INTRODUCTION:

Paracetamol (PCM) chemically is N-acetyl-p-aminophenol or 4-hydroxy acetonitrile¹, it acts as antipyretic, analgesic by blocking the release of certain chemical messengers that cause fever and pain. It is formulated in oral dosage form and injectable form². Etoricoxib (ECB) chemically 5-Chloro-2-(6-methyl-3-pyridinyl)-3-(4-methyl-sulfonylphenyl) pyridine¹ is an NSAID and selective inhibitor of cyclo-oxygenase-2. It is used in the symptomatic relief of rheumatoid arthritis, osteoarthritis, spondylitis and acute gouty arthritis².

Thiocolchicoside (TCS) chemically N-(1, 2-dimethoxy-10-(methylthio)-9-oxo-3-((3, 4, 5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy)-5, 6, 7, 9-tetrahydrobenzo heptalen-7-yl) acetamide³ is a muscle relaxant and has GABA-mimetic and glycinergic action used in lower back pain with spasms².

All these drugs are official in Indian Pharmacopoeia⁴, while paracetamol is described under monograph in British Pharmacopoeia⁵. Chemical structures of these drugs are shown in (Fig No 1).

Literature survey revealed that many techniques have been reported for the estimation of these drugs individually or along with other drugs. Various analytical methods have been reported for estimation of PCM such as UV spectrophotometric method alone⁶, along with TCS or ECB⁷^,8, chemometric method for 3 component⁹, chromatographic HPLC method^10,11, with TCS or other^12,13, UPLC/Q-TOF-MS¹⁴, HPTLC method ¹⁵ and clinical related method, hepatoprotective activity of polyherbal against PCM study^16,17.

Literature survey revealed that for estimation of ECB lonely UV analytical methods^18,19, or in combination with other^20,21, with TCS^22,23, chromatographic RP-HPLC methods along with TCS^24,25 and HPLC/stability indicating with PCM^26-29, stability indicating RP-UFLC method³⁰ and efficacy with PCM³¹ have been stated in literatures.

For estimation of TCS spectroscopic methods^32,33, UV methods along with Diclofenac^34,35, with other^36,37, RP-HPLC methods with ECB^38,39 and with other drugs⁴⁰, bio analytical LC method⁴¹, review on TCS⁴² and efficacy of combination therapy⁴³ have been described in literatures.

Fig No 1: Chemical structure of Drug molecule

MATERIALS AND METHODS:

Instrumentation:

Analysis was performed with a Shimadzu Double beam UV-Visible spectrophotometer (Shimadzu, Kyoto, Japan) with spectral bandwidth of 2nm and wavelength accuracy of ±1nm with 10mm matched Quartz cells was used. Electronic balance Afcoset balance (The Bombay Burmah Trading corpo Ltd) with accuracy ±0.1mg Model No. ER 200A was utilised for weighing and for degassing the solution Digital Ultrasonic cleaner 1.8 Ltr (Labman scientific Instruments Chennai) was used.

Reagents and Chemicals:

Pharmaceutically pure samples of PCM, ECB and TCS were procured as a gift sample from Macleods Pharmaceuticals Ltd, Mumbai Maharashtra, ethanol 90 % available in laboratory was used as solvent and the commercial formulation containing paracetamol, etoricoxib and thiocolchicoside was procured from the local market.

Solvent selection:

PCM is freely soluble in ethanol and acetone, sparingly soluble in water and very slightly soluble in dichloromethane and in ether. ECB is freely soluble in tetrahydrofuran, DMSO and in DMF, soluble in methanol and in acetone and sparingly soluble in ethanol; and TCS soluble in water very slightly soluble in ethanol 95%.

Although the solubility of the procured drugs were studied in alcohol 90%, 0.1 N HCl and 0.1 N NaOH separately; and found that in water, 0.1 N NaOH and 0.1 N HCl ECB was insoluble/sparingly soluble, paracetamol is soluble in alkaline solution and thiocolchicoside soluble in water. Hence alcohol (90%) was selected as common solvent for making stock solution. Each solution with known conc of analyte was scanned in UV range of 400nm to 200nm. The recorded spectra in solvent are shown in Fig No 2, 3 and 4. It was found that suitable solvent is 90% ethanol with respect to average cost, robust and precise in producing result.

Preparation of stock solutions and standard solutions:

10 mg each of drug PCM, ECB and TCS were separately and accurately weighed; and transferred into separate 25 ml volumetric flasks. Dissolved into 90% alcohol and volume was made to 25ml with solvent. Subsequent standard solution of each drug with conc 80μg/ml was prepared by diluting aliquot 5ml of stock solution to 25 ml into 25ml capacity volumetric flask.

Selection of wavelength and conc range:

Standard solution of PCM with conc 10μg/ml was prepared and scanned in the spectrum mode from 400 nm to 200nm.

From UV spectra (Fig No 5) it was found that PCM has measurable absorbance at 248.5nm (λ_max)and less interference was observed by ECB and TCS; similarly, ECB has maximum absorbance at 287 nm (λ_max) and less interference by PCM and TCS was accounted; For TCS the (λ_max) was selected as 380.5nm where both drugs PCM and ECB has negligible absorbance. Chemometric method using simultaneous equation method was applied and which was reasonable remedy to overcome interference at each other’s absorbance. From the nature of spectra to study linearity, working conc range 1 to 14μg/ml for PCM, 1 to 24μg/ml for both ECB and TCS was selected. Also combined drug solution was prepared simulated to marketed formulation. Selected critical parameters based upon above discussion, observations were listed in Table No 1 and by using these; method was validated as per ICH guidelines and by analysing marketed preparations⁴⁴.

Fig No 2: UV Spectra of Paracetamol

Fig No 3: UV Spectra of Etoricoxib

Fig No 4: UV Spectra of Thiocolchicoside

Fig No 5: Overlain Spectra of PCM, ECB and TCS in spectrum form

Table No 1: Selected parameters for UV-VIS analytical method of PCM, ECB and TCS

Parameter	Paracetamol	Etoricoxib	Thiocolchicoside
Wavelength	248.5	287	380.5
Solvent	90 % Alcohol	90 % Alcohol	90 % Alcohol
Scan speed	Medium Fast	Medium Fast	Medium Fast
Sampling interval	0.5 nm	0.5 nm	0.5 nm

Experimental Method for estimation:

From the overlain spectra simultaneous equation method was applicable for estimation of these analytes from the combined dosage form.

Chemo metric assisted Simultaneous equation method for estimation of drugs:

PCM was shown maximum absorbance i.e., λ_maxat 248.5 nm where less interference by ECB and TCS found and ECB has maximum absorbance i.e., λ_maxat 287 nm where measurable interference by TCS and PCM observed. Hence these two wavelengths 248.5 and 287 nm were considered as 1 and 2 and 380.5 nm where TCS can be estimated directly due to insignificant absorbance by PCM and ECB for the said method. The equation A= abc was applied for all these three components viz. X (PCM), Y(ECB) and Z (TCS) determination from the laboratory mixture and combined formulation. Working standard solutions of PCM, ECB and TCS containing 10 μg/ml conc were separately prepared and used for the method.

Absorbance of mixed drugs at 1 wavelength may be expressed as

A₁ = ax_1. b. C_x + ay₁. b. C_Y+ az_1.b.C_Z

Similar equations are resulted at 2 and 3 wavelengths, on rearranging the equations conc of each drug in mixed solution of formulation can be obtained by applying below formulae.

Where

C_PCM C_ECBC_TCS= Concentration of paracetamol, etoricoxib and thiocolchicoside in

Tablet / mixed solution

A₁, A₂and A3 = absorbance of sample solution at λ₁, λ₂and λ₃ wavelength

az₁, az₂andaz₃= absorptivity of TCS at λ₁, λ₂and λ₃ wavelength of standard solution

ay₁,ay₂anday₃= absorptivity of ECB at λ₁, λ₂and λ₃ wavelength of standard solution

ax₁ax₂andax₃= absorptivity of PCM at λ₁, λ₂and λ₃ wavelength of standard solution

A_s= absorbance of sample solution at λ₃ wavelength

Validation of the Method:

Selected critical parameters should meet the performance characteristics of the analytical method so as to attain analytical target profile of the method. An ICH guideline Q2 R1 was applied to study methods performance with critical parameters in order to implement anticipated approach. The method was validated as per ICH guidelines.

System suitability:

System suitability is studied to demonstrate the suitability of the developed procedure under consideration for the analytical method. Six replicates of working standard solutions with conc 10μg/ml each of PCM, ECB and TCS were prepared separately and absorbance was recorded, and SD and %RSD of the response was calculated.

Linearity:

The linearity of an analytical method is its ability to obtain response i.e., absorbance which is directly proportional to the conc of analyte. Series of working standard solutions were prepared in conc. range of 1-14 μg/ml for PCM and 1-24μg/ml for both ECB and TCS; and scanned in 200 to 400nm range in spectrum mode of the spectrophotometer, absorbance of the standard solutions were recorded at their respective wavelength; i.e., 248.5 for PCM, 287nm for ECB and 380.5nm for TCS in spectrum order. Microsoft office excel software tool was used to obtain the standard regression curve and its analysis as slope, intercept, and correlation coefficient.

Assay of formulation by simultaneous equation method:

Assay was carried out by proposed methods and assay was validated by statistical parameters. Tablet powder equivalent to 15mg PCM, 5mg ECB and 0.2mg TCS was weighed and transferred into 50ml volumetric flask. Dissolved into 90% ethanol and volume was made to 50 ml with solvent. Solution was filtered through what man filter paper and aliquots of solution were further diluted to obtain tablet solution. Solution was scanned in the range of 200 to 400nm to obtain absorbance of tablet solution at 248.5, 287 and 380.5nm in spectrum order. Obtained absorbance were utilised to estimate unknown conc of formulation; and results were statistically validated to obtain % of nominal conc, standard deviation and % of RSD.

Accuracy and Precision:

The accuracy of an analytical method expresses the closeness of an agreement between test result and true result. Accuracy study was performed by recovery study i.e., standard addition method; diluted sample solutions of PCM, ECB and TCS were prepared and standard solutions added in 80,100 and 120% proportionate to the tablet solution. Three replicates at each of these three levels were prepared and measured and % of conc, SD and RSD were calculated. The precision study was carried out by performing assay of tablet six times; also, the reproducibility in result was studied by inter day and intraday precision.

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ of these analytes by the proposed method were determined using calibration graph method and calculated as 3.3σ/s and 10σ/s for LOD and LOQ respectively; σ is the standard deviation of calibration curve and s is the slope of regression line.

Robustness and Ruggedness:

It is measure of capacity of analytical procedure to remain unaffected by small but deliberate variations in method parameter.

RESULTS AND DISCUSSION:

Method development comprises numerous steps, and of which solvent selection, selection of method for measurement are significant one. Uses of aqueous solvents, eco-friendly solvents like hydrotropic have got remarkable weightage due to low cost, readily available and environmentally sound. Drugs underlying analysis must have appreciable solubility in the selected solvent. Chemical structure of the drug and physico-chemical properties available in the literature guides about use of appropriate solvent in the method. From UV spectra three wavelengths were selected as 248.5nm (λ_maxof PCM), 287nm (λ_max of ECB) and 380.5 nm (λ_max of TCS) for calculation of all these three drugs in combined solution.

System Suitability:

The absorbances of six replicates of standard solutions (10μg/ml) are reported in Table No 2. The SD and % RSD was found for PCM, ECB and TCS and meets the system suitability requirements indicates method was suitable for analysis.

Table No 2: System suitability study

Sr No

Conc in μg/ml

Absorbance of PCM

Absorbance of ECB

Absorbance of TCS

10 μg/ml

0.7918*

0.6953*

0.2625*

RSD

0.88586

0.91536

0.99547

0.99367

1.28962

1.64925

*Mean of 6 replicates

Linearity:

The calibration curve of all three drugs was found to be linear in the conc range of 0-14μg/ml for PCM, 0–24 μg/ml for ECB and 0-24μg/ml for TCS as shown in Fig No 6. The regression equation of line and its parameters slope, r²value and intercept are tabulated in Table No 3, which proved the linear relationship between conc and obtained response.

Table No 3: Parameters of regression equation obtained in Microsoft excel

Parameters	PCM	ECB	TCS
Detection wavelength	248.5	287	380.5
Beer’s law limit (μg/ml)	0-14μg/ml	0–24μg/ml	0–24μg/ml
Correlation coefficient (r²)	0.9971	0.9987	0.9987
Regression equation (y = mx + c)	Y=0.0827 X+0.0027	Y=0.0712 X+0.0112	Y=0.0261 X+0.0021

Assay:

The assay was carried out by the proposed method. The spectrum of formulation by method is shown in Fig No 7. The assay of formulation was carried out by proposed method and calculated % of nominal conc and RSD was found within acceptable limits are summarized in Table No 4. The results indicated applicability of the method for estimation of formulation.

Fig No 7: Spectra of formulation obtained in the assay

Table No 4: Results of assay of formulation by proposed method

Formu -lation	Drug	Label Claim (mg/ Tablet; n=6)	Amount found /mg	Drug Cont-ent	Std Devi-ation	% RSD
Method	PCM	300	307.98	102.66	0.5302	0.5163
	ECB	100	98.56	98.56	0.2345	0.2681
	TCS	4	3.92	98.02	0.3052	0.4628

Accuracy and Precision:

The spectrum obtained in accuracy study is shown in Fig No 8 and the results of accuracy are summarised in Table No 5, the obtained results were within acceptable limit; and methods accuracy was justified by calculating % drug content.

The precision study was carried out by performing assay of solutions; further the reproducibility in result was studied by interday and intraday precision. The values obtained SD and % RSD was shown methods precision and are summarised in Table No 5.

Fig No 8: Overlaid spectra obtained in accuracy study

Table No 5: Results of accuracy and precision

S. No.	Parameter	Level of study	Drug Name	S.D.	% RSD
1	Precision	Intraday Precision	PCM	0.5581	0.5382
			ECB	0.4424	0.4280
			TCS	0.1438	0.1446
		Inter day precision	PCM	1.6227	1.5409
			ECB	0.6975	1.0521
			TCS	0.3609	0.8553
2	Accuracy study of PCM, ECB and TCS	80%	PCM	0.2773	0.2882
		100%		0.2059	1.8277
		120%		0.1275	0.9759
		80%	ECB	0.1340	3.3638
		100%		0.7815	1.9348
		120%		0.2797	0.5548
		80%	TCS	0.7284	3.9601
		100%		0.3988	2.4826
		120%		0.5032	2.9516

Limit of Detection (LOD) and Limit of Quantitation (LOQ) and Robustness and Ruggedness:

The LOD and LOQ of PCM, ECB and TCS by the proposed method were found within acceptable limits. Robustness was studied and capacity of analytical procedure to measure analyte was remain unaffected by small but deliberate variations in method parameter. The analytical method was found rugged during development; similarity the result was produced by performing the analysis by different analyst.

CONCLUSION:

All these drugs were quantitatively estimated from the combined formulation by simultaneous equation method. Results were found within acceptable limits, statistical data obtained were shown rigidity of the method. The validated method was employed 90 % alcohol as unique solvent for all these analytes. The proposed method is precise, accurate, robust and reproducible hence can be routinely used for simultaneous estimation of paracetamol, etoricoxib and thiocolchicoside from combined dosage form.

CONFLICT OF INTEREST:

All Authors declared that there is no conflict of interest.

ACKNOWLEDGEMENT:

Authors are thankful to Macleods Pharmaceuticals Ltd, Mumbai Maharashtra, for providing paracetamol, etoricoxib and thiocolchicoside as a gift sample. Authors are thankful to Principal and Management SVPM’S College of Pharmacy Malegaon (BKII) Tal. Baramati Dist. Pune, Maharashtra, India for providing necessary facilities for research.

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Received on 29.11.2023 Modified on 19.03.2024

Asian J. Pharm. Ana. 2024; 14(3):135-141.

DOI: 10.52711/2231-5675.2024.00024