UHPLC Specific Method For Simultaneous Determination of Probable Impurities of Ezetimibe and Simvastatin in Combined Dosage Form

Method development, degradation impurities that may appear in Ezetimibe, Simvastatin tablets with Ultra High Performance Liquid Chromatography (UHPLC or UPLC) technique. Water’s Acquity High strength (HSS) T31.8µ, 100x2.1mm column is used for critical separation between closely eluting impurities originating from the combined dosage form. Perchloric acid buffer (1.0 mL/L)and acetonitrile are selected as Elution phase A, B. The resolution between polar, mid polar and highly polar impurities is achieved with flow of 0.5 mL per minute in step gradient mode with short run time. Detection was done at 238nm for quantification of both Ezetimibe and Simvastatin impurities. Method validation is performed as per compound requirement. The validated UHPLC method is specific and stability demonstrating for process or degradation impurities that may originate from drug product or peaks appears during stress degradation in Ezetimibe and Simvastatin tablets . The method was accurate, robust and validation is as per ICH guide lines. The results are more reliable, precise and reproducible.


Fig. 2. SIM Structure
EZE and SIM tablets are marketed with the name VYTORIN.It is available for oral administration.In the combined drug product EZE is present as 10mg only, but SIM is present as 10mg, 20mg, 40mg, and 80mg.The therapeutic nature of this combined drug is to lower the amount of bad cholesterol produced by liver and to enhance the good cholesterol.The combination of EZE and SIM is more effective in lowering the production of bad cholesterol, elevate good cholesterol and hence minimize the chances of heart stroke. 4 per the wide-ranging literature hunt, estimation of the EZE and SIM in its combined dose form is reported by Spectrophotometric determination [5][6][7] , by HPTLC [8][9] and by HPLC [10][11][12][13][14][15] .There are also HPLC methods cited for impurities quantification in their individual dosage forms [16][17][18] .There are HPLC methods reported to determine probable impurities in their combination dosage form 19 .LCMS method is also reported for quantification of EZE and SIM in human plasma 20 .Literature search indicates absence of method for quantification of probable impurities originating from EZE and SIM in combined dose combination using UHPLC.High variations are seen in the polarity nature of the impurities of EZE and SIM.Hence for quantification of probable impurities from EZE and SIM by ultraperformance liquid chromatographic technique (UHPLC) is the preliminary option to get shorter run time and to achieve specific and stability demonstrating so that number of samples can be analyzed in a day.The current method is more precise and robust when compared to Seshukumar et al., This method is capable to separate all the probable impurities and peaks formed during different stress conditions in forced degradation studies and meets the ICH requirement parameters [21][22][23][24][25] .Probable impurities that are formed from EZE is EZE impurity-1, EZE impurity-2, Fig. 3.For SIM is SIM Impurity-1, SIM Impurity-2a, SIM Impurity-2b, SIM Impurity-3,4, 5, 6 and 7 Fig. 4. SIM Impurity-2a and SIM Impurity-2b elute as split peak as both peaks are epimers.

Instruments
Waters acquity UPLC instrument with binary pump, auto injector, thermostated column controller and PDA detector used for evaluation of peak homogeneity, purity of analytes and for quantification purpose.Empower Pro software was used as interphase.Waters Acquity High strength silica (HSS) T31.8µ, 100×2.1mmcolumn was used for separation of components and the probable impurities originating from EZE and SIM.

Chemicals
Pure components (EZE and SIM), EZE and SIM impurities, Ezetimibe and Simvastatin tablets (VYTORIN 10/20 tablets) Acetonitrile, Perchloric acid Sodium dihydrogen orthophosphate dihydrateare sponsored by Aurobindo Pharma Limited.Milli Q water is used for all purposes.

Method development and optimization
EZE and its impurities are polar when compared to SIM and its impurities.Attaining specific and stability demonstrating with short run time method is difficult in HPLC.In order to develop a short run time method which is specific, the choice of separation for these impurities which are polar and highly non polar is likely dependent on low ID and low micron columns using UHPLC.
Trials were initiated using Acquity BEH C18, 1.7µ, 100x2.1mmcolumn using pH 3.0 sodium phosphate buffer along with acetonitrile in step gradient mode with 0.5 mL/min, at temperature 40°C.EZE, its impurities and SIM, its impurities were injected to observe the pattern of elution.EZE and its impurities eluted very closely and SIM, SIM Impurity-5 co-eluted.Trials were extended with the above conditions by modifying gradient programme, but satisfactory separation could not be achieved.
Trials were continued further using same above conditions by using the column High strength silica (HSS)-T3, 1.8µ, 100x2.1mm.In initial trials SIM Impurity-7 merged with an unknown peak and later in further trails SIM Impurity-2a, SIM Impurity-2b, SIM Impurity-3 co-eluted.Trials could not be succeeded by modifying gradients, column temperature and flow.
From the above experiments it could be understood that separation of impurities is favorable in lower pH only.The idea to select perchloric acid is, it acts as ion pair and less viscous in nature.Elution Phase~A is 0.1% Perchloric acid and phase~B is acetonitrile.
Step gradient mode elution is opted with High strength silica (HSS)-T3, 1.8µ, 100x2.1mmcolumn.Logical trials were initiated and the elution pattern of the components and its impurities were studied at 0.5 mLflow.
Finally chromatographic conditions were finalized with high strength silica (HSS)-T3,1.8µ,100x2.1mmcolumn kept at 40°C with 0.5mL.min - flow.Acetonitrile along with 20 mM sodium phosphate buffer mixed in the proportion of 40:60 is opted as diluent for preparing solutions.
To check the individual impur ities wavelength maxima between 220nm and 240nm and whereas SIM shows at about 280 nm (Fig. 3).To adopt a common quantification wavelength for all impurities, it is proposed to keep 238nm.5 µL injection volume showed satisfactory responses at 200µg/mL for EZE and 400 µg/mL for SIM.

Finalized chromatographic conditions
Finalized conditions are depicted in Table 1.
System suitability data have been summarized in Table 2.

Solution preparations Standard solution preparation
Pure dr ug solution concentration containing 0.20 and 0.60 µg/mL of EZE and SIM respectively is prepared.

Sample solution preparation
Take not less than 10 tablets and crush them to get finer power.Take sample powder corresponding to 10 mg of EZE or 20 mg of SIM and sonicated for 30 minutes.Test solution is mixed intermittently to get homogeneity of solution.15 mL of sample solution is taken into centrifuge tube and performed centrifugation at 5000 rpm for about 10 minutes.The upper clear solution filtered and sample concentration is about 200 and 400 µg/ mL of EZE, SIM.

Specificity and Stress degradation
Specificity of the optimized method was confirmed by injecting placebo, individual impurities.

Precision
Sample powder taken for analysis and prepared six individual test preparations, spiked with impurities at proposed specification level and loaded into UHPLC.Observed results were calculated for %w/w and %RSD and is assessed for each impurity.Similarly ruggedness experiment was repeated with another lot column, different UHPLC system on different day.

Sensitivity of the test method
LOD along with LOQ values were determined by loading series of injections ranging from (1 to 150) % of proposed specifications, using impurity stock solutions and precision was performed based on predicted concentrations derived from linearity curves.

Linearity and range
Linearity and range was established by injecting series of injections ranging from observed LOQ level of each impurity to 150% to the proposed specification limit.Slope, correlation coefficient and Y-intercept for each impurity were calculated individually from linearity curve.

Accuracy
Impurity stock solution was prepared and accuracy was proved by spiking them to the control sample at proposed LOQ, 50%, 100% and 150% concentration level.Based on %w/w observed results, calculated the accuracy values.Each spiking procedure was done in triplicate preparations.

Solution Stability
To establish solution stability, standard and sample solutions were periodically injected at refrigerated temperature (~6°C) at different time intervals.Values at different time points were extrapolated from initial freshly injected solutions of standard and sample.

Robustness
By performing deliberate changes in the proposed methodology, robustness of the method was assessed.Flow rate was from 0.45mL and 0.55mL per min, temperature is 35°C and 45°C, and wavelength is 233nm and 243nm.In each experiment one parameter was deliberately altered and rest of the parameters were kept unchanged.

Specificity and Stress studies
Comprehensive degradation data suggests the following behavior.Table 4 and Fig. 6 explain the data.

CONCLUSION
Developed method is suitable for quantification of EZE,SIM impurities present in combination drug product in a single injection.This method is robust, specific and stability demonstrating.There is no such method published earlier using UHPLC especially under these optimum conditions.Wavelength of 238nm is suitable for quantification purpose.Linearity data shows greater than 0.99 correlation coefficient.Accuracy, precision are obeyed and method followed as per ICH guide lines.The samples at different shelf life conditions can be tested using this method without any interference.Also, short run time of the assay method enables analysis of more number of samples.Hence the method is appropriate for estimation of impurities.

Fig. 6 .
Photolytic (10K Lux along 7 days ----No significant known and unknown impurities of with UV 200 watt hours/m 2 ) EZE and SIM were seen