ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2007, том 62, № 6, с. 598-603
^=ОРИГИНАЛЬНЫЕ СТАТЬИ =
УДК 543
VALIDATED SPECTROPHOTOMETRIC METHOD FOR THE QUANTITATIVE DETERMINATION OF SIMVASTATIN IN PHARMACEUTICAL FORMULATIONS AND HUMAN SERUM
© 2007 r. M. Saeed Arayne1, Najma Sultana2, Fida Hussain3, Syed Asif Ali2
department of Chemistry, University of Karachi Karachi-75270, Pakistan 2Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, University of Karachi
Karachi-75270, Pakistan 3Quality Control Department, Noa Hemis Pharmaceuticals, 154/23, Korangi Industrial Area
Karachi-74900, Pakistan Received 10.01.2006; in final form 01.08.2006
A simple ultraviolet spectrophotometric method for the determination of simvastatin in methanol has been devised and compared with the existing pharmacopoeial RP-HPLC method for the estimation of the drug. Analytical parameters such as stability, selectivity, accuracy and precision have been established for the method, using SIMS® tablets and human serum samples, and evaluated statistically to assess the application of the method. The method was validated under the ICH and USP guidelines and was found to comprise the advantages of simplicity, stability, sensitivity, reproducibility and accuracy for using as an alternate to the existing non-spec-trophotometric methods for the routine determination of the drug in pharmaceutical formulations and for pharmaceutical investigations involving simvastatin.
Simvastatin, 2,2-dimethyl-,1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)-ethyl]-1-naphthalenyl ester, [1S-[1a,3a,7p,8p(2S*,4S*),-8ap]] butanoic acid, is a lipid-lowering agent that is derived synthetically from a fermentation product of Aspergillus terreus and has been shown to lessen both normal and elevated LDL-C concentrations. The drug is official in the United States Pharmacopoeia 2004 and the official method of its determination is high performance liquid chromatography [1]. Various other methods have been reported such as second-derivative spectrophotometry [2], high pressure liquid chromatography [3-7], mass spectrometry [7, 8] and gas chromatography [8, 9]. Though an ultraviolet spectroscopic method [10] has been suggested as a method of analysis, no other analytical parameter except absorptivity has so far been reported. In this study, a simple ultraviolet spectrophotometric method for the determination of simvastatin in methanol has been reported. Analytical parameters for the method have also been established and compared with those established for the official pharmacopeial method.
EXPERIMENTAL
Apparatus. Shimadzu 1601 UV visible spectrophotometer with quartz cells of 10-mm path length; HPLC that comprised a pump (LC-10AT VP Shimadzu, Japan), a variable wavelength UV-VIS spectrophotomet-ric detector (SPD-10AT VP UV-VIS Shimadzu, Ja-
pan), and a communication Bus Module (102 Shimad-zu, Japan) for integration; Bath sonicator (Clifton, England); Analytical balance (AX-200 Shimadzu, Japan); Filtration assembly (Sartorius, Goettingen, Germany); Filter papers 0.45 |m, 13 mm (Sartorius, Germany); Sartorius Minisart RC 4 Syringe filters 0.45 |m (Hannover, Germany), Whatman 41 filter paper circles of diameter 125 mm (Whatman, Maidstone, England).
Materials. Simvastatin reference standard (99.17% potency) and SIMS® tablets (20 mg) were supplied by Noa Hemis Pharmaceuticals, Karachi, Pakistan and were used without any further purification; methanol, glacial acetic acid, and phosphoric acid were of analytical grade (Merck, Darmstadt, Germany); acetonitrile was of HPLC grade, (J.T. Baker, Deventer, Holland).
Stock solution of simvastatin (300 |g/mL) was prepared by dissolving 30 mg of simvastatin in 100 mL of methanol using a bath sonicator.
Preparation of calibration curve. Suitable ali-quots (3-5 mL) of the stock solution were pipetted into 100 ml volumetric flasks and the volume was made up to 100 ml with methanol. The solutions were shaken well for proper mixing and their absorbance was measured at 238 nm.
The above procedure was repeated six times. Mean absorbance values along with the regressed values (method of least squares) and statistical data for the method are shown in Table 1. The optical characteris-
Table 1. Mean absorbance values, regressed values and statistical data of the calibration curve for the estimation of sim-vastatin
Concentration (|g/mL) Mean ABSa (±S.E.) Regressed valuesb
09.0 0.54854 ± 0.00244 0.9693
10.5 0.64887 ± 0.00106 0.1362
12.0 0.73410 ± 0.00304 0.6405
13.5 0.83354 ± 0.00458 0.7404
15.0 0.92564 ± 0.00206 0.9809
a Mean of six values. b Using regression equation.
Regression equation statistical data. Intercept (a) = 1.296 x 10-2;
slope (b) = 6.26 x 10-2; correlation coefficient = 0.9997; n = 42.
tics for the solution of simvastatin in methanol are given in Table 2. Absorptivity scan over the UV wavelength range between 200 and 400 nm for 11 |g/mL solution of simvastatin in methanol is shown in Figure.
Stability. Stability of the solutions of simvastatin, used for preparing the calibration curves in the method, was ascertained by observing changes in the absor-bance at their respective analytical wavelengths over a period of 24 h.
Precision. In order to determine precision of the method, solutions containing known amounts of pure drug were prepared and analyzed in three replicates and absorbance readings were recorded in six replicates to get the mean. The analytical results obtained from these investigations for the methods are summarized in Table 3.
Accuracy and selectivity. The accuracy and selectivity of the method for the estimation of the drug in presence of various tablet excipients such as starch, lactose, gelatin, talc, and magnesium stearate were investigated.
A placebo comprising 46.2% w/w lactose, 46.1% w/w starch, 1.8% w/w gelatin, 0.28% w/w talc, and 0.085% w/w magnesium stearate was prepared. A 1 : 1 blend of drug and placebo was prepared. Drug was then extracted from this blend using methanol. The metha-
nol extract was filtered through Whatman 41 filter paper circles of diameter 125 mm (Whatman, Maidstone, England) and absorbance of the filtrates, appropriately diluted, was measured at 238 nm. The above procedure was carried out in triplicate and absorbance readings were recorded six times to get the mean. Results of these determinations are included in Table 4.
Comparison with pharmacopoeial method. Samples of pure drug and SIMS® tablets were estimated using the method detailed in the United States Pharmacopoeia [1]. Results of these estimations are given in Table 5.
Application of the method in human serum. Samples of pure drug were also estimated in human serum using the proposed method. Serum was coagulated by the acetonitrile and filtered through Whatman 41 filter paper circles of diameter 125 mm (Whatman, Maidstone, England). Stock solution of simvastatin (200 ^g/ml) was prepared by dissolving 20 mg of simvastatin in 100 ml of methanol using a bath sonicator. 1 ml of the stock solution was pipetted to a 10 ml volumetric flask and diluted with 4 ml of methanol and 5 ml of serum. The solution was shaken well for proper mixing and absor-bance was measured at 238 nm. The above procedure was carried out in triplicate and absorbance readings were recorded six times to get the mean. Results of these determinations are included in Table 6.
RESULTS AND DISCUSSION
Simvastatin in methanol yields a characteristic curve when scanned in the ultraviolet wavelength range between 200 and 400 nm. The scan (figure) shows absorption maxima at 230, 238 and 246 nm (Table 2) in close proximity of the maxima reported in the official RP-HPLC method of United States Pharmacopoeia (238 nm) [1]. The absorptivity at 238 nm was found to be 25511.71 mol-1 cm-1 and this wavelength was chosen as the analytical wavelength.
The ultraviolet spectra in this case can be attributed mainly to the lactone nucleus in the simvastatin molecule. Correlation coefficient was found to be 0.9997, signifying that a linear relation existed between the absorbance and concentration of the drug.
Table 2. Optical characteristics for simvastatin in reference standard and tablets
Characteristic Value in reference standard Value in tablets
Absorption maxima (nm) 230, 238a, 246 230, 238a, 246
Beer's law limitsb (|g/mL) 1-10 1-10
Apparent molar absorptivityb (l mol-1 cm-1) 25,511; 25121c; 18655d 25.625; 25121c; 18655d
a Analytical wavelength for proposed method. b At analytical wavelength. c At analytical wavelength 233 nm. d At analytical wavelength 243 nm.
A
Wavelength scan for simvastatin in methanol.
Beer's law was found to be obeyed between 1 and 10 |g/mL. Regression analysis was performed on the experimental data. The raw data along with the results of regression analysis (method of least squares) are shown in Table 1. Regression equation was y = 0.0626x + 0.01296.
2
The variance in the response variable, Syx, was calculated to be 1.15 x 10-4 (six degrees of freedom). This low value indicates the closeness of the experimental points to the least squares line. The fact is in concurrence with the low values of the standard error of the mean (S.E.M.) absorbances of the solutions used for preparing the calibration curve. The variance of the methods was compared using F' distribution to determine whether they were significantly different from
each other. The calculated F ' value was found to be 1.32 whereas the tabulated F' value was 4.29 for six and five degrees of freedom in the denominator and numerator, respectively. There is no significant difference in the variance and hence no difference exists in variability. This is supported by the narrower range in which Beer's law is obeyed (1-10 |g/mL) and the higher absorptivity of simvastatin at 238 nm (25.511 l mol-1 cm-1). The value of Sandell's sensitivity coefficient 8.17 x x 10-6 g cm-2 per 0.001 abs unit supports the above observation. The variance
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