEREST:
Authors do not have a conflict of interest.
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Received on 12.06.2021 Modified on 15.04.2022
Accepted on 07.01.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(9):4276-4280.
DOI: 10.52711/0974-360X.2023.00700