INTRODUCTION:

Simvastatin is lipid lowering agent, chemically 2, 2- Dimethyl butanoic acid (1S,3R,7S,8S,8a R)-1,2,3,7,8,8a hexahydro- 3,7- dimethyl-8- [2- [(2R,4R)- tetrahydro-4- hydroxy-6 oxo2H pyran-2yl]ethyl]1-napthalenyl ester used as a HMG CoA reductase inhibitors. It is derived synthetically from fermentation products of Aspergillus terreus. Simvastatin is HMG-CoA reductase inhibitors commonly called statins. All statins act by inhibiting 3-hydroxy-3-methyl glutaryl coenzyme (HMG-CoA). A HMG-CoA reductase, the rate limiting enzyme of the HMG-CoA reductase pathway, the metabolic pathway responsible for the endogenous production of cholesterol mainly used for the treatment of dyslipidemia and the prevention of cardiovascular diseases.¹

Figure No. 1 Structure of Simvastatin

Simvastatin is prodrug which is converted into its β- hydroxy which inhibits HMG CoA reductase (3-hydroxy-3- methyl glutaryl Coenzyme A) enzyme, responsible for catalyzing the conversion of HMG CoA to mevalonate a rate limiting step in the synthesis of cholesterol in liver. Literature survey revealed that there were few UV-visible methods have been reported. The drug is officially listed in the 2004 in United States Pharmacopoeia and the official method of its determination is high performance liquid chromatography.² Various other methods have been reported such as second derivative spectrophotometry³ high pressure liquid chromatography^4,5 mass spectrometry⁵and gas chromatography^6. Though an ultraviolet spectroscopic method has been suggested as a method of analysis, no other analytical parameter except absorptivity has so far been reported. The main purpose of this investigation is to develop and validate UV spectrophotometric method which is simple, rapid and precise for estimation of simvastatin from bulk. This method could also be easily used in routine analytical work and for dissolution studies at very low concentration of simvastatin.

MATERIALS AND METHOD:

Simvastatin was gifted by FDC limited Mumbai. The commercial methanol (AR Grade) was procured from Loba Chemicals Ltd, Mumbai, India.

Instrument and Apparatus:

Double beam Shimadzu model 1800 UV/Visible spectrophotometer with spectral width of 2nm, wavelength accuracy of 0.5nm and a pair of 10mm matched quartz cell was used to measure absorbance of all the solutions.

Method Development:

Methodology and Solvent selection (Solubility Studies):

These studies were carried out to find a suitable and compatible solvent in which drugs are completely soluble. Simvastatin is readily soluble in methanol. Therefore methanol was selected as solvent

Preparation of standard solution and λmax of Simvastatin:

100mg of simvastatin was accurately weighed and dissolved completely in 100ml methanol to get the concentration of 1000μg/ml (Stock A). From this stock solution A, pipette out 10 ml and it was further diluted with methanol to obtain the solution of 100μg/ml (Stock B) and scanned for λmax from 400 to 200nm on UV spectrophotometer. Scanning of simvastatin in methanol was shown in figure no. 2.

Calibration Curve of Simvastatin in Methanol:

From Stock Solution B, pipette out aliquots of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 and diluted with methanol in 10ml volumetric flask to get the concentrations of 2, 4, 6, 8, 10, 12, 14, 16 and 18μg/ml respectively. Absorbance of each was taken at λmax with methanol as blank and plotted against concentration and find out the value of slope, intercept and regression coefficient.

Method Validation^4,6:

Linearity:

The stock solution of simvastatin 100μg/ml was prepared and further diluted to obtain solution of range 2-18μg/ml with methanol. The plot obtained was integrated for various parameters were shown in figure no.3.

Precision:

The precision studies for the proposed method was carried out as per ICH guidelines. The precision of the system was determined by assay of six determinations at 100% test concentration (8µg/ml) and its relative standard deviation (% RSD) was calculated. The result of precison study was reported in terms of % RSD. The acceptance criteria should not be more than 2%.

Limit of Detection (LOD) and Limit of Quantification (LOQ):-

The limit of detection and the limit of quantification of the drug were derived by calculating the signal to noise ratio (S/N, i.e. 3.3 for LOD and 10 for LOQ) using the following equations as per ICH guidelines.

LOD = 3.3 × σ/S

LOQ = 10 × σ/S

Where,

σ = the standard deviation of the response and

S = slope of calibration curve.

Accuracy (Recovery Study):

The accuracy of the method was determined at three levels 50%, 100% and 150% of the method concentration 8μg/ml. The standard solution of simvastatin as 4, 8, 12μg/ml was prepared and were combined with 8μg/ml of sample solution that would yield a concentration of 12, 16, 20μg/ml. The mean recoveries of simvastatin expressed in terms of the percentage recovery and relative standard deviation (% R.S.D.) were determined. Mean recovery should be in the range of 98-102%.

RESULT AND DISCUSSION:

Spectral Analysis of Simvastatin (Determination of λ max):

The absorption maximum of the standard solution was scanned between 400-200 nm regions on Double beam UV/Visible spectrophotometer.

Figure No 2: Scanning of Simvastatin in Methanol

Selection of Wavelength:

A solution of 100μg/ml solution of drug was prepared in methanol and scanned in the range of 400-200nm, using the above solvent as blank. The maximum absorbance (λmax) was found to be 238nm.

Linearity and Range:

For the analysis of the linearity, the standard stock solution of simvastatin 100μg/ml was prepared in methanol and further diluted to obtain solution of range 2-18μg/ml with methanol. The absorbance was plotted against the concentrations to obtain the calibration curve.

Calibration Curve of Simvastatin in Methanol:

Calibration curve of simvastatin in methanol were shown in Table No 1.

Table No 1: Calibration Curve of Simvastatin in methanol

Sr. No.	Concentration (µg/ml)	Absorbance (nm)
01	0	0
02	2	0.103 ±0.012
03	4	0.202 ±0.008
04	6	0.301 ±0.015
05	8	0.393 ±0.006
06	10	0.485 ±0.015
07	12	0.584 ±0.018
08	14	0.688 ±0.017
09	16	0.794 ±0.003
10	18	0.891 ±0.018

Figure No 3: Calibration Curve of Simvastatin in methanol

Table No. 2 Linear Regression Data for calibration curve

Parameters	Results
λ max (nm)	238 nm
Linearity Range	2-18 μg/ml
Slope (m)	0.049173
Intercept (c)	0.001545
Correlation coefficient	0.999

Precision:

The precision of an analytical method was determined by assaying six determinations at test concentration (8µg/ml) and % Relative Standard Deviation (%RSD) calculated statistically. It was found to be less than 2%. (i.e.1.70%).

Table No.3 Precision Study

Sr. No.	Solution (µg/ml)	Absorbance (nm)	Average	SD*	% RSD
1.	8	0.383	0.392	0.0067	1.70
2.	8	0.397
3.	8	0.393
4.	8	0.402
5.	8	0.385
6.	8	0.396

Limit of Detection (LOD) & Limit of Quantitation (LOQ):

LOD and LOQ for the method were calculated separately based on the standard deviation response of the calibration curve and were shown in Tab No 4.

Table No. 4 LOD & LOQ of Simvastatin

Parameters	μg/ml
LOD (limit of detection)	0.44
LOQ (limit of quantitation)	1.36

Accuracy (Recovery Study):

Average % recovery for three different concentrations was found to be 99.47%. The results were in accordance with fixed limits from 98.0% to 102.0%, indicating the suitability of the developed method in quantifying the concentration of simvastatin.

CONCLUSION:

The method developed in this present study for the determination of simvastatin from bulk and formulation is simple, accurate, sensitive and reproducible and validated as per ICH guidelines in terms of linearity, precision, accuracy, LOD and LOQ. The proposed method utilizes inexpensive solvent and could be applied for routine analysis in quality control laboratories.

ACKNOWLEDGEMENT:

Authors are grateful to FDC limited Mumbai for providing the gift sample of Simvastatin. We are also thankful to the Bharati Vidyapeeth College of Pharmacy, Kolhapur as well as Ashokrao Mane College of Pharmacy, Peth-Vadgaon for providing the necessary facilities to carry out this work.

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Received on 08.05.2019 Modified on 21.06.2019

Research J. Pharm. and Tech. 2019; 12(12): 5745-5748.

DOI: 10.5958/0974-360X.2019.00994.6

Sample (μg/ml)	Ref. Std. Conc. added (μg/ml)	Total Conc. of Solution (μg/ml)	Conc. of Drug found (μg/ml)	Recovery %	% RSD (n=3)	Mean Recovery %
8μg/ml	4	12	11.8	98.33	1.12	99.47
	8	16	16.1	100.6	0.84
	12	20	19.9	99.50	0.96