Quim. Nova, Vol. 33, No. 4, 964-967, 2010
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*e-mail: hidekoy@iq.unesp.br

DETERMINATION OF 5-AMINOSALICYLIC ACID IN PHARMACEUTICAL FORMULATIONS BY SQUARE 
WAVE VOLTAMMETRY AT PENCIL GRAPHITE ELECTRODES

Carolina V. Uliana e Hideko Yamanaka*
Instituto de Química, Universidade Estadual Paulista “Júlio de Mesquita Filho”, CP 355, 14801-970 Araraquara - SP, Brasil
Gustavo S. Garbellini e Giancarlo R. Salazar-Banda
Instituto de Química de São Carlos, Universidade de São Paulo, CP 780, 13560-970 São Carlos - SP, Brasil

Recebido em 9/6/09; aceito em 24/11/09; publicado na web em 2/3/10

An analytical method for the determination of the anti-inflammatory drug 5-aminosalicylic acid (5-ASA) in pharmaceutical formulations 
using square wave voltammetry at pencil graphite electrodes was developed. After the optimization of the experimental conditions, 
calibration curves were obtained in the linear concentration range from 9.78 × 10–7 to 7.25 × 10–5 mol L–1 resulting in a limit of detection 
of 2.12 ± 0.05 x 10–8 mol L–1. Statistical tests showed that the concentrations of 5-ASA in commercial tablets and enemas obtained with 
the proposed voltammetric method agreed with HPLC values at a 95% confidence level.

Keywords: 5-aminosalicylic acid; pharmaceutical formulations; pencil graphite electrode.

INTRODUCTION

5-aminosalicylic acid (5-ASA, chemical structure is represented 
in Figure 1), also known as mesalamine or mesalazine, is a drug used 
to treat inflammation of the digestive tract (Crohn’s disease) and mild 
to moderate ulcerative colitis. As a derivative of salicylic acid, 5-ASA 
is also an antioxidant that traps free radicals, which are potentially 
damaging by-products of metabolism.1 This drug is a bowel-specific 
aminosalicylate that is metabolized in the gut and it has few systemic 
side effects.2,3 However, the determination of 5-ASA is important in 
pharmaceutical research for monitoring the quality control.

The levels of 5-ASA have traditionally been quantified using 
chromatographic techniques4-8 in pharmaceutical formulations and 
in biological matrices. Although routinely used, these techniques 
are relatively expensive and not free from drawbacks since they 
can often include lengthy, labor-intensive sample preparation and 
extended analysis time. 

Electroanalytical techniques using glassy carbon electrodes9-11 

have been also proposed for 5-ASA determinations, which contri-
buted for developing a cheaper and more rapid analysis. The pencil 
graphite electrode is another form of carbon electrode available for 
electroanalytical applications.12-14 This kind of electrode material 
presents high electrochemical reactivity, good mechanical rigidity, 
low cost, eases of renewal, and other interesting qualities.15

Among the more useful electroanalytical techniques, square wave 
voltammetry (SWV) has been successfully applied to the determina-
tion of pharmaceuticals.16-20 According to the available literature, there 
is no previous report of the determination of 5-ASA using SWV with 
pencil graphite electrodes. In the Brazilian Pharmacopeia,21 there is 

no procedure for the determination of 5-ASA in pharmaceuticals. In 
this sense, the present study reports a rapid and sensitive procedure 
for the routine quantification of 5-ASA in pharmaceutical formula-
tions such as tablets and enemas at a pencil graphite electrode using 
the SWV technique. 

In this study, very low concentrations of 5-ASA can be detected 
using the developed methodology. Moreover, the recovery results 
obtained for the 5-ASA in Brazilian commercial formulations using 
this electroanalytical methodology presented good correlation with 
the high-performance liquid chromatography (HPLC) method.

EXPERIMENTAL

Reagents

High-purity grade 5-ASA was supplied by Acros. The aqueous 
stock solution (1.0 × 10–3 mol L–1) was prepared by dissolving the 
pure powder in water followed by heating the solution to 60 ºC for 5 
min. The buffer used as supporting electrolyte was a Britton-Robinson 
(BR) 0.1 mol L–1 solution (pH 2.0), adjusted to the desired value by 
adding proper amounts of NaOH 1.0 mol L–1 solution. The water used 
was deionized using a Milli-Q (Millipore) system. Pharmaceutical 
tablets and enemas containing 400 mg per tablet and 3 g per sachet 
of 5-ASA, respectively, were purchased from a local drugstore. 

Instrumentation and experimental procedure

The electrochemical measurements were carried out using a 
PGSTAT 30 Autolab computer-controlled potentiostat. Pencil graphite 
electrode (geometric area: 3.18 mm2) as working electrode, platinum 
wire as auxiliary electrode and Ag/AgCl (KCl 3.0 mol L–1) as referen-
ce electrode were used. The working electrode was thoroughly rinsed 
with ethanol and deionized water and dried with a soft paper tissue 
prior to each electrochemical measurement. The pH of the solutions 
was measured using a Hanna Instruments HI 8417 pH electrode. 

Calibration curves (n = 4) were obtained by spiking the electrolyte 
with known quantities of the 5-ASA stock solution. The limit of 
detection (LOD = 3 S

B
/b) and quantification (LOQ= 10 S

B
/b) were 

determined using the standard deviation of the average value of the 
currents measured at the peak oxidation potential for eight voltammo-

Figure 1. Chemical structure of 5-ASA



Determination of 5-aminosalicylic acid in pharmaceutical formulations 965Vol. 33, No. 4

5-ASA at 4.75 × 10–5 mol L–1 using the pencil graphite electrode in a 
0.1 mol L–1 BR buffer solution from the values 2.0 to 12.0. The peak 
potential (Figure 3, E

p
 vs. pH) was strongly pH-dependent showing 

two linear regions that intercept at pH 5.65, a value very close to the 
pKa reported for 5-ASA in literature, which is 5.78.24 The graph of 
I

p
 vs. pH (Figure 3) revealed that the peak current decreases from pH 

2.0 to 5.0 and from 5<pH<12 there is no significant variation. The 
decreasing in magnitude is related to interaction between the electrode 
surface and the positive charge of amino group. After the pKa value, 
there is no charge and the intensity is almost the same from pH 5 
to 12.  At low pH values, the amino group of 5-ASA associates one 
proton, which become the oxidation process more difficult; it means 
higher potential is necessary for the oxidation as showed on Figure 3. 
Above pH 6, the potential is lower and it could be interesting to avoid 
interferences, but the current intensities are very low in comparison 
to pH 2. For the analytical purpose, all subsequent experiments using 
the pencil graphite electrode were carried out at pH 2.0.

The SWV parameters (f, a and ΔE
s
) were optimized in order to 

evaluate its influence on the peak current intensities of the 5-ASA 
voltammetric responses. First, a linear relationship (from 10 to 150 
Hz) between the I

p
 and the square root of the frequency was observed. 

According to the theoretical model proposed by Lovric et al.,25 this 
behavior indicates that the voltammetric oxidation of the 5-ASA is a 
diffusion-controlled electrode process. In addition, for larger values 
of f, their effect on the I

p
 was not significant. In the sequence, the 

grams of the blank solution (S
B
) and the slope (b) of the calibration 

curve, according to IUPAC definitions.22

The recovery experiments were carried out by adding known 
amounts of the target compound to the electrolyte followed by addi-
tions of the standard solution and then plotting the current intensity 
versus volume of standard solution. All measurements were perfor-
med in triplicate. The recovery efficiencies (R%) were calculated 
using the equation (R% = ([5-ASA] found / [5-ASA] added) × 100) 
where the value of “[5-ASA]

found
” refers to the concentration obtained 

by extrapolating the corresponding curve.

5-ASA assay in formulations 

The proposed methodology was applied for determination of 
5-ASA in commercial tablets and enemas. For any tablets pharmaceu-
tical formulation, the Brazilian Pharmacopeia21 recommends weigh 
twenty tablets and powdered them. Taking into account the labeled 
values, solutions of samples at 1.0 × 10–3 mol L–1 were prepared in 
Milli-Q water by heating at 60 ºC during 5 min. Aliquots of the sample 
solutions were transferred to the voltammetric cell containing 5 mL 
of the supporting electrolyte and the voltammograms were recorded.

Comparison method

The results obtained by the proposed methodology were com-
pared with HPLC method based on Gandini et al..8 A HPLC system, 
model LC 10AT from Shimadzu (Kyoto, Japan), equipped with 
UV/Vis detector and a STR ODS-II PEEK LC-18 column (250 × 
4.60 mm with 5 μm particle size) from Shinwa Chemical Industries 
(Kyoto, Japan). The mobile phase was composed of both methanol 
and an aqueous solution adjusted to pH 3.5 with acetic acid 0.1 mol 
L–1 (10:90 v/v). The flow rate was 1.0 mL min–1, the temperature of 
the column oven was 30 ºC, the injection volume was 20 μL and the 
UV detection was performed at 230 nm. Working solutions were 
prepared by dilution of the standard stock solution of the 1.0 × 10–3 
mol L–1 5-ASA. The concentration range for the calibration curve 
construction (n = 3) was the same as that used in the electrochemical 
studies, i.e. from 9.78 × 10–7 to 7.25 × 10–5 mol L–1. Solutions of the 
pharmaceutical formulations were prepared as 2.92 × 10–5 mol L–1 
5-ASA concentration. 

RESULTS AND DISCUSSION

5-ASA and medicine solution preparation

The 5-ASA is not promptly soluble in water. So the 5-ASA and 
the medicine samples were previously dissolved in water and then 
heated at 60 °C during 5 min, after that all the components were so-
lubilized and no filtration or sonicating were necessary. The solutions 
were stable at least until 8 h after the preparation. 

Development of the voltammetric methodology

The electrochemical oxidation of 5-ASA over the pencil graphite 
electrode obtained by SWV is shown in Figure 2. The intensity of the 
reverse current (curve 3) indicates that the electron transfer process 
is reversible23 at the peak potential of 0.55 V vs. Ag/AgCl.

The optimization of the analytical procedure, aiming to obtain 
the maximum analytical signal, involved a systematic study of the 
experimental parameters such as the pH value of the media and others, 
which affect the SWV response, namely the frequency (f), the pulse 
amplitude (a) and the scan increment (ΔE

s
). The effect of the pH on 

anodic peak potential (E
p
) and current intensity (I

p
) was examined for 

Figure 2. SWV response for 5-ASA at 4.75 × 10–5 mol L–1 in 0.1 mol L–1 BR buf-
fer solution (pH 2.0), using a pencil graphite electrode. Curves: (1) total; (2) 
direct and (3) reverse. SWV conditions: f = 100 s–1, a = 50 mV and ΔE

s
 = 2 mV

Figure 3. Influence of the pH on peak current () and peak potential (•) for 
the SWV experiments performed using the pencil graphite electrodes; [5-ASA] 
= 4.75 × 10–5 mol L–1 in 0.1 mol L–1 BR buffer solution, pH from 2.0 to 12.0, 
with f = 100 s–1, a = 50 mV and ΔE

s
 = 2 mV



Uliana et al.966 Quim. Nova

effect of the variation of pulse amplitude (from 10 to 80 mV) on the 
I

p
 values observed in voltammetric responses for a 5-ASA solution 

was evaluated. The results showed a nearly linear variation between 
I

p
 and amplitude, for amplitude values lower than 50 mV. At higher 

values of amplitude, no significant changes on I
p
 intensities were 

observed. Finally, the variation of the scan increment (from 2 to 10 
mV) did not promoted a considerable increase of the I

p
 for 5-ASA. 

Therefore, the value of 2 mV was chosen as ΔE
s
. The optimization 

of the SWV parameters revealed that the highest I
p
 signal for 5-ASA 

is obtained using, simultaneously, a frequency of 150 Hz, a pulse 
amplitude of 50 mV and a scan increment of 2 mV. 

After the optimization of the experimental parameters, the cali-
bration curve was constructed. Aliquots of the 5-ASA stock solution 
were added successively to the electrolyte in order to change the 
concentration of the analyte from 9.78 × 10–7 to 7.25 × 10–5 mol L–1.  
The resulting SWV profiles obtained for each concentration are 
presented in Figure 4 and the resulting calibration curve is shown in 
the inset, where the equation is I = 0.4528 ± 0.0114 A L mol–1 [5-
ASA] – 6.53 × 10–7 ± 1.40 × 10–7 A and the correlation coefficient is 
0.9967 ± 0.0012 (n = 4). 

The LOD and LOQ values calculated were, respectively, (2.12 ± 
0.05) × 10–8 mol L–1 (3.25 ± 0.08 μg L–1) and (7.07 ± 0.17) × 10–8 mol 
L–1 (10.8 ± 0.3 μg L–1). On the literature, the LOD values were 8.16 
× 10–7 mol L-1 (125 μg L–1)9 and 3.0 × 10-7 mol L-1 (46 μg L–1)10 that 
means the proposed methodology indicated significantly lower value.

The intra-day precision of the method was evaluated for ten 
independent determinations of 4.87 × 10–6 and 46.6 × 10–6 mol L–1 

solutions, yielding relative standard deviations of 2.93 and 2.30%, 
respectively. Recovery experiments were carried out for the quantifi-
cation of the analyte in electrolyte solutions following the procedure 
described in the experimental section. Solutions with a concentration 
of 2.92 × 10–5 mol L–1 were analyzed and satisfactory values (96.9 ± 
1.99% (n = 3)) for the recovery experiments were obtained.

Analytical determination of 5-ASA in commercial formulations

The performance of the proposed voltammetric method for the de-
termination of 5-ASA in commercial samples was evaluated initially 
by the interpolation of the current intensities values of each sample 
in the standard calibration curves. However, analyte concentrations 
obtained by this procedure disagreed to the labeled ones, possibly 
due to effects of the sample matrices on the 5-ASA voltammetric 
responses. The same behavior was presented with HPLC experiments. 

As a consequence, the standard addition method was used for the 
determination. SWV responses and calibration curve for enema 2 
are presented in Figure 5 to illustrate the determination of 5-ASA in 
medicine samples. Voltammetric measurements and calibration curves 
for others samples were very similar to those obtained for enema 2.

The results obtained by voltammetric and HPLC methods for all 
samples are shown in Table 1.

The statistical calculations for the assay results showed good 
precision of the voltammetric method. The results obtained were 
also compared by applying the F-test and t-test at a 95% confidence 
level.26 The calculated F or t values for all samples did not exceed the 
theoretical values (F

2.2 
= 39.00, t

4
 = 2.78), confirming that there are 

no considerable differences between the results obtained by proposed 
and HPLC methodologies. 

According to the Brazilian Pharmacopeia,21 for tablets containing 
over 0.250 g and enemas up to 60.0 g of drug, the weight variation 
limit are ± 5.0 and ± 10.0%, respectively.  In this case, all the samples 
analyzed using both methodologies, are according to the Brazilian 
regulations, except tablet 2.

CONCLUSIONS 

The developed voltammetric methodology for the determination 
of 5-ASA in pharmaceutical formulations is rapid, low cost and simple 
in comparison to HPLC method. Moreover, the SWV results showed 
detection limit values significantly lower than those electroanalytical 
methodologies reported in the literature. 

The satisfactory results of the recovery experiments with the small 
variation coefficients shown here and the simplicity of the methodolo-
gy allows us to recommend the proposed electroanalytical procedure 
for the quality control to 5-ASA in pharmaceutical formulations.

Figure 4. SWV response for different 5-ASA concentrations over the pencil 
graphite electrode: 0.0 (1); 9.78 × 10–7 (2) to 72.5 × 10–6 mol L–1 (11) in 0.1 
mol L–1 BR buffer, pH 2.0, f = 150 s–1, a = 50 mV, ΔE

s
 = 2 mV. Inset: linear 

dependence of the peak current intensities on 5-ASA concentrations

Figure 5. SWV responses in solutions containing the enema 2 (1) and this 
sample spiked with different amounts of 5-ASA: 4.87 (2) to 37.0 × 10–6 mol 
L-1 (6) in 0.1 mol L–1 BR buffer, pH 2.0, f = 150 s–1, a = 50 mV, ΔE

s
 = 2 mV. 

Inset: calibration curve

Table 1. Results of the determination of 5-ASA in Brazilian pharmaceutical 
formulations using both SWV and HPLC techniques

Sample Labeled value (g) SWV* (g) HPLC* (g)

tablet 1 0.400 0.385 ± 0.006 0.387 ± 0.007

tablet 2 0.400 0.377 ± 0.009 0.374 ± 0.008

enema 1 3.00 2.86 ± 0.06 2.84 ± 0.06

enema 2 3.00 2.87 ± 0.04 2.83 ± 0.05
* Result ± standard deviation (n = 3). 



Determination of 5-aminosalicylic acid in pharmaceutical formulations 967Vol. 33, No. 4

ACKNOWLEDGMENTS

The authors wish to thank CNPq (Proc.142930/2005–9, 
134868/2007–2 and 475577/2007-8) and FAPESP (Proc. 06/50692–2) 
for the scholarships and financial support for this work.

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