Densitometric Determination of Ezetimibe in the Presence of its Alkaline Degradation Product

Objectives: A simple, accurate, selective and sensitive densitometric method was developed for the determination of ezetimibe in the presence of its alkaline degradation product. Methods: TLC-densitometric separation of ezetimibe from its degradation products was carried out on silica gel plates using ethyl acetate: n-hexane (2:1 v/v) as a developing system. This method depends on quantitative densitometric evaluation of ezetimibe at 230 nm over a concentration range of 1–8 μg/spot. Ezetimibe and its alkaline degradation product were resolved with Rf values of 0.49 and 0.68 respectively. Results: The proposed method has been successfully applied to the analysis of ezetimibe in pharmaceutical dosage form without interference from additives and the results were statistically compared with the reported method. Conclusion: TLCdensitometric technique has provided a simple, straightforward method for separating ezetimibe and its alkaline degradation product simultaneously.

Reviewing the literature on the determination of (EZE) in the presence of its alkaline degradation product, revealed the lack of any quantitative determination method based on separating technique.
The aim of this work is to develop a simple, economic, rapid, sensitive, accurate and precise densitometric method for determination of (EZE) in the presence of its alkaline degradation product without sophisticated instruments or any pretreatment.

Theory of TLC-densitometric chromatography method
This technique offers a simple way to quantify separated drugs directly on the TLC plate by measuring the optical density of the separated bands.The amounts of compounds are determined by comparing to a standard curve from reference materials chromatographed simultaneously under the same condition [33][34]

Standard and sample solutions Standard solution:
A stock solution of (1000 µg/mL) for (EZE) was prepared by dissolving 100 mg of (EZE) in 100 ml methanol.10mL of this stock solution was transferred into 100-ml volumetric flask and diluted to the mark to give a working solution of (100 µg/mL). 5,3200 mg of EZE was dissolved in 50 mL (0.1 M methanolic sodium hydroxide), The solution was refluxed at 80º C for 30 minutes.The time required for complete degradation was followed by spotting on TLC plates at 10 minute intervals for 30 minutes.The plates were developed using ethylacetate: n-hexane (2:1 v/v), which indicate complete degradation with complete separation.The cooled solution was neutralized with 1M hydrochloric acid, then filtered.The filtered solution was evaporated under vacuum till dryness, the residue was dissolved by pure methanol and filtered (several times), evaporate the filtrate using Rota-vapor under vacuum, the residue was dissolved in 100 pure methanol to give a degradate stock solution of (1mg/mL).10 mL of this stock solution was transferred into 100-mL volumetric flask and then, diluted to the mark with methanol to give a working solution of (100 µg/mL) (Figure 1).

Pharmaceutical dosage form
Ten zetamibe ® tablets were accurately weighed, crushed and finely powdered in a mortar.A quantity of the powder equivalent to one tablet was accurately weighed and transferred to a 100-mL volumetric flask then 50 mL of methanol was added.The flask was sonicated for 15 min.Then filtered through Whatman filter paper no 41 into 100-mL volumetric flask, the residue was washed with methanol.All the filtrate and washings were collected and the volume was adjusted to 100 ml with methanol to obtain the solution claimed to contain 100 µg/mL of EZE.Filtration system was evaluated to ensure that the filter does not adsorb any of the drug.

Procedure Construction of calibration graph
The aliquots of stock solution of EZE (1000µg/mL) which equal to (1, 2, 3, 4, 5 and 6 µg/mL) were spotted as bands of 5mm width on TLC plates (20 ×10 cm).Bands were applied at 15 mm intervals and 15mm from the bottom and side edges of the plate.Linear ascending development was performed in a chromatographic tank which previously saturated with ethyl acetate: n-hexane (2:1 v/v) for 30 minutes at room temperature.EZE was scanned at 230 nm.A calibration curve relating the optical density of each spot to the corresponding concentration of EZE was constructed.The regression equation was computed for the studied drug and used for determination of unknown samples of ezetimbe (Figure 2a and b).portions equivalent to (6.0 -10 µg) of its alkaline degradate from its working solution (100 µg/mL) were added to the same flasks and then, diluted to the mark with methanol and mixed well.The procedure under 2.7.1.section was repeated with laboratory prepared mixtures which, give a high degree of resolution, (Figure 3 a and b).

Analysis of pharmaceutical dosage form
The procedure mentioned above.was repeated and the proposed method should be applied in different concentrations of EZE which, covering the concentration range.Tablet content of EZE was calculated using the corresponding regression equation.

RESULTS AND DISCUSSION
To the best of our knowledge, the literature survey revealed that there is no separating technique was developed for the determination and quantification of EZE in the presence of its alkaline degradation product.It was found that the complete degradation of EZE proceeds optimally in alkaline media and at elevated temperature.
The structure of the alkaline degradate was elucidated and confirmed by using 1 H NMR technique where, the 1 H NMR of intact ezetimibe (Figure 4), showed a characteristic proton of aliphatic (OH) at 4.86 ppm, aromatic (OH) at 9.40 ppm and aliphatic proton (CH2) at 1.75 ppm.While, 1 H NMR of degradate (Figure 5), showed disappearance of aliphatic (OH) at 4.86 ppm and appearance of characteristic proton of (CO-NH) at 9.98 ppm and aromatic OH still present at 9.06 ppm 4 .So, the above findings are in agreement with the reported mechanism of recyclization of ezetimibe under alkaline conitions 32 as shown in Figure 1.
The role of this method is to develop a simple, accurate and selective densitometric method for the determination of EZE in the presence of its alkaline degradation product.

Spectral characteristics
The absorption spectra of EZE and its alkaline degradation product were recorded over the range 200-400 (Figure 6). 34,35o improve separation of bands, it was necessary to investigate the effect of different variables.The study of the optimum parameters for maximum separation was carried out as follows:

Band dimension
Different band dimension was tested in order to obtain sharp and symmetrical separated peaks.The optimum bandwidth chosen was 5 mm and the interspace between bands was 15 mm.

Scanning wavelength
Different scanning wavelengths (251, 246, 240, 230 and 235 nm) were tried.Peaks at 230 nm were more sharp, symmetrical and minimum noise was obtained.

Slit dimension of scanning light beam
The slit dimension of the scanning light beam should ensure complete coverage of band dimensions on the scanned track without interference of adjacent bands.Different slit dimensions were tried, where 5 mm × 0.2 mm proved to be the slit dimension of choice which provides highest sensitivity.
This method is based on the difference in the Rf values of EZE (Rf = 0.49 ± 0.02) and its alkaline degradate (Rf = 0.68 ± 0.01) (Figure 5 a).

Method validation
The linearity range, limit of detection (LOD), limit of quantification (LOQ), selectivity, accuracy and precision of the proposed method was tested according to International Conference on Harmonization (ICH) guidelines 36 .

Linearity and range
The calibration graph for the proposed method was constructed by plotting the area under peak versus drug concentrations of EZE.The regression plot was found to be linear over the range of (1-8 μg/band).
The linear regression equations for the graphs were: Y = 3688.4X -17408 r = 0.9996 Where, y is the area under peak values, x is the drug concentration and r is the correlation coefficient.Linearity range, regression equation, intercept, slope and correlation coefficient for the calibration data are supplied at Table 1.

Limit of detection (LOD) and limit of quantification (LOQ)
LOD and LOQ were calculated according to ICH guidelines 49 from the following equations:  Where Sa is the residual standard deviation of a regression line.
LOD and LOQ values of EZE for the proposed method were listed in the Table 1.

Selectivity
The selectivity of the methods was achieved by the analysis of different laboratory prepared mixtures of EZE and its alkaline degradation product within the linearity range.Satisfactory results listed in the Table 2, and the results of the standard addition technique Table 3, prove that the proposed method can selectively analyze EZE without any interference from its alkaline degradate or the excipients.

Accuracy
The accuracy of the proposed method calculated as the mean percent recovery (% R), by applying the proposed procedure for triplicate determination of three concentration levels covering the specified range for EZE (3, 4 and 5 μg/band).The concentrations were obtained from the corresponding regression equations and the mean percent recoveries, shown in Table 1.The accuracy of the methods was further assured by the use of the standard addition technique.It was performed by the addition of known amounts of pure EZE to known concentrations of the pharmaceutical preparation and the resulting mixtures were assayed, and the results obtained were compared with the expected results Table 3.The good recoveries of the pure added of EZE suggested good accuracy of the proposed methods.

Precision
The Precision of the proposed method calculated as percent relative standard deviation (% RSD) of the percent recoveries, was checked by applying the proposed procedures for triplicate determination of three concentration levels covering the specified range for each drug (3, 4 and 5μg/band) in the same day (intra-day analysis) for repeatability and on three different days (inter day analysis) for intermediate precision.The results were listed in the Table 1.

Robustness
The robustness of the proposed method tested by applying deliberated changes in experimental parameters such as the working wavelengths (± 2 nm) and a mobile phase content (± 2%).These minor changes did not have any significant effect on the peak area or separation of EZE from its alkaline degradation products and % RSD of the responses were < 2 %, confirming the robustness of the procedures, as shown in Table 1.

System suitability
System suitability was checked by calculating different parameters including resolution, tailing factor, capacity factor and number of theoretical plates.The obtained values were found to be in the acceptable ranges when compared to USP reference values as shown in Table 4.

Application to the pharmaceutical preparation
The proposed method was applied for the determination of EZE in zetamibe ® tablets.The obtained results Table 3 assure that the excipients did not interfere with the proposed method.

Statistical analysis
Statistical analysis of the results obtained by the proposed method and the reported method (ratio subtraction) of EZE in pharmaceutical form using student's t-test and F-value revealed that there is no significant difference between the proposed and the reported method 5 regarding the accuracy and precision Table 5.

CONCLUSION
TLC-densitometric technique has provided a simple, straightforward method for separating ezetimibe and its alkaline degradation product simultaneously.Its major advantage that it can be run using a small quantity of the mobile phase, thus lowering the analysis time and the cost with higher sensitivity rather than other described methods.

Figure 1 .
Figure 1.Degradation pathway of ezetimibe under the alkaline condition.Laboratory prepared mixturesAccurate aliquots equivalent to (20 -70 µg) of EZE are transferred into a series of 10-mLvolumetric flasks from its working solution (100 µg/mL) and

Figure 5 . 1 H
Figure 5. 1 H NMR of alkaline degradation product of ezetimibe in DMSO.

Table 1 . Regression and validation data for the determination of EZE by the densitometric method Parameters EZE
*Average of three determinations of three concentration levels.

Table 3 . Determination of EZE in Zetamibe ® tablets by the densitometric methodand application of standard addition technique
a Average of three determinations.bAverage of the recoveries for five different concentrations.ISSN:

Table 5 . Statistical comparison between the results obtained by densitometric method and the reported method for the determination of EZE 5 in pharmaceutical form
*The value in the parenthesis are the corresponding theoretical values of t and F at (P=0.05).