Aswini Kumar Parida1*, K. Srinivasa Rao 2, Ajay Kumar Patnaik3
1Ph.D Research Scholar, SPER, Berhampur University, Berhampur-760007, Odisha
2Department of Pharmacy, Indira Gandhi National Tribal University, Amarkantak-484887,
Madhya Pradesh, India.
3Department of Chemistry, Ravenshaw University, Cuttack-753003, Odisha, India.
*Corresponding Author E-mail: aswini.parida18@gmail.com
ABSTRACT:
A reverse phase high performance liquid chromatographic (RP-HPLC) method was developed and validated for the estimation of Ticagrelor in pharmaceutical dosage forms. The chromatographic separation of Ticagrelor was achieved on a Symmetry C18 column (250mm×4.6 mm, 5µm particle size), Agilent LC1220 HPLC system with UV detection (VWD detector) at 256nm. The optimized mobile phase was consisted of Methanol: Phosphate buffer (PH adjusted to 4 with orthophosphoric acid) (75:25 v/v). The flow rate was 1ml/min and effluents were monitored at 256nm. Chromatogram showed the main peak at a retention time of 2.750min. The method was validated for linearity, accuracy, precision, and limit of detection, limit of quantitation, robustness and ruggedness. The linearity was found in the concentration range of 5-50µg/ml. The Correlation coefficient was 0.999. The regression equation was found to be Y = 10290x+3252. The limit of detection and limit of quantitation for estimation of Ticagrelor was found to be 0.4 µg / ml and 1.28 µg / ml respectively. Recovery of Ticagrelor was found to be in the range of 99.57-99.97%. Proposed method was successfully applied for the quantitative determination of Ticagrelor in pharmaceutical dosage forms as per ICH guidelines.
KEYWORDS: Ticagrelor, RP-HPLC, VWD detector, ICH guidelines.
INTRODUCTION:
Ticagrelor is an orally administered platelet aggregation inhibitor used in the treatment of patients with acute coronary syndrome and myocardial infarction. It is an antagonist of the P2Y12 receptor [1]. Ticagrelor is chemically known as (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-Difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl]-5-(2 -hydroxyethoxy)cyclopentane-1,2-diol was shown in Figure 1. Literature review tells that very few analytical methods have been reported for the determination of Ticagrelor which includes UV-Spectrophotometry [2], High performance liquid chromatography [3-9] and Liquid chromatography-Mass spectroscopy method [10-11].
The present study was aimed to develop a novel, simple, economic and validated RP-HPLC method for the estimation of Ticagrelor according to ICH guidelines [12].
Figure 1: Chemical structure of Ticagrelor
MATERIALS AND METHODS:
Chemicals and Reagents:
Ticagrelor bulk drug were kindly provided as gift sample by Laurus Lab, Hyderabad, India. Potassium dihydrogen phosphate (Merck Chemical Company, GR-Grade), Orthophosphoric acid (Merck Chemical Company, GR-Grade), Water (Merck Chemical Company, HPLC-Grade) and Methanol (Merck Chemical Company, HPLC-Grade) were used in the study. Brilinta® tablet contain Ticagrelor 90mg is obtained from a local pharmacy manufactured by Astrazeneca, India.
Instrumentation:
The chromatography was performed on Agilent LC1220 HPLC system, equipped with VWD detector and EZ Chrome software, Symmetry C18 column (250mm×4.6 mm, 5µm particle size) was used as stationary phase. All weights were taken on electronic balance (Model: CA123, Make: Contech), pH Meter (Model: 3 Star, Make: Global) and Sonicator (Model: UCB 70, Make: Life care) were used in the study.
Chromatographic conditions:
In this work a reverse phase Symmetry C18 column with 250 × 4.6 mm i.d. and 5 µm particle size was chosen as stationary phase and mobile phase consisting of mixture of Methanol: Phosphate buffer (PH adjusted to 4 with orthophosphoric acid) (75:25 v/v) was delivered at a flow rate of 1.0ml/min and detector wavelength at 256 nm. Injection volume was 20μl. The run time was 5min and the retention time of Ticagrelor was found to be 2.750min.
Chromatographic Parameters:
|
Equipment: |
Agilent LC1220 HPLC system, equipped with VWD detector |
|
Column: |
Symmetry C18 (250mm×4.6 mm, 5mm particle size) |
|
Flow rate: |
1ml/min |
|
Wavelength: |
256 nm |
|
Injection volume: |
20 ml |
|
Column oven: |
Ambient |
|
Run time: |
5 Minutes |
Preparation of mobile phase:
Solution A: Accurately weighed about 1.368g of Potassium di-hydrogen phosphate (KH2PO4) was taken into 1000ml beaker and dissolved to 1000ml with HPLC grade water and degassed in ultrasonic water bath and filtered through 0.45µm filter using vacuum filtration and the pH was adjusted to 4 with orthophosphoric acid.
Solution B: Methanol HPLC-Grade
Mobile Phase:
Volume of Solution (A) and solution (B) taken in ratio 25:75 (v/v) and mixed well and filtered through 0.45µm membrane filter and degas for 10 minutes.
Preparation of diluent:
Mobile phase was used as diluent.
Preparation of Standard Stock Solution:
An accurately weighed quantity of Ticagrelor 100mg was transferred to 100ml volumetric flask, dissolved in 100ml distilled water, the final volume was made with distilled water to obtain standard solution having concentration of 1000μg/ml. These stock solutions were used to prepare further dilutions.
Preparation of Sample Solution:
Ticagrelor is available as tablets containing 100mg of Ticagrelor. Ticagrelor is available in the local market with brand names BRILINTA® (90mg, Astrazeneca, India). Twenty tablets of Ticagrelor were taken and made into a fine powder of the tablets and the powder equivalent to 100mg of Ticagrelor was weighed accurately and transferred into a 100ml standard volumetric flask. The contents were dissolved in mobile phase and sonicated for 30 Minutes. This entire solution was filtered through 0.45 micron Whatmann filter paper (No. 41) and the final solution was made with mobile phase to get the solution of 1000μg/ml. 10ml of this solution was transferred to 100ml volumetric flask, volume was made with methanol. It gives 100 µg/ml. 2ml of the solution were pipetted out separately into 10ml volumetric flask and make up to the mark to give 20μg/ml concentration. The sample solution 20µl was injected and chromatographed and the peak areas were measured for Ticagrelor was shown in Figure 2 and 3 respectively. The % Assay was calculated by comparing the peak area of standard and sample chromatogram by using the formula given below and the assay result was shown in Table 1.
|
Assay% |
AT |
× |
WS |
× |
DT |
× |
P |
× |
Avg. Wt |
×100 |
|
AS |
DS |
WT |
100 |
LabelClaim |
Where:
AT = Average peak area of sample preparation
AS= Average peak area of standard preparation
WS = Weight of standard taken in mg
WT=Weight of sample taken in mg
P = Percentage purity of working standard
DS= Dilution factor for standard preparation
DT=Dilution factor for sample preparation
Optimization of RP-HPLC method:
For the method optimization, different mobile phases were tried, but acceptable retention times, theoretical plates and good resolution were observed with Methanol: Phosphate buffer (75:25 v/v), pH was adjusted to 4 with orthophosphoric acid using Symmetry C18 (250mm×4.6 mm, 5mm particle size).
System Suitability:
At first the HPLC system was optimized as per the chromatographic conditions. One blank followed by six replicates of a single calibration standard solution of 20µg/ml of Ticagrelor was injected to check the system suitability. To ascertain the system suitability for the proposed method, the parameters such as retention time, theoretical plates and peak asymmetry were taken and results were presented in Table 2.
Figure 3: Sample chromatogram of Ticagrelor
Figure 5: Chromatogram of Placebo
Table 1: Assay of marketed formulation of Ticagrelor
|
Drug |
Brilinta® Label Claim (mg) |
Amount Found (mg) |
% Label Claim ± % RSD (n=3) |
|
Ticagrelor |
90 |
91.1 |
101.2±0.43 |
Table 2: System suitability test parameters for Ticagrelor
|
Parameter (n=6) |
Ticagrelor |
|
Retention Time (Mins) |
2.750 |
|
Theoretical plates |
2788 |
|
Tailing factor |
1.1 |
Specificity:
The effect of excipients and other additives usually present in the dosage form of Ticagrelor in the determination under optimum conditions was investigated. The specificity of the RP-HPLC method was established by injecting the blank and placebo solution into the HPLC system. The representative chromatogram of blank and placebo was shown in Figure 4 and 5 respectively.
Linearity:
Linearity was performed by taking from stock solution (100µg/ml) aliquots of 0.5, 1, 2, 3, 4, 5 ml were taken in 10ml volumetric flasks and diluted up to the mark with diluent such that the final concentrations are in the range of 5-50 µg/ml. Each of these drug solutions (20μl) was injected into the chromatographic system for three times. The peak area and retention time were recorded and the mean values of peak areas were plotted against concentrations. The linearity data is presented in Figure 6 and Table 3. Acceptance Criteria: Correlation coefficient should be not less than 0.999
Figure 6: Linearity graph of Ticagrelor
Table 3: Linearity data for Ticagrelor
|
Linearity of Ticagrelor |
|
|
Concentration (µg/ml) |
Peak Area |
|
5 |
59987 |
|
10 |
105331 |
|
20 |
210114 |
|
30 |
308967 |
|
40 |
413465 |
|
50 |
519895 |
Accuracy studies:
The accuracy of the method was determined by calculating recovery of Ticagrelor by the method of standard addition. Known amount of standard solution of Ticagrelor at 50%, 100% and 150% was added to a pre quantified sample solution and injected into the HPLC system. The mean percentage recovery of Ticagrelor at each level was calculated and the results were presented in Table 4. Acceptance Criteria: The % Recovery for each level should be between 98.0 to 102.0%.
Table 4: Recovery study data of Ticagrelor
|
Sample name |
Amount added (µg/ml) |
Amount found (µg/ml) |
% Recovery |
Statistical Analysis |
|
S1:50% |
10 |
10.05 |
100.5 |
Mean-99.97 S.D-0.76 %RSD-0.76 |
|
S2:50% |
10 |
10.03 |
100.3 |
|
|
S3:50% |
10 |
9.91 |
99.1 |
|
|
S4:100% |
20 |
19.87 |
99.35 |
Mean-99.78 S.D-0.4 %RSD=0.41 |
|
S5:100% |
20 |
19.97 |
99.85 |
|
|
S6:100% |
20 |
20.03 |
100.15 |
|
|
S |
30 |
29.81 |
99.37 |
Mean-99.57 S.D-0.41 %RSD-0.41 |
|
S8:150% |
30 |
29.79 |
99.3 |
|
|
S9 :150% |
30 |
30.01 |
100.03 |
Table 5: Method precision data for Ticagrelor
|
Ticagrelor |
||
|
S. No. |
Concentration (μg/ml) |
% Assay |
|
1 |
20 |
101.2 |
|
2 |
20 |
101.8 |
|
3 |
20 |
101.1 |
|
4 |
20 |
100.2 |
|
5 |
20 |
99.5 |
|
6 |
20 |
101.6 |
|
Average |
100.9 |
|
|
SD |
0.88 |
|
|
%RSD |
0.87 |
|
TABLE 6: System precision data for Ticagrelor
|
Ticagrelor |
||
|
S.No. |
Concentration (μg/ml) |
Peak Area |
|
1 |
20 |
214125 |
|
2 |
20 |
213531 |
|
3 |
20 |
216543 |
|
4 |
20 |
215876 |
|
5 |
20 |
214517 |
|
6 |
20 |
217651 |
|
Average |
215373.8 |
|
|
SD |
1580.78 |
|
|
%RSD |
0.73 |
|
Precision studies for Ticagrelor:
Method precision (Repeatability):
Twenty tablets were accurately weighed and tablet powder equivalent to 100mg of Ticagrelor were taken into 100ml clean dry volumetric flask, diluent was added and sonicated to dissolve it completely and volume was made up to the mark with the same diluent and filtered through 0.45 μm nylon membrane filter. Further pipette out 0.2ml from the above Ticagrelor sample stock solution into a 10ml volumetric flask and diluted up to the mark with diluent to get the concentration of 20µg/ml of Ticagrelor. A homogenous sample of a single batch analysed six times and was checked whether the method is giving consistent results. The %RSD for the area of six replicate injections was calculated as mentioned in Table 5. Acceptance Criteria: The % RSD for the peak area of six sample injections should not be more than 2%.
TABLE 7: Ruggedness data for Ticagrelor
|
Ruggedness Data for Ticagrelor |
|||||||||||
|
Laboratory-1 (% Assay)-HPLC-1 |
Laboratory-2 (% Assay)-HPLC-2 |
||||||||||
|
|
Analyst-1 |
Analyst-2 |
Analyst-1 |
Analyst-2 |
|||||||
|
Conc. (μg/ml) |
Day-1 |
Day-2 |
Day-1 |
Day-2 |
Day-1 |
Day-2 |
Day-1 |
Day-2 |
|||
|
20 |
100.8 |
99.6 |
100.6 |
101.2 |
99.1 |
100.1 |
100.8 |
101.4 |
|||
|
20 |
100 |
100.2 |
101.4 |
100.6 |
99.9 |
99.7 |
101.2 |
101.2 |
|||
|
20 |
100.2 |
100.9 |
99.7 |
99.6 |
100.6 |
100.4 |
99.5 |
100.2 |
|||
|
20 |
100.1 |
101.2 |
100.4 |
100.2 |
100.3 |
100.1 |
100.6 |
99.8 |
|||
|
20 |
99.8 |
100.6 |
101.1 |
100.8 |
100 |
100.6 |
99.8 |
100.5 |
|||
|
20 |
100.4 |
99.8 |
100.1 |
100.4 |
99.4 |
100 |
101.6 |
101.3 |
|||
|
Average |
100.21 |
100.3 |
100.55 |
100.40 |
99.8 |
100.15 |
100.58 |
100.73 |
|||
|
SD |
0.889 |
0.627 |
0.628 |
0.715 |
0.556 |
0.3146 |
0.8060 |
0.6623 |
|||
|
%RSD |
0.847 |
0.625 |
0.628 |
0.795 |
0.5571 |
0.3147 |
0.80134 |
0.6574 |
|||
|
Intermediate precision within-laboratories variations (n=24) |
|||||||||||
|
Laboratory-1 (% Assay)-HPLC-1 |
Laboratory-2 (% Assay)-HPLC-2 |
||||||||||
|
Average |
100.35 |
Average |
100.42 |
||||||||
|
SD |
0.714 |
SD |
0.584 |
||||||||
|
%RSD |
0.72 |
%RSD |
0.58 |
||||||||
|
Reproducibility between laboratories (n=48) (% Assay) |
|||||||||||
|
Average |
100.30 |
||||||||||
|
SD |
0.443 |
||||||||||
|
%RSD |
0.44 |
||||||||||
Table 8: Summary of validation parameter for Ticagrelor
|
Parameters |
RP-HPLC method |
|
|
Ticagrelor |
||
|
Linearity range (µg/ml) |
5-50 |
|
|
Slope |
10290 |
|
|
Intercept |
3252 |
|
|
Correlation coefficient |
0.999 |
|
|
LOD (µg/ml) |
0.4 |
|
|
LOQ (µg/ml) |
1.28 |
|
|
Method Precision (% RSD, n=6) |
0.87 |
|
|
System precision (% RSD, n=6) |
0.73 |
|
|
Ruggedness (% RSD, n=24) |
Lab-1 |
Lab-2 |
|
0.72 |
0.58 |
|
|
Reproducibility (% RSD, n=48) |
0.44 |
|
|
% Accuracy |
99.57-99.97 |
|
|
Robustness (% RSD, n=6) |
Less Flow rate |
More Flow rate |
|
|
0.05 |
0.14 |
|
Less Organic phase |
More Organic phase |
|
|
0.02 |
0.02 |
|
Table 9: Summary of Robustness (Change in Flow Rate and mobile phase) for Ticagrelor
|
Parameters |
Mean peak Area(n=3) |
S.D |
%R.S.D |
RT |
Theoretical plates |
|
Flow rate 0.9ml/min |
305352 |
152.7525 |
0.05 |
3.157 |
2421 |
|
Actual flow rate 1ml/min |
296264 |
3487.6 |
1.2 |
2.665 |
2831 |
|
Flow rate 1.1ml/min |
287498 |
404.1452 |
0.14 |
2.610 |
2961 |
|
10% less organic(67:33) |
316111 |
57.73503 |
0.02 |
2.750 |
2521 |
|
Actual mobile phase(75:25) |
269264 |
3487.6 |
1.2 |
2.665 |
2831 |
|
10% more organic (83:17) |
282894 |
57.735 |
0.02 |
2.683 |
2573 |
System precision:
The system precision was carried out to ensure that the analytical system is working properly. The standard preparation concentration of 20µg/ml of Ticagrelor was injected six times into the HPLC and the %RSD for the area of six replicate injections was calculated as mentioned in Table 6. Acceptance Criteria: The % RSD for the peak area of six standard injections should not be more than 2%.
Intermediate precision/ruggedness:
The intermediate precision (also known as Ruggedness) of the method was evaluated by performing precision on different laboratory by different analyst and different days. The sample preparation concentration of 20µg/ml of Ticagrelor was injected six times into the HPLC and the %RSD for the area of six replicate injections was calculated as mentioned in Table 7. Acceptance Criteria: The % RSD for the peak area of six standard injections should not be more than 2%.
Limit of Detection (LOD) and Limit of Quantification (LOQ):
Limit of Detection (LOD) and Limit of Quantification (LOQ) were calculated as 3.3×SD/S and 10×SD/S respectively as per ICH guidelines, Where SD is the standard deviation of the response (Y-intercept) and S is the slope of the calibration curve. The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3). The LOD of Ticagrelor was calculated and shown in Table 8. The LOQ is the smallest concentration of the analyte which gives response that can be accurately quantified (signal to noise ratio of 10). The LOQ of Ticagrelor was calculated and shown in Table 8.
Robustness:
As part of the Robustness, deliberate change in the flow rate and mobile phase proportion of ±10% was made to evaluate the impact on the method. The results reveal that the method is robust. The results are summarized in Table 9.
RESULTS AND DISCUSSION:
To optimize the RP-HPLC parameters, several mobile phase compositions were tried. A satisfactory separation and good peak symmetry for Ticagrelor were obtained with a mobile phase containing a mixture of Phosphate buffer (pH adjusted to 4 with orthophosphoric acid) and Methanol (25:75, v/v) was delivered at a flow rate of 1ml/min to get better reproducibility and repeatability. Quantification was achieved with VWD detection at 256nm based on peak area. The retention time of Ticagrelor was found to be 2.750min. Linearity was established for Ticagrelor in the range of 5-50µg/ml with correlation coefficient 0.999 and mean accuracies were found to be is 99.57% to 99.97% for Ticagrelor, which indicates accuracy of the proposed method. The % RSD values of accuracy for Ticagrelor were found to be < 2 %. The % RSD value of method precision was 0.87% for Ticagrelor and % RSD value of system precision was 0.73% for Ticagrelor. The % RSD value of reproducibility is 0.44% for Ticagrelor reveal that the proposed method is precise. LOD value for Ticagrelor was found to be 0.4µg/ml and LOQ value for Ticagrelor were found to be 1.28µg/ml. The % RSD values of robustness studies were found to be < 2% reveal that the method is robust enough. These data show that the proposed method is specific and sensitive for the determination of Ticagrelor.
CONCLUSION:
RP-HPLC method for the estimation of Ticagrelor in their bulk and pharmaceutical dosage form was established and validated as per the ICH guidelines. Linearity was achieved for Ticagrelor in the range of 5-50µg/ml with correlation coefficient 0.999. The percentage recovery of drug was achieved in the range of 98-102% which was within the acceptance criteria. The percentage RSD was NMT 2 % which proved the precision of the developed method. The developed method is simple, sensitive, rapid, linear, precise, rugged, accurate, specific, and robust. Hence it can be used for the routine analysis of Ticagrelor in their bulk and pharmaceutical dosage form.
ACKNOWLEDGEMENT:
The authors are thankful to Laurus Lab, Hyderabad for providing the samples for research.
REFERENCES:
1. Jacobson KA., Boeynaems JM. P2Y nucleotide receptors: promise of therapeutic applications. Drug Discovery Today. 2010; 15: 570–578.
2. Ambasana MA, Kapuriya NP, Faldu NJ, Ladva KD. Development and validation of a UV-spectrophotometric method for the determination of Ticagrelor in bulk form. Der Pharma Chemica. 2014; 6: 237.
3. Uttam Prasad P, Reddy ASK. A novel validated RP-HPLC-DAD method for the estimation of Eluxadoline in bulk and pharmaceutical dosage form. Research J. Pharm. and Tech. 2015; 8: 1469-1476.
4. Kalyani L, Lakshmana Rao A. Validated stability-indicating HPLC method for determination of Ticagrelor in bulk and its formulation. International journal of pharmacy. 2013; 3:634-642.
5. Caren G, Rubia LP, Andreas Sebastian LM, Cássia Virginia G. Determination of the new antiplatelet agent Ticagrelor in tablets by stability-indicating HPLC Method. Current Pharmaceutical Analysis. 2015; 10: 279-283.
6. Eena J, Anu B, Delma D’cruz, Aneesh TP. Development and validation of RP- HPLC method for determination of Ticagrelor in pharmaceutical dosage formulation. Der Pharmacia Lettre. 2016; 8:206-212.
7. Kulkarni PR, Gajare GK. Development and validation of RP-HPLC method for estimation of Ticagrelor in bulk form. International Journal of Research in Pharmacy and Chemistry. 2016; 6: 733-737.
8. Rahul PC, Rajendra BK. Development and Validation of RP-HPLC Method for the Quantitation of Ticagrelor Using Box-Behnken Experimental Design. Am. J. PharmTech Res. 2016; 6: 388-402.
9. Kapil V, Rambabu K, Prasad K, Nagaraju D, Rajashekar N, Tirumaleswara Rao B, Challa S. Analytical method development and validation for the estimation of Ticagrelor in drug substance by RP-HPLC method. European Journal of Biomedical and Pharmaceutical Sciences. 2017;4: 268-272.
10. Sillen H, Cook M, Davis P. Determination of Ticagrelor and two metabolites in plasma samples by liquid chromatography and mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2010; 878: 2299-2306.
11. Prashant K, Yadavendra A, Gunjan S, Pravin P. Simultaneous quantification of ticagrelor and its metabolite deshydroxyethoxy Ticagrelor in human plasma by ultra-performance liquid chromatography electrospray ionization-tandem mass spectrometry. World J Pharm Sci. 2015; 3: 37-45.
12. Shabir GA. Validation of high-performance liquid chromatography methods for pharmaceutical analysis. Understanding the differences and similarities between validation requirements of the US Food and Drug Administration, the US Pharmacopeia and the International Conference on Harmonization. J Chromatogr A. 2003; 987:57-66.
Received on 29.10.2017 Modified on 24.11.2017
Accepted on 06.12.2017 © RJPT All right reserved
Research J. Pharm. and Tech. 2018; 11(3): 867-872.
DOI: 10.5958/0974-360X.2018.00161.0