Development and Validation of HPTLC Method for Determination of Edoxaban in Bulk and Tablet

 

Ms. Tarkeshwari K. Dhiware^1*, Mr. Paresh A. Patil ², Mr. Mahesh G. Salaraya³

¹C. K. Pithawalla Institute of Pharmaceutical Science and Research, Surat, (Gujarat) 395007 [India]

²Ahinsa Institute of Pharmacy, Dondaicha, Shindkheda, Dhule. (MS) 425408 [India]

 

ABSTRACT:

Objective- To develop as simple and sensitive, high- performance thin layer chromatography (HPTLC) method for the quantitative estimation of Edoxaban in bulk and tablet. Validation of developed methods for linearity, accuracy, precision, ruggedness as per ICH guidelines. Method- Sample of edoxaban was applied on precoated silica gel 60F₂₅₄ glass plate (20 × 10 cm with 200 μm thickness HPTLC, Merck) TLC plate under pure nitrogen stream by Camage Linomat automic sample applicator. Separation was carried out by using the mobile phase of toluene: methanol: triethylamine and ratio (7.5:1:0.2, v/v/v). Developed TLC plates were scanned by camag TLC scanner and detection was carried out at 230 nm. Result- R_f value of Edoxaban was found to be 0.6. linearity was found from 400-2400 ng/band. The mean percentage recovery was found to be 98.4% Materials-The drug was used without further purification. As the tablet formulation was not available in Indian market; tablet containing 15, 30, 60mg Edoxaban were prepared in-house using direct compression technique. Prepared tablets were used as pharmaceutical formulation for further analysis.  Conclusion: The present study represents first HPTLC method that deals with the estimation of Edoxaban. Validation results indicated that the developed method is simple, rapid, accurate, specific, sensitive and precise. The developed method was validated as per ICH Q2 (R1) guideline by studying various validation parameters like accuracy, precision, specificity, assay, LOD and LOQ. It can be concluded that the method can be used inroutine analysis of Edoxaban in tablet dosage form.

 

 

 

 

INTRODUCTION:

Edoxaban (Molecular formula- C₂₄H₃₀CIN₇O₄S M.W.-548.06  g/mol .) Chemically isN’-(5-chloropyridin-2-yl)-N-[(1S,2R,4S)-4-(dimethylcarbamoyl)-2-{5-methyl-4H,5H,6H,7H-[1,3]thiazolo[5,4-c]pyridine-2-amido}cyclohexyl]ethaniamide .

 

Used as an anti-coagulants, anti-thrombin and factor Xa inhibitors^. It is a new generation of oral anticoagulant drug that selectively inhibits coagulation factor Xa. [1]. It is used in thrombo prophylaxis in patients following total knee replacement surgery with a desired efficacy and safety profile [2]. FDA approved Edoxaban (savasya, Lixiana) on Jan 8, 2015 for the prevention of stroke and systemic embolism in patients with non valvular atrial fibrillation (AF) [3]. Edoxaban is not an official drug in any Pharmacopoeia. Literature survey reveals that some methods have been reported for their determination of Edoxaban by HPLC [4-6] and hyphenated techniques such as UPLC–MS/MS [7-8], LCMS [9-10], GCMS [11], either alone or in combination. Most of the reported methods are based on hyphenated techniques and overall cost of the analysis using these techniques is more as compared to high-performance thin layer chromatography [12-15]. There is no HPTLC method available yet for estimation of Edoxaban in tablets. This paper presents development and validation of a simple, accurate and precise HPTLC method for estimation of Edoxaban in bulk and tablets.

 

The detailed literature survey revealed that there was no HPTLC method reported, Hence, the attempts was made to develop and validate the HPTLC method on Edoxaban. The HPTLC method was more efficient and sensitive than UV-Spectrometric Method.

 

 

Selection and optimization of mobile phase:

Initially, single solvent was selected on the basis of drug polarity, then Cyclohexan: Methanol in varying ratios was tried. In order to obtain high resolution and reproducible peaks different mobile phase compositions were tested. The developed spot was diffused and tailing was observed. To the above mobile phase, 1.0 mL Triethanolamine was added. The peak was symmetrical in nature. The mobile phase Toluene: Methanol: Triethanolamine (3.5: 0.5: 1.0 v/v) gave good, sharp and symmetrical peak. The wavelength 290 nm was found to be optimal for highest sensitivity. Sharp and well resolved peak was obtained for Edoxaban at R_f of 0.61 ± 0.02 when the chamber was saturated with mobile phase for 15 min at room temperature.

 

Table 1. Optimization of mobile phase

Sr. No.	Solvent System	Composition (v/v/v)	Rf
1	Cyclohexane: Methanol	4:1	0.76 spreading
2	Toluene: Methanol	4 : 1	0.73 tailing
3	Toluene : Methanol:Triethylamine	2.5:1.5:1.0	0.71 tailing
4	Toluene: Methanol: Triethylamine	3.5 : 0.5: 1.0	0.61

 

 

HPTLC Chromatogram of Edoxaban-

Figure 1: HPTLC Chromatogram of Edoxaban (R_f = 0.61 ± 0.02)

 

 

 

Linearity study:

Edoxaban depicted good linear relationship of area against concentrations evaluated during HPTLC analysis. Linear regression equation for HPTLC was found to be y = 1.476x + 144.2 with regression coefficient (r² = 0.999) which is generally considered as evidence of acceptable fit. Table 2

 

Prepared the stock solution of 1000µg/mL from that for linearity take the 0.4, 0.8, 1.2, 1.6, 2.0, 2.4 pipette out and diluted upto 10mL. the concentration of that dilution was 400,800,1200,1600,2000,2400ng/mL and analysed in UV spectrophotometer.

 

Table 2: Linearity Study of Edoxaban

 

 

Fig 2. Calibration curve of Edoxaban

 

 

Fig.3.  3-D linearity chromatogram of Edoxaban

 

Validation of the Proposed Method:

The method was validated in compliance with ICH guidelines.

 

Accuracy:

As per ICH guidelines 80, 100 and 120 % solutions were analyzed. The concentrations of the drug were calculated from linear regression equation. The obtained % recovery were found to be in 98.4% (Table 3).

 

Table 3: Recovery Study

Initial amout	Amount added	area	Amount found	%amount Found	%recovry
1.2	9.6	1536	219428.6	14285.71	98.4
1.2	1.2	1650	183333.3	11111.11	98.4
1.2	1.4	1806	167222.2	9259.259	98.4

 

Precision:

The three 400, 1200, 2000 ng/band consequent solutions of Edoxaban were analysed three times in a day and in a continuously three days. The concentrations of the drug were calculated from linear regression equation. The obtained amount found and %RSD of intra-day and inter-day found to be , 1.60 and 1.40 respectively. The method was precise as the %RSD were less than 2 and there was not much difference in % RSD of intraday and interday was observed i.e. 1.23-1.60 and 0.65- 1.41 respectively.

 

Table 4: Data of Precision Study

Intraday precision:

Concentration			area		Amount found			%Amount found
400			614		532.0682			86.65606
1200			1462		1380.068			94.39591
2000			2312		2230.068			96.45624
Con	Mo.	Af.		Even		Mean	SD		% RSD
400	614	605		620		613	7.549834		1.231621
1200	1462	1500		1483		1481.667	19.03506		1.284706
2000	2312	2358		2387		2352.333	37.81975		1.607755

Con= Comcentration; Mo.= Morning; Af.= Afternoon; Even= Evening

 

On the basis of above result area taken at morning, afternoon and evening was found to be in the range and SD and % RSD was found to be less than 2%

 

Interday Precision:

Concentration	Area	Amount found	%amount found
400	598	515.4112	86.18917
1200	1475	1392.411	94.40076
2000	2304	2221.411	94.41542

 

Conc.	day 1	day 2	day 3	Mean	SD	%RSD
400	600	584.6	598.1	594.2333	8.396626	1.413018
1200	1460	1478.6	1458.2	1465.6	11.29425	0.770623
2000	2300	2330.5	2315	2315.167	15.25068	0.658729

 

On the basis of above result the area taken at 3 different day was found to be in the range and SD and % RSD was found to be less than 2% .

 

Repeatability:

The six dilutions of 1200 ng sample solution of Edoxaban were analyzed using HPTLC. The concentrations of the drug were calculated from linear regression equation. The % RSD of repeatability study was found to be 0.12 i.e. less than 2 which indicate that this passes parameter Table 5.

 

Table 5: Repeatability Study

Concentration	Peak area	Amount found	% Amount found
1200	1458	1360.304	93.29928
1200	1433	1335.304	93.18238
1200	1460	1362.304	93.30846
1200	1501	1403.304	93.49124
1200	1468	1370.304	93.34493
1200	1500	1402.304	93.4869
Mean	1869	1372.304	93.3522
SD	1869	26.37423	0.119232
%RSD	1869	1.921895	0.127723

 

Sensitivity:

For the LOD and LOQ 400, 500, 600, 700 and 800 ng/band concentration solutions were analysed. And LOD and LOD were calculated using equation LOD = SD/S x 3.3 and LOQ = SD/S x 10, where SD is the residual standard deviation of the peak areas of the drug (n = 6) and ‘S’ is the slope of the line The LOD and LOQ were found to be 1.95 and 5.93 respectively which indicates that method is sensitive. Table 6.

 

Table 6: Sensitivity

LOD (ng/band)	LOQ (ng/band)
1.95	5.93

 

 

Ruggedness:

The 1200 ng concentration sample solutions of Edoxaban from the homogenous slot using same operational and environmental conditions were analyzed in HPTLC by two different analysts i.e. analyst I and analyst II. The calculations were carried out and the resultant values of % amount found and %RSD by analyst I and analyst II were found to be 93.35, 1.26 and 93.48, 1.90 respectively. The result shows that there is not much difference in % amount found after changing the different analyst which indicates that method is rugged Table 7.

 

Table 7: Ruggedness Data

Analyst	% Amount found	% RSD (n = 6)
I	93.35	1.26
II	93.48	1.90

 

Robustness:

In Design Expert software includes four options viz. Factorial, Combined, Mixture, and response surface. Design of experiment is preferred and in that, Full Factorial design is chosen for four factors and two responses. It provides 16 runs to assess for Robustness studies. Robustness was performed by deliberately  introducing  a  variety  of  changes  in  the previous chromatographic conditions  viz.  Plate development, Activation time, and Saturation time the effects on the results were examined. Equations obtained from the model were with the forms:   

 

Retention factor (y1) =

0.21 + 0.019*A +0.019*B + 0.00375*C + 0.014*D + 0.00625*A*C + 0.00625*A*D (1)

 

Area (y2) =

0.73 + 0.015*A + 0.015*B – 0.0050*C +0.005*D – 0.0025*A*B + 0.0075*A*C (2)

 

Where, the mobile phase is Tolune: methanol: triethylamine (3.5:0.5:1.0) was used. As per the values of coefficients from the polynomial models and their Equations (1) - (2), factor A has negative influence on all respective responses while factor B also has positive influence over respective responses. Thus, increase in % of Tolune lead to increase in R_fand Area also. Like wise positive changes in saturation time also shows positive effect over R_f and Area.

 

Estimation of experimental error and measurement of validity of polynomial models (lack of fit) were obtained through repetition of experimental points (optimized level of variables). The ANOVA was used to obtain regression lack of fit. Robustness studies were performed using 10 µg/mL of Edoxaban.  The results are shown in Table .8 and Fig. 4.

 

 

 

 

Table 8. Robustness Design data from FFD design soft

 

Table 9. Robustness Design data from Design Expert Software Studies for HPTLC analysis of Edoxaban.

 

Table 10. Comparison of experimental and predictive values of different experimental runs under optimum conditions

 

 

 

A	B
C	D

Fig: 4: Three-dimensional response surface plot showing effect of factors on R_fand Area

A is Plate development, B is saturation time, C is Tolune D is Activation time.

(a) For variation in R_fof A and B factor, (b) For variation in R_f of A and D factor (c) For variation in area of B and C factor (d) For variation in area of C and D factor.

 

 

CONCLUSION:

Both the developed methods are economical, simple, accurate, precise and rugged, and can be used for the usual study of Edoxaban from its pharmaceutical formulations. The methods are developed for quantification of Edoxaban tablets. It is also used in routine quality control of the formulations containing Edoxaban.

 

REFERENCES:   

1.        Pinto DJ. Discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidine-1-yl) phenyl)-4, 5, 6, 7-tetrahydro-1 H-pyrazolo [3, 4-c] pyridine-3-carboxamide (Apixaban, BMS-562247), a highly potent, selective, efficacious and orally bioavailable inhibitor of blood coagulation factor Xa. J Med Chem. 2007; 50:5339-56.

2.        First market approval in Japan for LIXIANA (Edoxaban)". Press Release. Daiichi Sankyo Europe GmbH. 2011-04-22. Archived from the original on 2013-11-06

3.        Lassen MR. The efficacy and safety of Edoxaban, an oral, direct factor Xa inhibitor, as thrombo prophylaxis in patients following total knee replacement. J Thromb Haemostasis 2007; 5:2368-75.

4.        FDA approves Edoxaban for stroke prevention in non valvular AF-Medscape; 2015.

5.        Landge SB. Development and validation of stability indicating RP-HPLC method on core shell column for determination of degradation and process related impurities of Edoxaban-an anticoagulant drug. Am J Anal Chem 2015; 6:539-50.

6.        Prabhune SS, Jaguste RS, Kondalkar PL, Pradhan NS. Stability indicating high-performance liquid chromatographic determination of Edoxaban in presence of degradation products. Sci Pharm 2014; 82:777-85.

7.        Zhang W, Lou D, Zhang D, Zhang Y, Huang H. Determination of rivaroxaban, apixaban and edoxaban in rat plasma by UPLC MS/MS method. J Thromb Thrombolysis 2016; 42:205-11.

8.        Schmitz E. Determination of dabigatran, rivaroxaban and apixaban by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants. J Thromb Haemostasis 2014; 12:1636-46.

9.        Delavenne X, Mismetti P, Basset T. Rapid determination of apixaban concentration in human plasma by liquid chromatography/tandem mass spectrometry: application to pharmacokinetic study. J Pharm Biomed Anal 2013; 78-79:150-3.

10.      Vaghela D, Patel P. High-performance thin layer chromatographic method with densitometry analysis for determination of rivaroxaban from its tablet dosage form. Int J Pharm Pharm Sci 2014; 6:383-6.

11.      Rathinam S, Lakshmi KS. High-performance thin layer chromatographic method for the simultaneous estimation of camylofin dihydrochloride and mefenemic acid in a pharmaceutical tablet. Int J Pharm Pharm Sci 2014; 6:585-9.

12.      Patel DJ, Desai SD, Savaliya RP, Gohil DY. Simultaneous HPTLC determination of lopinavir and ritonavir in combined dosage form. Asian J Pharm Clin Res 2011; 4:59-61.

13.      Aher A, Jain HK. Development and validation of HPTLC method for determination of gliclazide in API and pharmaceutical dosage form. World J Pharmacy Pharm Sci 2014; 4:1485-93.

14.      ICH Harmonized-Tripartite Guidelines. Validation of Analytical Procedure: Text and Methodology. November Q2(R1); 2005.

 

 

 

 

 

 

 

Received on 28.06.2019                 Accepted on 21.07.2019

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2019; 9(3):161-166.