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Introduction
Paracetamol and caffeine appear to be associated in many
commercial formulations because caffeine increases the
analgesic character of paracetamol.
Paracetamol (acetaminophen, N-acetyl-p-aminophenol, 4-
acetamidophenol) is used as analgesic and antipyretic agents.
Its action is similar to aspirin, and is a suitable alternative for
patients who are sensitive to aspirin.
1
Numerous methods have
been reported for the determination of paracetamol in
pharmaceuticals based on different techniques: volumetry,
2
spectrophotometry,
3
–
5
spectrofluorometry,
6
high-performance
liquid chromatography (HPLC) with photometric
7
–
9
and FTIR
10
detection, thin-layer chromatography,
11
Raman spectrometry,
12
near infrared reflectance spectrometry,
13
electroanalytical
14,15
and FI-FTIR
16
methods.
Caffeine (7-methyltheophylline, 1,3,7-trimethylxantine), a
xanthine alkaloid, is a powerful stimulant of the central nervous
system. Several methods have been reported for its
determination: spectrophotometry,
17,18
HPLC
19,20
and gas
chromatography (GC).
21
There are many methods for the simultaneous determination
of paracetamol and caffeine, including spectrophotometric,
22
–
25
electroanalytical,
26,27
HPLC
28
–
30
and GC techniques.
31
HPLC and GC methods require expensive instrumentation and
are relatively highly time-consuming. Although,
spectrophotometric methods are simpler and faster, the
simultaneous determination of both analytes is not possible by
conventional direct UV absorption measurements, because of
the spectral overlap. To resolve this problem, a derivative
22,23
absorbance ratio technique
25
and PLS calibration
24
have been
used.
In this paper, we propose a simple, fast and inexpensive
spectrophotometric continuous-flow sensor for the simultaneous
determination of paracetamol and caffeine based on the use of
C
18
silica gel as an active solid phase. This biparameter sensor
is based on the strong retention of caffeine in a column filled
with C
18
silica gel (placed on line just before the cell). The
paracetamol passes through it while developing an analytical
signal in the solid phase placed into the flow cell (also C
18
silica
gel). Then, caffeine is conveniently eluted from the column and
also carried to the cell in the detector. In both cases, the
intrinsic UV absorbance of the analyte is used as an analytical
signal. Thus, the solid phase acts as a dual sensing zone that
responds successively to the two analytes. Thus, a temporary
discrimination in the detection is carried out to achieve a
satisfactory resolution of the mixture with this simple UV
spectrophotometric flow-through biparameter optosensor. It
was successfully applied to the determination of these analytes
in pharmaceuticals.
Experimental
Reagents
All solutions were prepared from analytical reagent-grade
chemicals using deionized water.
Paracetamol (Fluka) and caffeine (Merck) stock solutions
1241ANALYTICAL SCIENCES NOVEMBER 2002, VOL. 18
2002 © The Japan Society for Analytical Chemistry
Simultaneous Determination of Paracetamol and Caffeine by
Flow Injection
–
Solid Phase Spectrometry Using C
18
Silica Gel
as a Sensing Support
P. O
RTEGA-BARRALES, R. PADILLA-WEIGAND
, and A. MOLINA-DÍAZ
†
Department of Physical and Analytical Chemistry, Faculty of Experimental Sciences, University of Jaén,
Paraje Las Lagunillas, E-23071 Jaén, Spain
A continuous and simple UV-photometric flow-through biparameter-sensing device has been developed for the
simultaneous determination of paracetamol and caffeine at 275 nm. The sensor is based on temporary sequentiation in
the arrival of the analytes to the sensing zone by on-line separation using C
18
bonded phase beads (the same as that used
in the sensing zone) placed into a minicolumn just before the flow cell. The sample containing these compounds is
injected into the carrier solution; paracetamol is determined first because it passes through the minicolumn, while caffeine
is strongly retained in it. Then, caffeine is conveniently eluted from the precolumn and develops its transitory signal.
Using 200 µl of a sample and deionized water as a carrier, the analytical signal showed a very good linearity in the ranges
of 10
–
160 µg ml
–1
and 3.5
–
50 µg ml
–1
with detection limits of 0.75 and 0.56 µg ml
–1
for paracetamol and caffeine,
respectively. If deionized water with the pH adjusted at 12 was used as a carrier solution, these parameters were 25
–
400
and 4
–
55 µg ml
–1
with 2.0 and 0.50 µg ml
–1
as the detection limits, respectively. The biparameter optosensor was
satisfactorily applied to the simultaneous determination of these two analytes in pharmaceuticals.
(Received April 23, 2002; Accepted August 28, 2002)
†
To whom correspondence should be addressed.
E-mail: amolina@ujaen.es
(1000 mg l
–1
) were prepared by directly dissolving the drug in
deionized water. Only freshly prepared solutions of
paracetamol were used due to the low stability. The caffeine
solution was stable for at least four weeks at 4
–
5˚C. Work
solutions were prepared fresh daily by appropriate dilution with
deionized water.
The following carrier solutions used were: Carrier 1 (C
1
),
deionized water; Carrier 2 (C
2
), deionized water adjusted at pH
12 with NaOH (Panreac). A 10% (v/v) aqueous methanol
(Panreac) solution was used as an eluting medium.
C
18
bonded silica (Waters) with average particle sizes of 55
–
105 µm, packed both in a precolumn (1 mm i.d.) of 27 mm
length and in a Hellma 138-QS flow-through cell, was used to
measure the solid-phase UV light absorption in the sensing
zone.
Instrumentation
A Varian Cary 50 Spectrophotometer, equipped with a Hellma
138-QS flow cell (1-mm optical path length and 50 µl inner
volume), was used for absorbance measurements. It was
controlled by a microprocessor fitted with the WIN UV
software package.
A four-channel Gilson Minipuls-3 peristaltic pump with a rate
selector, teflon tubing of 0.8 mm i.d. and two Rheodyne Model
5041 injection valves were also used. One of them was the
injection valve, and the other was connected as a selection
valve.
Procedure
The continuous-flow diagram used is shown in Fig. 1. A
sample solution (200 µl) containing both paracetamol (10
–
160
µg ml
–1
) and caffeine (3.5
–
50 µg ml
–1
) was injected into the
carrier solution (C
1
) and pumped at a flow rate of 1.23 ml min
–1
.
Caffeine was retained on a solid support (C
18
) placed in the
precolumn, while paracetamol, which passed through it, was
carried to the flow cell and retained within. The paracetamol
retention signal was monitored at the working wavelength (275
nm).
When paracetamol was totally eluted by the carrier, itself, by
turning the selection valve, a 10% (v/v) aqueous methanol
solution was used as an eluting solution for caffeine retained in
the precolumn, carrying it, in turn, to the flow cell. Its
transitory retention signal was also monitored at 275 nm. Then,
by again turning the selection valve, the baseline was restored
and it became possible to make another sample injection.
The same procedure was carried out with carrier C
2
. In this
case, the sample solution inserted into the carrier solution
contained 25
–
400 µg ml
–1
of paracetamol and 4
–
55 µg ml
–1
of
caffeine.
Results and Discussion
Preliminary study
The spectral features of both analytes in homogeneous
solutions of paracetamol, caffeine and a mixture of both were
previously established; they are shown in Fig. 2. These spectra
were obtained using a cell of 1-mm optical path length. The
maximum absorbance wavelengths were 245 nm for
paracetamol and 275 nm for caffeine. Because the scans of the
analytes overlapped, it was impossible to conduct a
simultaneous determination by conventional spectrophotometric
measurements without significant errors.
Because caffeine is a minor constituent in pharmaceuticals (in
a ratio from 0.3 to 0.02 times that of paracetamol), we chose
275 nm as the wavelength for a simultaneous determination of
the analytes, the peak height being used as an analytical signal.
Optimization of variables
All of the variables were studied with deionized water (C
1
) as
the carrier solution.
In order to choose the most convenient solid support for both
analytes, several anionic-exchange resins (Sephadex QAE A-25
and DAE A-25), resins without exchangeable groups and no-
polar sorbents (C
18
silica gel) were tested
Caffeine was only retained in C
18
because of the absence of
functional ionic groups; its retention was found to be very
strong. Paracetamol was also retained in C
18
silica gel, but not
very strongly; however, it was not retained on Sephadex QAE
A-25 because at the carrier pH value it was not ionized.
7
C
18
gel
was selected as a solid support. Although the changes in the
positions of the absorption maxima were observed for both
analytes when the species were retained on the sorbent, the
analytical signals were about 36 and 43-times higher than that
obtained in solution for paracetamol and caffeine, respectively.
Level of the packing in the flow cell and amount of resin in the
packed precolumn
The level of support in the flow cell is a very important
variable. This level was just the necessary one to fill it up to a
sufficient height (15 mm), permitting a light beam to pass
completely through the solid layer.
With only C
18
silica gel in the flow cell, it is impossible to
1242 ANALYTICAL SCIENCES NOVEMBER 2002, VOL. 18
Fig. 1 Schematic diagram of the FIA system: C
1
and C
2
, carriers;
E, eluting; S, sample; PP, peristaltic pump; S, injection valve; SV
1
and SV
2
, selection valves; P, precolumn; D, detector; FC, flow cell;
W, waste; PC, computer.
Fig. 2 Scans in homogeneous solution: 50 µg ml
–1
of paracetamol
(1), 20 µg ml
–1
of caffeine (2) and mixture (3). They are made in
stopped-flow with a 1-mm path length.
simultaneously determine a mixture of both analytes for the
same reasons as mentioned above (in a preliminary study). We
tried to separate them on-line before they reached the detection
zone by means of a different retention-elution process of the
analytes in C
18
. We therefore used a precolumn filled with the
same solid support as that used in the flow cell (C
18
) just before
the cell in order to retain caffeine in it, while paracetamol was
carried to the flow cell.
The precolumn length (and consequently, the amount of silica
gel) was studied from 0.5 to 35 mm, using an i.d. of 1 mm.
Separation of the analytes was completed for 27 mm (Fig. 3).
We chose a precolumn length of 27 mm because it gave a
satisfactory and complete separation in the minimum possible
time.
Influence of the carrier pH, eluent nature and sample pH
The effect of the pH on the retention of both analytes in the
solid support was studied a) in the carrier and b) in the sample
by injecting each one of them alone. The single monochannel
manifold in Fig. 1 was used, but without a precolumn.
Deionized water with an appropriate concentration of HCl or
NaOH (pH ranging from 2 to 12) was used as carrier.
The obtained results are shown in Fig. 4. It can be seen that
the retention of caffeine was found to be independent of the pH
from 2 to 12, whereas the signal of paracetamol decreased
drastically at pH values above 10 due to dissociation of the
phenolic group (pK
a
= 9.5). In this way, analytical signals were
similar for both analytes at pH = 12. This is a very interesting
result: because of the usually higher concentration values of
paracetamol with respect to caffeine in pharmaceuticals (from 3
to 44), the simultaneous determination of both analytes in FIA
systems is usually not possible due to the great difference
between both signals. Thus, two different aliquots would have
to be injected in order to obtain concentrations appropriate for
the respective calibration line.
In the sensor developed here, a simultaneous determination
can be achieved just by choosing the appropriate carrier pH
value, according to the paracetamol/caffeine ratio found in the
pharmaceuticals. Therefore, in order to analyze mixtures of
both analytes in different proportions, deionized water (C
1
) and
aqueous NaOH (pH 12) (C
2
) were selected as carrier solutions.
Although paracetamol was easily eluted by the two carrier
solutions, the elution of caffeine from the solid support could
not be performed by any of them because of its strong retention
on it. Therefore, a study of the effect of different solvents as
eluting agents for caffeine had to be performed. Two hydro-
alcoholic solvent mixtures ranging between 5 and 25% (v/v)
from methanol and ethanol were tested. For this study, the
single monochannel manifold shown in Fig. 1 was used.
Both mixtures could elute caffeine when the alcoholic
concentration increased; both the elution time and the analytical
signal decreased. As for the nature of the eluting solution, for
the same concentration, the use of ethanol produced a decrease
in the analytical signal of 30% compared to the use of methanol,
whereas the decrease in the elution time was lower (only 10%);
10% aqueous methanol (v/v) was selected as the eluting
solution.
The absorbance value for caffeine was not influenced by the
sample pH in the tested range (2
–
12). The signal from
paracetamol decreased at sample pH values above 10, as was
also observed in a study of the influence of the carrier pH.
Hence, it was necessary to adjust the sample pH value to that of
the carrier solution only when the value of the carrier solution
pH was 12.
Optimization of FIA variables
A study of the influence of the flow system variable (flow rate
and sample volume) was performed.
The effect of varying the flow rate from 0.7 to 1.66 ml min
–1
is shown in Fig. 5. An increase in the flow rate did not
significantly influence the analytical signals for both analytes
(100 and 10 µg ml
–1
of paracetamol and caffeine, respectively);
however, it did produce a more significant decrease in the
elution time from 0.7 to 1.23 ml min
–1
. A flow-rate value of
1.23 ml min
–1
was chosen; a flow rate value beyond this could
cause excessive pressure in the system.
1243ANALYTICAL SCIENCES NOVEMBER 2002, VOL. 18
Fig. 3 FIAgram corresponding to the influence of the precolumn
length on the separation. (1) Without precolumn, (2)
–
(5): 5, 20, 23
and 27 mm of precolumn length, respectively.
Fig. 4 Influence of the carrier pH: (1) 100 µg ml
–1
of paracetamol;
(2) 10 µg ml
–1
of caffeine.
Fig. 5 Effect of the flow rate on the elution time. Inset, absorbance
signal vs. flow rate: (1) paracetamol, (2) caffeine.
By injecting in the flow system different volumes of a
solution containing both analytes, paracetamol (25 µg ml
–1
) and
caffeine (5 µg ml
–1
), the effect of this variable on the analytical
signal could be assessed (Fig. 6). The absorbance signal
increased linearly for paracetamol up to 1500 µl (A = 0.13 + 3.3
× 10
–4
v) with increasing injection volume (v, µl). Beyond this
volume, the signal increase was very low. This increase was
linear (A = 0.03 + 3.1 × 10
–4
v) for caffeine in all ranges tested
(from 40 to 2300 µl) due to the strong retention of caffeine on
the sensing zone.
One of the main advantages of the sensor is the potential
increase in the sensitivity as the sample volume taken for
analysis is increased. This makes it possible to select the most
appropriate volume of sample taking while considering the
concentrations of samples that are going to be analyzed.
Analytical features of the proposed method
Calibration graphs were obtained simultaneously for both
analytes by following the proposed method for both carrier
solutions. The analytical figures of merit for 200 µl sample
volume are given in Table 1. Very good linearity was found in
the concentration ranges a) 10
–
160 and 3.5
–
50 µg ml
–1
for
paracetamol and caffeine, respectively, using C1 as the carrier
solution, and b) 25
–
400 and 4
–
55 µg ml
–1
for carrier C
2
,
respectively. The detection and quantification limits were
estimated as the concentration of the analyte that produced an
analytical signal equal to three
32
and ten
33
-times the standard
deviation of the background absorbance, respectively. The
reproducibility was established for ten analyses of solutions
containing 50/5 µg ml
–1
of paracetamol/caffeine with C
1
and
250/8 µg ml
–1
with C
2
. The sampling frequency for the
simultaneous determination of both analytes was 15 and 20 h
–1
with C
1
and C
2
, respectively.
Effect of foreign species
In order to determine the effect of foreign species, a tolerance
study was performed with those compounds that are usually
found along with paracetamol and caffeine in pharmaceuticals.
The study was carried out with 100 and 20 µg ml
–1
of
paracetamol and caffeine, respectively, for both carriers.
Foreign species were added to the samples at concentrations
higher than those usually found in pharmaceutical preparations.
Also, an interference study in a conventional homogeneous
solution (without solid phase) was performed with carrier C1
and by using 500 and 50 µg ml
–1
of paracetamol and caffeine,
respectively.
The tolerance limit was established as the maximum
concentration of foreign species that caused a relative error of
±3% in the analytical signal. As can be seen, the tolerance to
the presence of foreign species (Table 2) is, in general, very
much higher than the amount in which these compounds are
usually found together with the analytes in pharmaceuticals. In
addition, the tolerance limits are very much higher than those
corresponding to the determination of the analytes by direct UV
measurements in the solution method (i.e. up to 40-times higher
for dimenhydrinate than in solution without a solid support).
This is due to the selectivity conditions stated concerning the
active solid support, which excludes from it and, consequently,
from the detection zone, all those species that can not be
retained on it in the working conditions. It should be
emphasized that the dimenhydrinate tolerance drastically
increases when using carrier C
2
. This makes possible the
determination of paracetamol and caffeine in commercial
preparation “Saldeva forte”, where the ratios
dimenhydrinate/paracetamol (15:500) and dimenhydrinate/
caffeine (15:50) are higher than the tolerance level when using
carrier C
1
(and, in turn, 40-times higher than the tolerance level
of the same method in solution without a solid support).
Application of the method
The proposed sensor was applied to the determination of
paracetamol and caffeine in pharmaceuticals using the standard
calibration graph method for an injection volume of 200 µl.
The results for this method (Table 3) are in a very good
concordance with the theoretical contents of both analytes given
1244 ANALYTICAL SCIENCES NOVEMBER 2002, VOL. 18
Fig. 6 Influence of the sample volume: (1) 25 µg ml
–1
of
paracetamol, (2) 5 µg ml
–1
of caffeine.
Table 1 Analytical parameters
Paracetamol
Caffeine
Parameter
C
2
C
2
C
1
C
1
0.014 – 0.003 – 0.005 0.002
5.5 × 10
–3
2.1 × 10
–3
1.72 × 10
–2
1.70 × 10
–2
10
–
160 25
–
400 3.5
–
50 4
–
55
0.9997 0.9998 0.9999 0.9992
0.75 2.0 0.56 0.50
2.5 6.7 1.9 1.7
0.5 2.1 3.1 1.8
Calibration line
Intercept (absorbance)
Slope (ml µg
–1
)
Linear dynamic range
(µg ml
–1
)
Correlation coefficient
Detection limit
(µg ml
–1
)
Quantification limit
(µg ml
–1
)
RSD, % (n = 10)
Sample volume (200 µl)
a. Maximum ratio tested.
Table 2 Interference study for caffeine and paracetamol
Tolerance level
(µg ml
–1
interfering species/µg ml
–1
analyte)
Caffeine
Paracetamol
Solid
phase
carrier
C
2
Solid
phase
carrier
C
1
Homo
geneous
solution
Homo
geneous
solution
Solid
phase
carrier
C
2
Solid
phase
carrier
C
1
Foreign species
Saccharose 3.5 4 1 0.8 1 0.2
Lactose > 50
a
30 2.5 > 10
a
61
Saccharin > 4
a
3 0.8 > 1
a
0.3 0.1
Salicylamide 2 2 0.4 0.4 1.6 0.06
Brompheniramine
maleate 0.5 1 0.05 2 0.5 0.03
Codeine 0.1 0.1 0.02 0.02 0.08 < 0.01
Dimenhydrinate 0.025 0.4 < 0.01 0.005 0.04 < 0.001
by the manufacturers. It should be noted that the simultaneous
determination of the analytes in “Melabón” and “Ilvico” (as
well as in “Saldeva forte”, too) could be possible by using
carrier C
2
due to the high ratios of paracetamol/caffeine in these
commercial preparations.
In order to check the accuracy of the proposed procedure, a
recovery study was also performed by adding different known
amounts of the analytes to four pharmaceuticals: two of them
with carrier C
1
and other two with carrier C
2
. The percentage of
recovery is given in Table 4.
Conclusions
The developed flow-injection solid-phase UV
spectrophotometric system is very simple. The C
18
bonded
phase silica gel beads packed in the cell respond alternately to
the analytes. Their arrival to the sensing solid support is time
discriminated: a precolumn strongly retains on-line one of them
(caffeine), whereas the other one reaches the sensing microzone.
The second analyte also develops a transitory signal after being
eluted from the precolumn. Therefore, the used solid support
(C
18
silica gel) performs three functions:
a) A transitory retention and preconcentration of the analytes in
the detection zone, itself;
b) An on-line separation of the analytes in the precolumn;
c) To drastically increase the selectivity (increase factors for
tolerance levels of 10, 20, and even 40 are achieved).
Finally, just by selecting the carrier solution C
2
(pH = 12) the
simultaneous determination (one only injection) of paracetamol
and caffeine at paracetamol/caffeine (w/w) ratios as high as 100
can easily be performed.
Acknowledgements
The authors are grateful to the Dirección General de Enseñanza
Superior (DGES) of the Ministerio de Educación y Cultura
(project No. PB98-0301) for financial support.
References
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1245ANALYTICAL SCIENCES NOVEMBER 2002, VOL. 18
a. Melabón: paracetamol 350 mg, caffeine 8 mg, propifenazone 200 mg. b. Ilvico: paracetamol 500 mg, caffeine 30 mg,
brompheniramine maleate 3 mg, saccharose 4.85 mg, sodium cyclamate 35 mg, sodium saccharin 7.5 mg. c. Saldeva forte: paracetamol
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2
.
**Determination by using carrier C
1
.
Table 3 Determination of paracetamol and caffeine in pharmaceutical preparations
Pharmaceutical
Recovery mean ± SD
(%)
Labeled
(mg/unit)
Ratio
Paracetamol
Caffeine Paracetamol/caffeine Paracetamol Caffeine
Melabón
a
* 350 8 44 99.9 ± 1 99.2 ± 0.3
Ilvico
b
* 500 30 17 99.6 ± 0.6 99.2 ± 0.2
Saldeva forte
c
* 500 50 10 101.2 ± 0.7 99.8 ± 0.5
Saridon
d
** 250 50 5 99 ± 1 100.7 ± 0.1
Hemicraneal
e
** 300 100 3 100 ± 1 102.1 ± 0.2
Apiretal
f
** 100 —— 100 ± 0.3 —
Duorol
g
** 500 —— 100.5 ± 0.8 —
Termalgin
h
** 500 —— 99.8 ± 0.5 —
Gelocatil
i
** 650 —— 100.2 ± 0.7 —
Durvitan
j
** — 300 ——98.9 ± 0.4
a. Carrier C
1
.
b. Carrier C
2
.
Table 4 Recovery study of paracetamol and caffeine in
pharmaceuticals
Paracetamol
Caffeine
Recovery ± SD,
%
Added
(mg/unit)
Recovery ± SD,
%
Added
(mg/unit)
Pharmaceutical
25 100.5 ± 0.6 5 99.3 ± 0.6
Hemicraneal
a
50 100.2 ± 0.1 10 100.2 ± 0.2
100 100.0 ± 0.2 20 99.9 ± 0.4
25 99.3 ± 0.3 5 100.6 ± 0.3
Saridon
a
50 99.5 ± 0.1 10 100 ± 1
100 99.7 ± 0.2 20 101.5 ± 0.2
50 98.3 ± 0.5 5 98.0 ± 0.4
Melabón
b
100 101.2 ± 0.1 10 99.6 ± 0.5
200 100.3± 0.1 20 100.1± 0.2
25 99.5 ± 0.5 5 98.7 ± 0.6
Ilvico
b
50 100 ± 1 10 99.0 ± 0.3
200 99.3 ± 0.2 20 99.9 ± 0.4
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