INTRODUCTION:
Statins, the 3 hydroxy-3methyl glutarate coenzyme A reductase inhibitor are the most effective cholesterol lowering drug which catalyze the conversion of HMG- CoA to mevalonate, an early and rate limiting step in the biosynthesis of cholesterol.1, 2
Chemically Atorvastatin calcium is [R-(R*, R*)-2-(4-Flurophenyl)-B, B dihydroxy 5 (1-methylethyl)-3-phenyl-4 (phenylamino) Carbonyl]-1H-pyrole-1-hepatanoic acid trihydrate.3
Nicotinic acid is known as niacin or Vitamin B3 which is chemically pyridine-3- carboxylic acid official in IP (2007) which is a colorless, water soluble solid. It is useful to reduce LDL cholesterol, Very low density lipoprotein cholesterol (VLDL) and Triglyceride (TG), it is also effective for increase High density lipoprotein cholesterol (HDL)4
Stability testing forms is the important part of the process of drug product development. The main purpose of stability testing is to provide evidence that how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors like temperature, humidity, and light, and enables recommendation of storage conditions, retest periods, and shelf lives to be established. The two main aspects of drug products that play an important role in shelf life determination are assay of active drug, and degradation products generated, during the stability study. The assay of drug product in stability test sample needs to be determined using stability indicating method, as recommended by the International Conference on Harmonization (ICH) guideline5
Detailed literature survey for Atorvastatin revealed that several methods based on different technique like extractive spectrophotometry6, HPLC7-11, HPLC12-14, for its determination in human serum, capillary electrophoresis15, Derivative spectrophotometric methods16, RP- HPLC method with other combination17
Similarly survey of literature of Nicotinic acid revealed various methods based on spectrophometry18, RP-HPLC19-21, tandem mass spectrophotometry22, some method are reported for determination Atorvastatin calcium and nicotinic acid in combination by RP–HPLC23 and stability indicating RP- HPLC method24 in tablet dosage form.
MATERIAL AND METHODS:
Chemical and reagent:
Atorvastatin calcium was the generous gift from Reine lifescience, Gujarat and Nicotinic acid is purchased from S S reagents and chemicals, Hyderabad, India Combination of these drug Products of ATR and NTA (TONACT PLUS Label claim: atorvastatin calcium equivalent to atorvastatin 10mg, and nicotinic acid 375 mg by Lupin Pharmaceuticals, India) purchased from local market. Acetonitrile, Acetic acid, Methanol and Water of used were of HPLC grade. Sodium hydroxide, Hydrochloric acid and Hydrogen peroxide were from Qualigens fine Chemicals.
RP-HPLC instrumentation and chromatographic conditions:
The HPLC system consisted of Shimadzu HPLC consisted of binary pump, Rheodyne universal injector and Shimadzu UV–Visible detector. The chromatographic separations were performed using Thermo ODS C18, 5μm, 150mm X 4.6 mm i.d. column, at ambient temperature, eluted with mobile phase at the flow rate of 1ml/min. The mobile phase consisted of acetonitrile and 0.1% acetic acid (70:30, v/v), apparent pH adjusted to 4.5±0.1 with NaOH and HCL solution, Filtered through 0.45μm nylon filter and degassed in ultrasonic bath prior to use. Wavelength was selected by scanning standard solutions of both drugs over 200 to 400nm wavelengths using JASCO MODEL V-530 double beam UV–visible spectrophotometer with a pair of 10mm matched quartz cells. Measurements were made with injection volume 10μL and ultraviolet (UV) detection at 216 nm, as both components shows reasonable good response at this wavelength.
Preparation of standard solutions:
(I) Stock solution:
A standard stock solution of Atorvastatin calcium and Nicotinic acid was prepared by dissolving 375mg of Nicotinic acid and 10mg of Atorvastatin calcium in 50 ml methanol. To get final concentration of about 200 µg/ml of Atorvastatin calcium and 7500µg/ml of Nicotinic acid.
II) Preparation of working standard:
2ml of stock solution was taken and volume was taken and dissolved in mobile phase and volume was made up to 50ml mark to get final concentration of about 300 µg/ml of Nicotinic acid and 8 µg/ml of Atorvastatin calcium.
III) Sample Preparation:
To determine the content of atorvastatin calcium and nicotinic acid in pharmaceutical formulation, twenty tablets were weighed accurately, finely powdered and powder equivalent to 10mg of Atorvastatin calcium and 375mg of Nicotinic acid was weighed accurately in six replicate separately. the powder was transferred 50ml flask and containing 10ml of methanol, shake for 5 min, the solution was sonicate for 20 minute, allowed the solution to cool to room temperature and then volume made up to the mark with methanol, the resultant solution was filtered through Whatman filter paper # 41.2ml of stock solution was taken and volume was taken and dissolved in methanol and volume was made up to 50ml mark to get final concentration of about 300 µg/ml of Nicotinic acid and 8 µg/ml of Atorvastatin calcium.
Procedure For Forced Degradation Study:
Forced degradation of drug product was carried out under thermolytic, relative humidity, acid/base hydrolytic and oxidative stress conditions. Thermal and relative humidity degradation of drug product were carried out in solid state. After the degradation stock solutions were prepared by dissolving in methanol. From these solutions aliquots were diluted with mobile phase to achieve a concentration of 8μg/ ml of ATR and 300μg/ml of NTA based on the labeled strength. For thermal stress, samples of drug substances and drug product were placed in a controlled-temperature oven at 60°C for 24 hr. Acid hydrolysis of drug substance and drug product in solution state was conducted with 0.1N hydrochloric acid at reflux for 8 hr. Alkali hydrolysis of drug product was conducted by 0.1N sodium hydroxide solution at reflux for 8 hr.For oxidative stress, sample solutions of drug product in 6% hydrogen peroxide were kept at 50°C for 8 hr.
Analytical Method Validation:
Specificity:
A mobile phase was injected and chromatogram showed no interfering peaks at retention time of the two drugs. The chromatogram of AST and NA extracted from tablet dosage form were compared with those acquired from AST and NA standard, correlation was good indicates specificity of method.
Precision:
System Precision:
Was determined by five replicate injection and five time measurement of working standard of Atorvastatin calcium and Nicotinic acid and percentage relative standard deviation (%RSD) was calculated
Method Precision:
Method precision was determined with the product. An amount of the product equivalent to Atorvastatin calcium and Nicotinic acid was accurately measured and assayed (Six replicate sample was prepared and analyzed), and percentage relative standard deviation (% RSD) was calculated.
Accuracy:
A study of Accuracy was conducted. Drug Assay was performed in triplicate as per test method with equivalent amount of Atorvastatin calcium andNicotinic acid pharmaceutical dosage forminto each volumetric flask for each spike level to get the concentration of Atorvastatin calcium and Nicotinic acidequivalent to 50%, 100% and 150% of the labeled amount as per the test method. The average % recovery of Atorvastatin calcium and Nicotinic acidwas calculated.
Linearity:
Linearity for Atorvastatin calcium and Nicotinic acid was determined in the concentration range of 25-125% of working concentration that is at five concentration level, ranging from 2µg/ml-10µg/ml for Atorvastatin calcium and 75µg/ml - 375µg/ml for Nicotinic acid of each solution 10µg/ml was injected in triplicate under the operating chromatographic condition described above. The peak areas were plotted against the corresponding concentration to obtain the calibration graph.
Ruggedness:
A. System to system variability:
System to system variability study was carried out on different HPLC systems, under similar conditions at different times. Six samples were prepared and each sample was analyzed as per test method. Comparison of both the results obtained on two different HPLC systems, which shows that the assay test method are rugged for System to system variability.
Limit of Detection (LOD) and Limit of Quantification (LOQ):
The LOD and LOQ were calculated using following equation as per international conference on harmonization guideline
LOD= 3.3 x 
/s
LOQ= 10 x/s
Where is Standard deviation of the response and
S is the standard deviation of the y intercept of regression line.
RESULT AND DISCUSSION:
To develop a novel precise, accurate, rapid, specific and suitable stability indicating RP-HPLC method for the simultaneous estimation of ATR and NTA, different mobile phases were checked and proposed chromatographic condition was found appropriate for the quantitative determination in presence of degradation products. The optimum mobile phase consisted of acetonitrile and 0.1 acetic acid (70:30, v/v), apparent pH adjusted to 4.5with NaOH and HCL solution, selected because it was found to ideally resolve the peaks of ATR(tR_1.539 min) and NTA (tR_2.475 min), with clear line separation in presence of their degradation products at effluent flow rate of 1 ml/min. UV detection wavelength at 216 nm, injection volume 10μl, ambient temperature for column and HPLC system was found to best for analysis.
Validation:
Fig 1: Typical Chromatogram of std. of Atorvastatin calcium and Nicotinic acid at 245 nm
System suitability parameter:
System suitability parameter proved that the given method is suits for the Simultaneous determination of Atorvastatin and Nicotinic acid. After various trial performer it was found that chromatogram for the Atorvastatin and Nicotinic acid satisfactory by using mobile phase composition contain Acetonitrile and 0.1% Acetic acid (70:30) at pH 4.5 resolution of the given method was found satisfactory
Table 1: Study of system suitability
|
Sr. No |
Parameters |
AST |
NA |
|
1 |
Peak Area |
193376 |
2294197 |
|
|
SD |
485.688 |
10800.94 |
|
|
% RSD |
0.25 |
0.47 |
|
2 |
Theoretical plates |
28785 |
12656 |
|
3 |
Retention time (Min) |
2.477 |
1.54 |
|
4 |
Tailing Factor |
1.156 |
1.314 |
Specificity:
The method was found to e specific and no interference of chromatogram occur and correlation coefficient was good indicate the specificity of method
Precision:
The proposed method was found to be precise as indicated by percent RSD is not more than 2% as per ICH guideline for the system and method precision
Linearity:
The linear regression data state that a good linear relationship over the concentration range of 2-10 µg/ml for atorvastatin calcium and 75-375µg/ml for nicotinic acid with correlation coefficient(R2=0.9999) for Atorvastatin and correlation coefficient (R2=0.996) for the Nicotinic acid the result are shown in the figure
Fig 2: Linearity Plot (ConcentrationVsResponse) of Nicotinic Acid
Fig 3: Linearity Plot (ConcentrationVsResponse) of Atorvastatin
Table 2: Linear regression data of Atorvastatin calcium and Nicotinic acid
|
Sr. No |
Parameter |
AST |
Nicotinic Acid |
|
1 |
Detection wavelength |
254 nm |
254 nm |
|
2 |
Linearity range |
2-10 µg/ml |
75-375 µg/ml |
|
3 |
Correlation coefficient |
0.9999 |
0.996 |
|
4 |
Linear regression coefficient (Y= mx+c) |
Y= 49876x + 49442 |
Y=556345x + 503937 |
|
5 |
LOD |
0.032135 |
0.064067 |
|
6 |
LOQ |
0.097379 |
0.194141 |
|
7 |
Precision (% RSD) |
||
|
System precision |
0.25 |
0.47 |
|
|
Method precision |
1.32 |
0.8 |
Accuracy:
The proposed method when used for the determination of atorvastatin calcium and nicotinic acid from tablet dosage form after spiking with the standard affordable recovery of 102.32(50%),102.11(100%),101.31(150%) at different level for atorvastatin and 94.45(50 %),94.26 (100%), 94.14(150%) for the nicotinic acid resp.
Table 3 Recovery studies of Atorvastatin calcium
|
Excepted concentration |
|||
|
Level of recovery |
|||
|
50% |
100% |
150% |
|
|
12 ppm |
16 ppm |
20 ppm |
|
|
Peak Area |
297269 |
39498 |
4491015 |
|
Mean % recovery |
102.32 |
102.11 |
101.3 |
Table 4 Recovery studies of Nicotinic acid
|
|
Excepted concentration |
|
|
|
|
Level of recovery |
|
|
|
|
50% |
100% |
150% |
|
|
450 ppm |
600 ppm |
750 ppm |
|
Peak Area |
3237 404 |
25745 |
408015 |
|
Mean % recovery |
94.45 |
94.26 |
94.14 |
Limit of Detection and Limit of Quantitation:
The limit of detection` was found to be 0.032135 and 0.064067 for Atorvastatin calcium and Nicotinic acid resp. the Limit of quantitation was found to be 0.097379 and 0.194141 for Atorvastatin calcium and Nicotinic acid respectively.
Table 5: LOD and LOQ of Atorvastatin Calcium and Nicotinic acid
|
Parameter |
Atorvastatin Calcium |
Nicotinic acid |
|
LOD(µg/ml) |
0.032135 |
0.064067 |
|
LOQ(µg/ml) |
0.097379 |
0.194141 |
Forced degradation study:
Acid degradation study:
Fig. 4: Degradation study of Atorvastatin calcium and Nicotinic acid in 0.1N HCL
Oxidative degradation:
Fig 5: Degradation study of Atorvastatin calcium and Nicotinic acid in 30% Hydrogen Peroxide.
CONCLUSION:
The proposed HPLC method is simple and total run time for two component is less than 5min the method is accurate and precise proven by the recovery study. It can be concluded that the given Stability indicating HPLC has great important in Simultaneous determination of two component.
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Received on 25.07.2019 Modified on 20.08.2019
Accepted on 21.09.2019 © RJPT All right reserved
Research J. Pharm. and Tech 2020; 13(2):604-608.
DOI: 10.5958/0974-360X.2020.00114.6