International Journal of Innovative Pharmaceutical Sciences and Research

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1 International Journal of Innovative Pharmaceutical Sciences and Research A NEW RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF TICAGRELOR IN BULK AND FORMULATION AND ITS EXTENSION TO DISSOLUTION STUDIES Khatija Mohammed Bhameshan, S.H.Rizwan*, Arshiya Sultana, Mehnaaz Department of Pharmaceutical Analysis, Deccan School of Pharmacy, Hyderabad , Telangana, INDIA Abstract A simple, selective and reproducible Reversed-Phase High Performance Liquid Chromatographic (RP-HPLC) method has been developed and validated for the estimation of Ticagrelor in Bulk and tablet dosage form, the proposed method was later extended to develop a dissolution method for the analysis of Ticagrelor in immediate release tablets. Analysis was performed on an Agilent 1220 HPLC system using a Qualisil BDS C18 column (250mm 4.6 mm, 5 μm particle size) with the mobile phase consisting of Acetonitrile and Water in the ratio (80: 20 v/v) at a flow rate of 1 ml/min. UV detection of the analyte was performed at 254nm. The retention time for Ticagrelor for routine analysis and in dissolution study was found to be 4.30 minutes. The developed method obeyed the basic system suitability parameters i.e. number of theoretical plates, tailing factor and repeatability. The analytical procedure was validated for specificity, linearity, precision, accuracy, ruggedness, robustness, LOD and LOQ.The calibration curve was linear showing a correlation coefficient of Linearity was in the range of 20 µg/ml to 60 µg/ml. Accuracy of the method was in the range of to %. Precision was found to be 1.74%. The method showed good results when examined for both robustness and ruggedness. Later, the analytical procedure was successfully applied to dissolution studies using the USP Type 2 paddle apparatus with water as dissolution medium. Hence, the proposed research work was found to be a new and sensitive RP-HPLC analytical method for the determination and dissolution studies of the drug both in bulk and formulation dosage form. Keywords: Ticagrelor, RP-HPLC, Dissolution. Corresponding Author: S.H.Rizwan Associate Professor, Department of Pharmaceutical Analysis, Deccan School of Pharmacy, Dar-Us-Salam, Aghapura, Hyderabad , Telangana, INDIA visitrizwan@rediffmail.com Phone: Available online: September Issue 43

2 INTRODUCTION Ticagrelor is a platelet aggregation inhibitor produced by AstraZeneca under the trade name Brilinta in US and in EU as Brilique and Possia.It belongs to the class of Cytochrome P-450 CYP2C9 Inhibitors (moderate) chemically it s a(1s,2s,3r,5s)-3-(7-{[(1r,2s)-2- (3,4difluorophenyl)cyclopropyl]amino}-5-(propylsulfanyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin 3- yl)-5-(2-hydroxyethoxy)cyclopentane-1,2-diol [1].Ticagrelor blocks adenosine diphosphate (ADP) receptors of subtype P2Y 12. Unlike to the other antiplatelet drugs, the binding site of Ticagrelor is different from ADP, making it an allosteric antagonist, with reversible blockage.hepatic activation is not needed for Ticagrelor,which might work better for patients with genetic variants regarding the enzyme CYP2C19 [2] Fig. 1: Structure of Ticagrelor Literature survey has revealed many analytical methods for the quantitation of Ticagrelor in tablets using HPLC [3-8]. The objective of the present study was to develop a simple, less time consuming and, economical analytical method for estimation of Ticagrelor in bulk and its formulation. The proposed method was validated as per ICH guidelines [9-11] and was extended to dissolution studies. This paper described a new RP-HPLC method for the estimation of Ticagrelor in dissolution samples using simple mobile phase. MATERIALS AND METHODS Chemicals and reagents: Ticagrelor reference substance was obtained as a gift sample from Chandra labs, hyd. HPLC grade water and solvents were used for HPLC analysis. All the buffer solutions were prepared according to the procedure given in pharmaceutical guidelines [12]. Instrumentations: Agilent 1220 series integrated High Performance Liquid Chromatographic system equipped with quaternary pump, manual sampler, variable wavelength detector and column thermostat was used Available online: September Issue 44

3 for HPLC analysis. Qualisil BDS C18 column (250mm 4.6 mm, 5 μm particle size) analytical column was used as a stationary phase. Chromatographic data was acquired using EZ Chrome Software UV /Visible detector SPD 20A. All dissolution experiments were carried out using a dissolution instrument USP Dissolution apparatus type II (Labindia, Model: TDT-08L) METHOD DEVELOPMENT Determination of working wavelength: Accurately weighed 10mg of drug was transfer to 100ml clean dry volumetric flask the contents were dissolved using methanol and sonicated for 15 mins, later the volume was made up using methanol.further 1ml from above stock solution was pipette out into a 10 ml volumetric flask and diluted up to the mark with methanol (10 µg/ml). In setting up the conditions for development of dissolution method, the choice of detection wavelength was based on the scanned absorption spectrum for the UV-Spectrum of Ticagrelor, which was obtained by scanning the sample over the wave length range nm against blank as diluent. After thorough examination of the spectra, the wave length 254 nm was selected for further analysis. Fig. 1.1:UV-Spectrum of Standard 40µg/ml Preparation of mobile phase A mixture of Acetonitrile and water in the ratio of 80:20 was prepared than sonicated for 10 mins to remove gases. Preparation of Standard and Working Standard Solution 10mg of Ticagrelor was accurately weighed and taken in 10ml clean and dry volumetric flask containing diluent and then the solution was made up to the mark using diluent. This is Available online: September Issue 45

4 considered as standard stock solution A(400 g/ml).from stock solution A, 0.4ml was pipette out and made up to 10ml to get a concentration of 40µg/ml, treated as working standard, 100% target concentration for which RP-HPLC chromatogram was recorded.five working standard solutions were prepared for calibration by adding defined volumes of the standard stock solution and diluting with mobile phase to get concentration in the range of 20-60µg/ml. respectively and were used for recording chromatograms. Tablet sample preparation 20 tablets (each containing 90 mg) were taken in mortar and crushed to fine powder and uniformly mixed. Tablet stock solution of Ticagrelor was prepared by dissolving weight equivalent to 10 mg of Ticagrelor in 10ml mobile phase. The solution was sonicated for 5 mins,from the above 0.4ml was pipetted out and made up to 10ml using mobile phase to get a concentration of 40µg/ml. METHOD VALIDATION Table 1: Optimized chromatographic conditions Agilent series HPLC Instrument EZ Chrome Software UV /Visible detector SPD 20A UV-Visible Spectrophotometer Thermo nicolet evolution-100 Column Qualisil BDS C18 column (250mm 4.6 mm, 5 μm particle size) Mobile phase Acetonitrile and water 80:20 Flow rate 1ml/min Detection wavelength 254nm Run time 7mins Retention time 4.30 min The HPLC parameters were optimized based on trial and error method.the developed method was validated for precision, linearity, specificity, accuracy,limit of detection, limit of quantitation, robustness and ruggedness according to US-FDA and ICH (International Conference on Harmonization) guideline [13,14]. System suitability: System suitability was determined from six replicate injections of the standard solution before the analysis and the chromatograms were recorded. Set up chromatographic system, Allow the HPLC system to stabilize for 40 mins,inject blank preparation (single injecton) and standard preparation (six replicates),relative standard deviation (RSD) of peak area for six replicates of standard was calculated. System suitability parameters like symmetry, theoretical plate, and tailing factor were also recorded. The system suitability data is reported in table no 2. Available online: September Issue 46

5 Table 2: System Suitability Studies SST studies Parameters S.No Drug Retention time Peak area Theoretical plates Tailing factor 1 Ticagrelor *=average of 6 readings Specificity: Specificity is the ability to assess accurately the analyte in the presence of components which may be expected to be present in the sample matrix such as impurities or excipients. In practice,this can be done by spiking the drug substance or product with appropriate levels of impurities or excipients and demonstrating that the assay results are unaffected by the presence of these extraneous materials.there should not be any interference of the diluents or placebo at retention time of drug substances. Fig. 1.2: Chromatogram of Blank Fig.1.3: Chromatogram of standard Fig. 1.4: Chromatogram of sample Available online: September Issue 47

6 Table 3: Specificity results for standard and sample Observation: It is observed from the above data, diluents or excipient peaks are not interfering with the Ticagrelor peak. Linearity: Linearity was studied by analyzing five standard solutions covering the range of 20-60µg/ml.From the primary stock solution 0.2ml,0.3ml,0.4ml,0.5ml and 0.6ml of aliquots were pipette out into 10ml volumetric flasks and made up to the mark with the mobile phase to give concentration of 20(µg/ml),30(µg/ml),40(µg/ml),50(µg/ml) and 60(µg/ml)respectively. Calibration curve showing concentration versus peak area was plotted by injecting the above prepared solutions and the obtained data was subjected to regression analysis using the least square method.the calibration data is presented in table no4 and calibration curve is shown in fig 1.5. Table 4: Calibration data for Ticagrelor Actual concentration (µg/ml) Observation Peak area 20(µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) Fig. 1.5: Calibration of Ticagrelor The correlation coefficient for linear curve obtained between concentration vs. Area was found to Precision Precision of the method was carried out for both standard and sample solutions as described, results are given below. Intra day precision Parameters Ticagrelor standard Ticagrelor sample Retention time (min) Number Of Theoretical plates (N) Tailing factor (T) Area This study was performed for six injections of Ticagrelor on the same day for the standard solution of 40 µg/ml. The obtained data was calculated statistically for Mean, SD and % RSD. Available online: September Issue 48

7 The obtained results showed %RSD below 2%, indicating precision of the analytical study. Intraday precision results are tabulated below. Table 5: Intra-day results of Ticagrelor N RT Peak Area Tailing factor NTP Average STDEV %RSD DISCUSSION The %RSD for the area of 6 injections was found to be less than 2. Hence the results obtain were found to be satisfactory. Inter Day precision Interday precision was performed during 3 consecutive days at working concentration and recording of peak area at 254nm for sample at working concentration and calculating % RSD which was found to be less than 2, Inter-day precision results are tabulated below. Inter-day precision Day 1 Day 2 Day 3 Table 6: Inter-day precision results of Ticagrelor Average STDEV %RSD DISCUSSION RT Peak Area Tailing factor NTP The %RSD for the area of 6 injections was found to be less than 2.Hence the results obtain was found to be satisfactory Accuracy (recovery studies): Accuracy was determined by means of recovery experiments by the determination of % mean recovery of sample by absolute method at three different levels 50, 100% and 150%. At each Available online: September Issue 49

8 level, three determinations were performed. The amount of drug recovered was estimated results are shown in table no 7. Acceptance criteria: The % recovery of Ticagrelor should lie between 98% and 102%. Recovery level Amount taken (mcg/ml) 50% 20 mcg 100% 40 mcg 150% 60 mcg Observation Table 7: Results of accuracy Peak Area Average % Recovery The percentage mean recovery of Ticagrelor was found to be %. Limit of detection and limit of quantification % mean recovery The limit of detection(lod)and limit of quantification(loq)were separately determined based on standard devition of the y-intercept and slope of the calibration curve by using the following formulas. Limit of detection 3.3*standard deviation/slope Limit of quantificatio: 10*standard deviation/slope Robustness parameters limit of detection limit of quantification Table 7: LOD and LOQ values mg 5.58 mg/ml mg/ml Robustness is the measure of the capacity of the analytical method to remain unaffected by small but deliberate variation in the procedure. The robustness of the method was evaluated by analyzing the system suitability standard and evaluating system suitability parameter data after varying, individually, the HPLC pump flow rate (±0.2 ml/min),detection wavelength (±2 nm). Acceptance criteria: System suitability should pass as per test method at variable conditions. Available online: September Issue 50

9 Observation Table 8: Robustness results for Ticagrelor From the observation it was found that the system suitability parameters were within limit at all variable conditions. Ruggedness The method was studied for ruggedness by analyzing the sample and standard preparations by two analyst. The %RSD assay values between two analysts were calculated,the results are shown in table no.9 Acceptance criteria The % Relative standard deviation of Assay values between two analysts should be not more than 2.0%. Observation Table 9: Ruggedness results for Ticagrelor TICAGRELOR %Assay %RSD Analyst % 0.12% Anaylst % From the observation the %RSD between two analysts Assay values not greater than 2.0%, hence the method was rugged. Assay Parameter Flow Rate 0.8 ml/min 1ml/min 1.2 ml/min Wavelength 252nm 254nm 256nm TICAGRELOR Theoretical plates Tailing factor Table 10:Assay results No of injections Ticagrelor Standard area Sample area Injection-1 Injection-2 Injection-3 Injection-4 Injection-5 Injection Average area Assay(%purity) Available online: September Issue 51

10 DISSOLUTION STUDIES Optimization of dissolution study The dissolution characteristics of an oral formulation should be evaluated in the physiologic ph range of Ticagrelor solubility was determined using purified water, 0.1N hydrochloric acid, phosphate buffer (ph 6.8) dissolution testing was performed on tablet incompliance with USP 711 using paddle (USP-II).A calibrated dissolution apparatus (USP II) was used with paddles at 100 rpm and bath temperature maintained at 37 ± 0.5 ºC. 900 ml of water was used as the optimized dissolution medium. Dissolution samples were collected at 5, 10, 30, 45 and 60 min for immediate release tablets. At each time point, sample was removed from each vessel, filtered through a nylon filter (0.45 μm, 25 mm) into labeled glass tubes, and analyzed by RP-HPLC. The amount of Ticagrelor in the test samples was calculated, from the measured peak area for the test samples and standard solution. The results obtained were used to calculate the percentage release on each time of dissolution profile. Dissolution % = Where, AT & AS= absorbance of test and standard respectively, WS= Weight of Standard LC= Labeled Claim P= Potency Dissolution Conditions: Dissolution Medium : WATER Dissolution Volume : 900mL Apparatus : USP-2 (PADDLE) Rotations per Minute : 100 Sampling Time Point : 60 min (Q Point) Sampling Volume : 10mL Temperature : 37 ±0.5 C Working Concentration of Standard : 40µg/mL Working Concentration of Sample : 40µg/mL Detection Wavelength : 254nm Available online: September Issue 52

11 Chromatographic conditions: Mobile phase : Acetonitrile : water ( 80: 20) Column Wavelength Flow rate Pump mode : Phenomenex Luna C18 (250 X 4.6 mm;5μm) : 254 nm : 1ml/min : Isocratic Injection volume : 20µl Run time : 6mins Table 11: Dissolution results for Ticagrelor TIME AREA DRUG RELEASE 5min min min min min drug release RESULTS AND DISCUSSION A New RP-HPLC method has been developed and validated for the estimation of ticagrelor in bulk and its formulaion and has been extended to dissolution studies. The validation results showed that the method was specific, accurate, linear, precise, rugged, and robust. Mixture of acetonitrile and water in 80:20 ratio has been used as mobile phase at the flow rate of 1ml/min. The detector wavelength was found to be 254 nm in U.V spectroscopy. Retention time of Ticagrelor in was found to be 4.30 min with a run time of 7 min. The drug shows linearity in the range of 20-60µg/ml with a correlation coefficient of Further dissolution studies were carried out which gave satisfactory drug release of using water as dissolution medium. Available online: September Issue 53

12 Hence, this method can be routinely employed in quality control for analysis of Ticagrelor in pharmaceutical dosage form and dissolution studies. ACKNOWLEDGEMENT Authors are thankful to the Deccan School of Pharmacy for providing necessary facilities to complete this study. REFERENCES 1. Ticagrelor - DrugBank, 2. Ticagrelor - Wikipedia, 3. Caren Gobetti et al, Determination of the new antiplatelet agent Ticagrelor in tablets by stability-indicating HPLC method, Volume 10, 2014, K. Tabassum et al,analytical method development and validation studies of Ticagrelor tablets by RP-HPLC, int j appl pharm 2017;Volume 9(4): Eena Joshy et al, development and validation of RP-HPLC method for determination of Ticagrelor in pharmaceutical dosage formulation,scholars research library der pharmacia lettre, 2016, 8 (9): PR. kulkarni et al,development and validation of RP-HPLC method for estimation of Ticagrelor in bulk form, international journal of research in pharmacy and chemistry, ijrpc 2016, 6(4), BS Muddukrishna, method development and validation for the estimation of Ticargrelor in bulk and comparison with other published methods, journal of global pharma technology, volume 12 (8), Delma D Cruz et al,a LC-MS compatible RP-HPLC method for the determination of Ticagrelor in Bulk, int. j. of pharmacy and analytical research vol-6(2) 2017, ICH, Q2 (R1) validation of analytical procedure, test andmethodology, in Proceedings of the International Conference onharmonization, Geneva, Switzerland, ICH, Q2A. Validation of Analytical Procedures, ConsensusGuidelines, ICH Harmonized Tripartite Guidelines, ICH, Q2B. Validation of Analytical Procedures: Methodology,Consensus Guidelines, ICH Harmonized Tripartite Guidelines, Preparation of Buffer Solutions : Pharmaceutical Guidelineswww.pharmaguideline.com Available online: September Issue 54

13 13. USP General Chapter on Dissolution, United States Pharmacopeiaand National Formulary, United States PharmacopeialConvention, Rockville,Md, USA, FDA Guidance for Industry: Dissolution Testing of ImmediateRelease Solid Oral Dosage Forms, US Department of Healthand Human Services, Centre for Drug Evaluation and Research(CEDER), Food anddrugadministration, Rockville,Md,USA,1997. Available online: September Issue 55