INTRODUCTION:

Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) intended for use in painful and inflammatory rheumatic and certain non-rheumatic conditions.¹ For increase solubility of diclofenac it is available in different salt forms such as diclofenac sodium and diclofenac potassium. It is available in number of administration forms which can be given orally, rectally or intramuscularly.^2-3 The drug has a relatively short elimination half-life, which limits the potential for drug accumulation. As an analgesic, the diclofenac sodium has fast onset and duration of action.

The therapeutic index (ratio of gastrointestinal irritant and therapeutic dosages) of diclofenac is generally good in animals, but varies relative to other Non-steroidal anti-inflammatory drugs (NSAIDs) according to model used.^4-6 Diclofenac 75 to 150 mg daily administered orally or rectally has been well studied in controlled clinical trials in patients with rheumatoid arthritis, osteoarthritis and alkylosing spondylitis, showing similar analgesic and anti inflammatory efficacy to usual therapeutic dosages of other NSAIDs such as acemetacin, aspirin, carprofen, etc. Diclofenac exerts its action via inhibition of prostaglandin synthesis by inhibiting cyclooxygenase 1 (COX 1) and cyclooxygenase 2 (COX 2) with relative equipotency.^7-8 The level and activity of COX-1 are thought to be rather stable and participates in the maintenance of normal activity of platelets, blood flow into renal tissues, and protection of the gastric mucosa from harmful acidity, among other processes. COX-2 is an inducible enzyme that is overly expressed during times of tissue damage and in the presence of inflammatory mediators that also have nociceptive properties and induce pain. The various methods for quantification of diclofenac sodium were developed and validated on HPLC, gas chromatography and FTIR. So we can develop and validated the simple method for analysis of diclofenac sodium on UV visible spectrophotometer.^9-12

Figure 1: Structure of Diclofenac Sodium

Drug Profile:^13-15

Table 1: Diclofenac sodium drug profile

Name of drug	Diclofenac Sodium
Molecular formula	^{C14H10Cl2NNaO2}
Molecular weight	318.1g/mol
IUPAC name	Sodium2-((2,6-dichlorophenyl)amino) phenyl acetate
Category	Analgesic and antipyretic
Description	A white slightly yellowish, crystalline powder, slightly hygroscopic.

UV Visible Spectroscopy is concerned with the study of absorption of UV and visible radiations which ranges from 200-800nm. The analyze concentration in solution can be determined by measuring the absorbance at some wavelength and by applying the Beer Lambert's law.¹⁶

Beer law:

According to this law, when a beam of monochromatic radiation is passed through a solution of absorbing species, the intensity of beam of monochromatic light decreases exponentially with increase in concentration of absorbing species.¹⁷

Lambert law:

Lambert's law states that the rate of decrease of intensity of monochromatic light with the thickness of the medium is directly proportional to the intensity of incident light.^18-20

Analytical method validation:

Analytical method validation is the process of documentation or proving that as analytical method provides analytical data acceptable for the intended use. The main objective of an analytical procedure will give reproducible and reliable results that are adequate for the intended purpose.^21-23Validation characteristics are as follows:

1. Specificity

2. Linearity

3. Range

4. Accuracy

5. Precision

6. Intermediate precision

7. Robustness

8. Solution stability.

Specificity:

Specificity is the ability to analyze unequivocally the analyte in the presence of components which may be expected to be present. Typically these include impurities, degradants, matrix, etc.

Linearity:

The linearity of an analytical procedure is its ability to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.

Range:

The range of an analytical procedure is the interval between the upper and lower concentration of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.

Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found.

Precision:

The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility.

Intermediate precision:

Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc.

Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters.

Solution Stability:

The solution stability is stability of standard and extracted sample solution (ready for analysis) from the sample or matrix and analyzed as per specified method, and it should be stored properly in room temperature and refrigerated condition depending upon the stability of the sample and standard solution.^24-26

Chemicals and reagents:

Diclofenac Sodium manufactured and gifted by Research-lab fine chem industries Mumbai 400002(India), Distilled Water

Methanol- Mfg by: Research-lab fine chem industries Mumbai 400002 (India).²⁷

Instruments:

UV Visible double beam spectrophotometer with matched quartz cells (1cm) Model: Shimadzu UV- 1800.

Selection of solvent:

Based on solubility of diclofenac sodium, distilled water was used as solvent for dissolving diclofenac sodium.²⁸

Preparation of standard stock solution of diclofenac sodium:

10mg quantity of diclofenac sodium was accurately weighed and taken into 100ml volumetric flask and volume is made by the distilled water and followed concentration would be 100μg/ml.²⁹

Preparation of diclofenac sodium working standard solution:

10μg/ml of diclofenac solution was prepared using Distilled Water as diluents. It was made by taking 1ml from standard stock solution and volume was made up to 10ml by distilled water.³⁰

Preparation of sample:

10mg quantity of diclofenac sodium was accurately weighed and taken into 100ml volumetric flask and volume is made by the distilled water and followed concentration would be 100μg/ml. 10μg/ml solution of Diclofenac Sodium was made by taking 1ml from above solution into 10ml volumetric flask and volume was made with distilled water up to 10ml.³¹

Determination of λmax of diclofenac sodium:

10μg/ml solution of diclofenac sodium was scanned between 400nm-200nm. The absorption maxima (λmax) were found to be at 275nm. 275nm λmax was used for the whole analysis. The standard UV spectrum of diclofenac sodium is as shown in figure 2.³²

Figure 2: UV spectrum of diclofenac sodium standard solution

VALIDATION CHARACTERISTICS:

Specificity:

To check the interference of blank at the working wavelength blank was scanned between 400nm-200nm and the diclofenac sodium solution (10μg/ml) was also scanned between 400nm-200nm. The absorption maxima (λmax) were found to be at 275nm. 275nm λmax was used for the whole analysis. The spectrum of blank and diclofenac sodium solution were overlayed.³³

The specificity of the method was determined by taking the absorbance of blank and absorbance of diclofenac sodium solution (10μg/ml). The results are tabulated in table 2.

Linearity:

5 dilutions were made from standard stock solution of diclofenac sodium (100μg/ml)in 10ml volumetric flasks to obtain the concentrations of 8μg/ml, 10μg/ml, 12 μg/ml, 14μg/ml and 16μg/ml. Absorbance of these solutions were measured at 275nm.³⁴ Calibration plot was constructed against its absorbance on Y-axis and concentration in its X-axis given in figure 3. The absorbance values are given in table 3 and the results are tabulated in table 4.

Precision:

Precision was determined by analyzing the diclofenac sodium solution for 6 times on the same day at its 80%, 100% and 150% concentration level. That is the solutions of concentration 8μg/ml, 10μg/ml and 15μg/ml were analyzed and %RSD was calculated. The results of precision at 80%, 100% and 150% of concentration level are given in table 5, 6 and 7 respectively.³⁵

Intermediate Precision:

Intermediate precision was determined by analyzing the diclofenac sodium solution for 6 times at its 80%, 100% and 150% concentration level. That is the solutions of concentrations 8μg/ml, 10μg/ml and 15μg/ml were analysed by changing the day and the analyst for the analysis and %RSD was calculated. The results of intermediate precision at 80%, 100% and 150% of concentration level are given in table 8, 9 and 10 respectively. ³⁶

Accuracy:

The diclofenac sodium was spike into the blank solution to obtain the concentration of 8μg/ml, 10μg/ml and 15 μg/ml. The readings were taken three times and percent recovery was calculated by comparing the obtained result with the standard results of precision (Table 11).³⁷

Range:

The range was established by confirming that the analytical procedure provides an acceptable degree of linearity, accuracy and precision when applied to samples containing amounts of analyte within or at the extremes of the specified range of the analytical procedure.³⁸

Robustness:

Robustness was carried out by doing deliberate variation in method parameter (that is change of solvent and change of wavelength). Absorbance of 10μg/ml solution of diclofenac sodium was measured at three different wavelengths 273nm, 275nm, 277nm and absorbance of 10μg/ml solution of diclofenac sodium in methanol (change of solvent) was measured and %RSD was calculated. The results are given in table 12 (solvent change) and table 13 (Wavelength change).³⁹

Solution stability:

Solution stability was performed by taking absorbance of 10μg/ml solution of diclofenac sodium at room temperature at regular interval of time (1hour) and results were compared with the initial reading. The obtained results are tabulated in table 14.^40-41

RESULT AND DISCUSSION:

METHOD DEVELOPEMENT:

The existing methods for validation of diclofenac sodium by using UV visible spectroscopy involves the use of different organic solvents such as methanol, ethanol, acetate buffer, phosphate buffer, etc. which are expensive. So based on solubility of diclofenac sodium, distilled water was used as solvent for validation of diclofenac sodium.

METHOD VALIDATION:

Specificity:

Table 2: Evaluation data of specificity

Sr. No.	Dilutions	Concentration	Absorbance
1.	Blank	0 μg/ml	0.000
2.	Standard	10 μg/ml	0.289

Blank solution shows the zero absorbance at absorption maxima of diclofenac sodium, there is no interference of solvent in the absorbance of diclofenac sodium solution. Hence the developed method is specific.

Figure 3: UV spectrum of diclofenac sodium in standard solution

Linearity:

Table 3: Evaluation data of linearity

Sr. No.	Concentration	Absorbance
1.	8 μg/ml	0.231
2.	10 μg/ml	0.297
3.	12 μg/ml	0.346
4.	14 μg/ml	0.400
5.	16 μg/ml	0.449

Table 4: Results of linearity

Sr. No.	Parameter	Value
1.	Range	8-16 μg/ml
2.	Equation	0.027x+0.0212
3.	Slope	0.027
4.	Correlation coefficient (r²)	0.9969

The correlation coefficient (r²) obtained by plotting the linearity curve of diclofenac sodium solution in the concentration range 8-16 μg/ml is 0.9969. Hence the method is linear.

Figure 3: Linearity plot of diclofenac sodium

Precision:

Table 5: Evaluation data of precision of 80% concentration

Sr. No.	Sample	Concentration	Absorbance
1.	Sample 1	8 μg/ml	0.245
2.	Sample 2	8 μg/ml	0.243
3.	Sample 3	8 μg/ml	0.249
4.	Sample 4	8 μg/ml	0.242
5.	Sample 5	8 μg/ml	0.248
6.	Sample 6	8 μg/ml	0.250
Average			0.246
Standard Deviation			0.003312
% RSD			1.34%

Table 6: Evaluation data of precision of 100% concentration

Sr. No.	Sample	Concentration	Absorbance
1.	Sample 1	10 μg/ml	0.299
2.	Sample 2	10 μg/ml	0.298
3.	Sample 3	10 μg/ml	0.289
4.	Sample 4	10 μg/ml	0.286
5.	Sample 5	10 μg/ml	0.286
6.	Sample 6	10 μg/ml	0.291
Average			0.291
Standard Deviation			0.005753
% RSD			1.97%

Table 7: Evaluation data of precision of 150% concentration

Sr. No.	Sample	Concentration	Absorbance
1.	Sample 1	15 μg/ml	0.527
2.	Sample 2	15 μg/ml	0.519
3.	Sample 3	15 μg/ml	0.530
4.	Sample 4	15 μg/ml	0.523
5.	Sample 5	15 μg/ml	0.523
6.	Sample 6	15 μg/ml	0.525
Average			0.524
Standard Deviation			0.003782
% RSD			0.7%

According to precision study performed, %RSD is 1.34, 1.97 and 0.7% at 80%, 100%, 150 % concentration level of diclofenac sodium solution which is below 2%. According to ICH guidelines the method is precise.

Intermediate precision:

Table 8: Evaluation data of intermediate precision of 80%

Sr. No.	Sample	Concentration	Absorbance
Sr. No.	Sample	Concentration	Day and Analyst 1	Day and Analyst 2
1.	Sample 1	8 μg/ml	0.245	0.244
2.	Sample 2	8 μg/ml	0.243	0.242
3.	Sample 3	8 μg/ml	0.249	0.250
4.	Sample 4	8 μg/ml	0.242	0.243
5.	Sample 5	8 μg/ml	0.248	0.248
6.	Sample 6	8 μg/ml	0.250	0.246
Average			0.246	0.245
Standard Deviation			0.003312	0.003082
% RSD			1.34%	1.25%
Percentage Difference			0.4%

Table 9: Evaluation data of intermediate precision of 100%

Sr. No.	Sample	Concentration	Absorbance
Sr. No.	Sample	Concentration	Day and Analyst 1	Day and Analyst 2
1.	Sample 1	10 μg/ml	0.299	0.298
2.	Sample 2	10 μg/ml	0.298	0.296
3.	Sample 3	10 μg/ml	0.289	0.301
4.	Sample 4	10 μg/ml	0.286	0.295
5.	Sample 5	10 μg/ml	0.286	0.297
6.	Sample 6	10 μg/ml	0.291	0.295
Average			0.291	0.297
Standard Deviation			0.005753	0.00228
% RSD			1.97%	0.767%
Percentage Difference			2.0%

Table 10: Evaluation data of intermediate precision of 150%

Sr. No.	Sample	Concentration	Absorbance
Sr. No.	Sample	Concentration	Day and Analyst 1	Day and Analyst 2
1.	Sample 1	15 μg/ml	0.527	0.528
2.	Sample 2	15 μg/ml	0.519	0.521
3.	Sample 3	15 μg/ml	0.530	0.533
4.	Sample 4	15 μg/ml	0.523	0.530
5.	Sample 5	15 μg/ml	0.523	0.524
6.	Sample 6	15 μg/ml	0.525	0.530
Average			0.524	0.527
Standard Deviation			0.003782	0.00441
% RSD			0.72%	0.83%
Percentage Difference			0.5%

By changing the day and analyst, %RSD at 80%, 100%, 150% concentration level of diclofenac sodium solution is below 2%. And percentage differences between precision and intermediate precision at 80%, 100% and 150% concentration level are 0.4%, 2% and 0.5% so the method is precise.

Accuracy:

Table 11: Evaluation data of accuracy

Sr. No.	Concentration	Absorbance	Average	% Recovery
1.	8 μg/ml	0.231	0.235	95.52%
2.	8 μg/ml	0.237
3.	8 μg/ml	0.238
4.	10 μg/ml	0.289	0.287	98.62%
5	10 μg/ml	0.286
6.	10 μg/ml	0.286
7.	15 μg/ml	0.500	0.506	96.56%
8.	15 μg/ml	0.512
9.	15 μg/ml	0.508

Range:

From the data of Linearity study, precision study and accuracy study the range was established as 80% to 150%.

Robustness:

Solvent change: (methanol)

Table 12: Evaluation data of robustness (solvent change)

Sr. No.	Concentration	Absorbance
1.	10 μg/ml	0.382
2.	10 μg/ml	0.368
3.	10 μg/ml	0.368
4.	10 μg/ml	0.369
5.	10 μg/ml	0.373
6.	10 μg/ml	0.379
Average		0.373
Standard Deviation		0.006047
%RSD		1.62%

Wavelength change:

By changing the solvent for analysis and by changing the wavelength, % RSD is within limit according to ICH guidelines. So the method is robust.

Table 13: Evaluation data of Robustness (wavelength change)

Sr. No.	Concentration	Absorbance
Sr. No.	Concentration	At 273nm	At 275nm	At 277nm
1.	10 μg/ml	0.298	0.299	0.300
2.	10 μg/ml	0.298	0.303	0.303
3.	10 μg/ml	0.296	0.299	0.305
4.	10 μg/ml	0.299	0.300	0.302
5.	10 μg/ml	0.305	0.306	0.307
6.	10 μg/ml	0.309	0.309	0.309
Average		0.300	0.302	0.304
Standard Deviation		0.005037	0.004131	0.003327
%RSD		1.67%	1.36%	1.09%

Solution stability:

Table 14: Evaluation data of solution stability

Sr. No.	Time	Absorbance	%Difference
1.	Initial Absorbance	0.318
2.	After 1 hour	0.318	0%
3.	After 2 hour	0.317	0.3%
4.	After 3 hour	0.316	0.6%
5.	After 4 hour	0.319	0.3%
6.	After 5 hour	0.326	2.5%
7.	After 6 hour	0.335	5.3%

According to solution stability study performed, the solution of diclofenac sodium is stable up to 4 hours.

CONCLUSION:

The UV spectroscopic method for the analysis of Diclofenac sodium was found to be simple, precise, and accurate, rapid and economical. The developed method was validated in terms of accuracy, precision, linearity, robustness, solution stability and results will be validated according to ICH guidelines. Diclofenac sodium solution in distilled water was scanned between 400-200 nm wavelength ranges on UV Visible spectrophotometer, the maximum absorption was found to be at 275 nm. 275 nm was used as λmax for the whole study. The stock solution of Diclofenac sodium in distilled water was prepared of concentration 100 µg/ml. The working standard solution of 10µg/ml was made from stock solution. The linearity was established over the concentration range of 8, 10, 12, 14 and 16 µg/ml for Diclofenac sodium with correlation coefficient value of 0.9969. Precision studies showed that % relative standard deviation was within range of acceptable limits. The present analytical method was validated as per ICH Q2 (R1) guideline and it meets to specific acceptance criteria.

COMPETING INTERESTS:

All authors declare no competing interests.

ACKNOWLEDGEMENT:

The authors are thankful to Divine College of Pharmacy, Satana, Nashik, Maharashtra, India for providing experimental facilities to carry out this work.

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Received on 16.02.2023 Modified on 08.04.2023

Asian J. Pharm. Ana. 2023; 13(3):183-189.

DOI: 10.52711/2231-5675.2023.00030