The authors have declared that no competing interests exist.
The effectiveness of atorvastatin calcium in lowering cholesterol is dose-related. It is available in 10, 20, 40, and 80 mg film coated tablets. In order to ensure quality, safety and efficacy of tablets in formulations, the objective of this presented work was to develop a new high performance liquid chromatographic-UV method for quantitation of active atorvastatin calcium in pharmaceutical formulations. The method was based on reversed-phase high performance liquid chromatographic-UV separation of atorvastatin at detection wavelength of 246 nm using Acclaim 120 C18 reversed phase LC column (5 mm, 250×4.6 mm) with mobile phase of acetonitrile-dichloromethane-acetic acid (68.6: 30.6: 0.8 v/v/v) at a flow rate of 1.0 mL min-1 at 25°C. Different variables affecting chromatographic separation were carefully studied and optimized. The study results provided chromatogram of atorvastatin with retention time of 2.68 min. The calibration curve was linear over the concentration range of 15-300 mg mL-1. No interference was observed from common pharmaceutical excipients present in dosage forms. The proposed method was successfully applied to the determination of atorvastatin calcium in pharmaceutical formulations and proved to be significantly not different with reference method. The proposed can be used as an alternate method for routine quality control analysis of active atorvastatin in research, hospitals and pharmaceutical laboratories.
Atorvastain calcium trihydrate (CAS No. 344423-98-9; M.W. 1209) is a white powdery substance chemically known as Calcium (3R,5R)-7-(2-(4-fluorophenyl)-5-(1-methylethyl)- phenyl-4-(phenylcarbamoyl)-3,5-dihydroxyheptanoate trihydrate
Atorvastatin is an adjunct to diet for reduction of elevated total cholesterol. Atorvastatin selectively and competitively inhibits hepatic enzyme 3-hydroxyl- 3-methyl glutaryl-coenzyme A (HMGCoA) reductase, lowering plasma cholesterol levels by suppressing hepatic production of very low density lipoprotein and low density lipoprotein cholesterol. The effectiveness of atorvastatin in lowering cholesterol is dose-related. It is available in 10, 20, 40, and 80 mg film coated tablets. In order to ensure quality, safety and efficacy of tablets in formulations, it is important to develop new analytical method for quantitative analysis of drug in pharmaceutical formulations. Azmi and co-workers developed several analytical methods for the estimation and quality control analysis of cefixime
Solvents and reagents used were of high purity grade. Deionized water was obatined by double distillation and purification through milli-Q water purification system. Pure atorvastatin calcium reference drug was gifted by National Pharmaceutical Industries Company, Oman. 0.06% atorvastatin calcium was prepared in mobile phase of acetonitrile-dichloromethane-acetic acid (68.6: 30.6: 0.8 v/v/v) for proposed method. 0.02% atorvastatin calcium was dissolved in 0.005 M potassium dihydrogen phosphate-acetonitrile-methanol (39: 56: 5 v/v/v, pH 4.6) mobile phase for reference method. Both mobile phases were cleaned with nylon disc filter of 0.45 mm (Merck Millipore Ltd, Tullagreen, Ireland) and degassed by sonication before use for preparation of drug. Commercial tablets of Torvast 10 (National Pharmaceutical Industries Company, Oman) and Atorlip 10 (Cipla, India) were purchased from local market.
Hanna pH meter (USA) and Dionex-Ultimate 3000 high performance liquid chromatography (Thermo Scientific, Australia) were used for measuring pH and recording chromatogram.
Chromatography was performed in the isocratic mode on Dionex-Ultimate 3000 high performance liquid chromatography (Thermo Scientific, Australia) with a 20 µL sample injection loop (manual), HPG 3200 SD Pump, TCC 3000 SD column oven and WDM 3000 photodiode array UV-Visible detector. The output signal was monitored and integrated using Ver. 5.80 SR11 chromeleon Data System Software on an Acclaim 120 C18 reversed phase LC column (250 mm × 4.6 mm, i.d. 5 µm particle size).
A standard solution of atorvastatin calcium (0.6 mg mL-1) was prepared in a mobile phase of acetonitrile-dichloromethane-acetic acid (68.6: 30.6: 0.8 v/v/v). A HPLC column was cleaned and passed with said mobile phase. Into a series of 10 mL standard volumetric flask, different volumes (0.25-5.0 mL) of 0.06% atorvastatin calcium were pipetted and diluted up to the mark with said mobile phase. 20 μL of drug solution was injected into the sample port and mobile phase was pumped at a flow rate of 1.0 mL min-1 for a run of 5 min. The column temperature was maintained at 25̊C and the eluent was detected at 246 nm for HPLC separation of atorvastatin calcium. A calibration curve was constructed by plotting the corresponding peak area against the initial concentration of drug and the linear equation was obtained using Origin Pro6.1 software for the determination of active atorvastatin calcium in pharmaceutical formulations
Different volumes (0.25-5.0 mL) of 0.02% atorvastatin calcium prepared in a mobile phase of 0.005 M potassium dihydrogen phosphate-acetonitrile-methanol (39: 56: 5 v/v/v, pH 4.6) were transferred into 10 mL standard volumetric flask and diluted up to the mark with the said mobile phase. HPLC column was treated with the respective mobile so that the base line became flat. The drug solution (20 μL) was injected into the sample port and mobile phase was pumped at a flow rate of 1.0 mL min-1 for a run time of 10 min. The column temperature was maintained at 25̊C and the eluent was detected at 246 nm for HPLC separation of atorvastatin calcium. The peak area was plotted against initial concentration of atorvastatin calcium for calibration graph. The linear regression equation was generated and used to estimate atorvastatin calcium in commercial dosage forms.
10 commercially available tablets of Torvast 10 and Atorlip 10 were weighed and uniformly powdered. The solid equivalent to 60 mg for proposed method (or 20 mg for reference method) of atorvastatin calcium was weighed and completely dissolved in 100 mL mobile phase of acetonitrile-dichloromethane-acetic acid 68.6: 30.6: 0.8 v/v/v (or 0.005 M potassium dihydrogen phosphate-acetonitrile-methanol 39: 56: 5 v/v/v for reference method). The mixture was filtered using Whatmann No. 42 filter paper (Whatmann International Limited, Kent, UK), cleaned with 0.45 mm millipore nylon disc filter (Merck Millipore Ltd, Tullagreen, Ireland) and sonicated. The tablet solutions were assayed subjected to the procedure for assay of atorvastatin calcium by proposed and reference methods. The amount of atorvastatin calcium was calculated using the respective linear regression equation.
The proposed method has been validated as per the International conference on Harmonization guidelines
Linearity was investigated at different concentrations of atorvastatin calcium. Each concentration level was tested 5 times and the peak area of the eluted drug was recorded. The linear regression equation and other statistical parameters were generated using OriginPro 6.1 Software.
Limits of detection (LOD) and quantitation (LOQ) were calculated using the following expressions:
where S0 and b are standard deviation and slope of the calibration line, respectively.
The precision of the proposed method was tested at 2 concentration levels of 49.8 and 252.0 µg mL-1 atorvastatin calcium and independently analyzed 5 times repeatedly within a day (intra-day precision) and over 5 consecutive days (inter-day precision).
The specificity of the proposed method was studied at 60 µg mL-1 atorvastatin calcium along with excipients such as glucose, fructose, lactose, sodium benzoate, starch, povidone, methyl cellulose and micro crystalline cellulose added in different volumes at selected % concentration. The peak area was recorded within ±2% at each addition of excipients. The maximum tolerance limit of excipients was calculated using the following equation:
Mass/Volume (g L-1 or mg mL-1) = Volume taken (mL) × % concentration (3)
The robustness of the proposed method was investigated at 60 µg mL-1 atorvastatin calcium with small variations in the optimized experimental values of pH, flow rate, temperature of the column and mobile-phase ratio. The peak area was recorded within ±2% at each deliberate change of chromatographic conditions.
The accuracy of the proposed method was tested by standard addition technique. One volume of the fixed concentration of tablet solution was spiked individually with different concentrations of the reference atorvastatin calcium solution in 10 mL standard volumetric flask and diluted up to the mark with respective mobile phase. Each level was analyzed repeatedly for 5 times for the determination of active atorvastatin calcium in tablet
The accuracy of the proposed method was tested by direct method and compared with the reference method. The freshly prepared tablet solutions of Torvast and Atorlip containing atorvastatin calcium were independently analyzed 5 times at 60 µg mL-1 atorvastatin calcium by proposed and reference methods. The results of 2 methods (proposed and reference) were utilized to get experimental t and F-values at 95% confidence level and compared with tabulated t and F-values for significance of testing. If experimental t and F-values are less than tabulated t and F-values values
where and are mean recovery values at selected measurements, respectively. Sp is the pooled standard deviation and t
The UV visible absorption spectrum of atorvastatin calcium solution in mobile phases of acetonitrile-dichloromethane-acetic acid (68.6: 30.6: 0.8 v/v/v) and 0.005 M potassium dihydrogen phosphate-acetonitrile-methanol (39: 56: 5 v/v/v; pH 4.6) were recorded in the wavelength range of 190-400 nm. The λmax of 246 nm was recorded in each mobile phase and thus used as UV detection wavelength in HPLC system for HPLC separation of atorvastatin calcium.
The solution stability of atorvastatin calcium in both mobile phases was checked by UV-visible spectrophotometry for a period of 1 day. The results showed an absorption peak at 240 nm with no change in the absorbance. The solution stability of the standard drug solutions was also tested using TLC plates coated with silica gel G (Merck Limited, Mumbai, India) and developed in acetonitrile: dichloromethane: acetic acid (45:20:0.5 v/v/v) and acetonitrile: 0.005 M KH2PO4: methanol (28.5: 5: 2.5 v/v/v) solvent systems. The results showed a single spot with Rfof 0.66 (acetonitrile: dichloromethane: acetic acid - 45:20:0.5 v/v/v) and 0.75 (acetonitrile: 0.005 M KH2PO4: methanol - 28.5: 5: 2.5 v/v/v) indicated that the drug solutions were stable for at least 1 day. Hence, the prepared drug can be analyzed within the stability time period of 1 day.
Method validation was performed on the best determined stationary phase i.e. C18 column (250 mm × 4.6 mm; i.d. 5 µm particle size). Separation with good resolution and low tailing factor (less than 2) were advantages of using C18 column.
The basic chromatographic conditions were optimized for developing a new HPLC-UV method for determination of atorvastatin calcium after testing different conditions of HPLC such as column temperature, components of mobile phase, proportion of mobile phase components, detection wavelength, and flow rate.
The mobile phase of acetonitrile-dichloromethane-acetic acid (68.6: 30.6: 0.8 v/v/v) was selected after preliminary trials using different compositions of mobile phase. The chromatogram obtained showed better eluted peak, less retention time and low tailing factor (
Under theoptimized experimental conditions
|
|
|
---|---|---|
246 | 246 | Maximum wavelength (nm) |
5-100 | 15-300 | Linear dynamic range (µg mL-1) |
A= 0.101 + 0.669 C | A= 0.077 + 0.156 C | Linear regression equation |
0.08 | 0.106 | Standard deviation of intercept, Sa (µg mL-1) |
1.0×10-3 | 6.3×10-4 | Standard deviation of slope, S |
0.9999 | 0.9999 | Correlation coefficient (r) |
0.018 | 0.028 | Variance (S02) |
0.135 | 0.168 | Standard deviation of calibration line (S0) |
0.67 | 3.57 | Limit of detection (µg mL-1) |
1.96 | 10.82 | Limit of quantitation (µg mL-1) |
1.76 | 1.81 | Tailing factor |
6868 | 4418 | Theoretical plate |
7.57 | 2.68 | Retention time (min) |
Intra (within day) and inter day (between days) precisions were tested by determining the concentration of atorvastatin calcium at lower and upper concentration levels for 5 repeated times within the same day and on five consecutive days, respectively. The results are summarized in
|
|
|
49.8 | 49.6 ± 0.085; 0.171 | 48.80 ± 0.089; 0.182 |
252 | 251.5 ± 0.090; 0.036 | 251.0 ± 0.094; 0.037 |
Mean for five independent analysis
The selectivity of the proposed method was investigated by testing the interference from common excipients such as glucose, fructose, lactose, sodium benzoate, starch, povidone, methyl cellulose and micro crystalline cellulose at 60 mg mL-1 of atorvastatin calcium. The peak area was recorded at varying concentrations of excipients and the results are summarized
Excipients | Tolerance amount (mg mL -1) |
Glucose | 3.50 |
Fructose | 3.50 |
Sodium benzoate | 1.44 |
Lactose | 5.56 |
Starch | 0. 12 |
Povidone | 0. 12 |
The ruggedness of the proposed method was examined with a small change in the optimized data in the following manner.
volume of acetonitrile-dichloromethane-acetic acid (68.6: 30.6: 0.8 v/v/v, pH 4.5) (± 0.2 mL)
column temperature, 25 ˚C (± 1.0 ˚C)
flow rate, 1 mL min-1 (± 0.2 mL min-1)
In the above conditions, tablet solution of atorvastatin calcium (Torvast) at 60.0 µg mL-1 atorvastatin calcium was analyzed by the proposed method. The results showed mean % recovery of 100.04. Hence the proposed method was rugged and considered to be reliable for determination of active drug in tablet formulations.
The accuracy of the proposed method was tested by performing recovery experiments through standard addition technique. For this purpose, known portions of pure atorvastatin calcium were spiked with definite amount of Torvast solution (unknown) and the peak area was recorded. Standard addition graph was obtained by plotting the peak area versus concentration of added standard drug solution (
The value of was found to be 1.038 µg mL-1. The confidence limit for the concentration of atorvastatin calcium in Torvast was calculated by at n - 2 degrees of freedom and found to be 59.86 ± 1.038 µg mL-1. The most attractive feature of the proposed method by standard addition technique was its relative freedom from pharmaceutical additives and excipients and hence did not interfere with the determination process.
The applicability of the proposed method for the determination of atorvastatin calcium in Torvast and Atorlip has been tested. The results of the proposed method were statistically compared with those of the reference method using point and interval hypothesis tests. t- and F-values at 95% confidence level were calculated using point hypothesis test and found to be in the range of 0.769-1.581 and 1.617-2.275, respectively. Both cases, t and F values were found to be less than the theoretical t and F values at 95% confidence level and hence proved that both methods were accurate and having no significant difference between them. The results are summarized in
|
|
|
|
|
|
|
||
|
|
|
|
|
|
|
|
|
---|---|---|---|---|---|---|---|---|
|
|
|
|
|
|
|
|
|
Torvast | 99.88 | 0.371 | 100.10 | 0.291 | 1.564 | 2.83 | 0.996 | 1.008 |
Atorlip | 99.81 | 0.311 | 100.05 | 0.235 | 1.366 | 1.75 | 0.995 | 1.007 |
Mean for 5 independent analyses.
Theoretical
A bias, based on recovery experiments, of ± 2% is acceptable
The performance of the proposed HPLC method was compared with other existing HPLC methods (
Mobile phase | Temperature (⁰C) & UV detection wavelength (nm) | Linear range (μg mL-1) | Flow rate (mL min-1) & retention time (min) | References |
Acetonitrile +dichloromethane + Acetic acid (68.6:30.6:0.8 v/v) | 25 & 246 | 15-300 | 1 & 2.68 | Proposed method |
Ammonium dihydrogen phosphate, pH 5 (acidified with acetic acid) + methanol (60:40 v/v) | 25 & 240 | 2-12 | 1 & 6.91 |
|
Potassium dihydrogen phosphate, 0.034 M (acidified with H3PO4 for pH 3.5) + Acetonitrile (30:70 v/v) | 25 & 254 | 10-50 | 1 & 4.8 |
|
Potassium dihydrogen phosphate, 0.02 M (acidified with H3PO4 for pH 3.3) + Acetonitrile (30:70 v/v) | 30 & 280 | 5-30 | 1 & 3.2 |
|
Ammonium acetate, 0.01M (pH 3) + Acetonitrile (50:50 v/v) | 25 & 254 | 4-400 | 1 & 19.3 |
|
Potassium dihydrogen phosphate, 0.01 M (acidified with H3PO4 for pH 2) + Acetonitrile (72:28 v/v) | 25 & 247 | 20 to 200 | 0.6 & 1.92 |
|
Sodium lauryl sulphate, 0.13 % (acidified with 0.06mL H3PO4) + Acetonitrile (72:28 v/v) | 55 & 210 | 20-160 | 1 & 2.15 |
|
H3PO4, 0.1 % + Acetonitrile (55:45 v/v) | 25 & 230 | 3.2-12.8 | 0.35 & 2.89 |
|
HClO4, 0.1 % (pH 2.5) + Acetonitrile (80:20 v/v) | 35 & 215 | 5-20 | 0.6 & 1.78 |
|
Triethylamine buffer, 0.1 % (acidified with H3PO4 for pH 3) + Acetonitrile (40:60 v/v) | 45 & 240 | 56-104 | 0.8 & 1.62 |
|
Ammonium acetate, 0.01M (pH 6.7) + Acetonitrile (42:58 v/v) | 40 & 245 | 50-150 | 0.2 & 0.68 |
|
Potassium dihydrogen phosphate, 0.02 M + Acetonitrile + methanol (10:40:50 v/v) | 30 & 236 | 5-60 | 1.1 & 9.1 |
|
The proposed method was successfully applied for the determination of atorvastatin calcium in pharmaceutical formulations in the presence of excipients. The separation of the drug was achieved in 2.68 min. The proposed method was more specific, selective and rapid. It followed system suitability parameters such as tailing factor of 1.81 and theoretical plate of 4418. The ease of operation, sensitivity and reproducibility of the proposed method made it more suitable for routine quality control analysis of drug in research laboratories, hospitals and pharmaceutical industry.
The authors are highly grateful to the Ministry of Manpower, Sultanate of Oman for providing necessary research facilities and to the Dean, Heads of the Applied Science Department and Chemistry Section, Higher College of Technology, Muscat for their continuous support and encouragement. The authors wish to express their gratitude to M/s National Pharmaceutical Industries Company, Oman for providing the gift sample of pure atorvastatin calcium.