analytical Method Validation of Ranolazine tablets by RP-HPLC
Ratan Bahadur Mahatara1, Sajan Maharjan2*, Roshni Thapa1, Uttam Budhathoki1
1Department of Pharmacy, School of Science, Kathmandu University, Dhulikhel, Nepal.
2Department of Pharmacy, CiST College, New Baneshwor, Kathmandu, Nepal.
*Corresponding Author E-mail: maharjansajan02@gmail.com
ABSTRACT:
Ranolazine is a novel anti-anginal drug used in the treatment of chronic stable angina in adults. The present study was designed with an objective to develop and validate a simple, fast, precise, selective and accurate Reverse phase high performance liquid chromatography (RP-HPLC) method for quantitative estimation of Ranolazine in bulk drugs and in selected dosage forms. The efficient chromatographic separation was achieved on a SunShell C18 column (150mm X 4.6mm, 2.6μm particle size) as stationary phase with a mobile phase consisting of buffer (0.01M Ammonium acetate pH adjusted to 5.0 with diluted acetic acid) and the methanol in the ratio of 40:60 v/v at a flow rate of 1.0ml/min and the detection wavelength was 273nm. The method was statistically validated for linearity, accuracy (recovery), precision, specificity, robustness, and forced degradation study. The method was successfully applied for analysis of a tablet formulation.
KEYWORDS: Ranolazine, Eco-friendly method, RP-HPLC, Analytical method validation.
INTRODUCTION:
The analytical procedure is used to measure the quality of pharmaceutical products. It is necessary to assure that the performance characteristics of the developed analytical procedure meet the requirements for the intended analytical application. The procedure which provides assurance for the same by the means of laboratory studies is defined as method validation. Method validation is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity, strength and quality, for the quantification of the drug substances and drug products.
Method validation has received considerable attention in the literature and from industrial committees and regulatory agencies. The U.S. FDA CGMP states for validation for the test methods employed by the firm1. The U.S. FDA has also proposed industry guidance for Analytical Procedures and Methods Validation2. ISO/IEC 17025 includes a chapter on the validation of methods with list validation parameters3. The ICH has developed a consensus text on the validation of analytical procedures4. ICH also developed guidance with detailed methodology. The ICH document on validation methodology recommends accuracy should be established across the specified range of the analytical procedure.
Ranolazine, a novel anti-anginal agent, has been widely used in the treatment of cardiovascular diseases, including arrhythmias, myocardial infarction. It has been reported that Ranolazine improves the myocardial oxygen balance between the supply and demand of the ischemic heart, by increase in the coronary blood flow5. The main aim of the study was to optimize the easy, accurate, precise, specific and economic RP-HPLC conditions and validate the method.
MATERIALS AND METHODS:
Working standard used:
Ranolazine manufactured by Trichem Life Science and supplied by Quest Pharmaceuticals Pvt. Ltd. was used as working standard.
Reagents and chemicals used:
Table 1: List of reagents
|
S.N |
Reagents |
Company |
Grade |
|
1 |
Methanol |
Qualigens |
HPLC |
|
2 |
Water |
Qualigens |
HPLC |
|
3 |
Ammonium Acetate |
Fischer Scientific |
Lab Grade |
|
4 |
Glacial Acetic acid |
Merck |
HPLC |
|
5 |
Formic Acid |
Merk |
HPLC |
|
6 |
Acetonitrile |
Fischer Scientific |
HPLC |
Analytical method:
Optimization:
Column chemistry, solvent selectivity (solvent type), solvent strength (volume fraction of organic solvent in the mobile phase), additive strength, detection wavelength and flow rate were varied to determine the chromatographic conditions giving the best separation. The mobile phase conditions were optimized so the peak from the first eluting compound did not interfere with those from the solvent and excipients. Other criteria, i.e. time required for analysis, appropriate k values for eluted peaks, better theoretical plates number, tailing factor (peak asymmetry), assay sensitivity and buffer solubility for solvent type were also considered. After each change of mobile phase the column was re-equilibrated by passage of at least ten column volumes of the new mobile phase.
Wavelength selection:
To investigate the appropriate wavelength, the solution containing 100g/ml of Ranolazine in 0.1M hydrochloric acid was scanned by UV-visible spectrophotometer in the range of 200-400nm and the region of maximum absorbance was taken as 273nm.
Also from the RP-HPLC, in specificity test for method validation, the blank solution, placebo solution and the standard solution in diluents were injected and the chromatogram were recorded at 215nm, 245nm and 273 nm. From the chromatograph data, suitable wavelength was selected for monitoring the drug in HPLC system.
Mobile phase selection:
Different ratios of Acetonitrile and methanol in combination with water, 0.01% Formic Acid solution and 0.01M Ammonium Acetate buffer at different pH were analyzed and chromatographic data were evaluated. Based on the system suitability results such as Theoretical plates, resolution, analysis time, tailing, were recorded and best option was selected. Similarly, other factors such as solvent toxicity, ready availability, buffer solubility capacity for solvent etc. were also taken in consideration for optimization of chromatographic conditions.
Standard Stock solution preparation:
167mg of Ranolazine working standard was accurately weighed and transferred into a 100ml volumetric flask and dissolved in methanol to volume with the diluents to produce a 1.67mg/ml of Ranolazine. The stock solution was stored in refrigerator until analysis. The stock solution was diluted to suitable concentrations for spiking placebo samples for recovery analysis.
System suitability test:
System suitability was evaluated by replicate (n=6) injections of the same standard solution containing about 200μg/ml of Ranolazine. The RSD (%) of retention time, peak area, number of theoretical plates, resolution, capacity factor and tailing factor for Ranolazine was within 2% indicating the suitability of the system. The number of theoretical plates and the tailing factor were within the acceptance criteria of N>2000 and T≤2 respectively indicating good column efficiency and optimum mobile phase composition.
Validation of the Optimized Analytical Method:
Validation is a process which involves confirmation or establishment by laboratory studies that a method/ procedure/system/analyst can give the required accuracy, precision, sensitivity, robustness, etc. Validation of the method was carried out after the development of the HPLC methods. Validation of analytical method was carried out by optimum coverage of ICH (Q2) guide line for method validation6-8.
a. System suitability test
b. Specificity test
c. Linearity
d. Range
e. Accuracy
f. Precision
g. Robustness
RESULTS AND DISCUSSION:
Optimization of chromatographic conditions:
Wavelength selection9:
When Ranolazine solution was scanned in UV-visible spectrophotometer in the range of 200-400nm, the maximum absorbance was selected at 273nm wavelength for monitoring the drugs in HPLC system which gives well resolved peak.
Also from specificity, at 240 and 215nm small impurities peak were detected at the place near where active peak was retained at time about 3.6 and 3.8 minutes, this was not observed at 273nm, so 273nm was selected for monitoring Ranolazine in HPLC system.
Figure 1: UV absorbance spectra of Ranolazine in 0.1 N HCl
Figure 2: HPLC chromatogram for placebo at 273, 240 and 215nm
Figure 3: HPLC chromatogram for Ranolazine active at 273, 240 and 215nm.
Mobile phase selection:
The chromatograms were recorded on different mobile phase and compositions. Methanol was selected for its better Capacity factor and theoretical plates than Acetonitrile and it is also relatively cheaper and nontoxic with good buffer solubility.
When Formic Acid in 0.01% v/v was used with methanol in ratio 50:50, increased tailing and low number of theoretical plates were observed. When 0.01 M Ammonium acetate buffer in different pH values (pH 3, 4 and 5) was used with methanol in about 50:50 ratios better results were obtained with 50:50 with buffer pH 5.0 (K and values), but increased retention times, similarly when ratio was changed to 35:65 the retention times were reduced but K, and N were also reduced. So pH 5.0, buffer: methanol in the ratio 40:60 was selected.
Table 2: Chromatograph summary report on C18 (150 X 4.6mm i.d, 2.6m).
|
Mobile phase |
TR |
TF |
R |
K |
|
N |
|
70% Acetonitrile |
1.83 |
1.536 |
1.61 |
0.406 |
2.85 |
829 |
|
70% Methanol |
3.48 |
1.404 |
12.01 |
1.36 |
-- |
3518 |
|
50% Acetonitrile n 0.01% FA |
1.30 |
1.895 |
-- |
-- |
2.30 |
1500 |
|
50% MeOH n 0.01% FA |
2.18 |
2.645 |
2.83 |
0.395 |
|
1500 |
|
40% Aceto n 0.01M Acetate pH 5.02 |
2.39 |
1.690 |
4.72 |
1.024 |
1.99 |
5117 |
|
65% MeOH n 0.01M Acetate pH 5.02 |
3.46 |
1.491 |
5.45 |
1.584 |
1.95 |
4871 |
|
50% MeOH n 0.01M Acetate pH 5.02 |
10.00 |
1.543 |
18.5 |
5.004 |
2.84 |
11164 |
|
55% MeOH n 0.01M Acetate pH 4.05 |
3.11 |
1.866 |
1.76 |
1.135 |
1.34 |
2047 |
|
55% MeOH n 0.01M Acetate pH 2.95 |
2.43 |
1.806 |
0.88 |
1.263 |
1.12 |
1834 |
Where; TR=Retention time, TF=Tailing factor, R=Resolution, K=Capacity factor, =separation factor/selectivity, N=Theoretical plates/meter
The results from C18 columns were found better than that with C8 column.
Optimized chromatographic conditions
|
Apparatus |
: |
UFLC XR Shimadzu |
|
Column |
: |
C18, 2.6µm particle size, Dimensions 150 x 4.6 mm (Sunshell) |
|
Flow rate |
: |
1 ml/minute |
|
Injection volume |
: |
20 µl |
|
Run time |
: |
6.0 minutes |
|
Oven temperature |
: |
350C |
|
Detector |
: |
PDA set at 273 nm |
|
Diluent |
: |
Same as mobile phase |
|
Mobile phase |
: |
Filtered and degassed mixture of buffer pH 5.0 and methanol in the ratio of 40:60 v/v respectively |
Validation of Developed Method:10-12
Estimation of the drug in dosage forms was carried out using optimized chromatographic conditions. The following parameters were considered to test for validation of the optimized method.
System suitability:
Six repeat injections of standard solution in about 194.48 g/ml concentration were injected in HPLC system and chromatograms were recorded. The %RSD of area was found to be 0.091. The %RSD of retention time was found to be 0.038. The average tailing factor was found to be 1.25. The average resolution was found to be 5.41. The average capacity factor was found to be 2.18. The average of theoretical plates were found to be 6738.8
Acceptance criteria for %RSD, tailing factor and resolution should be less than 2% and the theoretical plates should not be less than 2000.
Specificity:
Specificity of the method was evaluated by injecting diluting solution, placebo mixture prepared in diluents, placebo spiked with standard Ranolazine (from Recovery anlaysis) and degradation sample subjected to oxidation, the samples were injected into HPLC system and chromatograms were recorded. These were compared with the chromatograms obtained from standard solutions and each chromatogram was tested for any interference. The chromatography provided was free from significant interfering excipient components at the retention time of the selected drug and some additional peaks were observed in the chromatogram recorded with stressed degradation sample. These peaks, however, did not interfere with the drug peaks.
These observations show that the developed assay method was specific and selective.
Figure 4: HPLC Chromatogram of diluent.
Figure 5: HPLC Chromatogram of placebo mixture.
Figure 6: HPLC Chromatogram of Ranolazine active
Linearity:
The linearity of an 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 Ranolazine solutions ranging from the concentration of 150g/ml to 260g/ml (70 to 120 % of target concentration, 200g/ml) were injected and the responses were plotted in graph. The correlation coefficient (r) was found to be 0.99982, which indicates that the responses were linear within the intended range13.
Figure 7: Linearity curve of Ranolazine.
|
Correlation coefficient, r |
0.999910679 |
|
Slope of regression line, m |
7382.13275 |
|
Std. deviation of slope |
49.337 |
|
Determination coefficient, r2 |
0.99982137 |
|
Fisher F statistic |
2896.797 |
|
Regression SS, SSR |
66434884.570 |
|
Y- intercept |
10341.68212 |
|
Std. deviation of y-intercept |
10339.945 |
|
Std. deviation of y, Sy |
151.439 |
|
Degree of freedom |
4.000 |
|
Detection limit |
4.6222 |
|
Quantitation limit |
14.0067 |
Range:
The minimum specified ranges for the assay of a drug substance or a finished product should be considered from 80 to 120 percent of the test concentration.
Accuracy (Recovery analysis):
Placebo mixtures as prepared above were spiked with Ranolazine in known concentration in 80%, 100% and 120% of the label content equivalent is injected in duplicate and the recovery was calculated with respect to the standard.
Table 3: Observation of Recovery of Ranolazine:
|
Mean |
100.059 |
|
SD |
0.93427 |
|
%RSD |
0.93372 |
|
Variance (σ2) |
0.4396 |
|
Minimum value |
99.50 |
|
Maximum value |
101.74 |
For acceptance of the criteria, the percentage recovery of samples should be 100+2%. The above data shows the criteria can be accepted as per criteria.
Precision14:
The precision of an analytical method is defined as the closeness in agreement between independent test results obtained under stipulated conditions, and is a measure of the extent of agreement between repeated injections of a homogenous sample. The precision of a method is usually expressed as the standard deviation or as percent relative standard deviation.
Repeatability:
Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision.
a) Injection Repeatability:
Six consecutive injections were done at 100 % of the test concentration of recovery test. The % RSD of repeated injections was found to be 0.11 % which is less than 2% (acceptance level). Thus it was conformed to be repeatable.
b) Analysis Repeatability:
Nine determinations were analyzed covering the range of analytical method developed. Known amount of standard was spiked in placebo.
The % RSD for analysis repeatability of 9 different determinants was found to be 0.9337 (Limit=NMT 2%), hence the analysis was confirmed to be repeatable.
Intermediate precision:
Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc.
a) Different days:
The %RSD of Recovery analysis was determined to be 0.69064% on different days analysis for intermediate precision, which was within the acceptance region (NMT 2%)
b) Different equipments:
The %RSD of Recovery of samples in different equipments was determined to be 0.662%, and this was in the acceptance limit (NMT 2%), which demonstrate the method was precise for intended procedure.
Robustness:
To determine the robustness of the developed method, experimental conditions such as column oven temperature, flow rate of the mobile phase, pH of the buffer used, ratio of methanol and buffer volume, of buffer: methanol ratio and different lots of Column. Effect of column temperature was studied by varying ±5°C, flow rate was changed to ±3 units (0.7 and 1.3 ml/min), pH of the buffer was changed to 4.7 and 5.2 (more than ±0.2 units), buffer: methanol ratio was changed to 50:50 and 30:70 from 40:60, similarly two different C18 colums from SunShell were used to study the robustness study.
The average recovery of Ranolazine was found to be 99.16% and %RSD was 0.665 on pH variations.
The average recovery and %RSD of Ranolazine was found to be 99.26% and 0.579 respectively on mobile phase composition variation
The average recovery of Ranolazine was found to be 99.79% and RSD was found to be 0.1616% on different column (different lots/suppliers). Although the recovery was good, the peak shape was not good in reduced column length.
The average recovery and %RSD of were found to be 100.00% and 0.0217% respectively on temperature variation of column oven:
The average recovery of Ranolazine was found to be 100.09 % and %RSD was found to be 0.0292 % on flow rate variation.
CONCLUSION:
Present study was successful to develop a good alternative for estimation of Ranolazine in bulk and pharmaceutical dosage form. It was very useful, fast, precise, accurate, simple, specific, easy, and economic and eco-friendly method based on RP-HPLC using silica based C18(150mm length, 4.6mm internal diameter, 2.6 µm particle size) column as stationary phase. The method using mobile phase using 0.01M ammonium acetate buffer pH 5.0 and methanol in the ratio 40:60 was validated for parameters like Linearity, Accuracy, Precision, Range, Specificity, and Robustness etc.
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Received on 25.01.2024 Revised on 20.07.2024 Accepted on 05.11.2024 Published on 28.02.2025 Available online from March 04, 2025 Asian Journal of Pharmaceutical Analysis. 2025; 15(1):7-12 DOI: 10.52711/2231-5675.2025.00002 ©Asian Pharma Press All Right Reserved
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