INTRODUCTION:

Atorvastatin is chemically ((3R,5R)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid¹. It is a dihydroxy monocarboxylic acid that is a member of the drug class known as statins, used primarily for lowering blood cholesterol and for preventing cardiovascular diseases². Atorvastatin is official in IP, USP and BP. Literature survey reveals Septrophotometric^3-4methods for estimation of Atorvastatin in pharmaceutical formulations. The present communication describes simple, sensitive, accurate, precise and economical visible spectrophotometric method using MBTH Reagent [3-Methyl-2-Benzothiazolinone Hydrazone] for the estimation of Atorvastatin in bulk formulation.

Figure 1. Atorvastatin structure

MECHANISM OF ACTION:

Atorvastatin is a statin medication and a competitive inhibitor of the enzyme HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase, which catalyzes the conversion of HMG-CoA to mevalonate, an early rate-limiting step in cholesterol biosynthesis. Atorvastatin acts primarily in the liver, where decreased hepatic cholesterol concentrations stimulate the up regulation of hepatic low-density lipoprotein (LDL) receptors, which increases hepatic uptake of LDL. Atorvastatin also reduces Very-Low-Density Lipoprotein-Cholesterol (VLDL-C), serum triglycerides (TG) and Intermediate Density Lipoproteins (IDL), as well as the number of apolipoprotein B containing particles, but increases High-Density Lipoprotein Cholesterol (HDL-C)⁵.

MATERIALS AND METHODS:

Apparatus:

A Shimadzu model T60 double beam UV/Vis Spectrophotometer with spectral width of 2nm wave length accuracy of 0.5nm and a pair of 10mm matched quartz cells was used to measure absorbance of the resulting solutions. Shimadzu analytical balance, an ultra sonic cleaner were used in the study.

Reagents and Materials:

Atorvastatin drug was gifted from Fugen Laboratories Pvt. Ltd. [Choutuppal, Hyderabad, Telanagana]. MBTH Reagent [3-Methyl-2-Benzothiazolinone Hydrazone] and Ferric chloride was purchased from loba chemie and SD Fine Chemicals Pvt. Ltd. [Mumbai Maharashtra] was used in the study.

Preparation of Reagent and Working standard stock solution:

2% Ferric Chloride: The solution was prepared by dissolving 2gm of ferric chloride in 100ml of milli-Q water.

Standard Stock Solution: Accurately weighed the 10mg of Atorvastatin API and transferred in 100ml of volumetric flask. To it 5mL of methanol was added sonicated to dissolve. Later made upto100mL with milli-Q water. (100µg/mL) or (100ppm).

Reaction Mechanism:

From Standard stock solution 30ppm or 30µg/mL concentration of solution was prepared and scanned in the range of 800-400nm against reagent blank solution as reference, and the maxima peak was observed at 566nm. The results obtained in this method were based on the reaction of MBTH with carbonyl group of Atorvastatin and gives a greenish blue colour species which was measured at 566nm shown in the Figure 2.

Figure 2: Reaction mechanism (λ max)

Preparation of Standard Solution:

From the Standard stock solution 2mL was pipetted out and transferred in to 20mL of volumetric flask. To it 1 mL of MBTH reagent and 2mL of 2% ferric chloride was added and volume was made up to 20mL with milli-Q water. (10µg/mL) or (10ppm).

Methodology:

From the Standard stock solution different concentration ranging from 5-25µg/mL of Atorvastatin were transferred into series of volumetric flask. To it 1mL of MBTH reagent and 2mL of 2% ferric chloride was added and volume was made up to 20mL with milli-Q water. The contents of the each flask was mixed well and allowed to stand at room temperature for 10 minutes. The absorbance of coloured species was measured at 566 nm against reagent blank solution. The amount of drug present in the sample solution was computed from the calibration curve.

Method Validation⁶:

Linearity:

Five points calibration curve were obtained in a concentration range from 5-25µg/ml for Atorvastatin. The response of the drug was found to be linear in the investigation concentration range and the linear regression equation was y=0.032x+0.001 with correlation coefficient 0.999 results are tabulated in table No.1 and Figure 3.

Table No.1 – Result for Linearity

Concentration (µg/ml)	Absorbance
5	0.162
10	0.328
15	0.486
20	0.632
25	0.810
Correlation	0.999
Intercept	0.001
Slope	0.032

Figure 3: Linearity graph

Precision:

Precision of the analytical method is ascertained by carrying out the analysis as per the procedure and as per normal weight taken for analysis. Repeat the analysis six times. Calculate the %assay, mean assay and %RSD. The developed method was found to be precise as the %RSD values for the repeatability and intermediate precision studies were 0.38% and 0.36%, respectively shown results are tabulated in table No.2.

Table No.2 – Result for Precision

Sample No.	% Assay
Sample No.	Intra day	Inter day
1	100.92	100.85
2	100.72	100.74
3	100.11	100.53
4	99.91	100.47
5	100.32	99.99
6	100.21	100.02
Mean	100.36	100.43
% RSD	0.38	0.36

Accuracy:

Accuracy of the method is ascertained by standard addition method at 3 levels. Standard quantity equivalent to 50%, 100% and 150% is to be added in sample. The result shown that best recoveries (101.04 - 100.06%) of the spiked drug were obtained at each added concentration, indicating that the method was accurate and results are tabulated in table No.3.

Table No.3 – Result for Accuracy

% Recovery Level	Amount Added(mg)	Amount Found(mg)	% Recovery	Mean(%)	% RSD
50%	5.04	5.10	101.04	101.04	0.40
	5.03	5.07	100.62
	5.04	5.13	101.45
100%	10.04	10.04	100.00	100.41	0.41
	10.03	10.07	100.41
	10.05	10.13	100.82
150%	15.03	14.95	99.45	100.06	0.54
	15.02	15.08	100.34
	15.04	15.11	100.41

Robustness:

Measure of the capacity of an analytical method to remain unaffected by small intentional variations in the operational parameters and provide an assurance of its reliability during the normal usage. It was determined by carrying out the analysis at different wavelength (±2nm). Results are tabulated and indicated by %RSD in table No.4.

Table no.4 – Result for Robustness

Parameter (±2nm)

566nm

(As per Method)

564nm

568nm

% RSD

0.177%

0.178%

0.179%

Sensitivity:

Limit of Detection and Quantitation (LOD and LOQ)

From the linearity data calculate the limit of detection and quantitation using the following formula.

LOD = 3.3σ / S

σ = Standard deviation of response.

S = Slope of the calibration curve of the analyte.

LOQ = 10σ / S

σ = Standard deviation of response.

S = Slope of the calibration curve of the analyte.

The results are tabulated in Table No.5

Table No.5 Result for LOD and LOQ

LOD (µg/ml)	0.103
LOQ (µg/ml)	0.313

RESULTS AND DISCUSSION:

The analytical method was developed by studying different parameters. The method was validated for all validation parameters as per ICH guidelines. The lambda max of Atorvastatin was found to be 566nm. Linearity was found with the concentration range 5-25 µg/ml and correlation coefficients found to be 0.999 indicate good linearity between concentration and slope area. Beer’s law was obeyed by the fundamental spectrum. This method was found to be simple, sensitive, accurate, precise and economical for routine analysis for the estimation of Atorvastatin in Bulk form. Recovery studies was found to be close to 100 % at each added concentration which indicates the method was observed to be accurate and precise. The values of LOD and LOQ were found to be 0.103(µg/ml) and 0.313(µg/ml) respectively which indicates the sensitivity of the method. The method was also found to be robust as the % RSD value was less than 2.0%

CONCLUSION:

The proposed visible spectrophotometric method was found to be simple, sensitive, accurate, precise and economic for determination of Atorvastatin in bulk formulation. Hence it can be conveniently adopted for routine quality analysis of drug in pharmaceutical dosage form.

ACKNOWLEDGMENTS:

The authors are thankful to the Head of the Pharmaceutical Analysis Department and my guide for his moral support and encouragement during the work and to the Fugen Laboratories Pvt. Ltd, loba chemie and SD Fine Chemicals Pvt. Ltd. Mumbai Maharashtra, India for providing the necessary facilities to carry out this research.

REFERENCES:

1. United States Pharmacopeia 2020 Volume-I, USP 43, 414.

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

3. Shyni Bernard and Molly Mathew-Spectrophotometric method of estimation of Atorvastatin using sulfo-phospho-vanillin reaction. Journal of Applied Pharmaceutical Science.

4. Gowari Sankar D., Raju M S M., Sumanth, Kalyan S., Latha P-Estimation of Atorvastatin by high performance liquid chromatography in pure and pharmaceutical dosage form. Asian J. Chem. 2005; 17 (4): 2571.

5. https://go.drugbank.com/drugs/DB01076.

6. ICH Q2B, Text on Validation of Analytical Procedures. Geneva (1994) 1-5.

Received on 21.08.2023 Modified on 07.09.2023

Asian J. Pharm. Ana. 2023; 13(4):236-238.

DOI: 10.52711/2231-5675.2023.00038