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Abstract: Quantitative estimation of poorly water-soluble drugs involves use of organic solvents. Major drawbacks of
organic solvents include high cost, volatility and toxicity. In the present investigation, hydrotropic solubilization is
employed to enhance the aqueous solubilities of poorly water-soluble drugs like Atorvastatin Calcium in tablet dosage
forms. This method utilizes 2.0 M urea solution as, hydrotropic solubilizing agent. In the urea solution Atorvastatin
Calcium shows maximum absorbance at 240 nm. The 2.0 M urea solution does not show any interference with the
sampling wavelength. Another method is formation of green color complex between the drug Atorvastatin Calcium and
0.3 % w/v ferric chloride and 0.02 % w/v potassium ferricyanide. The green colored complex shows the maximum
absorbance at 787 nm. The hydrotropic agent and additives used in the manufacture of tablets did not interfere in the
analysis. The results of tablet analysis were found be in range of 99.26 to 100.12% with standard error values of 0.2728
and 0.2082 by Hydrotropy and Colorimetry respectively. The results of analysis of both methods were validated
statistically following ICH Q2A (R1) guidelines. Both methods were found to be useful for accurate, sensitive, selective,
precise and robust analysis of Atorvastatin from marketed formulations. After optimization, methods will be useful for
analysis of Atorvastatin in biological fluids.
Key Words: Atorvastatin Calcium, Urea, Hydrotropy, Colorimetry.
Introduction
Atorvastatin Calcium is chemically described as [R- water-soluble drugs involves use of organic solvents.
(R*, R*)]-2-(4-fluorophenyl)-β, ∂ dihydroxy-5-(1- Major drawbacks of organic solvents include high
methylethyl)-3-phenyl-4-[(phenyl amino) carbonyl]- cost, volatility and toxicity. In the present
1H-pyrrole-1-heptanoic acid calcium salt trihydrate is investigation, hydrotropic solubilization is employed
an anti hyperlipoproteinemic agent act by inhibiting to enhance the aqueous solubilities of poorly water-
HMG - CoA reductase (1-2). Many analytical soluble drugs like Atorvastatin Calcium in tablet
methods like UV spectroscopy, HPLC were reported dosage forms. In a proposed method this problem has
for determination of Atorvastatin Calcium alone and been solved by using 2 M urea solution. Highly
combination with other antihypertensive drugs (3-8). sensitive and economic colorimetric method is not
In the analysis of Atorvastatin, major problem is reported for determination of Atorvastatin. Hence in
solubilization of Atorvastatin in most of solvents this communication we have reported new UV
during analysis (9). Quantitative estimation of poorly spectrophotometric methods for determination
Swapnil D. Jadhav et al /Int.J. PharmTech Res.2010,2(3) 1949
Atorvastatin Calcium using hydrotropy and the urea used does not interfere with the sampling
colorimetry. wavelength. Therefore 2.0 M urea is used for the
solubilization of drug (11). Different aliquots of the
Experimental standard solutions were taken in series of volumetric
Materials: flasks to prepare the concentrations ranging from 5-45
Instrument: μg/ml and volume was made up to mark with water.
Spectrophotometric analysis was carried out on a These concentrations were scanned in the range 200-
JASCO UV-spectrophotometer 530 using a 1 cm 400 nm range absorbances of each concentration taken
quartz cell. The instrument settings was zero order at 240 nm. Calibration curve was prepared by plotting
derivative mode and band width of 2.0 nm in the absorbance against concentration.
range of 200–350 nm and 400-800 nm. Procedure for analysis of tablet formulation:
Reagents and Chemicals: Twenty tablets were weighed and ground to a fine
Atorvastatin Calcium supplied by Cipla Ltd. India. All powder. Tablet powder equivalent to 30 mg
chemicals were analytical grade obtained from SD fine Atorvastatin Calcium was weighed and transferred to a
chemicals. Water purified by glass distillation 100 ml volumetric flask 70 ml of 2.0M urea solution
apparatus. was added to the flask and stirred for 15 min to
Preparations of Standard Drug Solutions and dissolve the drug and the final volume was made up to
Reagents: 100 ml with distilled water. The solution was filtered
For hydrotropic solubilization 20 mg of pure through Whatmann fitter paper No. 41 and the first
Atorvastatin Calcium was dissolved in 50 ml of 2.0 M few ml were rejected. The filtrate was diluted suitably
urea solution and stirred for 15 minutes and the final with distilled water to get 10 μg/ml of Atorvastatin
volume of both solutions was made up to 100 ml with Calcium. The absorbance at 240 nm was measured and
distilled water (10). The solution was filtered through the amount of drug present in the sample solution was
Whatmann filter paper No. 41. This solution was obtained from the slope and intercept values obtained
further diluted with distilled water to prepare working from the calibration curve (Table 1). From these,
concentrations of 100 μg/ml of Atorvastatin Calcium. concentrations, the composition of the tablet was
For the colorimetric method standard solution of obtained. The results of analysis of tablet formulations
Atorvastatin Calcium was prepared in solvent system are recorded in Table 2. After 48 hours, the solutions
methanol: water (60:40) about 10 mg of drug was were reanalyzed to determine chemical stability and
dissolved in 70 ml of solvent system sonicated for 10 precipitation, if any.
minutes and final volume was made up to 100 ml to
get working solution of 100 μg/ml. Ferric Chloride Method 2: Spectroscopic method employing
was prepared by dissolving 300 mg in 100 ml of 0.5 M colorimetric method.
hydrochloric acid and potassium ferricyanide was Overlain spectra of reagent and the drug reagent colour
prepared by dissolving 20 mg in 100 ml of distilled complex is shown in Figure 2, which shows that
water. change in the colour is observed by addition of drug to
reagent. The figure shows maximum absorbance at
Methods: 787 nm which is selected as sampling wavelength.
Preliminary Solubility Studies of Drugs: Different aliquots of the standard drug solutions were
Solubility of Atorvastatin Calcium was determined at taken in series of 10 ml volumetric flasks to prepare
28±1°. An excess amount of drug was added to 2M the concentrations ranging from 4-24 μg/ml to each
urea solution in vials. The vials were shaken flask 2 ml of 0.3 % w/v of ferric chloride and 2 ml of
mechanically for 12 h at 28±1°, in a mechanical 0.02 % w/v potassium ferricyanide was added and
shaker. These solutions were allowed to equilibrate for volume was made up to mark with water after 30
the next 24 hours, and then centrifuged for 5 minutes minutes the green coloured stable complex was
at 2000 rpm. The supernatant of each vial was filtered formed. These solutions were analyzed in 400-800 nm
through Whatmann filter paper No. 41. The filtrates range and spectras were recorded. The absorbance of
were diluted suitably, and analyzed each concentration at 787 nm is plotted against
spectrophotometrically against corresponding solvent concentration which gives calibration curve.
blank.
Procedure for analysis of tablet formulation:
Method 1: Spectroscopic method employing Twenty tablets were weighed and ground to a fine
hydrotropic solubilization. powder. Tablet powder equivalent to 10 mg
From the overlain spectra of the drug Atorvastatin Atorvastatin Calcium was weighed and transferred to a
Calcium and 2.0 M urea (Figure 1), it was found that 100 ml volumetric flask to this 70 ml of solvent system
Swapnil D. Jadhav et al /Int.J. PharmTech Res.2010,2(3) 1950
methanol: Water (60:40) was added. This solution was calibration curve (Table 1). The results of analysis of
sonicated for 10 min and volume was made up to mark tablet formulation are reported in Table 2.
then solution was filtered through the Whatmann fitter
paper No. 41 first few ml is rejected. This solution was Method Validation:
further diluted to obtain the concentration of 10 μg/ml The method was validated according to ICH Q2B R1
of Atorvastatin Calcium. To this solution 2 ml of 0.3 guidelines for validation of analytical procedures in
% w/v of ferric chloride and 2 ml of 0.02% w/v of order to determine the linearity, sensitivity, precision,
potassium ferricyanide was added solution was kept robustness and accuracy for the analyte (12). Accuracy
aside, after 30 minutes, stable green colored complex and specificity of analysis was determined by
was formed. Then solution was analyzed in 400-800 performing recovery studies by spiking different
nm range and from absorbance at 787 nm was concentrations of pure drug in the preanalyzed tablet
recorded. Concentration of solution was calculated sample. Results of validation parameters are reported
from the slope and intercept values obtained from in Table 3.
Intraday Precision
Hydrotropy 99.48± 0.29 -0.6 0.1674 0.29
Colorimetry 99.51± 0.24 -0.5 0.1385 0.25
Inter Day Precision
Hydrotropy 99.55± 1.20 -0.5 0.6939 1.20
Colorimetry 99.51± 0.24 -0.6 0.6939 0.36
S.D.: Standard Deviation; CV: Coefficient of Variance; SE: Standard Error.
Analyst 1
Hydrotropy 99.69± 0.72 0.4157 0.72
Colorimetry 99.46± 0.45 0.2598 0.45
Analyst 2
Hydrotropy 99.22± 0.66 0.3810 0.66
Colorimetry 99.95± 0.28 0.6634 0.28
Figure 1: Overlain Spectra of 5 µg/ml of Atorvastatin Calcium in Urea and 2.0 M Urea Solution.
Swapnil D. Jadhav et al /Int.J. PharmTech Res.2010,2(3) 1952
Results Discussion
Quantitative estimation of poorly water-soluble drugs It is evident from results of validation studies for both
involves use of organic solvents. Major drawbacks of methods that methods are accurate, sensitive, selective,
organic solvents include high cost, volatility and precise and robust for spectroscopic estimation of
toxicity. In the present investigation, hydrotropic Atorvastatin. Moreover the method is economic,
solubilization is employed to enhance the aqueous simple and rapid, hence can be employed for routine
solubilities of poorly water-soluble drugs like analysis in quality control laboratories for estimation
Atorvastatin Calcium in tablet dosage forms. The of Atorvastatin form marketed formulations. After
results of solubility studies indicated that enhancement optimizing these methods to estimate Atorvastatin
in aqueous solubility of Atorvastatin Calcium in 2.0 M from biological fluids, these methods can be used in
urea solution was more than 6-7 folds as compared to Clinical and Bioequivalence studies.
their solubilities in distilled water. Therefore, this
solution was employed to extract Atorvastatin Calcium Acknowledgement
from the fine powder of tablet formulation and thus The authors are very thankful to Cipla India Pvt. Ltd.
analysis will become easier one. for providing gift sample of Atorvastatin Calcium and
The colorimetric method was optimized for to Tatyasaheb Kore College of Pharmacy,
colorimetric reagents selection, their concentration, Warananagar and Bharati Vidyapeeth College of
sequence of additions, time for color development etc Pharmacy, Morewadi, Kolhapur for provision of
(13-14). Addition of ferric chloride and potassium facilities for this research work.
ferricyanide to sample of Atorvastatin leads to
development of highly intense green colored complex. List of Non-standard Abbreviations Used
The result of analysis of tablet formulation by both · rpm: revolutions per minute
methods showed % relative standard deviation values · ATR: Atorvastatin Calcium
in the range of 0.5859 to 0.9992 for ATR, which · Abs: Absorbance
indicates repeatability of the method. The results · Conc.: Concentration
indicated excellent recoveries ranging from 98.52 to · S.D.: Standard Deviation
101.1 % for ATR. Recoveries obtained for the drug do
· CV: Coefficient of Variance
not differ significantly from 100 %, which showed that
· SE: Standard Error.
there was no interference from common excipients
used in the formulation. The results of limits of · R.S.D.: Relative Standard Deviation.
detection and quantitation were within limits. · n:Number of times analysis is repeated
Swapnil D. Jadhav et al /Int.J. PharmTech Res.2010,2(3) 1953
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