Development and validation of an analytical method for the simultaneous estimation of Etoricoxib and Thiocolchicoside in tablet dosage form by

UV Spectrophotometric method

 

Nisha kumari1, Amit Chaudhary2, Kapil Kumar Verma2

Kritika Verma2, Geetanjali Saini4, Manish Vyas3, Bhupendra Singh4*

1Abhilashi College of Pharmacy, Mandi, HP.

4College of Pharmacy, Teerthankar Mahaveer University, Moradabad, UP.

2School of Pharmacy, Abhilashi University, Mandi H.P, India.

3School of Pharmaceutical Sciences, Lovely Professional University Phagwara.

*Corresponding Author E-mail: bhupendratomar81@gmail.com

 

ABSTRACT:

A simple, accurate, precise, and rapid UV spectrophotometric method was developed for simultaneous estimation of Etoricoxib and Thiocolchicoside in the tablet dosage form. Etoricoxib showed maximum absorbance at 270 nm, while Thiocolchicoside showed maximum absorbance at 260 nm. These two wavelengths were selected for further analysis. The developed method was validated as ICH guidelines including parameters as linearity, range, precision, LOD, LOQ, and accuracy. The absorbance range for Etoricoxib (5-30 µg/ml) was found to be 0.210-0.799. These aliquots obeyed Beer-Lambert’s law with regression of 0.995. The absorbance range of Thiocolchicoside (5-30 µg/ml) was found to be 0.147-0.468 with a regression of 0.998. The method of precision (interday, intraday) was found within limits (RSD>2). The % recovery was found to be within the range. As per the ICH Q2 R1 guideline, the result was validated statistically and was found to be satisfactory.   

 

KEYWORDS: Simultaneous estimation, Etoricoxib, Thiocolchicoside, method development, UV Spectroscopy.

 

 


INTRODUCTION:

Chemically Etoricoxib is 5-chloro-3-(4-methanesulfonylphenyl)-2-(6-methylpyridine-3-yl) pyridine has the chemical formula C18H15ClN2O2S.[1-4] It is a new COX-2 selective inhibitor. COX-2 inhibitor blocks the production of prostaglandin (PGI2) without affecting platelet TXA2 synthesis.5-7 Etoricoxib is mainly used in the treatment of osteoarthritis, rheumatoid arthritis, acute dental surgery pain, and gout.8-9 It is also used to reduce swelling and joint stiffness.10-11

 

Fig. 1: Chemical structure of Etoricoxib

Chemically Thiocolchicoside is 2, 14-di-(demethoxy)-2-glucosidoxy-14- methylthiocolchicin has the chemical formula C27H33NO10S.[13-14] It is a synthetic sulfur derivative of Colchicoside. It has potent for GABA receptor and it acts on muscular contracture by inhibitory of GABA.15 In the human cortex, GABA is the main inhibitory neurotransmitter. It also has a binding for inhibitory glycine receptors. Glycine acts as an allosteric site of the NMDA (N-methyl-D-aspartate) receptor. 16 It is also an inhibitory neurotransmitter. It has a muscle relaxant with an anti-inflammatory and analgesic effect. It is used in the treatment of painful muscular conditions. This combination of etoricoxib and thiocolchicoside can be used safely in the prevention of cardiac attacks and strokes due to bad cholesterol. 17-18

 

Figure.2 Chemical structure of Thiocolchicoside

 

In our deep literature survey and as per the present knowledge reveals that not a single analytical method is reported for the determination of these drugs in combination and also during the research not following standard guidelines. Based on the above reasons I have selected the topic of the simultaneous estimation of Etoricoxib and Thiocolchicoside in the UV spectrophotometric method.

 

Instrumentation and Solution Preparation:

Instrumentation:

UV1800 (Shimadzu, Japan) was used for the development and validation of a simultaneous estimation method. The system was composed of a UV double beam spectrophotometer connected to a computer system having UV-probe version 2.70 and two quartz cuvettes of 1 cm path length.

 

Preparation of standard stock solutions:

Accurately weighed 10 mg each of Etoricoxib and Thiocolchicoside were separately dissolved in 1 ml methanol and the volume was made up to 10 ml to obtain solutions of 1000 µg/ml each. These solutions were used as standard stock solutions for further analysis.

 

Selection of analytical wavelength:

From the standard stock solutions, appropriate dilutions were made to obtain final concentrations of 10 µg/ml Etoricoxib and Thiocolchicoside. The prepared solutions were then separately scanned in the wavelength range of 200 – 400 nm. Etoricoxib showed maximum absorbance at 270 nm, while Thiocolchicoside showed maximum absorbance at 260 nm. These two wavelengths were selected for further analysis.

 

The method was developed and validated concerning linearity, range, accuracy, precision, the limit of detection, and the limit of quantitation as per ICH guidelines.

 

Linearity and range:

For each drug, appropriate aliquots were pipette out from standard stock solution into a series of 10ml volumetric flasks. The volume was made up to the marks with methanol. The prepared aliquots for Etoricoxib (5-30 µg/ml) were scanned for absorbance at 270 nm. The absorbance range was noted and determined if these aliquots followed Beer-Lambert’s low or not. Similarly, aliquots for Thiocolchicoside (5-30 µg/ml) were prepared and scanned for absorbance at 260 nm. The regression coefficients for Etoricoxib and Thiocolchicoside were determined.

 

Determination of absorptivity at analytical wavelength:

The absorbances obtained for each of the prepared aliquots were divided by their respective concentrations (g/100ml) to obtain their absorptivity values.

 

Analysis of standard mixture:

The standard mixture was made in the ratio of 20:15 (Etoricoxib and Thiocolchicoside) from the working stock solution. The standard mixture prepared was then scanned over the range 200 – 400 nm. The absorbance was measured at 270 nm and 260 nm. The concentrations of each drug were calculated by putting the absorbance and absorptivity values in the following equations:

 

Where,

Cx = Concentration of Etoricoxib (g/100ml)

Cy = Concentration of Thiocolchicoside (g/100ml)

A1 = Absorbance of mixture at 270 nm

A2 = Absorbance of mixture at 260 nm

ax1 = Absorptivity of Etoricoxib at 270 nm

ax2 = Absorptivity of Etoricoxib at 260 nm

ay1 = Absorptivity of Thiocolchicoside at 270 nm

ay2 = Absorptivity of Thiocolchicoside at 260 nm

 

Precision:

The precision of the method was determined with the prepared aliquotes. The precision of the method was verified by repeatability (intraday) and intermediate precision (inter-day) studies.

 

Repeatability:

Repeatability studies were performed by analysis of 20 µg/ml standard of Etoricoxib and 15 µg/ml of Thiocolchicoside. Method repeatability was achieved by repetition a similar procedure six times on a similar day for intraday exactness.  The repeatability of the sample application and measurement was expressed in terms of relative standard deviation (% RSD) and standard error.

 

Intermediate precision:

The intermediate (inter-day) exactness of the method was checked by activity the same procedure on completely different days under the same experimental conditions.

 

Limit of detection (LOD) and limit of quantitation (LOQ):

Limit of detection is defined as the lowest amount of analyte that can be detected but may not get quantified. The limit of quantitation is the lowest amount of analyte that can be determined quantitatively with high accuracy and precision. To estimate the LOD and LOQ, individual standard deviations were determined of the six replicates for 20 µg/ml of Etoricoxib and 15 µg/ml for Thiocolchicoside. The LOD and LOQ were then calculated by dividing the standard deviations of intercepts with slopes of their respective calibration curves.

 

Where,

LOD = Limit of detection

σ = Standard deviation of the intercept

S = Slope of the calibration curve

 

Where,

LOQ = Limit of quantitation

σ = Standard deviation of the intercept

S = Slope of the calibration curve

Where,

n = number of observations

The standard error was obtained from regression analysis.

 

Accuracy:

The accuracy of the method was validated by preparing three dilutions containing 80%, 100%, and 120% of the actual concentration. Triplicate determinations at each concentration level were executed and the obtained outcomes were compared with the expected outcomes.

 

RESULT AND DISCUSSION:

Identification of Etoricoxib and Thiocolchicoside:

Melting point study:

The melting point of Etoricoxib and Thiocolchicoside were carried out by capillary method using melting point apparatus. The observed melting point value was compared with reported value.

 

FT-IR Spectroscopy:

FT-IR spectrum of any compound or drug gives information about the group present in that particular compound. FT-IR spectra and interpretation of Etoricoxib and Thiocolchicoside are shown in figure 3 and figure 4.

 

 

Figure 3: IR spectra of Etoricoxib

 

 

Figure 4: IR spectra of Thiocolchicoside

 

Development and validation of simultaneous estimation method for the simultaneous estimation of Etoricoxib and Thiocolchicoside using UV spectrophotometric method.

Linearity and range:

Standard samples of Etoricoxib and Thiocolchicoside were analyzed to determine their respective absorption maxima (λmax). The λmax is depicted in Figures 5 and 6.    

 

The absorbance range for Etoricoxib (5-30 µg/ml) was found to be 0.210-0.799. These aliquots obeyed Beer-Lambert’s law with regression of 0.995. The absorbance range of Thiocolchicoside (5-30 µg/ml) was found to be 0.147-0.468 with a regression of 0.998. The calibration curves are shown in Figures 7-8.

 

Figure 5: λmax of Etoricoxib at 270 nm

 

Figure 6: λmax of Thiocolchicoside at 260 nm

 

 

Figure 7: Linearity curve of Etoricoxib at 270nm

 

Figure 8: Linearity curve of Thiocolchicoside at 260nm

 

Determination of absorptivity at analytical wavelength:

The data of absorptivity values for simultaneous estimation of Etoricoxib and Thiocolchicoside has been tabulated as below:

 

Table 1: Absorptivity values of Etoricoxib

S. No.

Concentration (µg/ml)

270nm

260 nm

Absorptivity

270 nm

260 nm

1.                     

5

0.210

0.104

42.0

20.8

2.                     

10

0.322

0.231

32.2

23.1

3.                     

15

0.419

0.446

27.9

29.7

4.                     

20

0.586

0.588

29.3

29.4

5.                     

25

0.691

0.722

27.6

28.8

6.                     

30

0.799

0.841

26.6

28.03

 

 

 

 

ax1= 30.93

ax2= 26.63

 

Table 2: Absorptivity values of Thiocolchicoside

S. No.

Concentration (µg/ml)

270nm

260 nm

Absorptivity

270nm

260 nm

1.                     

5

0.059

0.147

11.8

29.4

2.                     

10

0.112

0.205

11.2

20.5

3.                     

15

0.222

0.273

14.5

18.2

4.                     

20

0.341

0.327

17.05

16.35

5.                     

25

0.449

0.398

17.96

15.92

6.                     

30

0.511

0.468

17.03

15.6

 

 

 

 

ay1= 14.92

ay2= 19.32

 

Analysis of standard mixture:

Analysis of standard mixture gave values close to 100%. This affirmation was further confirmed by accuracy and precision studies.

 

Table 3: Data of standard mixture analysis

Drug

Concentration of drug taken (µg/ml)

Concentration of drug observed (µg/ml) ± S.D.

% Recovery

% RSD

Etoricoxib

20

19.89±0.398

99.49359

1.94

Thiocolc hicoside

15

14.789±0.622

98.59649

0.811513

 

Precision:

Intra-day and inter-day relative standard deviation values were determined and the data are shown below:

 

Intermediate precision:

Table 4: Results of intermediate precision of Etoricoxib and Thiocolchicoside

Drug

Etoricoxib

Thiocolchicoside

Concentration of drug taken (µg/ml)

20

15

Concentration observed (µg/ml)

Day 1

19.85±0.301

14.74±0.506

Day 2

19.75±0.098

14.58±0.087

Day 3

19.54±0.132

15.11±0.321

Mean

19.71

14.81

% Recovery

98.49

98.77

Standard deviation

0.15

0.27

%RSD

0.80

1.83

Standard error

0.018

0.015

Values represent mean±SD (n=6)

%RSD of Etoricoxib    = 0.60 (intra-day) and 0.80 (inter-day).

%RSD of Thiocolchicoside = 0.92 (intra-day) and 1.83 (inter-day).

 

The low relative deviation value (<2) obtained from the simultaneous estimation of Etoricoxib and Thiocolchicoside indicated good precision of the method.

 

Limit of detection (LOD) and limit of quantitation (LOQ)

The LOD and LOQ of the developed method were found to be as follows:

 

Table 5: Results of LOD and LOQ of Etoricoxib and Thiocolchicoside

Drug

Etoricoxib

Thiocolchicoside

Number of observations

6

6

Standard error

0.010

0.0042

Standard deviation of intercept

0.026

0.0102

LOD

3.82

2.25

LOQ

11.58

6.83

 

The percentage recoveries of three concentrations were found to be close to 100%, which is indicative of high accuracy.

 

Table 6: Results of recovery studies of Etoricoxib

Level of % recovery

Concentration taken (µg/ml)

Concentration observed

(µg/ml)

% Recovery

% RSD

Low (80%)

15

14.95±0.081

99.71

0.013

Middle (100%)

20

20.07±0.389

100.36

0.020

High (120%)

25

25.86±0.574

103.44

0.021

Recovery studies of Thiocolchicoside.

Low (80%)

10

9.82±0.087

98.22

1.03

Middle (100%)

15

15.04±0.729

100.29

0.25

High (120%)

20

19.93±0.885

99.66

0.66

Values represent mean±SD (n=3)

 

Table 7: Optical characteristics of simultaneous estimation method

Parameters

Etoricoxib

Thiocolchicoside

Absorbance maxima (λmax)

270 nm

260 nm

Beer’s law limit (µg/ml)

5-30

5-30

Correlation coefficient (R2)

0.995

0.988

Slope

0.024

0.019

Absorptivity

ax1= 30.93, ax2= 26.63

ay1= 14.92, ay2= 19.32

Repeatability

%RDS=0.60

%RDS=0.92

Intermediate precision

%RDS=0.80

%RDS=1.83

LOD

3.82

2.25

LOQ

11.58

6.83

 

Accuracy

%RSD(Low)= 0.013

%RSD(Low)= 1.03

%RSD(Middle)= 0.020

%RSD(Middle)= 0.25

%RSD(High)= 0.021

%RSD(High)= 0.66

 

CONCLUSION:

A simple, accurate, precise, and rapid UV spectrophotometric method was developed for simultaneous estimation of Etoricoxib and Thiocolchicoside in the tablet dosage form. The results of validation tests were found to be satisfactory and therefore, these methods can be applied successfully for routine quality control. The developed method can be conveniently used for the assay determination of Etoricoxib and Thiocolchicoside in the tablet dosage form. FTIR spectrum, solubility study, and melting point determination (133-137°C and 192-195°C for Etoricoxib and Thiocolchicoside respectively) confirmed the identity and purity of the drug. The results of these studies suggested that both drugs were pure and good in quality for the method development and validation. The calibration curve of Etoricoxib and Thiocolchicoside was found to be linearly regressed with an r2 value of 0.995 and 0.998. It can be concluded that the reported method could find practical application as an economical and rapid quality-control tool with good separation for the simultaneous analysis of the two drugs from tablet dosage forms in both research and industrial quality-control laboratories.

 

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Received on 30.10.2020           Modified on 15.04.2021

Accepted on 25.07.2021         © RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(7):3051-3056.

DOI: 10.52711/0974-360X.2022.00510