INTRODUCTION:

Rosuvastatin^[1-2] chemically is (3R,5S,6E)-7-[4-(4-fluorophenyl)-2-(N-methylmethanesulfonamido)-6-(propan-2-yl) pyrimidin-5-yl]-3,5-dihydroxyhept-6-enoic acid. It belongs to drug class of statins, used in combination with exercise, diet and weight-loss to treat high cholesterol and related conditions and to prevent cardiovascular disease. Rosuvastatin is a competitive inhibitor of HMG-CoA reductase. HMG-CoA reductase catalyzes the conversion of HMG-CoA to mevalonate, an early rate-limiting step in cholesterol biosynthesis. Rosuvastatin acts primarily in the liver. Decreased hepatic cholesterol concentrations stimulate the upregulation of hepatic low-density lipoprotein (LDL) receptors which increases hepatic uptake of LDL.

Rosuvastatin also inhibits hepatic synthesis of very low-density lipoprotein (VLDL). The overall effect is a decrease in plasma LDL and VLDL.

Fig.1. ROSUVASTATIN

Fig.2. EZETIMIBE

Ezetimibe ^[3-4] chemical name is (3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl) azetidin-2-one. It is a drug that lowers plasma cholesterol levels. It acts by decreasing cholesterol absorption in the small intestine. It may be used alone when other cholesterol lowering medications are not tolerated, or together with statins, when statins alone do not control cholesterol. Ezetimibe is recommended as second line therapy for those intolerant statins or unable to achieve target LDL cholesterol levels. The exact mechanism is not known, but it appears that ezetimibe blocks the critical mediator of cholesterol absorption, the Niemann-Pick C1-Like 1 (NPC1L1) protein on the gastrointestinal tract epithelial cells as well as in hepatocytes. Ezetimibe is also used in combination therapy with HMG-CoA reductase inhibitors. Ezetimibe has a mechanism of action that differs from those of other classes of cholesterol-reducing compounds (HMG-CoA reductase inhibitors, bile acid sequestrants, fibric acid derivatives, and plant stanols).

Literature survey ^[5-10] revealed that there were methods reported for estimation of RST and EZE in combined dosage forms and for individual drugs using UV, HPLC and HPTLC. Here is an attempt made to develop a spectrophotometric method for simultaneous estimation of RST and EZE using water as a solvent which is very economic.

EXPERIMENTAL:

INSTRUMENTS USED:

Shimadzu double beam UV/Visible spectro-photometer model UV1800s was employed with a spectral band width of 1nm and a wavelength accuracy of 0.3nm (with automatic wavelength correction with a pair of 1cm matched quartz cells). SHIMADZU Electronic balance model AX 200 and Ultra Sonicator (Fast clean) model 2k811056 were also used during the analysis.

CHEMICALS AND REAGENTS:

Pure drugs of Rosuvastatin and Ezetimibe were obtained from KP labs (Hyderabad, India). Razel-EZ10 tablets manufactured by Glenmark Pharmaceuticals LTD., purchased from local pharmacy was used for the analysis. The label claim states that this formulation contains 10mg of Rosuvastatin and 10mg of Ezetimibe.

METHOD:

SELECTION OF SOLVENT AND WAVELENGTH:

The UV spectra of Rosuvastatin (RST) and Ezetimibe (EZE) differ in different solvents like ethanol, methanol and distilled water were recorded. The two drugs showed good absorbance when dissolved in distilled water. Hence, distilled water was selected as the solvent for the method. In this the two drugs showing good absorbances at wavelengths of 223nm and 229nm, hence selected as λ_max of RST and EZE respectively. Simultaneous equation method was developed for the estimation of RST and EZE in the combined dosage form.

PREPARATION OF STANDARD STOCK SOLUTIONS:

Standard stock solution of Rosuvastatin and Ezetimibe were prepared by dissolving 100 mg of each drug separately in a 100ml volumetric flask and both the drugs were dissolved in distilled water to get a concentration of 1000µg/ml.

PREPARATION OF WORKING STANDARD SOLUTIONS:

The working standard solutions of Rosuvastatin and Ezetimibe were prepared by diluting 1 ml each of the standard stock solution to 10 ml with distilled water in a 10ml volumetric flask to get the concentration of 100µg/ml.

Fig.3: Overlain Normal spectra of Rosuvastatin and Ezetimibe in distilled water

The absorptivity values were determined at the two selected wavelengths. The concentration of two drugs in the sample was calculated using the following equation.

C_RST = A₂ay₁-A₁ay₂/ax₂ay₁-ax₁ay₂

C_EZE = A₁ax₂-A₂ax₁/ax₂ay₁-ax₁ay₂

Where C_RST, C_EZE are the concentrations of RST and EZE in sample solutions. A₁, A₂ are the absorbances of sample at 223nm and 229nm respectively, ax₁, ax₂ are the absorptivity values of RST at 223nm and 229nm, ay₁, ay₂ are the absorptivity of EZE at 223nm and 229nm respectively.

PREPARATION OF SAMPLE SOLUTIONS:

Twenty Razel-EZ10 tablets each containing 10 mg of both Rosuvastatin and Ezetimibe were weighed, average weight was calculated and powdered. A quantity equivalent to 100 mg of EZE and RST was weighed and transferred into 100 ml volumetric flask. It was dissolved in distilled water. The volumetric flask was sonicated for about 5mins to affect the complete dissolution of the drugs and the solution was made up to the volume with distilled water to obtain concentration of 1000µg/ml and filtered. This solution was further diluted with distilled water to get a solution having concentration of 10µg/ml of both RST and EZE.

Fig.4: UV Spectra of Rosuvastatin and Ezetimibe in formulation (Razel EZ10)

Fig: 5 Linearity of Rosuvastatin.

Fig: 6 Linearity of Ezetimibe

Fig. 7 Calibration graph of Rosuvastatin at 223 nm

Fig. 8 Calibration graph of Rosuvastatin at 229 nm

Fig. 9 Calibration graph of Ezetimibe at 223 nm

Fig. 10 Calibration graph of Ezetimibe at 229 nm

Fig:11 Accuracy Studies

Table 1: Analysis of marketed formulation

Drug	Amount labelled (mg)	Amount found (µg/ml)	% Assay
Rosuvastatin	10.0	10.32	103.2%
Ezetimibe	10.0	9.86	98.6%

Table 2: Absorbance values of Rosuvastatin with distilled water

S. No.	Concentration (µg/ml)	Absorbance
S. No.	Concentration (µg/ml)	At 223nm	At 229nm
1	4	0.215	0.15
2	8	0.377	0.272
3	12	0.537	0.37
4	16	0.679	0.488
5	20	0.828	0.603
6	24	1.029	0.73
7	28	1.173	0.85
8	32	1.317	0.96

Table 3: Absorbance values of Ezetimibe with distilled water

S. No.	Concentration (µg/ml	Absorbance
S. No.	Concentration (µg/ml	At 223nm	At 229nm
1	4	0.14	0.144
2	8	0.237	0.275
3	12	0.361	0.404
4	16	0.445	0.523
5	20	0.534	0.629
6	24	0.651	0.753
7	28	0.776	0.894
8	32	0.889	0.987

Table 4: Absorbance values of Precision at intraday

S. No.	Absorbance
S. No.	At 223nm	At 229nm
1	0.396	0.4
2	0.398	0.402
3	0.399	0.403
4	0.4	0.404
5	0.401	0.405
6	0.404	0.404

Table 5: Absorbance values of Precision at interday

S. No.	Absorbance
S. No.	At 223nm	At 229nm
1	0.396	0.4
2	0.389	0.395
3	0.395	0.389
4	0.387	0.385
5	0.383	0.387
6	0.379	0.391

Table 6: Intraday and interday precision

S. No.		Mean		Standard deviation		%RSD
S. No.		223nm	229nm	223nm	229nm	223nm	229nm
1	Intraday	0.399	0.403	0.0027	0.0017	0.683	0.443
2	Interday	0.388	0.391	0.0066	0.0055	1.712	1.413

Table 7: Accuracy results for Rosuvastatin and Ezetimibe

S. No.	Amount of marketed formulation (µg/ml)	Amount of API mixture added (µg/ml)	Total amount of both the drugs (µg/ml)	Absorbance		Total amount of drug found (µg/ml)		% Recovery
S. No.	Amount of marketed formulation (µg/ml)	Amount of API mixture added (µg/ml)	Total amount of both the drugs (µg/ml)	223nm	229nm	223nm	229nm	223nm	229nm
1	10µg/ml	8µg/ml	18µg/ml	1.099	0.983	17.89	17.97	99.38%	99.83%
2	10µg/ml	10µg/ml	20µg/ml	1.215	1.086	19.83	19.80	99.15%	99.0%
3	10µg/ml	12µg/ml	22µg/ml	1.332	1.189	21.82	21.58	99.18%	98.0%

Table 8: LOD and LOQ values for Rosuvastatin at 223 nm

S. No.	Concentration (µg/ml)	Absorbance at 223 nm
1	4	0.215
2	8	0.377
3	12	0.537
4	16	0.679
5	20	0.828
6	24	1.029
7	28	1.173
8	32	1.317
Parameters:
Linearity range (µg/ml)	4-32
Correlation coefficient	0.997
Slope	3.5
Standard deviation	0.040
LOD (µg/ml)	0.014004
LOQ (µg/ml)	1.1

Table 9: LOD and LOQ values for Ezetimibe at 229 nm

S. No.	Concentration (µg/ml)	Absorbance at 229 nm
1	4	0.144
2	8	0.275
3	12	0.404
4	16	0.523
5	20	0.629
6	24	0.753
7	28	0.894
8	32	0.987
Parameters:
Linearity range (µg/ml)	4-32
Correlation coefficient	0.998
Slope	0.030
Standard deviation	0.010743
LOD (µg/ml)	1.1
LOQ (µg/ml)	3.5

RESULTS AND DISCUSSION:

METHOD DEVELOPMENT:

The analytical method was developed using simultaneous equation method by taking 223nm and 229nm as λ_max of RST and EZE respectively. For the tablet dosage form the %assay was found to be 103.2 for RST and 98.6 for EZE.

METHOD VALIDATION:

The analytical method was validated with respect to parameters such as linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ).

LINEARITY AND RANGE:

Linearity was established by least squares linear regression analysis of the calibration curve. The calibration curves were linear over the concentration range of 4-32µg/ml for both RST and EZE.

Absorbance was plotted against respective concentrations and linear regression analysis was performed on the resultant curves. Correlation coefficients were found to be 0.998 at 223nm and 0.999 at 229nm for RST and 0.997 at 223nm and 0.998 at 229 nm for EZE.

PRECISION:

The precision of the analytical method was studied by multiple sampling of the homogenous sample. The precision was done at two levels (intraday and interday). Intraday precision was done by analyzing the intermediate concentration of each drug (RST 10µg/ml and EZE 10µg/ml) for six times. Interday precision was measured over six consecutive days for the same drug concentrations. The %RSD values were calculated for each of them and the low RSD values indicate that the method is precise. The results were found within 2% limit.

ACCURACY:

Recovery studies were carried out by spiking the sample solution with standard solutions of RST and EZE at a level of 80, 100 and 120%. At each level % recovery was determined three times. The results were found within the limit (98-102%).

SENSITIVITY:

LOD and LOQ decide about the sensitivity of the method. LOD is the lowest detectable concentration of the analyte by the method while LOQ is the minimum quantifiable concentration. For these LOD and LOQ linearity values were taken and standard graphs were drawn. From the standard graphs, standard deviations and slope were calculated, then LOD and LOQ values were calculated using the following formulas.

LOD = 3.3 S_a/b

LOQ = 10S_a/b

S_a = Standard deviation

b= slope of calibration curve

CONCLUSION:

The elevation of obtained values suggests that the proposed UV spectrophotometric method provide simple, precise, accurate and economical quantitative analytical method for determination of RST and EZE in tablet dosage form. After validating proposed method as per ICH guidelines and correlating the obtained values with the standard values, satisfactory results were obtained. The sample recoveries in all formulations were in good agreement with their respective label claims and they suggested no interference of formulation excipients in the estimation. Hence, the method can be easily and conveniently adopted for routine estimation of RST and EZE in tablet dosage form.

ACKNOWLEDGEMENT:

We are very much thankful to Anurag Group of Institutions (Formerly Lalitha College of Pharmacy), Hyderabad, for giving permission to carry out my work.

REFERENCES:

1. https://www.drugbank.ca/drugs/DB01098

2. https://pubchem.ncbi.nlm.nih.gov/compound/Rosuvastatin

3. https://www.drugbank.ca/drugs/DB00973

4. https://pubchem.ncbi.nlm.nih.gov/compound/Ezetimibe

5. Vishal V. Rajkondwar, Pramila Maini and Monika Vishwakarma; Characterization and method development for estimation and validation of Rosuvastatin Calcium by UV – visible spectrophotometry; International Journal of Theoretical and Applied Sciences; 2009; 1(1); pg.no. 48-53.

6. Alka Gupta, P. Mishra and K. Shah; Simple UV Spectrophotometric Determination of Rosuvastatin Calcium in Pure Form and in Pharmaceutical Formulations; ECJHAO E-Journal of Chemistry; 2009; 6(1); pg.no. 89-92.

7. Gajjar Anuradha K and Shah Vishal D; Simultaneous UV Spectrophotometric Estimation of Rosuvastatin and Ezetimibe in their combined dosage forms; International Journal of Pharmacy and Pharmaceutical Sciences; 2010; 2(1); pg.no. 131-138.

8. Anuradha K. Gajjar and Vishal D. Shah; Development and Validation of a Stability-Indicating Reversed-Phase HPLC method for Simultaneous Estimation of Rosuvastatin and Ezetimibe for their Combination Dosage Forms; Eurasian Journal of Analytical Chemistry; 2010; 5(3); pg.no. 280-298.

9. Varghese, Sushell John, Ravi, Thengungal Kochupappy; Determination of Rosuvastatin and Ezetimibe in a Combined Tablet Dosage form using High-Performance Column Liquid Chromatography and High-Performance Thin-Layer Chromatography; Journal of AOAC International; 2010; 93(6); pg.no. 1222-1227.

10. Hasumati A. Raj, Sadhana J. Rajput, Jayant B. Dave and Chaggan N. Patel; Development and Validation of Two Chromatographic Stability-Indicating Methods for determination of Rosuvastatin in pure form and Pharmaceutical preparation; International Journal of Chem Tech Research; 2009; 1(3); pg.no. 677-689.

Received on 10.04.2017 Accepted on 28.05.2017

Asian J. Pharm. Ana. 2017; 7(3): 135-140.

DOI: 10.5958/2231-5675.2017.00021.7

S. No.	Parameters	Rosuvastatin	Ezetimibe	Acceptance criteria
1	Linearity	R² = 0.998	R² = 0.998	Correlation coefficient (R² = 0.997-0.999)
2	Precision	Intraday: % RSD: 0.683 Interday: % RSD: 1.712	Intraday: % RSD: 0.443 Interday: % RSD: 1.413	%RSD = <2%
3	Accuracy	99%-102%	98%-102%	98%-102%
4	LOD	1.1 µg/ml	1.1 µg/ml	-
5	LOQ	3.5 µg/ml	3.5 µg/ml	-