A simple and easy method of analysis for diclofenac sodium is reported. A spectrofluorometric method for the microdetermination of diclofenac sodium has been developed through its reaction with cerium(IV) in an acidic solution and measurement of the fluorescence of the Ce(III) ions produced. Under the optimum experimental conditions for the oxidation reaction, 1.0 M H 2 SO 4 with 90 min of heating time (100°C), the range of application is 124.3 - 600 ng mL -1 and the limit of detection is 72.7 ng mL -1 . The proposed method was applied to the determination of diclofenac sodium in pharmaceutical tablets. The results of the analysis show a good agreement with those obtained by the official USP 27 HPLC method.Facultad de Ciencias Exacta

Bruzzone, Liliana

Castillo, Marcela A.

Analytical Sciences

English

SEDICI - Repositorio de la UNLP

431ANALYTICAL SCIENCES MARCH 2006, VOL. 22 
2006 ©The Japan Society for Analytical Chemistry
Indirect Fluorometric Determination of Diclofenac Sodium
Marcela A. CASTILLO*,** and Liliana BRUZZONE*† *
† To whom correspondence should be addressed.
E-mail: bruzzone@quimica.unlp.edu.ar
*División Química Analítica, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 
Calle 47 esq. 115, 1900 La Plata, Argentina
**CIDEPINT, 52 e/121 y 122, 1900 La Plata, Argentina
A simple and easy method of analysis for diclofenac sodium is reported. A spectrofluorometric method for the 
microdetermination of diclofenac sodium has been developed through its reaction with cerium(IV) in an acidic solution 
and measurement of the fluorescence of the Ce(III) ions produced. Under the optimum experimental conditions for the 
oxidation reaction, 1.0 M H2SO4 with 90 min of heating time (100˚C), the range of application is 124.3 –600 ng mL–1 and 
the limit of detection is 72.7 ng mL–1. The proposed method was applied to the determination of diclofenac sodium in 
pharmaceutical tablets. The results of the analysis show a good agreement with those obtained by the official USP 27 
HPLC method.
(Received June 29, 2005; Accepted July 25, 2005)
Introduction Experimental
In pharmacologic studies,
Diclofenac, [o-[(2,6-dichlorophenyl)amino]phenyl] acetic acid 
belongs to a class of drugs called nonsteroidal anti­
inflammatory drugs (NSAIDs).
diclofenac has shown anti-inflammatory, analgesic, and 
antipyretic activity. As with other NSAIDs, its mode of action 
is not known; its ability to inhibit prostaglandin synthesis, 
however, may be involved in its anti-inflammatory activity, as 
well as contributing to its efficacy in relieving pain related to 
inflammation and primary dysmenorrhea. With regard to its 
analgesic effect, diclofenac is not a narcotic.
Diclofenac is used in treating osteoarthritis, rheumatoid 
arthritis, and ankylosing spondylitis. Due to its low solubility,1 
it is commercially available as its sodium salt. A number of 
analytical methods have been developed for the quantitative 
determination of this drug in dosage forms and in biological 
samples. Among these are spectrophotometry,1,2 fluorometry,3–5 
chromatography6–9 and, recently, some electrochemical sensors 
have been developed.10,11 USP 2712 describes a reversed-phase 
HPLC method based on UV detection for its determination.
The aim of this study is to elaborate a simple and not 
expensive method for the assay of diclofenac in pharmaceutical 
preparations. The method is based on oxidation of the drug by 
an excess of Ce(IV) with a subsequent fluorometric 
measurement.
Ce(IV) is a well-known oxidizing agent, and has been used for 
the fluorometric determination of As(III), Fe(II), oxalate,13,14 
hydrogen peroxide,15 uranium16 and some drugs, such as 
phenothiazines,17 ritodrine,18 and isoxsuprine.19
The method may be affected by coexisting substances, which 
may be oxidized with Ce(IV). Examples of the different 
interferences have been reported.13,14,16–18
Apparatus
The absorbance was measured on a Shimadzu UV-240 
recording spectrophotometer. All recordings of uncorrected 
fluorescence spectra and fluorometric measurements were 
carried out on a Perkin-Elmer LS-50B luminescence 
spectrometer equipped with a pulsed xenon lamp, an R928 
photomultiplier tube and a computer working with FL Winlab 
software. All of the measurements took place in a standard 10 
mm path-length quartz cell with 10 nm bandwidths for the 
excitation and emission monochromators.
Reagents and chemicals
Diclofenac sodium was supplied by Unifarma S.A. 
(Argentina), the content was determined by the USP 27 method 
and was found to be 99.4%. Dosage forms containing diclofenac 
sodium were purchased from a local market. All other reagents 
were of analytical grade. All solutions were prepared with double­
distilled water. A 1 × 10–2 M stock solution of Ce(IV) was 
prepared from cerium ammonium sulfate in 1.0 M sulfuric acid. 
Diclofenac standard solution. A 20 µg mL–1 working standard 
solution of diclofenac sodium was freshly prepared in double­
distilled water.
Procedure
General procedure. A 1 × 10–2 M stock solution of Ce(IV) was 
diluted with 1.0 M sulfuric acid to give a 1.7 × 10–4 M Ce(IV) 
solution; 3.0 mL of this solution were transferred into separate 12 
mL capped glass vials. To each vial, adequate aliquots (0 –300 
µL) of diclofenac stock solution and enough water to complete a 
3.3 mL final volume were added. The vials were sonicated for 
1 min, and then heated in a thermostated water-bath at 100˚C 
for 90 min. The reaction was stopped by cooling the vials in a 
water-ice bath for at least 15 min. The vials were left standing 
at room temperature, and the total volume was adjusted to 10 mL 
by the addition of water. The relative fluorescence intensity of 
the solutions was measured at λem = 356 nm with λex = 250 nm.
432 ANALYTICAL SCIENCES MARCH 2006, VOL. 22
Fig. 1 Effect of the heating time at 100˚C on the reaction yield 
(using 300 µL of a 20 µg mL–1 diclofenac standard solution).
Fig. 2 Effect of the H2SO4 concentration on the reaction yield 
(using 300 µL of a 20 µg mL–1 diclofenac standard solution). 
Heating time at 100˚C: (a) 90 min; (F) 40 min.
Procedure for tablets. Ten tablets were weighed and ground in 
a mortar. Twenty milliliters of water were added to a quantity 
equivalent to 4 mg of diclofenac. The solution was sonicated 
for 30 min and filtered through a 0.22 µm nylon membrane. 
The filtrate was placed into a 100.0 mL volumetric flask and 
diluted with water.
To each vial containing 3.0 mL of a 1.7 × 10–4 M Ce(IV) 
solution in 1.0 M sulfuric acid, a 40 µL aliquot of the sample 
solution was added. Adequate aliquots (0–260 µL) of 
diclofenac stock solution and enough water to complete a 3.3 
mL final volume were added. These solutions were analyzed 
according to a general procedure, described previously.
Results and Discussion
Cerium(IV) is a powerful oxidizing agent and is non-fluorescent 
under acid conditions, while its reduced form exhibits native 
fluorescence. The latest property has been used for the indirect 
determination of several drugs. The oxidation of diclofenac 
sodium with Ce(IV) is the basis of the present analytical 
procedure developed for diclofenac determination. The increase 
in the fluorescence intensity due to Ce(III) ions formed after the 
addition of the drug to an acid Ce(IV) solution was measured at 
λem = 356 nm with λex = 250 nm.
Optimization of experimental variables
A series of investigations were carried out to establish the 
optimum experimental conditions for the oxidation. The 
optimized parameters included the Ce(IV) concentration, 
temperature and heating time, acidity of the medium. 
Cerium(IV) concentration. The concentration of the Ce(IV) 
was chosen to avoid any inner filter effects when measuring the 
fluorescence of the final solutions. The absorbances must lie 
under 0.05 at the excitation and emission maxima.
The optimum Ce(IV) molar concentration to the diclofenac 
molar concentration ratio must be over 25.
Effect of temperature and heating time. At room temperature 
the oxidation reaction of diclofenac sodium with Ce(IV) 
proceeds slowly. Completeness of the reaction was reached 
after 24 h. The reaction was monitored by absorbance 
measurements using high concentrations of both Ce(IV) in 1.0 
M sulfuric acid and diclofenac sodium. The reaction rate 
increased when heating the solution.
The heating temperatures were studied in the 80 – 100˚C range 
over 80 min using 300 µL of a diclofenac stock solution by 
monitoring the fluorescence intensity. A 36% increment in the 
signal was observed at 100˚C with respect to 80˚C. Therefore, 
the recommended condition was to heat at 100˚C in a 
thermostated water bath.
Then, different periods of heating time at 100˚C were 
analyzed, from 20 to 140 min (Fig. 1). Although constancy was 
reached at 80 min of heating time, 90 min was chosen in order 
to assure completeness of the reaction.
Acidity of the medium. The effect of the sulfuric acid 
concentration on the reaction yield was examined over the range 
of 0.5 M to 3.0 M H2SO4. Although no substantial changes in 
the fluorescence signal were observed after heating at 100˚C for 
90 min, a slight decrease was produced in the 0.5 – 1.0 M H2SO4 
range and above 2.5 M H2SO4. A lesser heating time produced 
minor and scatter signals that increased up to 2.0 M H2SO4 
concentration, with a posterior decrease at higher concentrations 
(Fig. 2). Therefore, 1.0 M H2SO4 with 90 min of heating time 
was selected to avoid working with high concentrations of the acid.
Quantitative performance
Results obtained with diclofenac sodium standard. The 
optimized procedure was applied to 0–300 µL aliquots of a 35 
µg mL–1 diclofenac sodium standard solution.
The final concentration of the standards (Ci) ranged from 18 
to 958 ng mL–1. The response standard deviations at each 
analyte level (si) were estimated from the replicate values of the 
fluorescence intensity (Fi); the homogeneity of the data was 
confirmed by means of Cochran's test.20
A fit of Fi to Ci was carried out by means of a least-squares 
regression analysis. The F-test for the lack of a fit,20 at a 0.05 
significance level, was used to estimate the linear range of the 
calibration plot.
The results of the regression analysis through points below the 
upper limit of the linear range and the values estimated for those 
limits are gathered in Table 1.
The detection limit was estimated from the prediction bands, 
following calculation procedures given by Zorn and co- 
workers.21 Its value is compared in Table 1 with that calculated 
as the analyte concentration, giving a signal equal to the blank 
signal plus three standard deviations of the blank.22 No 
significant difference between both values could be detected by 
means of a t-test.
The quantitation limit was calculated as the analyte level at 
which the relative standard deviation (RSD = si/Fi) is 0.10.23
An RSD of 2.1% was obtained by measuring 10 replicate 
samples containing 351 ng mL–1 of diclofenac sodium.
Application
The proposed method was applied to the determination of 
diclofenac sodium in three pharmaceutical formulations 
(tablets). They were treated as described under Experimental.
The slopes of the standard calibration line and the standard 
addition line were compared by means of a t-test.20 Two of the 
formulations showed significant differences with the standards,
ANALYTICAL SCIENCES MARCH 2006, VOL. 22 433
Table 1 Results of the least-squares regression of fluorescence intensity (Fi/a.u.) against the diclofenac sodium concentration (Ci/ng 
mL–1), upper limit of the linear range, detection limit and quantitation limit
a. Number of calibration data points. b. Standard deviation. c. Residual standard deviation. d. Correlation coefficient. e. Upper limit of the linear 
range. f. Detection limit. g. Calculated from the least squares regression analysis. h. Calculated from the classic approach. i. Quantitation limit.
Na Intercept ± sb Slope ± sb sF/Cc rd U.L.e
L.D.f
L.Q.i
LSRg C.A.h
65 76.338 ± 4.036 0.8561 ± 0.0116 18.278 0.9953 600 72.7 64.0 124.3
ReferencesTable 2 Determination of diclofenac sodium in 
pharmaceutical preparations
1.
Sample Nominal content(mg/tablet)
Proposed 
methoda
Reference 
methodb
A 50 51.4 ± 1.8 51.0
B 50 51.6 ± 3.1 51.8
C 75 76.9 ± 4.7 76.5
2.
3.
a. Content calculated from the standard additions and Youden plots. 
b. HPLC method (USP 27).12
which indicates the presence of matrix effects. Calculation 
methods that enable corrections for these effects must be used 
for the quantitative evaluation of diclofenac sodium in 
pharmaceutical formulations.24 Therefore, five different
portions of the ground tablets were carefully weighed and used 
to prepare the aqueous solution indicated under Experimental. 
Then, 40 µL aliquots of this solution were treated according to 
the optimized procedure. The intercepts of the plots of 
fluorescence intensity against the sample weight (Youden plots) 
were statistically different from those obtained by the standard 
addition method.
The contents of diclofenac sodium in each formulation were 
roughly estimated by means of the corresponding calibration 
equation. Then, the standard addition method was applied, as 
indicated under Experimental. Aliquots of 40 µL of the sample 
solutions were spiked at 0, 50, 100 and 150% of the roughly 
estimated levels for diclofenac sodium. The contents of 
diclofenac were calculated from the ratio of the difference 
between the intercepts of the standard additions and the Youden 
plots to the slope of the standard additions one. The obtained 
results are listed in Table 2 together with those obtained by the 
official method (USP 27)12 and the nominal contents. A t-test 
revealed that there was no statistical difference between the 
results obtained using the proposed method and the official one 
at the 95% confidence level.
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Conclusion
18.
19.
20.
A very simple and easy method for the analysis of diclofenac 
sodium has been reported. The developed method is suitable 
for the routine assay and quality control of pharmaceuticals. 
Furthermore, this method is less time consuming than the 
proposed HPLC official method. Due to its sensitivity, the 
indirect fluorometric method could be applied for the assay of 
diclofenac in biological fluids.
21.
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Indirect fluorometric determination of diclofenac sodium

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Indirect fluorometric determination of diclofenac sodium

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