TLC- Densitometric Method for Determination of some Cholesterol

In the developed TLC-Densitometric method EZ, AT and SIM were quantitatively separated on 60F254 silica gel plates using ethyl acetate: hexane: glacial acetic acid (5.5:4.5:0.1 by volume) as a developing system with UV detection at 254 nm. Factors affecting the chromatographic separation have been studied, moreover the method has been validated as per ICH guidelines and it has been successfully applied for determination of the studied drugs in their different dosage forms without interference from excepients. Results obtained by the developed TLCDensitometric method were statistically compared with those obtained by the reported spectrophotometric method and no significant difference was found between them.

Reviewing the literature in hand, none of the recommended pharmacopeias has been determined EZ and AT or EZ and SIM mixtures. EZ and AT have been determined by some techniques including HPLC [18], HPTLC-Densitometric [19] and spectrophotometric methods [20]. On the other hand EZ and SIM have been determined in their association by HPLC [21][22][23], HPTLC-Densitometric [24] and spectrophotometric methods [25].
Only one method [26] has been published for determination of EZ/ AT and EZ/SIM combinations which depend on using first derivative of ratio spectra spectrophotometric method (1DD) for EZ /AT and EZ / SIM mixtures and measuring each of the two mixtures in separate steps using different instrumental conditions. From the previous literature review, no TLC-Densitometric method has been developed for simultaneous determination of the studied mixtures and no methods have been used for their determination without preliminary separation steps.
This work aims to develop selective, sensitive and accurate TLC-Densitometric method for simultaneous determination of the three studied drugs using the same solvent system, scanning wavelength and the same run hence it is time and cost effective and it can be used as alternative method to the high cost RP-HPLC method in quality control laboratories. The developed TLC-Desitometric method has the advantage of being more selective than the published spectrophotometric and HPTLC methods because it is able to separate the three components without interference from each other or from tablets excipients, moreover it does not need any sophisticated apparatus or high cost solvents compared to the published RP-HPLC methods.

Chemicals and Solvents
All chemicals and solvents used throughout this work were of analytical grade and were used without purification. Ethyl acetate, hexane, glacial acetic acid and methanol (El-Nasr Pharmaceutical Chemicals Co. Abu-Zabaal, Cairo, Egypt)

Construction of the calibration curves
Accurate volumes (4-40 µl),(4-31 µl) and (5-29 µl) of EZ, AT and SIM, respectively were accurately transferred from their standard working solutions (0.1 mg/mL), applied in triplicates on the prewashed TLC plates in the form of bands and the procedure under the chromatographic conditions was followed. The area under peak was then recorded and the calibration curve for each drug was constructed by plotting the mean integrated peak area versus the corresponding concentration.

Application to pharmaceutical formulations
The content of ten tablets of Atoreza® (10/10), Zocozet® (10/10), Lipitrin® (10/10 and 10/20) and Alkorplus® (10/20 and 10/40) tablets were separately weighed and then finely powdered. Accurate amounts each of the powdered tablets equivalent to 1 mg EZ (and the corresponding concentration of either AT or SIM) were separately weighed, dissolved in 75 mL methanol and sonicated for about 15 minutes. The prepared solutions were then filtered, transferred quantitatively to four separate 100 mL volumetric flasks and the volume was then completed to the mark with methanol. Appropriate dilutions of the prepared solutions were made to prepare their working solutions (0.1 mg/mL) and the developed method was then followed.

Results and Discussion
Ezetimibe is cholesterol lowering drug that is co-formulated with both AT and SIM which are used in cases of high cholesterol level. Hence, they play an important role in the treatment of some serious diseases such as heart disease [26]. Instrumental planar chromatography with precise application of the samples, and computer controlled evaluation and quantification of the developed chromatograms has been considered as reliable for quality control and quantitative drug mixture with either AT or SIM in their bulk powder and in their combined pharmaceutical dosage forms using one and the same developing system and scanning wavelength with satisfactory precision for Good Analytical Practice (GAP).

Method optimization
Experimental conditions such as developing system composition, band dimensions, scanning wavelength and slit 000dimension were optimized in order to provide accurate, precise and reproducible chromatographic separation. The first step is to test all the published TLC-Densitometric developing systems [19,24] [in the first method the mobile phase was composed of chloroform: benzene: methanol: acetic acid (6:3:1:0.1 by volume) and the detection of the developed spots was carried out at 250 nm while in the second method the mobile phase was composed of n-hexane: acetone (6:4 v/v) and the detection of the developed spots was carried out at 234 nm]. Unfortunately, none of them was able to separate all the EZ/AT and EZ/SIM mixtures together using one developing system. Using the first and second systems resulted in very bad resolution between the studied drugs. Replacing chloroform with hexane in the third one slightly enhanced the resolution but with tailed asymmetric peaks. On the other hand addition of glacial acetic acid in the last system enhanced both the chromatographic resolution and the peaks symmetry. So developing system consisted of ethyl acetate: hexane: glacial acetic acid (5.5:4.5:0.1 by volume) was used as the developing system.
Different band dimensions were tested in order to obtain sharp and symmetric peaks. The optimum band width chosen was 4 mm and inter-space between bands was 8.9 mm. Moreover Different scanning wavelengths were tried such as 210, 220 and 254 nm where scanning at 254 nm gave the best sensitivity for all the separated components. After method optimization the R f values of AT, SIM and EZ are 0.2, 0.4 and 0.59 respectively as shown in Figure 1. The slit dimensions of 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 3 mm × 0.45 mm proved to be the slit dimension of choice which provides highest sensitivity.

Method validation
Validation was performed according to ICH guidelines [28]. Where Y 1 , Y 2 and Y 3 are integrated peak area ×10 -4 , C 1 , C 2 and C 3 are the corresponding concentrations of EZ, AT and SIM in µg/band, respectively, r 1 , r 2 and r 3 are the corresponding correlation coefficients. testing [27].
The main task of this work is to establish sensitive and selective TLC-Densitometric method for determination of EZ in its binary Good linearity is evident from the high value of correlation coefficient and low value of intercept as shown in Table 1.
Accuracy: Accuracy of the method was checked by applying the proposed method for determination of different blind samples of pure EZ, AT and SIM. The concentrations were calculated from the corresponding regression equations and the results are presented in Table 1. Accuracy of the method was further assured by applying the standard addition technique on different pharmaceutical dosage forms where good recoveries were obtained revealing no interference from excipients, Tables 5,6.

Precision
A) Repeatability: Three concentrations of EZ, AT and SIM (0.6, 1.2 and 1.8 µg/band) were analyzed three times intra-daily using the proposed method. Good % RSD was obtained, confirming the repeatability of the method as shown in Table 1.
B) Intermediate precision: The previous procedure was repeated inter-daily on three different days for the analysis of the three chosen concentrations. Acceptable % RSD was obtained and given in Table 1.

Specificity:
Specificity of the method was tested by how accurately and specifically the analytes of interest are determined in the presence of other components (e.g.: co-formulated drugs, excipients, impurities, degradation products, etc). This is evident from TLC-Densitograms in Figure 1.
The good recovery percentages obtained by applying the proposed method on pharmaceutical dosage forms, Tables 2,3 also proved the specificity of the proposed method as shown in Figure 3. [26] Spectrophotometric determination of EZ and ATR using 1 DD at 289.5 and 288 nm for EZ and AT, respectively and methanol as solvent. [26] Spectrophotometric determination of EZ and SIM using 1 DD at 299.5 and 242.5 nm for EZ and AT, respectively and methanol asa solvent.

Robustness:
The robustness meaning is the capacity of the method to remain unchanged upon intended small change in method parameters e.g.: changing PH ± 0.1, changing mobile phase composition, changing saturation time ± 5 min and changing the scaling wavelength ± 1 nm. The low value of % RSD shows that the method is robust and that deliberate small changes in the studied factors do not lead to significant changes in R f values, area or symmetry of the peaks.
System suitability: In order to validate the suggested TLC-Densitometric method, an overall system suitability testing was done to determine if the operating system are preformed properly. Parameters including resolution (R S ), peak symmetry, and selectivity (α) were calculated where good results were obtained and the peak information is given in Table 4.
After method optimization and validation it has been applied for the determination of EZ, AT and SIM in different pharmaceutical dosage forms where acceptable percentage recoveries have been obtained and shown in Tables 2,3. Furthermore, its validity was assessed by applying the standard addition technique which showed that tablet excipients did not interfere (Table 5,6).
On the other hand, statistical comparison of the results obtained by the developed method with those obtained by the reported spectrophotometric one [26] using F and Student's t-tests showed no significant difference. The developed method has the advantages of being economic, reproducible, and accurate and can be easily applied in quality control laboratories.

Conclusion
In the present work sensitive and selective TLC-Densitometric method for the determination of EZ, AT and SIM in their pure form and in different dosage forms has been developed and validated.
The developed TLC-Densitometric method is considered superior to the reported spectrophotometric methods of being more selective and sensitive. On the other hand it can be used as alternative method to the published HPLC methods in laboratories lacking the facilities of HPLC. The developed TLC-Densitometric method is time effective since several samples can be run simultaneously using a small quantity of the developing system, which lowers the analysis time and cost. Finally we can conclude that the suggested method can be used in routine analysis of the studied drugs without any preliminary separation step.