Analytical Method Development and Validation of Ticagrelor from Bulk and Formulation

Anand Gupta*, Vaishali Jadhav, Ashish Jain

Shri. D. D. Vispute College of Pharmacy & Research Center, New Panvel

 

ABSTRACT:

Ticagrelor is a Platelet Aggregation Inhibitor which is white to off-white, highly crystalline and non-hygroscopic powder, having High Solubility and Low Permeability. A Simple, Rapid, Selective, Precise and Accurate UV and HPLC Method have been developed for the Estimation of Ticagrelor in bulk drugs and its Tablet dosage form. The max was found to be λmax 255 nm. As per the solution stability experiments, this drug was stable in the solvent composition of ACN:Methanol (85:15 v/v) and hence used as a diluent for sample preparation. The developed method was optimized to get reproducible results with minimum run time. The Stationary Phase was C18 (250 x 4.6 mm i.d., 5μ) Mobile Phase was ACN:Methanol (85:15 v/v), Flow rate 1.0 ml/min, Injection volume 10 µl, PDA detection at λmax 255nm and Run time 7 min. It provides a linear response over the Conc. Range of 5-25 µg/ml with Correlation Co-efficient of 0.999 and LOD and LOQ was found to be 0.20 μg/ml and 0.61 μg/ml respectively. %Recovery was found to be 99.06, 99.77, and 100.99% for the levels of 80, 100, and 120% respectively.  Both the Method was found to be Robust with better accuracy and Precision having % RSD value less than 2.

 

 

 

1. INTRODUCTION:

Ticagrelor ((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) is Platelet Aggregation Inhibitor which Ticagrelor blocks adenosine diphosphate (ADP) receptors of subtype P2Y12. In contrast to the other antiplatelet drugs, ticagrelor has a binding site different from ADP, making it an allosteric antagonist, and the blockage is reversible.

 

 

 

Moreover, the drug does not need hepatic activation, which might work better for patients with genetic variants regarding the enzyme CYP2C19 (although it is not certain whether clopidogrel is significantly influenced by such variants). It absorbed quickly from the gut and the bioavailability is 36%. The drug is metabolized principally by Cytochrome P-450 (CYP) isoenzyme 3A4 to an active metabolite that has similar antiplatelet activity as the parent drug. Plasma concentrations of ticagrelor and its active metabolite increase in a dose-dependent manner with peak concentrations achieved within approximately 1.5 and 2.5 hours, respectively. The primary route of ticagrelor elimination is hepatic metabolism. When radiolabeled ticagrelor is administered, the mean recovery of radioactivity is approximately 84% (58% in faeces, 26% in urine). The most common side effects are shortness of breath (dyspnea, 14%) and various types of bleeding, such as hematoma, nosebleed, gastrointestinal, subcutaneous or dermal bleeding. Ticagrelor should be administered with caution or avoided in patients with advanced sinoauricular disease. They are decreased in patients that described themselves as ‘coloured’ and those with severe renal impairment. These differences are considered clinically irrelevant.

 

 

Fig 1: Chemical Structure of Ticagrelor

 

2. MATERIALS AND CHEMICALS:

2.1 Instrumentation:

Table No 1: Instruments Used

Sr No.	Name of instrument	Make and Model
1.	HPLC	Jasco Extrema LC System - 4000
2.	UV-Vis Spectrophotometer	Shimadzu 1800
3.	FTIR	Simadzu, IR Affinity – 1S, No. A219652
4.	Weighing Balance	Aczet (Sensitivity 0.0001 gm) Shimadzu (Sensitivity 0.001 gm)
5.	pH Meter	Lab India
6.	Sonicator	Life Care Equipment
7.	HPLC Column	C18 (250 x 4.6 mm i.d., 5µ)
8.	Digital pH Meter	Equiptronics EQ 610

 

2.2 Chemicals and reagents:

Table No 2: Chemicals Used

Sr No.	Name of Material	Manufacturer	Grade
1.	Acetonitrile	Research lab fine chem, Mumbai	HPLC
2.	Methanol	Research lab fine chem, Mumbai	HPLC
3.	Water	Research lab fine chem, Mumbai	HPLC
4.	o-phosphoric acid	Research lab fine chem, Mumbai	AR
5.	Disodium hydrogen phosphate	Research lab fine chem, Mumbai	AR
6.	Potassium dihydrogen phosphate	Research lab fine chem, Mumbai	AR
7.	Glacial acetic acid	Research lab fine chem, Mumbai	AR
8.	Membrane filter paper (0.45μ)	Pall Life Sciences, Mumbai	HPLC
9.	Syringe filter (0.45μ)	Research lab fine chem, Mumbai	HPLC

 

2.3   Preparation of Solutions:

2.3.1 Preparation of Standard Stock solution:

·      Weigh 10 mg of drug and transfer in 10 ml volumetric flask and add 5 ml of diluents in the flask. Shake well for 2 min and make up the volume up to the mark with diluent. This gives solution of 1000 µg/ml.

·      Pipette out 1 ml of above solution and transfer it to 10 ml volumetric flask and dilute it upto the mark with diluent to get concentration of 100 µg/ml

 

2.3.2 Preparation of Sample Stock solution:

·        Weigh and powder 10 tablets. Weigh a quantity of powder equivalent to 10 mg of Ticagrelor i.e. 34.54 mg of powdered drug and transfer in 10 ml volumetric flask and add 5 ml of diluent in the flask. Shake well and Sonicate for 10 min and make up the volume up to the mark with diluent. This gives solution of 1000 µg/ml.

·        Filter the above solution with suitable filter paper and pipette out 1 ml of solution and transfer it to 10 ml volumetric flask and dilute it upto the mark with diluent to get concentration of 100 µg/ml.

 

 

 

 

3. RESULTS AND DISCUSSIONS:

3.1 Drug Identification:

 

Fig 2: IR Spectra of Ticagrelor

 

 

 

The spectra of Ticagrelor shows the following groups at their frequencies and interpretation is given in Table 3,

 

Table 3: Interpretation of IR Spectra of Ticagrelor

Sr No.	Wavenumber (cm-1)	Interpretation
1.	1273.02	C=N
2.	1195.87	C-F
3.	1327.03	C-O (stretch) Ester
4.	1624.06	C=C (stretch) Aromatic
5.	1425.04	CH2 (bend)
6.	671.23	C-H (out-of-pane bend)

3.2 Selection of Detection Wavelength:

The standard solution of 10 µg/ml of Ticagrelor were prepared and scanned over the range of 400-200 nm. After scan was completed it shows highest absorbance and peak at wavelength of 255 nm. So, 255 nm was selected as the detection wavelength.

 

3.3 Assay Method:

Six replicates of Standard and Sample solutions were prepared and %Assay was calculated

 

 

Fig 3: UV Spectra of Ticagrelor

 

 

Fig 4: Chromatogram of Standard Ticagrelor

 

 

Fig 5: Chromatogram of Sample Ticagrelor

Observation Table:

Table 4: Assay Results of Ticagrelor by HPLC Method

Sr No.	Conc. (µg/ml)	Standard Area	Sample Area
1	10	337150	337150
2	10	335462	325632
3	10	332589	334598
4	10	345698	351420
5	10	332154	339856
6	10	332147	342152
Average		335866.66	338468
Tab. Avg Wt.		207.2 mg
Std. Wt.		10 mg
Sample Wt.		34.54 mg
Label Amount		60 mg
Std. Purity (%)		99.7
Assay (%)		98.91

 

Observation:

The amount of Ticagrelor present in taken dosage form was found to be 98.91%.

 

3.4 ChromatographicConditions:

a.      Column: C18 (250 x 4.6 mm i.d., 5μ)

b.      Wavelength: 255 nm

c.      Oven Temperature: 30^oC

d.      Flow rate: 1.0 ml/min

e.      Injection Volume: 10 ml

 

3.5 Method Development and Validation:

3.5.1 Selection of Suitable Diluent:

The diluent was selected on the basis of its solubility studies performed on different solvents. So depending on Solubility study, Mobile Phase composition and Reproducible peaks diluent were selected. ACN: Methanol in the ratio of 85:15% v/v was selected as a diluent.

 

3.5.2 Selection of Mobile Phase:

A range of solvents were screened in an effort to get a well resolved, sharp and symmetrical peak for the drug. Hence, various numbers of trails were carried out to select a suitable mobile phase for the HPLC method development.

 

 

 

Fig 6: Chromatogram of Ticagrelor

 

3.5.3 Validation:

3.5.3.1 Specificity:

 

Fig 7: Blank Chromatogram for Specificity

 

 

Fig 8: Chromatogram of Ticagrelor for Specificity

 

Observation:

No interference of mobile phase, diluents, and impurities is observed and also no change in Retention time was observed and hence, the method was found to be Specific.

 

 

 

 

3.5.3.2 Linearity:

The linearity of Ticagrelor was found to be in the range of 5-25 µg/ml.

 

Table 5: Linearity Data of Ticagrelor by HPLC Method

Sr no.	Conc. (µg\ml)	Area
1.	0	0
2.	5	164266
3.	10	336735
4.	15	502576
5.	20	665644
6.	25	836735
Correlation Co-efficient		0.999
Y-Intercept		Y = 33449x-458.5

 

 

Observation:

The Correlation Co-efficient for linear curve obtained between Concentrations vs. Area for standard preparations of Ticagrelor was found to be 0.999. The relationship between the concentrations of Ticagrelor was linear in the range examined since all the points lie in a straight line and Correlation Co-efficient was well within limits.

 

 

Fig 9: Linearity graph of Ticagrelor by HPLC Method

Observation Table:

Table 6: Accuracy data of Ticagrelor by HPLC Method

 

Table 7: Precision data of Ticagrelor by HPLC Method

Table 8: Robustness data of Ticagrelor by HPLC Method

Sr no.			1.	2.	3.	Mean	SD	%RSD
Flow rate	0.8 ml/ min	Area	444323	445689	447855	445955	1781.03	0.39
		Rt	3.87	3.85	3.85	3.85	0.01151	0.29
		NTP	5586	5464	5562	5537	12.49	0.22
	1.2 ml/ min	Area	292750	298569	289987	293768	1488.286	0.50
		Rt	2.61	2.62	2.61	2.61	0.0057	0.22
		NTP	4675	4658	4596	4646	41.581	0.89
Wavelength	252 nm	Area	353501	356985	356696	355727	1933.458	0.54
		Rt	3.10	3.15	3.11	3.12	0.0264	0.84
		NTP	5323	5333	5454	5370	16.921	0.31
	258	Area	333335	335623	336562	335173	1659.829	0.49
		Rt	3.10	3.11	3.15	3.12	0.0264	0.84
		NTP	5323	5323	5322	5322	0.5773	0.01
Temperature	25oC	Area	349033	344585	348956	347524	2546.11	0.73
		Rt	3.15	3.15	3.18	3.16	0.0173	0.54
		NTP	5423	5444	5623	5496	16.70	0.30
	35oC	Area	365142	365656	359865	363554	3205.376	0.88
		Rt	3.05	3.08	3.05	3.06	0.0173	0.56
		NTP	5164	5112	5123	5133	27.404	0.53

 

 

 

3.5.3.3 Accuracy (Recovery):

The recovery studies were carried out three times, Chromatogram was recorded and %Recovery and Mean %Recovery was calculated.

 

Observation:

%Recovery was found to be 99.06, 99.77, and 100.99% for the levels of 80, 100, and 120% respectively.

 

3.5.3.4 Precision:

Six replicates of Standard solution were prepared and %RSD was calculated.

 

Observation:

The %RSD for System and Method Precision was found to be 0.29 and 0.77% respectively.

 

3.5.3.5 Robustness:

The Three replicates of Standard Solution were prepared by varying the Wavelength (±3 nm), Flow rate (±0.2 ml/min), and Temperature (±5ºC) and effect was checked for system suitability.

 

Observation:

The System Suitability was found to be within limit at different Wavelengths, Flow rates and Temperatures for Robustness study conducted.

 

3.5.3.6 LOD and LOQ:

LOD and LOQ were found to be 0.20 µg/ml and 0.61 µg/ml respectively.

 

3.5.3.7 System Suitability Test:

Injected single injection of blank and five replicate of Standard Solution. The %RSD of five replicate.

 

%RSD should not be more than 2.0%. The USP Tailing Factor for Ticagrelor peaks should not be more than 2.0. The USP Plates should not be less than 2000.

 

Table 9: Result of System Suitability Test

Sr. No	Parameter	Result
1.	Tailing Factor	1.03
2.	NTP	5789
3.	Retention time	3.12
4.	%RSD	0.77

 

Observation:

The system suitability was found to be within limits.

 

4. CONCLUSION:

Based on trial experiments conducted for Method Development, this drug was stable in the solvent composition of ACN: Methanol (85:15 v/v) and hence used as a diluent for sample preparation. The developed method was optimized to get reproducible results with minimum run time. The Stationary Phase was C18 (250 x 4.6 mm i.d., 5μ) Mobile Phase was ACN: Methanol (85:15 v/v), Flow rate 1.0 ml/min, Injection volume 10 ml, PDA detection at λmax 255nm and Run time 7 min. It provides a linear response over the Conc. Range of 5-25 mg/ml with Correlation Co-efficient of 0.999 and LOD and LOQ was found to be 0.20 μg/ml and 0.61 μg/ml respectively. %Recovery was found to be 99.06, 99.77, and 100.99% for the levels of 80, 100, and 120% respectively. Both the Method was found to be Robust with better accuracy and Precision having % RSD value less than 2. The proposed Method has been successfully used for the Routine analysis for the determination of assay in Ticagrelor Tablet.

 

5. REFERENCES:

1.     https://reference.medscape.com/drug/brilinta-ticagrelor-999674 (assessed on 1/10/2018)

2.     https://pubchem.ncbi.nlm.nih.gov/compound/Ticagrelor (assessed on 1/10/2018)

3.     http://www.thwink.org/sustain/glossary/AnalyticalMethod.html (accessed on 2/11/2018)

4.     https://www.quora.com/What-is-the-basic-principle-of-high-performance-liquid-chromatography-HPLC (assessed on 07/11/2018)

5.     http://www.chromatographyonline.com/mobile-phase-optimization-strategies-reversed-phase-hplc-0 (assessed on 14/11/2018)

6.     https://www.ich.org/products/guidelines/quality/article/quality-guidelines.html (assessed on 14/11/2018)

7.     Rajendra B. Kakde, et al. Method Development, Validation and Impurity Profiling Of Ticagrelor By Acid Degradation Method. IJPRIF. 2017, 10 (4), 225-236

8.     Md. ShabanaSulthana, et al. Development Of A Validated HPLC-PDA Method For Stability Indicating Study Of Ticagrelor: A Novel Anti-Platelet Agent (P2y12-Adp Receptor Blocker) IJPCBS. 2017, 7(1), 36-42

9.     Muddukrishna BS, et. al., Ticagrelor in bulk and comparison with published methods, Journal of Global Pharma Technology, 2016, 12(8), 01-06

10.   P R. Kulkarni, et al. Development and validation of RP-HPLC method for estimation of Ticagrelor in bulk form, IJRPC 2016, 6(4), 733-737

11.   Livia Maronesi Bueno, et al. HPLC method for simultaneous analysis of ticagrelor and its organic impurities and identification of two major photo degradant products, European Journal of Pharmaceutical Sciences 2017, 97, 22–29

12.   Vegesna Swetha, et al. Analytical method development and validation of stability indicating assay method of Ticagrelor tablets by using RP-HPLC, WJPMR, 2017, 10, 235-241.

 

 

 

 

Received on 14.03.2019        Accepted on 10.04.2019

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Asian J. Pharm. Res. 2019; 9(3):141-146.