Development and Validation of a Derivative Spectrophotometric Method for Simultaneous Determination of Simvastatin and Ezetimibe

A combination of simvastatin and ezetimibe with complementary mechanisms of action is used for treating high levels of cholesterol in the blood. The aim of this study was to develop a rapid and sensitive derivative spectrophotometric method for analysis of these drugs in combined dosage forms. A first order derivative spectrophotometric method was developed for simultaneous determination of simvastatin and ezetimibe using zero-crossing technique. The measurements were carried out at 219 and 265 nm for simvastatin and ezetimibe respectively. The described method was found to be linear (r>0.999) over the range of 2-40 μg/mL for simvastatin in the presence of 10 μg/mL ezetimibe at 219 nm and in the range of 1-20 μg/mL of ezetimibe in the presence of 20 μg/mL of simvastatin at 265 nm. The within-day and between-day precision values for both drugs were less than 3% (CV). Also, good recoveries were obtained with both synthetic mixtures and commercial tablets. The proposed method was successfully applied for simultaneous determination of simvastatin and ezetimibe in a pharmaceutical dosage form without any interference from excipients.


Introduction
Simvastatin is a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-COA) reductase which involve in the conversion of HMG-COA to mevalonate, an early and rate-limiting step in cholesterol biosynthesis.Simvastatin is widely used for the treatment of hypercholesterolemia. Simvastatin in higher doses can also reduce triglyceride levels 1 .
Ezetimibe selectively inhibits cholesterol absorption by enterocytes through binding to the Niemann-Pick C 1 -like 1(NPC 1 L 1 ) protein in the small intestine and effectively blocks the intestinal absorption of dietary and biliary cholesterol 2 .As cholesterol absorption reduced a reduction in the cholesterol into chylomicrons will occur which may decrease atherogenesis.Ezetimibe can also reduce the LDL-C levels and fasting triglycerides 1 .
A combination of simvastatin and ezetimibe have complementary action with each other and has been shown to be significantly more effective than simvastatin or ezetimibe alone for reducing plasma concentration of cholesterol 1,3 .
Literature survey showed few methods for the determination of simvastatin in dosage forms or human plasma using spectrophotometry methods 4,5 , HPLC 6,7 or voltammetry 8 .Also there are some reports for the determination of ezetimibe using HPLC [9][10][11] or LC-MS/MS 12,13 .Direct HPLC methods are also recommended in the USP and BP for determination of simvastatin in tablets 14,15 , but no pharmacopoeial method is reported for determination of ezetimibe alone or in combination with simvastatin in pharmaceutical dosage forms.There is also few reported HPLC methods for simultaneous determination of simvastatin and ezetimibe in drug products [16][17][18] .HPLC methods are more complex, time consuming and expensive than spectrophotometric methods.Direct spectrophotometric methods could not usually be used for determination of combined drugs because of overlapping of absorption bands.Derivative spectrophotometric methods provide a powerful tool for quantitative analysis of multi-component mixtures without previous separation.No spectrophotometric method has been reported yet for simultaneous determination of simvastatin and ezetimibe.The purpose of this study was to develop a derivative spectrophotometrc method for determination of simvastatin and ezetimibe in combination dosage forms.

Experimental
Ezetimibe was from Hangzhou Storshine Pharmaceutical Co (Hang Zhou, China) and simvastatin was from Samco Life Sciences (Maharashta, India).Methanol was of analytical grade and obtained from Merck (Darmstadt, Germany).

Apparatus
Spectrophotometric analysis was carried out in 1cm quartz cells using a Shimadzu UV-160 double beam UV visible spectrophotometer (Shimadzu, Kyoto, Japan) with a fixed bandwidth (2 m).The zero order absorption spectra were recorded over the range of 200-300 nm against a solvent blank.The derivative spectra were obtained over the same wavelength range at different slit width (∆λ).

Standard solutions
Stock standard solutions of ezetimibe and simvastatin were prepared separately in methanol to obtain a concentration of 100 µg/mL and 200 µg/mL of ezetimibe and simvastatin respectively.Standard solutions of 10 µg/mL ezetimibe and 20 µg/mL simvastatin were prepared by subsequently diluting of stock solutions.
Volumes of the standard stock solutions were also transferred into two sets of 100 mL calibrated flasks.The first series contained constant concentration of ezetimibe (10 µg/mL) and varying concentrations of simvastatin (2-40 µg/mL).The second series contained a constant concentration of simvastatin (20 µg/mL) and varying concentrations of ezetimibe (1-20 µg/mL).

Spectrophotometric measurements
Zero order spectra of standard solutions of ezetimibe (10 µg/mL) and simvastatin (20 µg/mL) were reported in the range of 200-300 nm.The first order derivative spectra of standard solutions were obtained in the same range.The first order derivative amplitudes (D 1 ) were recorded at 219 nm and 265 nm for determination of simvastatin and ezetimibe respectively.
The calibration curves for determination of ezetimibe (1-20 µg/mL) in the presence of simvastatin (20 µg/mL) and simvastatin (2-40 µg/mL) in the presence of ezetimibe (10 µg/mL) were also constructed at the metioned wavelengths using D 1 values at different concentrations.

Accuracy and precision
To establish the accuracy and precision of the proposed method, two series of solutions containing 2,10 and 40 µg/mL of simvastatin plus 10 µg/mL of ezetimibe and 1,2 and 20 µg/mL of ezetimibe plus 20 µg/mL simvastatin were prepared respectively and analyzed as discussed above.Each analysis was done in triplicate and for three consecutive days and the precision was calculated by within-day and between-day variations.The accuracy of the method was measured as percentage of deviation between added and measured concentrations.

Recovery studies
To check the relative recovery of the developed method and to study the interference of formulation additives, standard addition method was used.Ten tablets of simvastatin + ezetimibe were weighed and finely powdered.An accurately weighed amount of the powder equivalent to the averaged weight of one tablet transferred to a 100 mL volumetric flask and 50 mL methanol was added.After sonication for 15 min, the solution adjusted to the mark with methanol and centrifuged at 3000 rpm for 15 min.To four volumetric flasks 3 mL of the clear solution (30% of one tablet) and 0, 3, 5 and 7 mL of stock standard solutions of ezetimibe (100 µg/mL) and simvastatin (200 µg/mL) was added to reach 30%, 50% and 70% each of the standard drugs.The concentration of ezetimibe and simvastatin were measured using the above method and compared with the concentration of drugs in another series of solutions prepared at the same concentrations without the tablet solution.

Application of the method
The solution of tablets containing ezetimibe (10 mg) and simvastatin (20 mg) was prepared according to the method mentioned in recovery studies and analysed after 10 times dilution.

Derivative spectrophotometric method
Zero order absorption spectra of simvastatin and ezetimibe showed overlapping spectra which prevent the direct simultaneous determination of these drugs (Figure 1).Derivative spectra were recorded for standard solutions of ezetimibe and simvastatin at different orders (one to four) and ∆λ values and the recorded spectra was examined to select a suitable spectra to be used for determination of drugs.The best results were obtained by using the first order derivative spectra traced with ∆λ=36 nm (N=9).Zero crossing points for ezetimibe (265 nm) and simvastatin (219 nm) as presented in Figure 2 were used for simultaneous determination of these compounds.

Linearity
Calibration curves (five repetitions) of the synthetic standard solutions of ezetimibe and simvastatin were constructed under the optimized conditions.The statistical data are summarized in Table 1.The linearity of the calibration curves were validated by the high value of the correlation coefficient and value of intercept on ordinate which was close to zero.

Accuracy and precision
The results of accuracy and precision tests are illustrated in Tables 2 and 3.The within-day and between-day variations showed coefficient of variation (CV %) values less than 2% and 2.6% for ezetimibe and simvastatin respectively in all three selected concentrations.The relative error values were also less than 1.3% and 2% for ezetimibe and simvastatin.

Recovery studies
The results for recovery of ezetimibe and simvastatin according to the developed method are showed in Table 4.The relative recoveries of both compounds in different concentrations in the presence of tablet excipients were in the range of 98-105%.

Sensitivity
The limit of quantification with CV<2.6% was found to be 1 µg/mL and 2 µg/mL for ezetimibe and simvastatin respectively.

Application
The proposed method was used for the simultaneous determination of ezetimibe and simvastatin in tablets (label claim: 10 mg ezetimibe and 20 mg simvastatin) without prior separation of the excipients.The results shown in Table 5 indicates that the proposed technique can be used for simultaneous determination and routine quality control analysis of these two drugs in binary mixture in pharmaceuticals.Comparison with a reported HPLC method 16 showed no significant difference between the HPLC and proposed spectrophotometric method.

Conclusion
The proposed and validated first order derivative spectrophotometric method for simultaneous determination of ezetimibe and simvastatin is simple, specific, reproducible, rapid and cost effective which is suitable for routine pharmaceutical analysis.The proposed method can be used without any prior separation of drugs from the tablet excipients.

Table 1 .
Statistical data of calibration curves of ezetimibe and simvastatin in mixtures with different concentrations using first-order derivative

Table 2 .
Accuracy and precision data of determination of ezetimibe (1-20 µg/mL) in the presence of simvastatin (20 µg/mL) by first order derivative spectrophotometry

Table 3 .
Accuracy and precision data of determination of simvastatin (2-40 µg/mL) in the presence of ezetimibe (10 µg/mL) by first order derivative spectrophotometry

Table 4 .
Relative recovery of ezetimibe and simvastatin obtained by the first order derivative

Table 5 .
Results of the analysis of commercial product containing ezetimibe and simvastatin by first order derivative spectrophotometry