INTRODUCTION:

Rosuvastatin calcium (RSV), a member of the class of statins, is the calcium salt of (E)‐7‐[4‐(4‐fluorophenyl)‐6‐isopropyl‐2‐ [methyl(methylsulfonyl)amino] pyrimidi n‐5‐yl] (3R,5S)‐3,5‐dihydroxyhept‐6‐enoic acid. RSV is used to treat hypercholesterolemia and related conditions and to prevent cardiovascular disease. It increases the number of hepatic Low Density Lipoprotein (LDL) receptors on the cell surface to enhance uptake and catabolism of LDL. RSV also inhibits hepatic synthesis of Very Low Density Lipoprotein (VLDL) which reduces the total number of VLDL and LDL¹. RSV acts by inhibiting the activity of 3‐hydroxy‐3‐ methylglutaryl‐coenzyme A (HMG‐CoA) reductase, the rate‐limiting enzyme that converts 3‐hydroxy‐3‐methylglutaryl coenzyme A to Mevalonate, a precursor of cholesterol².

Figure 1. Rosuvastatin calcium and Glimepiride

Glimepiride (GLM) is an oral blood-glucose-lowering drug which is a derivative of sulfonylurea class³, which is chemically known as 1-[[p-[2-(3-ethyl-4-methyl-2-oxo-3-pyrroline-1carboxamido) ethyl]phenyl]sulfonyl]-3-(trans-4-methylcyclohexyl)urea⁴. Glimepiride is widely used as antidiabetic drug in patient with type-2 diabetes (non-insulin-dependent diabetes).

Rosuvastatin calcium is used for the treatment of hypercholesterolemia and glimepiride is used as hypoglycemic agent for type-2 diabetic patient. It has been observed that patient with high level of cholesterol may develop diabetic problem later. So combination preparation containing these two drugs in a single dosage form may be helpful for this kind of patient. But no such preparation is available in the market as well as no method is yet reported for simultaneous estimation of these two drugs from combined pharmaceutical dosage form. Therefore, it is highly required to develop and validate an analytical method for the simultaneous estimation of rosuvastatin calcium and glimepiride in combine dosage form.

Literature survey revealed that HPLC⁵, Capillary Zone Electrophoresis⁶, Spectrophotometry⁷ are available for rosuvastatin analysis in single formulation. Simultaneous UV Spectrophotometric estimation of rosuvastatin and ezetimibe in their combined dosage form is also reported⁸. Similarly, a survey of the analytical literature for glimepiride revealed methods based on UV Spectrophotometric ^{9, 10} HPLC ^11-12 for determination in pharmaceuticals either single or combine with other drugs.

Till date, none of the reported analytical procedures describes a simple and satisfactory UV spectrophotometric method for simultaneous determination of RSV and GLM in their combined dosage forms. So the present work was undertaken to develop and validate an economic, rapid spectrophotometric method for combination drug products containing RSV and GLM that allows the analysis of a large number of samples in a short period of time.

MATERIALS AND METHOD:

Materials:

Rosuvastatin calcium and glimepiride were gift from Eskayef Bangladesh Ltd. Methanol and sodium hydroxide were analytical-reagent grade and purchased from E. Merck, Darmstadt, Germany. Water was deionised and double distilled. Rosuvastatin calcium tablet 10 mg and glimepiride tablet 4 mg were purchased from local drug store in Dhaka city after checking their manufacturing license numbers, batch numbers, production and expiry dates.

Instrumentation:

A double‐beam Shimadzu (Kyoto, Japan) UV‐Visible spectrophotometer, Model UV‐1601 PC, equipped with 1 cm quartz cells, with a fixed slit width (1 nm), wavelength accuracy of +0.5 nm (with automatic wavelength correction) was used. The drug analyses data were acquired and processed using UV Probe software (Version 2.0, Shimadzu, Japan) running under Windows XP on a Pentium PC. For scanning, the wavelength range selected was from 400 nm to 200 nm with medium scanning speed.

Preparation of standard solution:

Stock solution of rosuvastatin (100µg/ml) and glimepiride (100µg/ml) were prepared by dissolving 10 mg drug in 100 ml 0.1N sodium hydroxide separately. Several aliquots of standard solutions of rosuvastatin (100µg/ml) and glimepiride (100µg/ml) diluted to get standard solution across the range of 2-22µg/ml. Solution containing mixture of rosuvastatin and glimepiride (10, 12, 15, and 18 µg/ml rosuvastatin along with 10 µg/ml glimepiride and vise versa) were also prepared by diluting standard solutions.

Preparation of sample solution:

Average weight of rosuvastatin tablet and glimepiride tablet were calculated. Then the tablets were grinded separately to fine powder with the help of mortar and pestle. Then, powder containing 5 mg rosuvastatin and 2 mg glimepiride was dissolved in 0.1 N sodium hydroxide, shaken for about 10 minutes and filtered through filter paper. The filtered solution was further diluted to make the final concentration of working sample equivalent to 100% of target concentration.

Methods development and optimization:

Simultaneous equation method was used to determine rosuvastatin and glimepiride. The wavelength was selected from the overlain spectra. Solutions containing 10 μg /ml of rosuvastatin and 10 μg/ml of glimepiride were scanned separately in the range of 200-400 nm to determine the wavelength of maximum absorption for both the drugs. Rosuvastatin and glimepiride showed absorbance maxima at 241 nm and 231nm respectively. Individual and overlain spectra for both the drugs are shown in Figure 2 to 4.

The absorbances of various standard solutions were recorded at the selected wavelengths and the absorptivity values were determined for rosuvastatin and glimepiride. Absorptivity values determined for rosuvastatin at 241 nm and 231 nm were 369.83 and 325.93 while respective values for glimepiride were 365.82 and 507.25.

Figure 2. Spectrum of rosuvastatin calcium

Figure 3. Spectrum of glimepiride

Figure 4. Overlain spectrum of combined mixture, RVS and GLM

From the absorptivity values of rosuvastatin and glimepiride the simultaneous equation was derived from the following equation 1 and 2 for determination of rosuvastatin and glimepiride in pure drug mixed standards and in its pharmaceutical formulation.

C_RSV = A2 ay1 – A1 ay₂ / ax₂ay₁-ax₁ ay₂ ………….(1)

C_GMR= A1 ax2 –A2 ax₁ / ax₂ay₁-ax₁ ay₂ …………..(2)

Where, A1 and A2 are absorbance of sample solution at λmax of RSV(241 nm) and λmax of GLM(231nm) respectively; ax₁ and ax₂ are the absorptivities of RSV at 241nm and 231 nm respectively and ay₁and ay₂ are the absorptivities of GLM at the two wavelengths respectively.

Validation of method:

Present study was conducted to obtain a new, affordable, cost-effective and convenient method for spectroscopic determination of rosuvastatin and glimepiride in solid dosage form. The method was validated for the parameters like linearity, accuracy, precision and robustness as per ICH guidelines ¹³.

Selectivity of the method was determined by comparing the spectrum of standard sample with that of market product.

The linearity of an analytical method is its ability to elicit that test results are proportional to the concentration of analyte in samples within a given range. This was determined by means of calibration graph using increasing amounts of standard solutions (10-22µg/ml). These standards were tested six times in agreement to the International Conference on Harmonization (ICH)¹³. Calibration curves were constructed and the proposed method was evaluated by its correlation coefficient and intercept value calculated in the corresponding statistical study (ANOVA) (p < 0.05). Characteristic parameters for regression equation (y = a + bx) of the method were obtained by least squares treatment of the results and these parameters were used to confirm the good linearity of the method.

Accuracy indicates the deviation between the mean value found and the true value. The accuracy studies were carried out at different concentrations by spiking a known concentration of standard drug to the pre‐analyzed sample. Accuracy was calculated from the test results as the percentage of the analyte recovered by the assay.

The precision of the method was investigated with respect to repeatability, ruggedness (intermediate precision) and reproducibility (by means of an inter laboratory trial). Repeatability was determined by performing three repeated analysis of the four standard solutions (10, 12, 15, 18 µg/ml rosuvastatin along with 10 µg/ml glimepiride and vise versa) of standard mixture solution on the same day, under the same experimental conditions. Ruggedness (intermediate precision) of the method was assessed by carrying out the analysis of standard solutions on three different days (inter-day) in the same laboratory. For reproducibility the procedure repeated in the Quality Control Laboratory of another lab by using equipment (Simardzu spectrophotometer model1240). The relative standard deviation (% RSD) was determined in order to assess the precision of the method.

The robustness of the method was assessed by altering the solvent composition of the experiment.

RESULTS AND DISCUSSION:

Selectivity:

UV spectroscopic method for rosuvastatin and glimepiride analysis was found selective as spectrum of standard rosuvastatin and glimepiride coincide with that of market product indicating that excipients has no noticeable effect on the effectiveness of the method.

Linearity:

Linearity of the method was evaluated from the correlation coefficient of calibration curves that were constructed from average absorbance of drugs at different concentration level (10-22µg/ml). Correlation coefficient was 0.997-0.9985 for rosuvastatin and 0.998 for glimepiride (Table 1)

Table 1. Data showing linearity of the developed method

Validation parameters		Rosuvastatin		Glimepiride
Validation parameters		231nm	241nm	231nm	241nm
Range (µg/ml)		10-22	10-22	10-22	10-22
Slope	Mean ±SD	0.0274 ± 0.00037	0.0341 ± 0.00057	0.0508 ± 0.00068	0.0372 ± 0.00022
Slope	% RSD	1.35	1.67	1.34	0.59
intercept	Mean ±SD	0.0465 ± 0.002	0.0247 ± 0.0033	0.00145 ± 0.00035	0.00537 ± 0.00278
Correlation co-efficient	Mean ±SD	0.997 ± 0.001	0.9985 ± 0.000886	0.998±0.0022	0.998 ±0.0062
Correlation co-efficient	% RSD	0.10	0.09	0.22	0.62

Table 2. Data showing accuracy and precision of the developed method

Parameters		Rosuvastatin	Glimepiride
Accuracy	% Recovery (mean ±SD)	99.04 ± 0.45	100.94 ± 0.431
Accuracy	% RSD	0.454	0.427
Repeatability	% Recovery (mean ±SD)	98.97 ± 0.537	100.8 ± 0.256
Repeatability	% RSD	0.543	0.254
Ruggedness	% Recovery (mean ±SD)	99.02 ± 0.545	100.77 ± 0.219
Ruggedness	% RSD	0.550	0.217
Reproducibility	% Recovery (mean ±SD) (UV 1605)	99.0 ±0.309	100.8 ± 0.256
	% RSD	0.312	0.254
	% Recovery (mean ±SD) (UV 1200)	99.14 ± 0.192	100.9 ± 0.164
	% RSD	0.312	0.163

Table 3. Results of robustness

Solvent Composition	Drug in standard solution (μg/ml)		Measured conc. (μg/ml)		% Recovery
Solvent Composition	RSV	GLM	RSV	GLM	RSV	GLM
0.1 N NaOH	10	10	9.89	10.11	98.90	101.10
	10	10	9.76	10.09	97.60	100.90
	10	10	9.87	10.14	98.70	101.40
97% 0.1 N NaOH + 3% Methanol	10	10	9.80	10.01	98.00	100.10
	10	10	9.91	10.20	99.10	102.00
	10	10	9.88	10.16	98.80	101.60

Table 4: Results of analysis of tablet dosage forms containing RSV and GLM

Drug	Label claim (mg) (n=6)	Observed amount (mg) (n=6)	Potency (%)	SD	% RSD
Rosuvastatin	10.00	9.88	98.80	0.92	0.93
Glimepiride	4.00	4.05	101.25	1.62	1.60

Accuracy and Precision:

Results of accuracy and precision (repeatability, ruggedness and reproducibility) are summarized in table 2. Accuracy is generally assessed by analyzing samples with known concentration and comparing the measured value with the true value .The measured value was obtained by recovery test. % recovery was found 99.04 ± 0.45 for rosuvastatin and 100.94 ± 0.431 for glimepiride with % RSD value 0.454 and 0.427 respectively. All the results indicate that the method is highly accurate.

The measurement for repeatability was done from 9.00 am to 9.00 pm. Three determinations of four concentrations across the intended range (10-18µg/ml) were included in the study. % RSD was calculated for various run. The method is highly precise as % RSD for repeatability, ruggedness and reproducibility was less than 2%.

Robustness:

Robustness study was performed by making slight variations in solvent composition. No significant effect was observed in the recovery of drugs. % recovery was 98% to 102%. So we can say that the method is robust.

Analysis of market products:

The proposed method was used to determine the potency of commercially available tablets (rosuvastatin tablet 10 mg and glimepiride tablet 4 mg). Six replicate determinations (n=6) were carried out and the results are summarized in Table 4.

CONCLUSION:

The validation study shows that the developed UV method is accurate, rapid, precise, reproducible and inexpensive with acceptable correlation co-efficient, RSD (%) and standard deviations which make it versatile and valuable for simultaneous determination of rosuvastatin and glimepiride in bulk or pharmaceutical dosage form (individual or combine). The advantages lie in the simplicity of sample preparation and the low costs of reagents used. The proposed method is simple and do not involve laborious time-consuming sample preparation. So this method can be used in the quality control department for potency and dissolution study.

ACKNOWLEDGEMENTS:

The authors are thankful to Eskayef Bangladesh Ltd. for providing API as gift.

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Received on 05.11.2011 Accepted on 29.11.2011

Asian J. Pharm. Ana. 1(4): Oct. - Dec. 2011; Page 74-78