[Espectrofotometria] Análise de AAS por método FIA

Prévia do material em texto

Flow injection spectrophotometric determination of
acetylsalicylic acid in tablets after on-line
microwave-assisted alkaline hydrolysis
Airton Vicente Pereira, Clezio Aniceto and Orlando Fatibello-Filho*
Grupo de Quı´mica Analı´tica, Departamento de Quı´mica, Centro de Cieˆncias Exatas e de
Tecnologia, Universidade Federal de Sa˜o Carlos, Caixa Postal 676, CEP 13.560-970, Sa˜o Carlos,
SP, Brazil. E-mail: doff@power.ufscar.br
The proposed method is based on the on-line
microwave-assisted alkaline hydrolysis of acetylsalicylic
acid (ASA) to salicylic acid (SA) that reacts with FeIII to
form a complex that absorbs at 525 nm. Samples merged
with NaOH were passed continuously through a domestic
microwave oven in order to accelerate the hydrolysis of
ASA. Under the best analytical conditions, the linearity of
the calibration equation for ASA ranged from 25 to 250
mg ml21. The precision for ten successive measurements
of 200 mg ml21 ASA presented a relative standard
deviation of 0.40%. The detection limit was 4.0 mg ml21
and recoveries of 99.1–101.0% were obtained for ASA. No
interference was observed from the common excipients of
tablets and other active substances such as ascorbic acid
and caffeine. The proposed FI method is adequate for a
large number of samples because it is not time consuming
and permits the determination of ASA in 90 samples per
hour.
Keywords: Acetylsalicylic acid; flow injection;
microwave-assisted hydrolysis; spectrophotometry
Acetylsalicylic acid (ASA) is widely used in the relief of
headaches, fever, muscular pains and inflammation due to
arthritis or injury. In solution, the rate of decomposition of ASA
to salicylic acid (SA) is dependent on the pH. At pH 11–12,
ASA is immediately hydrolysed; at neutral and acid solution
(pH 4–8), the hydrolysis rate is slow, and the maximum stability
is attained at pH 2–3.1
The conventional back-titration method2 for ASA determi-
nation is simple and economical, but requires heating over
reflux for 10 min. It has been replaced by HPLC in the more
recent editions of USP.3 UV–VIS spectrophotometry,4–6 fluori-
metry,7,8 amperometry9 and HPLC10–13 methods were reported
for the determination of ASA in pharmaceuticals. HPLC is
preferred over other methods because of the possibility of
simultaneous determination of ASA and SA. However, this
technique is time-consuming and requires sophisticated equip-
ment. Juhl and Kirchhoefer14 developed a semiautomated-UV
detection method for determination of ASA in tablets but a
complex system was used because extraction with chloroform
was necessary. 
The FeIII–salicylate reaction has been used for quantitative
determination of SA in ASA samples and appropriate condi-
tions for the reaction were established. The maximum colour
intensity was obtained at pH 2.5–3.5.15 Lopes-Fernandez
et al.16 proposed an asymmetrical FIA with dual injection for
the simultaneous determination of SA and ASA, which is on-
line hydrolysed to SA in a longer channel and two peaks were
obtained by complexation between FeIII and SA and the
resulting coloured product was measured at 520 nm. The linear
range for ASA varied from 300 to 1800 mg ml21 and 30 samples
were analysed per hour.
Microwave oven pre-treatment of samples has gained
widespread application in different areas and samples.17–20
However, few applications in pharmaceutical analysis have
been reported.21 FIA combined with microwave-assisted alka-
line hydrolysis has been applied to the spectrophotometric
determination of paracetamol in pharmaceuticals.22 On-line
microwave-assisted alkaline hydrolysis of vitamin A to retinol
and its detection at 325 nm was also reported.23
This paper reports the application of a FI spectrophotometric
method for determination of ASA using a microwave-assisted
hydrolysis incorporated in the system in order to minimize the
time of analysis and increase the sensitivity of the method. In
the method presented, it is unnecessary to perform two
determinations, one before and one after hydrolysis, to obtain a
value for the ASA concentration in the samples. The proposed
method is based on the on-line microwave-assisted alkaline
hydrolysis of acetylsalicylic acid to salicylic acid that reacts
with FeIII in acid medium to form a complex that absorbs at 525
nm.
Experimental
Apparatus
The flow injection manifold is shown in Fig. 1. A Panasonic 600
W (Manaus, Brazil) model NN 5556 B domestic microwave
oven equipped with a magnetron of 2.450 MHz was used. A
twelve-channel Ismatec (Zurich, Switzerland) model 7618-50
peristaltic pump supplied with Tygon pump tubing was used to
pump all solutions. The manifold was constructed with
polyethylene and PTFE tubing (0.8 mm id). Sample and reagent
solutions were injected manually into the carrier stream using a
laboratory-constructed three-piece injector–commutator made
of Perspex, with two fixed side bars and a sliding central bar,
that is moved for sampling and injection. A Femto (Sa˜o Paulo,
Brazil) spectrophotometer model 435 equipped with a glass
flow-cell (optical path 1.0 cm) was used for the absorbance
measurements. Peaks were recorded using a Cole Parmer
(Chicago, IL, USA) two-channel strip-chart recorder model
1202-0000.
Reagents and solutions
All reagents used were of analytical-reagent grade and all
solutions were prepared with water obtained from a Millipore
(Bedford, MA, USA) Milli-Q system (model UV Plus Ultra-
Low Organics Water). 
A stock solution of ASA (2.0 mg ml21) was prepared by
dissolving 500 mg of ASA (Synth) in 250 ml of water. Solutions
of desired concentrations were obtained by diluting the stock
solution with water. All solutions to be analysed were prepared
just before injection into the FIA system in order to avoid ASA
hydrolysis to SA.
The solution containing 0.50% (w/v) ferric nitrate in 0.4
mol l21 nitric acid was prepared by adding Fe(NO3)3·9H2O
Analyst, May 1998, Vol. 123 (1011–1015) 1011
Pu
bl
ish
ed
 o
n 
01
 Ja
nu
ar
y 
19
98
. D
ow
nl
oa
de
d 
on
 3
0/
06
/2
01
3 
13
:4
3:
18
. 
View Article Online / Journal Homepage / Table of Contents for this issue
(Riedel, Hannover, Germany) and an appropriate volume of
concentrated nitric acid to a 500 ml calibrated flask and diluting
to volume with water.
Methods
Analysis of pharmaceutical samples
To prepare commercial samples, a known number of tablets
were ground to a fine power and an accurate mass correspond-
ing to about 500 mg of ASA was transferred to a 250 ml
Erlenmeyer flask and stirred with about 200 ml of water. These
solutions were filtered through a filter paper into a 250 ml
calibrated flask and made up to volume with water. This
solution was used for determination after dilution with water to
the desired concentration. A volume of 250 ml of sample was
injected immediately into the carrier stream to prevent errors
due to the conversion of ASA into SA. 
Reference method
In order to compare the results obtained by the FI procedure, the
back-titration method described for aspirin tablets in the British
Pharmacopoeia2 was carried out with minor modifications. An
accurate amount of powder from the tablets was treated with 0.5
mol l21 NaOH and heated in a microwave oven for 3 min at
lower power level to ensure complete hydrolysis and to avoid
boiling. After cooling, the solution was titrated with standard-
ized 0.501 mol l21 hydrochloric acid solution, using phenol-
phthalein as indicator.
Flow injection procedure
The flow injection system for on-line ASA hydrolysis is shown
in Fig. 1. When the central bar of the injector is moved to the
injection position, sample (L1, 250 ml) and R1 reagent (0.2
mol l21 NaOH; L2, 250 ml) are injected as individual zones into
the water carrier streams (C1 and C2; both flowing at 3.4
ml min21) which merged downstream synchronously. At
confluence point x, the water carrier streams merged so that
alkaline hydrolysis of ASA occurs in the PTFE tube reaction
coil B1 (200 cm, 0.8 mm id) placed inside the microwave oven
turned on at the maximum power level (600 W). Forsafety
reasons, these PTFE tubes were passed through the walls of the
microwave oven using the external oven air vents. The
hydrolysed sample passed through the PTFE coil B2 (100 cm,
0.8 mm id), immersed in a water bath. To avoid the presence of
bubbles in the detector, produced during the passage of the
sample in the coiled reactor inserted in the microwave oven, a
T-shaped glass debubbler (aspiration rate, 0.2 ml min21) was
incorporated between the water bath and the confluence point y.
Then, the sample zone is mixed with the R2 reagent [0.50%
(w/v) Fe(NO3)3 in 0.4 mol l21 nitric acid, flowing at 1.3
ml min21] in the reactor coil B3 (100 cm, 0.8 mm id). The
absorbance resulting from the FeIII–salicylate complex,
measured at 525 nm, is proportional to the ASA concentration
of the sample. To avoid damage in the magnetron a beaker
containing 500 ml of water was placed inside the microwave
cavity. The volume of water that evaporated during the
hydrolysis step was continuously replaced by pumping water in
at a flow rate of 4.8 ml min21 (C3 channel).
Results and discussion
ASA can be hydrolysed rapidly in an alkaline medium to its
constituent salicylic and acetic acids.1 In the conventional
analytical procedure,2 complete hydrolysis is attained after 10
min heating at reflux, followed by back-titration with standard-
ized acid solution.
After hydrolysis the SA content is proportional to the ASA
concentration present in the sample. SA can be quantitatively
determined by complexation with FeIII ions and the resulting
colour read at 525 nm. In this work, a domestic microwave oven
was exploited as the energy source to accelerate the hydrolysis
of ASA (about 20 s) in order to minimize the time of analysis
and increase the sensitivity of the detection.
Study of chemical parameters
Initially, the FI manifold was used to investigate the conditions
of ASA hydrolysis. In all preliminary experiments, the coil
lengths of B1, B2 and B3 were 300, 200 and 100 cm,
respectively. The L1 and L2 loop volumes were 250 ml and fixed
flow rates of 3.0 ml min21 were used for C1 and C2 and 3.9
ml min21 for R2 reagent. The maximum power (600 W) of the
microwave oven was used.
The acidity of the R2 reagent is important because the nitric
acid concentration should be sufficient to neutralize the sample
zone and to give an acid pH in which the reaction between FeIII
ions and SA occurs. Increment of NaOH (R1 reagent)
concentration requires an increase in the R2 reagent acid
concentration. 
The effect of the alkalinity on the baseline stability was
studied replacing the sample (L1) by water. Within the
concentration range 0.05–0.25 mol l21 NaOH studied no
baseline drift was observed maintaining a concentration ratio of
2 : 1 (HNO3, R2 : NaOH, R1). A ratio lower than 2 : 1 could not
be used because it caused baseline noise, due to incomplete
neutralization of the injected alkaline zone.
The effect of NaOH concentration (R1 reagent) on the ASA
hydrolysis was evaluated over the concentration range from
0.05 to 0.25 mol l21. In this experiment, the nitric acid
concentration of R2 reagent [1% (w/v) Fe(NO3)3] was twice that
of NaOH (R1). ASA standards in the concentration range from
50 to 400 mg ml21 were used. Fig. 2 shows that the absorbance
intensity increased with an increase in NaOH concentration up
to 0.20 mol l21, above which it increased only slightly. In this
Fig. 1 Schematic diagram of the FI system for spectrophotometric determination of ASA. The three rectangular pieces represent a scheme of the sliding
bar injector. S, sample or standard solutions; L1, sample loop (50 cm, 250 ml); L2, R1 reagent (0.2 mol l21 NaOH) loop (50 cm, 250 ml); C1, C2 water carriers
streams, flowing at 3.4 ml min21; C3, water auxiliary channel pumped at 4.8 ml min21; B1, B2 and B3, reactor coils (200, 100 and 100 cm, respectively);
R2 reagent solution, 0.5% (w/v) Fe(NO3)3 in 0.4 mol l21 nitric acid, flowing at 1.3 ml min21; Db, glass T-debubbler, aspiration at the top 0.2 ml min21; D,
spectrophotometer at 525 nm; x and y, confluence points; W, waste. The numbers in parentheses are the flow rates in ml min21.
1012 Analyst, May 1998, Vol. 123
Pu
bl
ish
ed
 o
n 
01
 Ja
nu
ar
y 
19
98
. D
ow
nl
oa
de
d 
on
 3
0/
06
/2
01
3 
13
:4
3:
18
. 
View Article Online
work, a 0.2 mol l21 NaOH solution and Fe(NO3)3 in 0.4 mol l21
nitric acid solution were chosen for the R1 and R2 reagents,
respectively.
Study of microwave oven conditions
The efficiency of hydrolysis of the ASA is influenced by the
microwave oven power level (Fig. 3). The microwave oven has
five power levels corresponding to 60–600 W output. Increas-
ing the power level (60–600 W) gave continuous increase in the
sensitivity followed by irreproducible results (probably due to
the pulsed way that the energy is supplied by the magnetron)
was observed up to 420 W. The best hydrolysis efficiency and
more reproducible results were obtained using maximum power
(600 W). However, when maximum power was used bubble
formation occurred and caused problems in the measurements.
Efficient removal of the bubbles generated during the micro-
wave-assisted hydrolysis was obtained when a T-debubbler was
incorporated in the FI system maintaining a higher sensitivity.
Therefore, a maximum power level was selected for this work.
To obtain reproducible results, it is recommended that sample
injections are started 5 min after turning on the microwave oven.
Study of FI parameters
The effect of the reactor coil length B1 placed inside the
microwave was studied between 100–300 cm using PTFE
tubing (0.8 mm id) and maintaining constant all other
parameters as described above. Increasing the length of B1 coil,
a continuous increase of the sensitivity was observed (Fig. 4).
There was an increase of only 15% in sensitivity when B1 coil
length was increased from 200 to 300 cm. Therefore, a length of
200 cm was chosen as a compromise between sensitivity and
sample throughput.
The effect of the sample and R1 reagent volumes (L1 and L2)
was studied by changing the lengths of L1 and L2 loops in the
manual injector. The volumes were varied equally between 62.5
and 312.5 ml. The absorbance was found to increase with the
sample volume up to 250 ml, without meaningful increase to
312.5 ml. Thus, 250 ml volumes were chosen for the sample and
R1 reagent. 
The influence of the reactor coil lengths B2 and B3 on the
sensitivity of the FI method was studied in the range 100–300
and 50–200 cm, respectively. The results showed that the
absorbance decreased with increasing length due to the
dispersion of the sample zone. A length of 100 cm was chosen
for B2 in order to minimize the dispersion and maintain an
efficient cooling time. A length of 100 cm was chosen for B3 in
order to obtain ideal mixing of the hydrolysed sample with
Fe(NO3)3 solution in 0.4 mol l21 nitric acid and attain an
appropriate acid medium for the solution flowing through the
detector.
The C1 and C2 water carrier flow rates were optimized by
varying simultaneously these streams while keeping constant
the flow rate of R2 reagent stream at 3.9 ml min21. When flow
rates were increased from 1.5 ml min21 to 4.0 ml min21 it was
found that the sensitivity increased up to 3.4 ml min21, above
which it showed a slight decrease. Therefore, flow rates of 3.4
ml min21 were selected for both C1 and C2 water carrier
streams.
The effect of flow rate of ferric nitrate reagent solution on
sensitivity was studied using 1.0% (w/v) Fe(NO3)3 in 0.4
mol l21 nitric acid and varying the flow rate from 1.0 to 4.0
ml min21. A decrease was observed in the sensitivity with an
increase of the flow rate probably due to a decrease in the final
pH. The intensity of absorption of the FeIII–salicylate complex
depends on the pH value and it has been reported that maximum
intensity occurs at pH 2.5–3.0 and is five times higher than that
at pH 1.5.15 In all further experiments, a flow rate of 1.3 ml
min21 was selected for the R2 reagent. No baseline drift was
observed for blankruns (injecting water instead of ASA
solution).
Effect of the Fe(NO3)3 concentration
The influence of the Fe(NO3)3 concentration was studied in the
range 0.125 to 1.0% (w/v). The results showed that the
absorbance signals corresponding to 250 mg ml21 ASA
increased with increasing concentration of the R2 reagent up to
Fig. 2 Effect of the NaOH concentration (R1 reagent; L2, 250 ml) on the
determination of ASA (200 mg ml21; L1, 250 ml) using 1% (w/v) Fe(NO3)
in 0.4 mol l21 nitric acid (R2 reagent flowing at 3.9 ml min21). 
Fig. 3 Effect of the microwave power (%) on the efficiency of hydrolysis
of ASA (400 mg ml21) using NaOH (0.2 mol l21). Reactor coils B1, B2 and
B3 were 300, 200 and 100 cm, respectively. 
Fig. 4 Effect of the reactor coil B1 length (PTFE tubing, 0.8 mm id) placed
inside the microwave oven on the hydrolysis of ASA (400 mg ml21) with
reactor coils B2 and B3 of 200 and 100 cm, respectively. The other variables
were as described in Fig. 2.
Analyst, May 1998, Vol. 123 1013
Pu
bl
ish
ed
 o
n 
01
 Ja
nu
ar
y 
19
98
. D
ow
nl
oa
de
d 
on
 3
0/
06
/2
01
3 
13
:4
3:
18
. 
View Article Online
0.50% (w/v), above which it remained constant. Consequently,
a 0.50% (w/v) Fe(NO3)3 in 0.4 mol l21 nitric acid was
chosen.
Interferences and recovery studies
The selectivity of the proposed method was evaluated by
studying the effect of common excipients used in pharmaceut-
ical preparations and other active substances on the determi-
nation of 200 mg ml21 ASA. No interference in the flow
procedure was observed up to a tenfold excess of caffeine,
lactose, starch, sodium carbonate, sucrose, saccharin and
glucose. However, when 2.0 mg ml21 citric acid was added, the
absorbance decreased about 20% because of the complexation
of FeIII ions by this compound. Since FeIII is reduced to FeII by
ascorbic acid, a negative interference could be expected, but
ascorbic acid is easily decomposed at high pH even at room
temperature and produced a slight error in the determination
only when present in concentrations four times higher than
ASA.
The limit of free SA that could be present in ASA samples
due to hydrolysis was tested by adding standard salicylic acid
solution to ASA samples and results compared to unspiked
standard solution. ASA samples added with salicylic acid up to
0.3% (w/v) did not cause any interference in the proposed FI
procedure. Otherwise the content of SA as an impurity in plain
tablets is limited to 0.1–0.3% w/v. Thus, SA content correction
is not needed. Additionally, the effect of the interference caused
by SA was tested by processing ASA samples similarly but
without the hydrolysis step [replacing NaOH (L2) by water]. No
AS signals were obtained even at the lower acidity of R2 reagent
0.50% (w/v) [Fe(NO3)3 in 0.2 mol l21 nitric acid]. Therefore,
the interference from SA in the ASA determination was
negligible for all samples analysed. 
The accuracy of the proposed flow injection method was
evaluated by analysing tablets spiked with known amounts of
aspirin. In this study, 50 100 and 150 mg ml21 of ASA were
added to each tablet sample solutions (Table 1). Recoveries of
99.1–101.0% of ASA from four commercial tablets (n = 5)
were obtained using the FI procedure. This is good evidence for
the accuracy of the proposed FI procedure.
Calibration graph and applications
In order to demonstrate the applicability of the proposed FI
method to commercial samples it was applied to the determi-
nation of ASA in tablets. Fig. 5 shows typical transient signals
corresponding to a linear calibration graph for ASA and
injections of seven samples of tablets. The results of the analysis
of the tablets given in Table 2 compared favourably with results
obtained using the standard titration method (FI = 3.6 3 1021
+ 1.00 RM; r1 = 0.9997, where FI and RM are flow injection
and reference methods, respectively) and also agreed with those
declared on the labels (FI = 1.0 3 1021 + 0.99 DV;
r2 = 0.9996, where DV is the declared value) confirming the
accuracy of the FI spectrophotometric method with on-line
microwave-assisted alkaline hydrolysis. The calibration graph
for ASA was linear in the concentration range from 25 to 250
mg ml21 (A = 4.3 3 1022 + 3.1 3 1023 C; r = 0,9998, where
A is the absorbance and C the concentration of ASA in mg ml21)
with a detection limit (three times the signal blank/slope) of
4.0 mg ml21 ASA and a throughput of 90 samples h21. The
repeatability for ten successive measurements of 200 mg ml21
ASA presented a relative standard deviation of 0.40%.
Conclusion
The proposed FI method with on-line microwave hydrolysis of
ASA is simple, precise, accurate and sensitive enough to permit
the determination of ASA in tablets. The results obtained by the
FI method compared well with those obtained by a reference
method. However, while the back-titration method for the
determination of ASA requires heating over reflux for 10 min,
the proposed method is less time consuming increasing the
Table 1 Recovery of ASA from tablets spiked with three different
concentrations by the proposed FI method
ASA/mg ml21*
Recovery
Tablets Added Found (%)
Melhoral 50 49.8 99.6
100 99.8 99.8
150 149.7 99.8
Cibalena 50 49.5 99.1
100 99.3 99.3
150 150.3 100.2
Fontol 50 49.7 99.5
100 98.9 98.9
150 149.6 99.7
Bufferin 50 50.6 101.0
100 100.7 100.7
150 148.9 99.3
* n = 5.
Fig. 5 Transient absorbance signals obtained for ASA standards and
sample solutions of tablets. From left to right: triplicate signals for six
reference solutions (25–250 mg ml21) followed by six consecutive signals
for A, AAS; B, Melhoral; C, Doril; D, Aspirina; E, Cibalena; F, Bufferin; G,
Fontol and standard solutions again.
Table 2 Comparison of results obtained by reference and proposed FI
methods for ASA
ASA/mg tablet21 ± s
Relative
Flow error (%)
Label value/ Reference injection
Tablets mg tablet21 method* method† Re1 Re2
AAS 500 498.8 ± 3.9 503.5 ± 3.6 +0.9 +0.7
Melhoral 500 499.6 ± 2.1 494.4 ± 4.5 21.0 21.1
Doril 500 500.2 ± 2.4 498.2 ± 4.0 20.4 20.4
Aspirina 500 494.6 ± 4.4 495.9 ± 4.9 +0.3 20.8
Cibalena 200 198.4 ± 1.0 200.8 ± 1.3 +1.2 +0.4
Bufferin 500 —† 503.6 ± 3.3 —‡ +0.7
Fontol 650 646.7 ± 5.5 651.6 ± 5.7 +0.8 +0.2
* Average of three measurements back-titration method according to
BP 1980. † Average of five measurements. ‡ Buffered tablets containing
CaCO3 and MgCO3. Re1 FIA-spectrophotometric vs. back-titration method
value; Re2 FIA-spectrophotometric vs. declared value.
1014 Analyst, May 1998, Vol. 123
Pu
bl
ish
ed
 o
n 
01
 Ja
nu
ar
y 
19
98
. D
ow
nl
oa
de
d 
on
 3
0/
06
/2
01
3 
13
:4
3:
18
. 
View Article Online
speed of the sample pre-treatment, and therefore is useful for
routine analysis of the ASA in tablets. Additionally, it decreases
the possibility of interference caused by SA, the major
decomposition product of ASA, which is critical in ASA
determination. A sample throughput of 90 samples h21 was
obtained.
Financial support of FAPESP (Fundaça˜o de Amparo a` Pesquisa
do Estado de Sa˜o Paulo, Processes 91/2637-5 and 92/2637-5),
PADCT/CNPq (Process 62.0060/91-3) and also the scholarship
granted by CNPq to A.V.P. are gratefully acknowledged.
References
1 Connors, K. A., Amidon, G. L., and Kennon, L., Chemical Stability
of Pharmaceuticals, Wiley, New York, 1979, p. 151.
2 British Pharmacopoeia, HMSO, London, 1980, p. 733.
3 United States Pharmacopeia XXII, US Pharmacopeial Convention,
Rockville, MD, 1990, p. 113.
4 Levine, J., J. Pharm. Sci., 1961, 50, 506.
5 Mazzeo, P., Quaglia, M. G., and Segnalini, F., J. Pharm. Pharmacol.,
1982, 34, 470.
6 Verma, K. K., and Jain, A., Anal. Chem., 1986, 58, 821.
7 Lange, W. E., and Bell, S. A., J. Pharm. Sci., 1966, 55, 386.
8 Miles, C. I., and Schenk, G. H., Anal. Chem., 1970, 42, 656.
9 Ghosh, D., and Prasad, B. B., Chem. Anal., 1984, 29, 679.
10 Bevitt, R. N., Mather, J. R., and Sharman, D. C., Analyst, 1984, 109,
1327.
11 Verstraeten, A., Roets, E., and Hoogmartens, J., J. Chromatogr.,
1987, 388, 210.
12 Kirchhoefer, R.D., J. Pharm. Sci., 1980, 69, 1188.
13 Fogel, J., Epstein, P., and Chen, P., J. Chromatogr., 1984, 317,
507.
14 Juhl, W., and Kirchhoefer, R. D., J. Pharm. Sci., 1980, 69, 544.
15 Kelly, C. A., J. Pharm. Sci., 1970, 59, 1053.
16 Lopez-Fernandez, J. M., Luque de Castro, M. D., and Valcarcel, M.,
J. Autom. Chem., 1990, 12, 263.
17 Haswell, S. J., and Barclay, D., Analyst, 1992, 117, 117.
18 Arruda, M. A. Z., Fostier, A. H., and Krug, F., J. Braz. Chem. Soc.,
1997, 8, 39.
19 Morales-Rubio, A., Mena, M. L., and McLeod, C. W., Anal. Chim.
Acta, 1995, 308, 364. 
20 Burguera, M., and Burguera, J. L., Anal. Chim. Acta, 1986, 179,
351.
21 Martı´nez-Calatayud, J., Flow Injection Analysis of Pharmaceuticals,
Taylor & Francis, Hants., 1996, p. 130.
22 Bouhsain, Z., Garrigues, S., Morales-Rubio, A., and de la Guardia,
M., Anal. Chim. Acta, 1996, 330, 59.
23 Luque Pe´rez, E., and Haswell, S. J., Anal. Proc., 1995, 32, 85.
Paper 8/00036K
Received January 2, 1998
Accepted February 17, 1998
Analyst, May 1998, Vol. 123 1015
Pu
bl
ish
ed
 o
n 
01
 Ja
nu
ar
y 
19
98
. D
ow
nl
oa
de
d 
on
 3
0/
06
/2
01
3 
13
:4
3:
18
. 
View Article Online