UV Spectrophotometric and HPLC Method for Quantification of Ticagrelor in Bulk and Tablet Dosage Form

 

Ravinder Bairam¹, Hemant Kumar Tatapudi¹, Neelama Gajji², Shaik Harun Rasheed³

¹Department of Pharmaceutical Analysis, Srikrupa Institute of Pharmaceutical Sciences,

Siddipet-502 277 (TS), India.

²Department of Pharmacognosy, Srikrupa Institute of Pharmaceutical Sciences, Siddipet-502 277 (TS), India.

³Department of Pharmaceutics, Gurunanak Institute of Pharmacy, Khanapur, Telangana 501506 India.

 

ABSTRACT:

The aim of the present investigation was to develop, validate and compare a UV spectrophotometric and a high performance liquid chromatography method for estimating Ticagrelor in bulk and tablet dosage form. Spectrophotometry and high performance liquid chromatography were carried out using standard instrumental parameters, which were optimized. Both methods were validated in terms of linearity, accuracy, precision, robustness, ruggedness and stability according to the ICH guidelines. The optimized ratio of mobile phase in high performance liquid chromatography under low pressure gradient mode was 30:70 % v/v of acetonitrile:glacial acetic acid 1 % , which provide a sharp peak with a short retention time of 3.910 minutes. In UV spectrophotometric analysis iso-propyl alcohol as a solvent gave adequate molar absorptivity at a λ_max of 306 nm. Results indicated that both UV spectrophotometric and high performance liquid chromatography methods were linear, precise, accurate, rugged and robust with RSD values less than 2 % and percent recovery was within the standard limits (90-110 %). Both the methods were found to be statistically non-significant at 95 % confidence intervals (p<0.05) with respect to each other. The proposed methods were found to be highly effective and could be used for quantification of Ticagrelor in bulk and a tablet formulations for routine analysis.

 

 

 

INTRODUCTION:

Ticagrelor (1S,2S,3R,5S)-3-[7{[(1R,2S)-2- (3, 4 - difluorophenyl) cyclopropyl]amino}-5-(propylthio)-3H-[1,2,3]-triazolo[4,5d]pyrimidin-3-yl]-5-(2-hydroxyethoxycyclo-pentane-1,2diol) (Figure 1) is an orally administered and direct-acting reversible antagonist of the P2Y12 receptor¹. The mechanism of action of TCG occurs via the inhibition of ADP-induced platelet aggregation². Ticagrelor (TCG) displays a predictable pharmacokinetic profile after being rapidly absorbed following oral administration³. A multiple-dose pharmacokinetic study demonstrated that TCG reached its maximum plasma levels in 1.5–3.0 hours after the oral administration and its half-life ranged from 6.2 to 13.1 hours⁴. Several analytical methods; including high-performance liquid chromatography (HPLC)^5-13, UV-spectrophotometry^14-18 ,  HPLC-MS/MS^19-26  and UPLC²⁷  are reported in the literature to quantify TCG in biological fluids and pharmaceutical formulation.

 

Figure.1.  Structure of Ticagrelor

 

High performance liquid chromatography analysis is widely implemented for quality control purposes due to high sensitivity, specificity and precise determination of analytes in various biological and analytical media^28-38. On the other hand, spectrophotometric analysis is a simpler and inexpensive method of determining analytes in pharmaceutical dosage forms^40-53. However, no spectrophotometric/HPLC method in combination has been reported for Ticagrelor for analysis. An attempt was made to develop a UV and HPLC method for estimating Ticagrelor in bulk and in tablets and both methods were validated as per ICH guidelines⁵⁴.

 

MATERIALS AND METHODS:

Spectrophotometric studies were performed on on Elico SL 210 UV/Visible spectrophotometer connected to a computer, loaded with spectra treats software version 2.0 with spectral bandwidth of 2 nm and wavelength accuracy ±0.5 nm. The solvent used was isopropyl alcohol (100 % v/v) for preparing standard and serial dilutions of Ticagrelor bulk form. HPLC analysis was carried out using a reversed-phase column-based high performance liquid chromatographic method (Prominence, Shimadzu Asia Pacific Limited, Japan). The system consisted of Shimadzu LC-20 AD  binary pump, and a variable wavelength programmable SPD-20A detector. Rheodyne injector fitted with a 20 µL loop was used and data were recorded and analysed using LC solutions software version 5.0. An enable C18 G column (250 x4.6 mm i.d, 5- µm particle) was used for analysis. The isocratic mode containing mobile phases (acetonitrile and glacial acetic acid, 1 %) used at different concentrations were run at a constant flow rate of 1 ml/min to determine the optimized ratio for analysis. The prepared mobile phases were sonicated (Ultra Cleaner, Labpro International, India) and filtered with 0.22 μm filter membrane (Millipore, India) prior to analysis.

For UV spectrophotometry, working standard (primary stock) of concentration 1000 μg/ml was prepared by adding 100 mg of Ticagrelor in 100 ml of isopropyl alchol. An appropriate dilution (secondary stock) was made to obtain a working standard of 100 μg/ml and was scanned in the range of 200-400 nm to ascertain its λmax. Gradual replicates were prepared from this stock solution to prepare 10-60 μg/ml linear range, filtered using 0.45 μm filter membrane and quantified spectrophometrically at observed λ_max of the drug. For HPLC, working standard of 1000 μg/ml was prepared by dissolving 10 mg of Ticagrelor in 10 ml HPLC grade acetonitrile. Further dilutions were made with mobile phase and quantification was carried out at λ_max of drug. Each solution was filtered through nylon filter paper and sonicated prior to injection.

 

The calibration curve was generated using different concentrations in linear progression, a 10-60 μg/ml range for UV and 10-80 μg/ml for HPLC. The linearity was determined by linear regression analysis by auto zeroing the intercept at the vertices of slope. The acceptance criteria involved was that the correlation coefficient (r²) should not be less that 0.990 according to least square method of analysis⁵. Accuracy is the percent amount of given analyte recovered from a known added amount. The methodology for both spectrophotometric and HPLC studies involves the preparation of concentration ranges at three different levels (50, 100 and 150 %) against a nominal set range of UV (40 μg/ml) and HPLC (40 μg/ml). After injection, percent recovery of each prepared concentration was determined. Samples were prepared for both methods in triplicate and assayed⁶. To ascertain the reproducibility of the proposed method, precision studies (intra and inter day) for spectrophotometric studies were carried out by preparing replicates of three different test concentrations (20, 40 and 60 μg/ml) at 100 % level and the drug amount was quantified for intraday and interday precision. For HPLC studies, three different drug concentrations (20, 40 and 60 μg/ml) were analysed for intraday and interday precision⁷. Ruggedness defines the reproducibility of test results after giving variations in the laboratory test conditions like different analysts, different days and different reagents. For both spectrophotometric and HPLC studies, three replicates of different concentrations for two different analysts were prepared and analysed. The corresponding mean absorbance (UV) and peak area (HPLC) were noted and results were reported as % RSD with acceptable value of less than 2.

Robustness involve reproducibility of test results after passing through different temperature conditions. In spectrophotometric studies, experiments were performed at room temperature (25°) and cold temperature of 18°. For HPLC studies, experiments were carried out by varying the flow rate, run time and detection wavelength. The detection of lowest concentration of analyte in the sample defines lower limit of detection (LOD) and the upper concentration of sample that can be quantitatively determined defines upper limit of quantification (LOQ) and is calculated in accordance to the guidelines⁹ Ten tablets (marketed product) were accurately weighed and uniformly crushed and passed through sieve no. 21 to obtain a fine powder. For HPLC method, powder equivalent to 10 mg of Ticagrelor was transferred to a 10 ml volumetric flask containing mobile phase. This tablet powder was dissolved, sonicate for 15 min to complete solubility. The solution was made up to the mark with acetonitrile and filtered through 0.45 µ membrane filter. The solution was further diluted with mobile phase to get nominal concentration and injected. The peak area of the chromatogram was calculated and the drug content in the tablets was quantified using the regression equation obtained from the pure sample. For UV method powder equivalent to 10 mg of Ticagrelor was transferred to a 10 ml volumetric flask containing isopropyl alcohol. This tablet powder was dissolved, sonicated for 15 min for complete solubility. The solution was made up to the mark with isopropyl alcohol and filtered through whattman’s filter paper. The filtrate was further diluted with isopropyl alcohol nominal concentration. The drug content in the tables was quantified using the regression equation obtained from the pure sample.

 

RESULTS AND DISCUSSION:

The development of a spectrophotometric method for routine analysis of drugs with precise determination reduces tedious sample preparation and is cost effective following Beer Lambert’s law, Ticagrelor with specific chromophore (fig. 1) allows detection at a specific wavelength. The working standard scanned at a wavelength of 200-400 nm, presented with characteristic absorption spectra at λmax of 306 nm (Fig 2). The specified concentrations were prepared from working standard and the entire method was validated for its accuracy, precision, linearity, robustness, ruggedness as per ICH guidelines specified in the ICHQ2R1.

 

 

Figure 2:  UV spectra of ticagrelor standard solutions (a) and test solution (b)

 

The liquid chromatographic method was developed and optimized in order to provide reproducibility and specificity. The selection criterion for mobile phase was based upon their polarity. The mobile phase was modified with two different solvent systems (acetonitrile and glacial acetic acid) and optimized ratio was evaluated on the basis of peak symmetry and run time. The uniformity in flow rate is quite crucial as the longitudinal broadening is inversely proportional to the flow rate of mobile phase system. Too high or low flow rate affects the Gaussian peak and may cause defects in the overall peak symmetry. The optimized ratio of mobile phase was found to be 30:70 % v/v, which showed uniform peak symmetry at a flow rate of 1 ml/min (fig. 3). At this ratio, the retention time (RT) of eluted Ticagrelor in standard stock solution was found to be  3.910 min with no interference that permits rapid determination of drug in analytical media.

 

Figure .3:  Chromatogram from standard (a) and sample solution (b) of Ticagrelor

Linear correlation was observed in both spectrophotometric (concentration range: 10-60 μg/ml) and HPLC method (concentration range: 10-80 μg/ml) Beer’s law was well fitted in the developed linear concentrations in both analysis. The regression coefficient and Eqn. were found to be 0.9981, Y = 0.0177 x- 0.0266 and 0.9986, Y = 104737x+309702, for UV and HPLC methods, respectively. Furthermore, detection limit depends upon the instrument sensitivity as low detection limits give high sensitivity. The LOD/ LOQ in both analysis were found to be 0.321, 1.051 μg/ml (UV) and 0.021, 0.065 μg/ml (HPLC). The results concluded that developed method was linear according to the least square method of analysis. The % RSD values for both spectrophotometric and HPLC analysis was observed to be less than 2, indicating uniform reproducibility and statistically significant in different replicates of test concentrations. A negligible variation in interday (repeatability) and Intraday (reproducibility) studies between these developed analytical methods exhibited accurate precision for series of measurements (Table 1). Accuracy results displayed good reproducibility with % RSD values below 2. This was found to be accurate as percent recovery observed was high i.e. within the range of 99.305-100.375 % (spectrophotometric analysis) and 98.258-100.963 % (HPLC analysis, Table 2), suggesting that the proposed method showed good agreement between the standard and the observed values and demonstrate an adequate accuracy within the specified limits.

 

Table 1. Interday And Intraday Precision of UV And HPLC Methods

Interday precision
Method	Con. (µg/ml)	Day 1                                                           Day 2                                           Day 3
		(Absorbance±SD)	% RSD	Absorbance±SD)	% RSD	Absorbance±SD)	% RSD
UV method	20 40 60	0.3216 ± 0.0032 0.6751 ± 0.0066 0.9823 ± 0.0111	1.02 0.99 1.14	0.3429 ± 0.0039 0.6887 ± 0.0065 0.9981 ± 0.0129	1.15 1.88 1.69	0.3722 ± 0.0045 0.6754 ± 0.0095 0.9712 ± 0.0141	1.22 1.41 1.46
HPLC method	Con. (µg/ml)	(Peak Area ± SD)	% RSD	(Peak Area ± SD)	% RSD	(Peak Area ± SD)	% RSD
	20 40 60	2466411± 46121 4820985±79064 6384760±97686	1.87 1.64 1.53	2354621± 26371 4757865±55667 6290652±93730	1.12 1.17 1.49	2311102± 42524 4712473±62671 6213435±80153	1.84 1.33 1.29
Intraday precision
		Morning                                        Afternoon                                                    Evening
		(Absorbance±SD)	% RSD	(Absorbance±SD)	% RSD	Absorbance±SD)	% RSD
UV method	20 40 60	0.3311 ± 0.0040 0.6876 ± 0.0079 0.9909 ± 0.0119	1.22 1.16 1.21	0.3548 ± 0.0038 0.6933 ± 0.0088 0.9911 ± 0.0159	1.09 1.27 1.61	0.3282 ± 0.0037 0.6813 ± 0.0091 0.9937 ± 0.0130	1.14 1.34 1.31
HPLC method	Con. (µg/ml)	(Peak Area ± SD)	% RSD	(Peak Area ± SD)	% RSD	(Peak Area ± SD)	% RSD
	20 40 60	2312341± 25898 4745973±90648 6200941±116577	1.12 1.91 1.88	2392043± 35880 4737591±58746 6209862±78244	1.50 1.24 1.26	2339204±42339 4783888±84674 6228373±104013	1.81 1.77 1.67

 

TABLE 2.  Accuracy Of Spectrophotometric and HPLC Methods

Method	Nominal concentration (μg/ml)	Level of addition (%)	Concentration added (μg/ml)	Amount recovered±SD	% RSD	% Recovery
UV method	40 40 40	50 100 150	20 40 60	19.85±0.244 39.98±0.619 60.92±0.633	1.23 1.55 1.04	99.25 99.95 101.53
HPLC method	40 40 40	50 100 150	20 40 60	19.91±0.230 40.08±0.749 58.82±0.788	1.16 1.87 1.34	99.55 100.20 98.03

 

No major difference in % RSD was observed between analysts, instruments and environmental conditions in both spectrophotometric and HPLC analysis (Table 3), suggesting that the developed methods (UV and HPLC) are rugged in nature. Experimental findings from spectrophotometric analysis revealed that there was no effect of % RSD on different temperature conditions. Furthermore; in HPLC analysis, no significant difference in % RSD was observed by slightly changing the flow rate, run time and detection (Table 4). The analysis of standard drug in marketed tablets showed acceptable content in both UV and HPLC analysis (98.362 and 99.484 %, respectively) with a % RSD of less than 2 (Table 5). Thus, both UV and HPLC methods justified good agreement with the analysis of labelled claim for the tablets and were endorsed for rapid determination of Ticagrelor in routine analysis. Furthermore, the p-value for marketed product was greater than that the standard degree of freedom, implying that there is negligible difference in drug assay in both UV and HPLC methods, thus both methods were considered as statistically insignificant. Table 6 enlists the summary of all the parameters that were analyzed by both analytical methods. The developed UV and HPLC methods were found to be linear, precise and accurate. The cost effective, simple and low-cost reagents in spectrophotometric method allow routine use in pharmaceutical research. The overall results from both spectrophotometric and HPLC methods demonstrate rapid determination of Ticagrelor and is endorsed for routine analysis for quality control purpose.

 

Table 3. Ruggedness Of UV And HPLC Methods

Method	Analyst 1			Analyst 2
	Concentration (μg/ml)	Absorbance±SD	% RSD	Concentration (μg/ml)	Absorbance±SD	% RSD
UV method	20 40 60	0.3232 ± 0.0034 0.6221 ± 0.0103 0.9939 ± 0.0117	1.08 1.66 1.18	15 20 25	0.3134 ± 0.0036 0.6436 ± 0.0070 0.9912 ± 0.0152	1.17 1.09 1.54
HPLC method	Concentration (μg/ml	(Peak Area ± SD)	% RSD	Concentration (μg/ml	(Peak Area ± SD)	% RSD
	20 40 60	2347680± 26998 4733333±51593 6209891±73276	1.15 1.09 1.18	30 40 50	2310001± 38577 4738921±57814 6214235±68978	1.67 1.22 1.11

 

 

Table 4.  Robustness of UV And HPLC Methods

UV method

Room temperature (25°)

Temperature (18°)

Concentration (μg/ml)

Absorbance±SD

% RSD

Concentration (μg/ml)

Absorbance±SD

% RSD

20 40 60

0.3153 ± 0.0034 0.6364 ± 0.0108 0.9910 ± 0.0180

1.11 1.71 1.82

20 40 60

0.3251 ± 0.0035 0.6122 ± 0.0096 0.9825 ± 0.010

1.09 1.57 1.02

HPLC  method

Flow rate (±0.1 ml/min)

Detection wavelength (±2 nm)

Concentration (μg/ml)

+ 0.1 ml/min

- 0.1 ml/min

+2 nm

- 2 nm

(Peak Area)

% RSD

(Peak Area)

% RSD

(Peak Area)

% RSD

Peak Area

% RSD

20 40 60

2359719 4789955 6244435

1.33 1.48 1.12

2349890 4749859 6211111

1.61 1.24 1.42

2359393 4755583 6258439

1.61 1.88 1.63

2339835 4745858 6273897

1.66 1.82 1.19

 

Table 5.  Drug Assay By UV And HPLC Methods

 

Table 6.  Summary Of The Validation Parameters Of UV And HPLC Analysis

 

 

CONCLUSION:

The HPLC method and UV spectroscopy method developed and validated for the analysis of ticagrelor in tablets were found to be reliable, simple, fast, accurate and precise. The results of UV method showed no significant difference from the HPLC method. The purpose to develop the new spectroscopic method is not to replace the available methods for the content analysis of ticagrelor in tablet dosage forms, but to use as an alternative method where the advanced instruments like LC-MS and GC-MS are not available for routine analysis.

 

ACKNOWLEDGEMENT:

The authors are grateful to Management of Srikrupa Institute of Pharmaceutical Sciences for providing necessary facilities to carry out the above research work.

 

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54.      ICH Proceedings of the International Conference on Harmonisation of Technical Requirement of Registration of Pharmaceuticals for Human Use (ICH Harmonised Tripartite Guidelines). Validation of Analytical Procedures: Methodology, Q2B.

 

 

 

 

 

 

 

Received on 29.01.2022       Modified on 12.03.2022

Accepted on 02.05.2022   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2022; 12(3):159-165.