RP-HPLC method development and validation for hyperlipidemic agent Atorvastatin in pharmaceutical dosage form

 

Ashish A. Gawai, Kishor Charhate, Faisal Shaikh, Amir Khan, Aniruddha Chaubey, K. R. Biyani

Anuradha College of Pharmacy, Anuradha Nagar, Chikhli, Dist-Buldana, MS-443201, India.

 

ABSTRACT:

A simple rapid, accurate and precised RP-HPLC method was developed for determination of cardiovascular agent Atorvastatin in pharmaceutical dosage form. The method was validated and demonstrated a wide Linerity Accuracy, Precision, system suitability and Robustness. The proposed method has a simple and rapid procedure. In RP-HPLC method, the analyte were resolved using Methanol: Water (90:10 %v/v) mobile phase with0.7 ml/min flow rate on HPLC pump spectra system 600E with Phenomenex C18 150 × 4.6mm 5 μm column using autochro 3000 software detector for the study. The detection was carried out at 243 nm. This method gave the good resolution and suitable retention time.

 

 

 

 

1. INTRODUCTION:

Atorvastatin is a member of the drug class known as statins used primarily as lipid lowering agent and also group under cardiovascular diseases¹. The drug is marketed under the trade name Lipitor². The pharmacological action of it is HMG-CoA reductase inhibitor as an important enzyme found in liver tissue and works in production of cholesterol in the body³.Atorvastatin has [C₃₃H₃₅FN₂O₅]2Ca.3H₂O as a molecular formula and bears 1209.4 g/mol molecular weight⁴.The drug structure is shown in Figure 1. Atorvastatin lowers plasma cholesterol and lipoprotein levels by inhibiting HMG-CoA reductase and cholesterol synthesis in the liver and by increasing the number of hepatic LDL receptors on the cell-surface to enhance uptake and catabolism of LDL⁵.

 

Atorvastatin also reduces LDL production and the number of LDL particle; dose is given asan initial dose of 10 mg administered daily with maximum of 80mg daily⁶.

 

Structure:

 

Fig 1: Structure of Atorvastatin

 

MATERIAL:

Atorvastatin API was received as a gift sample from Lupin Pharmaceutical Pvt.Ltd. Nagpur, and from the literature and market survey, we selected Zydus Medica 10mg of formulation for analytical validation work. According to the physico-chemical features, RP-HPLC was selected as analytical technique for estimation of Atorvastatin.

Instruments or Equipment:

The analysis of the drug was carried out on a Younglin (S.K) Gradient System UV detector equipped with a reverse phase (Thermo) C8 column (4.6 mm x 150 mm; 5μm) and a SP930D pump. It has 20μl injection loop and a UV730D absorbance detector and running on Autochro-3000 software.

 

EXPERIMENTAL METHODS:

Solubility Studies:

This study was carried out to find an ideal solvent in which drugs are completely soluble. Various solvents were tried for checking solubility of Atorvastatin. From solubility studies it was concluded that Atorvastatin is soluble in methanol and water. Therefore methanol and water was selected as suitable solvent for further studies⁷.

 

Standard sample preparation: Stock solution of Atorvastatin:

Standard Atorvastatin 10mg was soluble in 10ml of methanol it gives 1000 µg/ml of Atorvastatin as stock-I. Then serial dilution were done to get 5, 10, 15, 20, 25 µg/ml concentration.

 

Selection of Analytical Wavelength:

UV detector was selected, as it is reliable and easy to set at constant wavelength. A fix concentration of analyte were analysed at different wavelengths. As per the response of analyte, 246 nm wavelength was selected.

 

Optimization of HPLC method / selection of mobile phase:

The optimization of HPLC method was done for the selection of proper mobile phase for method development. Pure drug products were injected and run in different solvent systems. In this, different trials are taken with different ratio of mobile phase. For trials, methanol and water at different flow rate and pH were used. Different combinations of mobile phases were tried for selections of proper mobile phase.

 

Chromatographic conditions for HPLC method:

The chromatographic conditions were established by trial and error and were kept constant throughout method. Column was of Agilent C­₈ (250mm X 4.6mm,5µm) with particle size packing 5m and stationary phase was C₁₈ (Water), mobile phase methanol (0.05 % OPA) (70:30) (MeOH:H₂O). The detection wavelength was selected at 243 nm with flow rate 0.7 ml/min. Temperature was ambient and sample size was 20 ml/ml.

 

Method validation:

Repetability:

System suitability was performed and calculated at the start of study of each validation parameter. The values of system suitability results obtained during the entire study were recorded⁸.

 

Linearity and range:

The linearity of analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity⁹.

 

Procedure:

The aliquot portions of standard stock solutions of Atorvastatin were diluted appropriately with 1M NaOH to get a series of concentration from 5-25mg/ml for drug. The absorbance of this drug was measured at 243nm respectively and calibration curve was plotted as concentration versus absorbance. The suitable aliquots was taken to obtain 5, 10, 15, 20, 25 µg/ml from Atorvastatin (1000µg/ml) stock solution.

 

Precision:

Precision of the method was verified by repeatability and intermediate precision studies. Repeatability studies (intra-day) were performed by analysis of 10, 15, 20 µg/ml concentration of Atorvastatin on the same day. Intermediate precision (inter-day) of the method was checked by repeating analysis of 10, 15, 20 µg/ml on three different days. Measurement of peak area for active compound was expressed in terms of % relative standard deviation (% R.S.D.) The intra-day and inter-day variation for Atorvastatin was carried out at concentration level 10, 15 and 20 µg/ml¹⁰.

 

Accuracy:

The accuracy of an analytical procedure expresses closnes of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. This is sometimes termed trueness¹¹.

 

Procedure:

For 80%of solution preparation, take 0.1ml tablet solution and add 8 µg/ml atorvastatin (0.1ml) and make up volume 10 ml with mobile phase. For 100%, take 0.1 ml tablet solution and add 10 µg/ml atorvastatin (0.1 ml) and make up volume 10 ml with mobile phase. For 120 %, take 0.1 ml tablet solution and add12 µg/ml of Atorvastatin (0.1 ml) and make up volume 10mlwith mobile phase.

 

Robustness:

As recommended in the ICH Guidelines, a robustness assessment was performed during the development of the analytical procedure. In addition, the robustness of the method was investigated under a variety of conditions including changes of flow rate, wavelength, mobile phase composition and column temperature¹².

 

Limit of Detection (LOD):

Limit of detection (LOD) is the lowest amount of analyte in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions. There are various approaches for determining LOD depending on whether the procedure is non instrumental or instrumental. The detection limit may be expressed as LOD = 3.3𝛼/S. Where, 𝛼 = Averaged the standard deviation of the response, S = the slope of the calibration curve¹³.

 

Limit of Quantitation (LOQ):

Limit of Quantitation (LOQ) is the smallest amount in a sample matrix that can be quantified with acceptable accuracy and precision under the stated experimental conditions. There are various approaches for determining LOD depending on whether the procedure is non instrumental or instrumental. It is expressed as LOQ = 10𝛼/𝑆.Where, 𝛼= Averaged of the standard deviation of the response. S = the slope of the calibration curve¹⁴.

 

RESULT AND DISCUSSION:

Selection of Analytical Method:

Analysis of analyte has shown response at 243 nm wavelength in UV detector as shown in figure 2 which indicates the selection of wavelength.

 

Fig 2: UV spectrum of Atorvastatin

 

Optimization of HPLC method:

Different trials of chromatographic conditions were tested. First Grace C18 (250 ×4.6mm, 5.0μ) column was tested with methanol: water as mobile phase with 0.05 % OPA at pH 3 at 243 nm wavelength with flow rate 0.7 ml. Injection volume was 20 ul/ml but sharp peak and accurate baseline was not obtained hence it was rejected. Then slightly changed the composition of mobile phase i.e 70.30 with pH 4 and 243 nm wavelength and rest of the conditions were maintained as same that showed sharp peak of the drug so finally this mobile phase was selected and was optimized for validation. Optimization was started with standard solution using MeOH: (0.05% OPA) (90:10) pH 3, 0.7 ml/min and drug was 20 μgm. The result obtained at first trial shows Retention Time (RT) at 1.91 min with area 2255.15 mV*s with 100 % and Therapeutic Plate (TP) is 979. 4, tailing Factor (TF) 2.31 with 0.00 resolution and chromatograph was as shown in figure 3.

 

Fig.3: Chromatogram of standard solution using MeOH: (0.05%OPA) (90:10) pH 3, 0.7 ml/min with sample 20ugm

 

The result obtained at second trial shows RT at 2.33 min with area 1440.65 mV*s with 100 % and TP is 1124.6, TF 1.96 with 0.00 resolution and chromatograph was as shown in figure 4.

 

Fig.4: Chromatogram of standard solution using MeOH: water(0.05%OPA) (80:20) pH 3, 0.7 ml/min20 ugm.

 

The result obtained for third trial shows RT at 4.80 min with area 1632.27 mV*s with 100 % and TP is 2800.9, TF 1.44 with 0.00 resolution and chromatograph was as shown in figure 5.

 

Fig.5: Chromatogram of standard solution using MeOH: water (0.05%OPA) (70:30) pH 4, 0.7 ml/min20 ugm.

 

The result obtained for third trial shows RT at 4.75 min with area 494.09 mV*s with 100 % and TP is 2173.7, TF 1.54 with 0.00 resolution and chromatograph was as shown in figure 6.

 

Fig.6: Chromatogram of standard solution using MeOH: water(0.05%OPA) (70:30) pH 4, 0.7 ml/min20 ugm

 

Method validation:

Repeatability:

Asymmetry of both the analytes peak in standard found to be not more than 2.0. Theoretical plates of both the analytes peak in standard found to be less than 2000. Relative Standard deviation for five replicates injections of both the standard preparation should not be more than 2.0% it indicates the system suitability. The result shown in table 1 and chromatograph obtained in figure 7 respectively.

Table 1:Data for Repeatability

 

Fig. 7:Chromatogram of Repetability

 

Tablet solution Preparation:

20 tablets of brand ZydusMedica procured and its weight was calculated and found 3.68 gms. Average weight of powder was found to be 0.184 gms per tablet. The equivalent weight for 10 mg was calculated as 184 mg. Then take 184 mg in 10 ml of methanol to make stock solution of 1000 ug/ml. For assay Take 25 µgm/ml Atorvastatin sample (0.25 ml from tablet stock and makeup 10 ml with mobile phase). The results obtained as shown in table 2 and chromatograph in figure 8.

 

Table 2: % Label claim

 

Fig. 8: Chromatogram for Marketed Formulation

 

Linearity:

The linear calibration plots of the proposed method obtained over concentration ranges from 5-25 ug/ml. Each solution was prepared in triplicate. The correlation of coefficient for atorvastatin was 0.99 as shown in figure 9 and chromatograph obtained as shown in Figure 9.

 

 

Table 3: Result of Linearity and range for Atorvastatin

Concn µg/ml	Average area	Parameters	Atorvastatin
5	72.33	Linearity range	µg/mL
10	137.99	Correlation coefficient	0.997
15	222.88	Slope	14.25
20	290.27	Intercept	1.376
25	352.58

 

Fig. 9: Standard Calibration Curve for Atorvastatin

 

3Fig. 10:Chromatogram of Linearity

 

Precision:

Inter-day and Intra-day precision was determined for 3 different concentration on same day and for 3 consecutive days. Standard deviation was calculated to present precision as shown in table 4. The percentage of amount found indicates high precision of the method.

 

Table 4: Result of Inter-day and Intra-day precision for Atorvastatin

*Mean of each 3 reading

 

 

Fig.11: Chromatogram of inter-day precision

 

Fig. 12: Choromatogram of Intra-day precision

 

Accuracy:

Accuracy was carried out at three levels of solutions (80,100 and 120 %) of analytical concentration. Percentage recoveries along with standard deviation and

 relative standard deviation for each analyte (n=5) were given in table 5. It indicated that the method is highly accurate and suitable for intended use.

 

Table 5: Result of Recovery data for Atorvastatin

 

 

Fig. 13: Chromatogram for Accuracy (80%)

 

Fig.14: Chromatogram for Accuracy (100%)

 

Fig. 15: Chromatogram for Accuracy (120%)

 

Robustness

The degree of reproducibility of the results obtained as a result of small deliberate a variations in the method parameters has proven that the method was robust. The result has shown in table 11 and chromatograph obtained in fig from 16-22.

 

 

Table 11: Data of Robustness study

 

 

Fig. 16: Chromatograms of change in flow rate 0.6 ml/min

 

Fig.17: Chromatograms of Change in Flow Rate 0.8 ml/min

 

 

Fig.18: Chromatograms of Change in wavelength± 1 units, wavelength 242nm

 

Fig.19: Chromatograms of change in wavelength 244 nm

 

Fig 20: Change in organic composition of mobile phase ±1% (Methanol 69:Water31)

 

Fig. 21: Chromatograms of change organic composition of mobile Phase -10% Methanol

 

Fig. 22: Chromatograms of change organic composition of mobile phase(Methanol71: Water29)

 

Limit of Detection and Limit of Quantitation:

The limit of detection and Quantitation were determined by making serial dilution as per previous method and used for analysis of Atorvastatin at very low concentration. It was found to be as LOD = 3.3 x1.54/14.25 = 0.36 µg/ml and LOQ = 10 x 1.54/14.25 = 1.08 µg/ml respectively.

 

CONCLUSION:

All the data obtained after RP-HPLC validation of Atorvastatin we can conclude that the proposed method is precise and accurate. It can be used for quantitative estimation of Atorvastatin in pharmaceutical dosage form. The advantages of proposed method are simple and with wide lineartiy range over exiting other methods.

 

ACKNOWLEDGEMENT:

The corrosponding author highly acknowledge Prof. Anil Vispute sir, Reliable Analytical Laboratory, Jalgaon for their support and kind co-operation.

 

CONFLICT OF INTEREST:

Author has no conflict of Interest

 

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