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Validated Simultaneous Derivative Spectrophotometric Estimation of Diflunisal and Lignocaine in Bulk and Pharmaceutical Formulation

Manish. A. Raskar*¹, Shweta. S. Mungse¹, Prajkta. A. Kate¹, Ganesh. R. Godge²

¹Department of Pharmaceutical Chemistry, Dr Vithalrao Vikhe Patil Foundation’s College of Pharmacy, Vilad Ghat, Ahmednagar

²Department of Pharmaceutics, Dr Vithalrao Vikhe Patil Foundation's College of Pharmacy, Vilad Ghat, Ahmednagar

Article Info:

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Article History:

Received 19 May 2023

Reviewed 03 July 2023

Accepted 17 July 2023

Published 15 August 2023

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Cite this article as:

Raskar MA, Mungse SS, Kate PA, Godge GR, Validated Simultaneous Derivative Spectrophotometric Estimation of Diflunisal and Lignocaine in Bulk and Pharmaceutical Formulation, Journal of Drug Delivery and Therapeutics. 2023; 13(8):51-55

DOI: http://dx.doi.org/10.22270/jddt.v13i8.6166 _____________________________________________

*Address for Correspondence:

Prof. Manish. A. Raskar, Department of Pharmaceutical Chemistry, Dr Vithalrao Vikhe Patil Foundation’s College of Pharmacy, Vilad Ghat, Ahmednagar, Maharashtra

Abstract

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Two simple, sensitive, accurate and precise spectrophotometric methods were developed and validated for the quantitative determination of Diflunisal (DIF) and Lignocaine (LIG) in bulk and pharmaceutical dosage form. Method A is based on the first-order derivative (D1), and Method B is based on the second-order derivative (D2) spectrophotometric method. In Method A, absorbance was measured at 224nm and 264nm being the zero crossing points for DIF and LIG, respectively. In Method B, absorbance was measured at 232nm and 273nm being the zero crossing points for DIF and LIG, respectively. For Method A, the two drugs obeyed Beer's law in the concentration range of 3-39µg/ml for DIF and 4-40 µg/ml for LIG with correlation coefficients 0.999 and 0.995, respectively. For Method B, the two drugs obeyed Beer’s law in the concentration range of 3-30µg/ml for DIF and 4-48µg/ml for LIG with correlation coefficients 0.998 and 0.996, respectively. Both methods can be used for routine analysis of these two drugs in their pharmaceutical dosage form. Results for analysis of both methods were tested and validated for various parameters according to ICH guidelines.

Keywords: Derivative, Diflunisal, Lignocaine, first order, second order

Diflunisal (DIF) chemically is 2́, 4́ -Difluoro-4hydroxybiphenyl-3-carboxylic acid. Diflunisal is a salicylic acid derivative nonsteroidal drug with analgesic, anti-inflammatory and antipyretic properties, and it is a peripherally-acting non-narcotic analgesic drug. It is used for symptomatic treatment of mild to moderate pain accompanied by inflammation (e.g. musculoskeletal trauma, post-dental extraction, post-episiotomy), osteoarthritis, and rheumatoid arthritis. It is official in British Pharmacopoeia¹. The most commonly used techniques for the determination of DIF in pharmaceutical dosage form are derivative spectrophotometry², HPLC-UV detectionby chemometric spectrophotometry³, synchronous fluorescence spectrometry^4-5, HPLC-DAD⁶, HPLC-fluorescence detection⁷, TLC-densitometry⁸, LC-DAD⁹, LC-DAD-MS¹⁰, differential-pulse polarography¹¹, differential-pulse and square-wave stripping voltametry¹², capillary electrophoresis with luminescence detection¹³.

Lignocaine chemically is 2-(Diethylamino)-N-(2, 6-dimethylphenyl), 2-(Diethylamino)-2́, 6́-acetoxylidide. Lignocaine is an amide-type local anaesthetic commonly used in injectable dosage forms or designed for local application in mucous membranes. It is official in Indian Pharmacopoeia¹⁴. The most commonly used techniques for the determination of LIG in pharmaceutical dosage form are UV spectrophotometry¹⁵, RP-HPLC¹⁶, HPTLC¹⁷, UPLC¹⁸, LC-MS/MS¹⁹, GC-FID²⁰, capillary electrophoresis with electrochemiluminescence detection²¹, partial least squares multivariate calibration²².

As per our knowledge, no derivative UV spectrophotometric method has been reported for simultaneous estimation of Diflunisal and Lignocaine from their formulation. Hence we have developed two derivative spectrophotometric methods for simultaneous estimation of these drugs from bulk and pharmaceutical formulation.

Lignocaine was purchased from Balaji Drugs, Surat (Gujarat). Diflunisal was purchased from Dolphin Pharmacy Instruments Pvt. Ltd (Mumbai). The ointment for combination of Diflunisal and Lignocaine was prepared in the laboratory by using chemicals such as liquid paraffin, carboxymethyl cellulose sodium and white petroleum jelly. AR grade methanol was used throughout the analysis.

A double-beam UV-Visible Spectrophotometer (Jasco, Model V-630) was employed with a pair of 1cm quartz cells for all analytical work.

For both drugs, methanol was used as a common solvent for developing spectral characteristics by assessing the solubility in various solvents.

The standard stock solutions of Diflunisal and Lignocaine were prepared by dissolving 10 mg of each drug in 40 ml of methanol. The final volume was adjusted with methanol to get a 100µg/ml solution of each drug. To select the analytical wavelength, a standard solution of 20 µg/ml of each Diflunisal and Lignocaine was prepared separately by appropriate dilution of standard stock solution with methanol and scanned in the UV range of 200-400 nm. The spectral data were processed to obtain each drug's first-order derivative spectrum, and the above process was repeated for the second-order derivative method.

Each pure drug’s first-order derivative (D1) overlain spectra showed zero crossing points (ZCP). They assisted in their simultaneous estimation, as shown in Figure 1. The first order derivative wavelength considered for DIF was 264 nm, at which LIG shows zero absorbance. Similarly, the analysis of LIG was carried out at 224nm, at which the DIF showed zero absorbance. Calibration curves were plotted between absorbance observed at D1 for two drugs at selected wavelengths against the concentration range 3-39µg/ml and 4-40µg/ml for DIF and LIG, respectively.

Figure1. First Order Derivative Overlain Spectra of Diflunisal and Lignocaine

The second order derivative (D2) overlain spectra of each pure drug were found to show zero crossing point (ZCP) and assisted in their simultaneous estimation, as shown in Figure 2. The second order derivative wavelength considered for DIF was 273nm, at which LIG shows zero absorbance. Similarly, the analysis of LIG was carried out at 232nm, at which DIF show zero absorbance. Calibration curves were plotted between absorbance observed at D2 for two drugs at selected wavelengths against the concentration range 3-30 µg/ml and 4-48 µg/ml for DIF and LIG, respectively.

Figure2. Second Order Derivative Overlain Spectra of Diflunisal and Lignocaine

The oral ointment containing DIF and LIG was prepared and analysed as follows^23-27:

0.5 gm of each DIF and LIG pure drugs were ground in a mortar, and then 0.5 ml of liquid paraffin was added and mixed well to make a suspension. Then about 18 gm of white petroleum jelly was gradually mixed in 5 gm of carboxymethyl cellulose sodium (CMC-Na). After that above suspension of drugs was added into base and mixed well, subsequently more base was added to make 25 gm of ointment. Afterwards 1 gm of this ointment was dissolved in 40 ml of methanol, sonicated for 10 minutes and diluted up to 200 ml with methanol. Then sample solution was filtered through Whatman filter paper (no. 41). After appropriate dilutions, the absorbance of sample solutions was recorded at corresponding wavelengths, and the results were recorded as shown in Table 1.

The methods were validated according to International Conference on Harmonization (ICH) Q2B guidelines for validation of analytical procedures to determine the linearity, precision and accuracy of each analyte²⁴. Both precision and accuracy were determined with standard samples prepared in triplicates at different concentration levels covering the entire linearity range.

The linearity was determined at concentration range 3-39µg/ml and 4-40µg/ml for DIF and LIG, respectively for the first order derivative method. For the second order derivative method, linearity was determined at concentration range 3-30µg/ml and 4-48µg/ml for DIF and LIG, respectively.

Precision was determined by studying repeatability and intermediate precision. The experiment was repeated three times a day for intra-day and on three different days for inter-day precision. The results of the precision study are presented in Table 2. S.D. in intra-day and inter-day precision study for both methods was found to be not more than 2.0%, which indicates excellent repeatability and intermediate precision.

Recovery studies by standard addition method assessed the validity and reliability of the proposed methods. The results are shown in Table 3. The SD for the mean of % recovery values was <2.0 for both methods.

The proposed UV spectrophotometric derivative methods for simultaneous estimation of DIF and LIG are found to be accurate and precise. The results obtained were found to be within the acceptable limit. The proposed methods are simple, rapid and easy to perform, and these methods are applicable for simultaneous estimation of DIF and LIG in pure and pharmaceutical dosage form. The good validation criteria of the proposed methods allow their use in quality control laboratories.

The author would like to thank all his mentors and our Principal, Dr V. V. P. Foundation's College of Pharmacy, Ahmednagar. The authors acknowledge the immense help they have received from researchers whose articles are cited and included in the references of this manuscript. The authors also thank the authors/editors/publishers of all articles, journals and books from which the literature for this article was reviewed and discussed.

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Parameters	Method A		Method B
Parameters	DIF	LIG	DIF	LIG
%Drug Content	99.05	100.37	99.55	96.90
SD*	0.013	0.02	0.041	0.015
%RSD	0.014	0.023	0.046	0.016

Parameters	DIF		LIG
Parameters	Method A	Method B	Method A	Method B
Working Wavelength (nm)	264	273	224	232
Beer-Lambert’s Law range (µg/ml)	3-39	3-30	4-40	4-48
Precision* i) Intraday precision (SD)	0.59	0.06	1.5	0.08
ii) Interday precision (SD)	0.15	0.064	0.17	0.10
LOD(µg/ml)*	0.3	0.4	0.5	0.4
LOQ(µg/ml)*	0.95	1.3	1.7	1.2
Regression Values
Slope	0.045	0.062	0.045	0.036
Intercept	0.008	0.007	0.001	0.001
Regression Coefficient(R²)	0.999	0.998	0.995	0.996

Drug	Recovery Level	%Recovery ± SD*
Drug	Recovery Level	Method A	Method B
DIF	50%	100.13±0.06	100.37±0.073
LIG	50%	99.44±0.05	99.05±0.04
DIF	100%	99.62±0.03	99.55±0.04
LIG	100%	101.07±0.02	99.90±0.05
DIF	150%	99.04±0.04	101.2±0.03
LIG	150%	100.85±0.05	100.3±0.07