INTRODUCTION:

High (LDL) low density cholesterol is a common problem for people with diabetes. Also, referred to as dyslipidemia, this condition greatly increases the risk of developing cardiovascular disease. EZE is selective cholesterol absorption inhibitors which is most effective at lowering LDL. Chemically, it is (3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl) azetidin-2-one. and GLM is second generation sulfonylurea class. Chemically it is, 3-Ethyl-4-methyl-N-[2-(4-{[(trans-4-methylcyclohexyl)carbamoyl] sulfamoylphenyl)ethyl]-2-oxo-2,5-dihydro-1H-pyrrole-1-carboxamide[1], which lowers blood sugar by causing the pancreas to produce insulin and helping the body use insulin efficiently[2]. This combination is used for treatment of Type 2 diabetes mellitus.[3]

Literature survey revealed RP-HPLC methods[4-6] and Stability indicating methods[7, 8] reported for simultaneous estimation of EZE and GLM in tablet dosage form.

The aim of the present work was to develop a simple,accurate,economic RP-HPLC method for simultaneous estimation of EZE and GLM in tablet formulation.

MATERIALS AND METHODS:

Chemical and reagent:

EZE and GLM working standards were received as gift samples from Bluecross Pharmaceutical Ltd, Nashik. Methanol, ortho phosphoric acid (HPLC grade) and Potassium dihydrogen orthophosphate (AR grade) were used throught these expeiments and obtained from SD fine chemical Mumbai,Maharashtra.Freshly prepared double distilled water used was prepared by All Glass Double Distillation assembly,purchased from Borosil, Mumbai,Maharashtra and further used after filtering through 0.45 mmembrane filter papers purchased from Millipore (India) Pvt. Ltd., Bengaluru, Karnataka. Tablets containing 10mg of EZE and 1 mg of GLM were prepared in house.

Instrumentation and chromatographic condition:

Chromatographic separation was carried out using an HPLC system (JASCO Corporation, Tokyo, Japan) equipped with dual PU 2080 plus pumps, multichannel UV detector, UV-2075 and injection loop (20 µL capacity), Rheodyne manual loop injector 7725i.Borwin Chromatography software (version 1.5) was used for data collection. The mobile phase was composed of methanol: 20mM Phoshpate buffer (pH 3) 80:20 %v/v. Isocratic elution was carried out on a Phenomenex Kinetex C18 column (250´4.6mm,5m) at a flow rate of 1 mL/min. The wavelength was fixed at 230 nm.

Preparation of Standard Stock solution:

Accurately weighed and transfer 10 mg of EZE and GLM in separate 10 mL of clean dry volumetric flasks and made up to the volume with methanol. The resulting solutions were of 1000µg/mL of EZE and GLM respectively.

Preparation of System Suitability Standard solution:

System suitability standard solutions were prepared daily by appropriate diluting standard stock solutions with mobile phase to get 10 µg/mL for EZE and 1 µg/mL GLM respectively.

Estimation of EZE and GLM in Tablets:

Tablets containing 10mg of EZE and 1mg GLM each, were prepared in house. Twenty tablets were weighed and taken into mortar and crushed to uniform powdered. A quantity of powder equivalent to 10mg of EZE and 1mg of GLM was transferred to 100mL volumetric flask and made up the volume up to the mark with methanol and shaken for 10min. Further, the solution was filtered through Whatman filter paper No. 1.

From the above solution, suitable aliquot was diluted with mobile phase to get concentrations of 10µg/mL of EZE and 1µg/mL of GLM, respectively and subjected to chromatographic analysis under mentioned chromatographic condition.

Validation of method:

The developed HPLC method was validated in according to ICH Q2(R1) guidelines for validation of analytical procedure for different validation parameters. The method was validated for its linearity, accuracy, precision specificity, limit of detection (DL) and limit of quantitation (QL)[9]

Linearity:

Five different calibration curve were prepared for EZE 20, 40, 60, 80 and 100 µg/mL and GLM 2,4,6,8 and 10µg/mL. Each calibration curve standards were analyzed in triplicates and the peak areas were plotted against corresponding drug concentrations and were subjected to regression equation. The slope, intercept, and co-efficient of regression were noted.

Accuracy and Precision:

Accuracy was performed by preparing three different concentrations (corresponding to 50%,100%,150% of the sample solution) by addition of known amounts of standard solution across the analytical range for EZE and GLM. Three replicates were prepared for the intra-day precision and on three successive days for the intermediate precision. The percent recovery of added concentration and % RSD were taken as measures of accuracy and precision, respectively. Also, the results obtained were subjected to one-way ANOVA and the between – day mean square compared to the within – day mean square by F – test.

Specificity:

The specificity studies were performed by preparing the placebo containing all excipients except the drug. According to that sample solution was prepared. Absence of peaks in the chromatographic run was taken as indication of specificity.

Limit of detection:

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected. The DL can be calculated based on standard deviation of the response and the slope of the calibration curve.

DL=3.3σ/S

σ=standard deviation of the intercept of the regression lines.

S=slope of the calibration curve.

Limit of Quantitation:

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

QL=10σ/S

RESULT AND DISCUSSION:

Various mobile phases were tried, and a mobile phase with composition of methanol: 20mM phosphate buffer (pH 3) 80: 20 %v/v flow rate 1mL/min was found to resolve EZE from GLM. The optimum wavelength for detection was 230 nm. The retention time of EZE and GLM were observed at 4.183 and 5.292 mins, respectively (Figure 1)

System suitability test verifies that the reproducibility and resolution of the chromatographic system are adequate for the analysis to be conducted. All critical parameters tested met the USP acceptance criteria (Area % RSD < 2, asymmetry < 2 and No. of theoretical plates > 2000).

In calibration studies, it was found that EZE was linear in the range of 20-100µg/mL and GLM 2-10µg/mL. The calibration curves with their respective calibration curve equations and regressions are depicted in Figure 2 and Figure 3, respectively.

Figure: 1 Representative chromatogram of EZE (4.183 min) and GLM (5.29 min)

Figure 2: Calibration curve of EZE

Figure 3: Calibration curve of GLM

When tablets prepared on lab scale were analyzed using the developed method, the results obtained were in good agreement with nominal amount of the drug. The drug content was found to be 99.82 ± 1.94 of the added amount.

The results obtained for accuracy and precision are summarized in Table 1 and Table 2, for EZE and GLM, respectively. Mean values of concentration found were close to the concentration added and low values of % RSD indicates the acceptable accuracy and precision of the method.

Table 1: Accuracy and Precision studies of EZE:

Concentration added (µg/mL)	Amount Found ((µg/mL)			Within mean square	Between mean square	F-value
Concentration added (µg/mL)	Day 1	Day 2	Day 3	Within mean square	Between mean square	F-value
(40+20) 60(µg/mL) 50%	59.52	59.4	59.56	0.022033	0.021944	0.995965709
	59.63	59.77	59.81
	59.7	59.53	59.83
Mean	59.61666667	59.56667	59.73333
SD	0.090737717	0.187705	0.150444
%RSD	0.15220193	0.315118	0.251859
(40+40) 80(µg/mL) 100%	79.2	78.1	78.79	0.399011	0.443011	1.110272618
	79.3	78.2	78.89
	78.89	78.98	77.85
Mean	79.13	78.4266	78.51
SD	0.7973079	0.481802	0.57376
%RSD	1.007592	0.614335	0.730811
(40+60) 100(µg/mL) 150%	99	98.1	98.4	0.363178	0.591078	1.627516368
	99	98.12	97.15
	99	98.9	98.88
Mean	99.03333	98.37	98.14333
SD	0.057735	0.540278	0.893103
%RSD	0.058298	0.54923	0.90999

Table 2: Accuracy and Precision studies of GLM:

Concentration added (µg/mL)	Amount Found ((µg/mL)			Within mean square	Between mean square	F-value
Concentration added (µg/mL)	Day 1	Day 2 Day 3		Within mean square	Between mean square	F-value
(4+2 ) 6(µg/mL) 50%	6.01	6.1	6.2	0.008689	0.008611	0.991048593
	6.03	6.01	6.25
	6.11	6.19	6.1
Mean	6.05	6.1	6.183333333
SD	0.052915026	0.09	0.076376262
%RSD	0.874628533	1.475409836	1.235195605
(4+4) 8(µg/mL) 100%	8	8.07	8	0.005444	0.007144	1.312244898
	8	7.95	8.23
	8.01	8.1	8.1
Mean	8.003333333	8.04	8.11
SD	0.005773503	0.079372539	0.115325626
%RSD	0.072138726	0.987220638	1.422017583
(4+6) 10(µg/mL) 150%	9.88	10.1	10.1	0.083867	0.108344	1.291865395
	10	9.98	10.2
	10.02	10.1	10.19
Mean	9.966666667	10.06	10.16333333
SD	0.075718778	0.069282032	0.055075705
%RSD	0.75972018	0.688688194	0.541905925

Also, when the results of intra-day and inter-day were subjected to one-way ANOVA and F values were calculated at each QC level, the F values were found to be less than the tabulated F values. This indicated that there was no significant difference between intra-and inter-day variability, suggesting good intermediate precision.

When blanked tablets were analyzed as per the mentioned chromatographic conditions, no peak was obtained at the retention times of EZE and GLM which indicates that the method was specific for the analysis of analytes in their dosage form.

Figure 4: Representative specificity of EZE and GLM

DL and QL of EZE and GLM:

Sr No.

Drug

Standard deviation

(S.D)

Slope

Ezetimibe

13534

51477

0.86µg/ml

2.62µg/ml

Glimepride

13224

65202

0.66µg/ml

2.00µg/ml

REFERENCES:

1. Anthony C Moffat, M.David Osselton and Barian Widdop, Clarke's Analysis of Drug and Poision.3rd edition 2: p.1080.

2. Indian Pharmacolopoeia. (2014). 7 ed: Indian Pharmacopoeial Commission 2014.

3. Saito, I., et al., A randomized, double-blind, placebo-controlled study of the effect of Ezetimibe on glucose metabolism in subjects with type 2 diabetes mellitus and hypercholesterolemia. Lipids in Health and Disease, 2015,14(1): p. 40.

4. B.Siddartha and Radhika Malipelli, Simultaneous Estimation and Validation of Ezetimibe and Glimepride in Bulk and Pharmaceutical Dosage Form By RP-HPLC International journal of Research and Development in Pharmacy and Life Sciences, 2014, 3(6)(3): p. 1280-1286.

5. T. Sonwjanya Jyothi, P.Nagaraju, Simultaneous Quantification and Validation of Glimepride and Ezetimibe By RP-HPLC in Bulk and Pharamcetutical Dosage Form, 2(3): p.1-5.

6. Sudheer Kumar N, Shilpa K, Ajitha A , Uma Maheswara Rao V Method Development and Validation of Simultaneous Estimation of Ezetimibe and Glimepiride By RP-HPLC International journal of Pharmaceutical Research and Analysis, 2016, 6(1): p.47-52.

7. Pavani, P., A. Srilekha, and B. Sreedhar, Stability indicating RP-HPLC method development and validation for simultaneous estimation of glimepiride and ezetimibe in bulk and tablet dosage form. International Journal of Pharmaceutical Sciences and Research, 2015, 6(3): p. 1066.

8. Hanifa Begum, S.H. Rizwan, Khaled Bin Sayeed Stability Indicating Analytical Method Development and Validation For Estimation of Ezetimibe and Glimepiride Using RP- HPLC Method in Bulk Drugs and Marketed Fromulation.Indo American Journal of Pharmaceutical Research, 2014,Vol 4, (Issue 10).

9. Validation of Analytical Procedures: Text and Methodology, Q2R1, International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use: ICH Harmonized Triplicate Guidelines 2005.

Received on 31.05.2017 Accepted on 28.07.2017

Asian J. Pharm. Ana. 2017; 7(3): 185-188.

DOI: 10.5958/2231-5675.2017.00029.1