Indian Journal of Chemistry

Vol. 48A, June 2009, pp. 812-816

Simultaneous kinetic determination of
paracetamol and caffeine using Cu(II)-
neocuproine in presence of dodecyl sulfate
by H-point standard addition method
H Tavallali* & M Sheikhaei
Payame Noor University (PNU), Shiraz, 71365-944, Iran
Email: tavallali@pnu.ac.ir, tavallali@yahoo.com
Received 6 Aug 2008; re-revised and accepted 18 May 2009
A simple, feasible and selective kinetic spectrophotometric
method for simultaneous determination of paracetamol and
caffeine using H-point standard addition method is described. The
method is based on difference in the rate of oxidation of the
compounds with Cu(II)-neocuproine system and formation of
Cu(I)neocuproine complex, which is monitored at 453 nm and at
pH 5.0 in the presence of sodium dodecyl sulfate. Experimental
conditions such as pH, reagents concentrations, ionic strength and
temperature have been optimized. Paracetamol and caffeine can
be determined in the range 1.5-7.0 and 0.1-3.0 g ml
-1
respectively. The proposed method has been applied for the
determination of paracetamol and caffeine in pharmaceutical
samples with satisfactory results.
Keywords: Analytical chemistry, Spectrophotometry, H-point
standard addition, Paracetamol, Caffeine, Neocuproine
IPC Code: Int Cl.
8
G01N21/25
Paracetamol (4-acetamidophenol) is one of the most
common drugs used in combination with aspirin an
analgesic. Caffeine (1,3,7 trimethylxanthine) is
mainly ingested by drinking coffee, cola-beverages,
and tea and acts both as diuretic and stimulant to the
central nervous and cardiovascular system. The use of
the mixture of paracetamol and caffeine as an
analgesic and antipyretic is well established in
pharmaceutical formulations. In order to achieve
better curative effect and lower toxicity, it is very
important to control the amounts of paracetamol and
caffeine in pharmaceutical preparations.

Various methods like spectrofluorimetric
determination in solid phase using partial least
squares multivariate calibration
1
, flow injectionsolid
phase spectrometry using C
18
silica gel as a sensing
support
2
, flow-injection spectrophotometry
3
and high
performance liquid chromatography (HPLC)
4,5
, have
been reported for the determination of paracetamol
and caffeine in various biological and pharmaceutical
preparations. Capillary electrophoresis (CE)
6-8
and
1
H-NMR spectroscopy
9
have been developed
for simultaneous determination of a few drugs
in their formulations. Reports on simultaneous
spectrophotometric determination of paracetamol and
caffeine are rare, and sensitivity
10,11
. Thus, there is a
need for a simple, accurate and sensitive method for
simultaneous determination of paracetamol and
caffeine.


H-point standard addition method (HPSAM) is a
modification of the standard addition method and
permits direct correction of both proportional and
constant errors produced by sample matrix. The
HPSAM has been applied with analytical
spectroscopy to resolve mixtures of two components
with extensively or fully overlapped spectra
12
.
HPSAM can remove errors resulting from the
presence of an interferent and blank reagent
13-15
.
Application of H-point standard addition method to
kinetic data has been proposed for the simultaneous
determination of binary mixtures. In the present study,
a variant of the HPSAM is used which is based on the
assumption that only analyte X evolves with time and
the other species Y or interference does not affect the
analytical signal with time. In this case the
variables to be fixed are two time values, t
1
and t
2,
at
which the species Y, which does not evolve
with time or over the range between these times, has
the same absorbance
16
. The method is very simple,
selective, precise, accurate, and is a low cost
procedure for simultaneous spectrophotometric
determination of paracetamol and caffeine. The
method is based on the difference in the rate of the
reactions of paracetamol and caffeine with Cu(II) in
the presence of neocuproine. The ability to measure
trace amounts of Cu(I) in the presence of an excess of
Cu(II) is used for indirectly quantifying substances
that reduce Cu(II)-Nc reagent at a suitable pH and in
the presence of surfactant SDS. The colored Cu(I)-Nc
chelate has been measured at 453 nm as a function of
time. The method is also applied to commercial
formulations.
NOTES


813
Experimental
All chemicals and reagent (Merck) used were of
analytical grade. Doubly distilled water was used
throughout. Stock solution of Nc, (3.010
-2
M), was
prepared by dissolving 0.1562 g of Nc in 25 ml of
ethanol. Copper(II) stock solution (1.010
-1
M), was
prepared by dissolving 0.6040 g of Cu(NO
3
)
2
.3H
2
O
in 25 ml of water.
Acetic-acetate buffer pH 5.0 was prepared by
adding 1.0 M sodium hydroxide to 1.0 M acetic acid
and adjusting to pH 5. Stock solution of sodium
dodecyl sulfate (SDS, 0.1 M) was prepared by
dissolving 0.2884 g of SDS in 10 ml of water. Stock
solutions of paracetamol and caffeine (1000 g ml
-1
)
were prepared separately by dissolving 25 mg each in
25 ml of water. Most of the solutions were
prepared daily.
UV-vis absorbance spectra were recorded on a
Perkin Elmer Lambda 2 (Federal Republic of
Germany) scanning spectrophotometer which was
equipped with a 1 cm path length glass cell and
spectrophotometer attached to a Pentium 200 MHz
computer. JENWAY (model 3510) pH meter with a
combined glass electrode was used for pH
measurements.
The reagent solution was prepared in 10 ml
volumetric flask by mixing stock solutions of 2 ml of
Nc solution, 0.6 ml of Cu(II), 2 ml of buffer (pH 5.0,
1.0 M) and 50 l of SDS solution, and diluting up to
the mark with water. For each measurement, 3 ml of
reagent solution was transferred to the
spectrophotometric cell and the absorbance of this
solution was set to read zero at 453 nm. Then, an
appropriate amount of paracetamol and/or caffeine in
the concentration ranges 1.5-7.0 and 0.1-3.0 g ml
1
,
respectively, was injected into the cell using a 100 l
syringe and stirred manually for a few seconds and the
variation of the absorbance versus time was recorded
immediately. The absorbance was measured at 453 nm
with 1 s time interval for each sample. Simultaneous
determination of paracetamol and caffeine with
HPSAM was performed by measuring the absorbances
at 10 and 200 s after initiation of the reaction.

Results and discussion
Spectrophotometery based methods in the visible
region involve redox reactions in which a colored
compound is formed as result of the reaction. The
present method is based on the reaction of Cu(II) in
the presence of Nc and SDS in buffered medium. The
Cu(I)-Nc complex formed was determined
spectrophotometerically. In principle, the Cu(II)-Nc
systems allows the spectrophotometric determination
of a reducing agent, A
red
, provided the redox reaction,
nCu
2+
+ 2n Nc +A
red
n [Cu (Nc)
2
]
+
+A
ox
, is
complete with the formation of an equivalent amount
of [Cu(Nc)
2
]
+
with respect to the n-electron reductant,
A
red
. Copper (II) acts as a strong oxidizing agent only
when its reduction product, Cu(I), is stabilized by a
strong complex-forming ligand, e.g., Nc. Weak
reductants should be determined either by masking
the excess of Cu(II) or by using a dilute solution
of Cu(II)
17
.
Difference in kinetic behavior of paracetamol and
caffeine accompanied with mathematical treatment of
data using HPSAM permitted simultaneous analysis of
the two compounds. Figure 1 shows the applicability of
HPSAM to the simultaneous analysis of paracetamol
and caffeine by the proposed system. The reaction rate
of paracetamol with Cu(II)-Nc system was fast, while
the reaction of caffeine was very slow.
The sensitivity and rate of reduction and complex
formation are dependent on pH of medium. The effect
of pH on the rate of Cu(I)Nc complex formation in
presence of paracetamol and caffeine was studied
over the range 2.0-6.0. Increase in pH up to 5.0,
caused an increase in the reaction rates for
paracetamol and thereafter decreased with further
increase in pH 5.0. However, over the studied range
of pH, no significant change in the reaction rate of


Fig 1Absorbance changes of Cu (II)Nc system versus time in
the reaction with (a) caffeine (1.0 g ml
-1
), (b) paracetamol
(2.5 g ml
-1
) and (c) a mixture of caffeine (1.0 g ml
-1
) and
paracetamol (2.5 g ml
-1
) at 453 nm.
INDIAN J CHEM, SEC A, JUNE 2009


814
caffeine with Cu(II)-Nc system was observed.
Therefore, for achieving the appropriate condition for
applying HPSAM, pH 5.0 was selected as the
optimum pH.
Use of micelles has frequently been made in
catalytic
18-20
and non-catalytic
21,22
reactions. The rate
enhancement in such reactions is due to increased
reactant concentration in the micellar pseudo phase.
This concentration results in increase in sensitivity.
The effect of micelles on the system was studied
under optimum pH, using surfactants such as sodium
dodecyl sulfate (SDS), cetyltrimethyl ammonium
bromide (CTAB) and Triton X-100 (a non-ionic
surfactant). SDS was found to be the best surfactant
for HPSAM as it increased the rate of paracetamol
reaction but had no effect on the reaction rate of
caffeine. This may be due to reactant concentration in
the surfactant medium
23
. Therefore, 5.010
-4
M SDS
was selected as the optimum concentration of
surfactant in this work.
A similar behavior of Cu(II) and neocuproine
concentrations was observed. Both increased the rate
up to 6.010
-3
M and decreased thereafter for
paracetamol. The reagent concentrations had no effect
on the reaction rate of caffeine. Therefore, the reagent
concentrations were kept at 6.010
-3
M each.
As expected, increasing temperature above 20C
increased the rate of reaction of paracetamol and
caffeine, the rate of paracetamol being faster than
caffeine. For ease of operation, reaction was carried out
at 25C.
The ionic strength of reaction mixture was adjusted
by addition of up to 0.7 M KNO
3
. The ionic strength
of solutions did not have any effect on the
reaction rates of paracetamol and caffeine with
Cu(II)-Nc system.
For selection of appropriate time period for
applying HPSAM, the principle followed was that the
analyte signals must be linear and interferent signal
must remain equal, over the range of concentration of
the analytes. The analytical signals of mixture of
analyte and interferent should be equal to the sum of
individual signals of the two compounds. In addition,
the difference of the slopes of the two straight lines
obtained at t
1
and t
2
must be as large as possible to
achieve good accuracy. It was possible to select
several pairs of time periods in the determination of
paracetamol. The pair of time periods which gave the
highest accuracy, greatest slope increment, and the
lowest error for analytes was selected.
The following pairs of time periods were studied:
10-150, 10-200, 15-180, 20-180, 30-200, 50-200 s,
and the
2 1
t t
A

was plotted versus C

added
variant
(Fig. 2). This yielded the concentration of
paracetamol directly from the intercept on Y-axis.
The time period pair that gave the maximum
absorbance change at 453 nm and the highest
accuracy corresponding to paracetamol concentration
was 10-200 s. Plot of Hpoint standard addition
method for paracetamols shown in Fig. 3. The

Fig 2Abs versus added paracetamol concentration at different
time intervals at 453 nm for synthetic mixtures containing
1.5 g ml
-1
paracetamol and 1.0 g ml
-1
caffeine. [1, 10-150 s;
2, 10-200 s; 3, 15-180 s; 4, 20-180 s; 5, 30-200 s; 6, 50-200 s].

Fig 3HPSAM plots for simultaneous determination of
paracetamol (1.5 g ml
-1
) and caffeine (1.0 g ml
-1
). [1, 10s;
2, 200 s].
NOTES


815
absorbances corresponding to paracetamol at the two
selected times of 10 and 200 s were selected to
calculate paracetamol concentration in the range
1.5-7.0 g ml
1
in the presence of caffeine. The
concentration of caffeine in the range 0.13.0 g ml
1

was calculated in each sample by obtaining the
ordinate values of the H-point (A
H
). The calibration
graph for caffeine was plotted using ordinate values
of H-points (A
H
) versus corresponding caffeine
concentration.
According to the theory of HPSAM at H-point
(-C
H
, A
H
), C
H
(concentration of paracetamol at
H-point) is independent of the concentration of
interferent (caffeine) (Fig. 4) and so A
H
, the
absorbance value at H-point, is also independent of
the analyte concentration. Table 1 gives the results
obtained from employing the modified HPSAM on a
mixture of paracetamol and caffeine. The results
obtained by this procedure are in good agreement
with the actual concentration.
The reproducibility of the method was determined
by five replicate analysis of paracetamol (1.5 g ml
1
)
and caffeine (1.0 g ml
1
) (r
2
= 0.9998).
Synthetic mixtures of paracetamol and caffeine in
different concentration ratios were analyzed. As
evident from the results in Table 2, the accuracy and
precision of the method are within acceptable limits.
Limits of detection, calculated as LOD=C
H
+3S
CH
,
where C
H
and S
CH
are the mean and standard
deviation of five replicated measurements of a blank
sample, were 0.80 and 0.05 g ml
-1
respectively for
paracetamol and caffeine.
To study the selectivity of the proposed method
several interferants were tested for their possible
interferences. Paracetamol (2.0 g ml
-1
) and caffeine
(0.5 g ml
-1
) were analyzed in the presence of
interfering species such as sucrose, glucose, starch,
urea, saccharin, riboflavin and ibuprofen at different
concentrations (maximum concentration tested was
300 g ml
-1
). A species was considered as interfering,
when its presence showed a variation in the change of
absorbance of the sample (time period of 200 s)
greater than twice the standard deviation did interfere
on the simultaneous determination of paracetamol and
caffeine in the system. The results show that above
species do not interfere in the determination of
caffeine and paracetamol.
To evaluate the applicability of the proposed
method, it was applied to simultaneous determination
of paracetamol and caffeine in commercially available
preparations in the form of tablets and capsules, viz.,


Fig 4HPSAM plots for fixed caffeine concentration
(0.8 g ml
-1
) and varying concentrations of paracetamol.
[1, 1.5 g ml
-1
; 2, 2 g ml
-1
; 3, 2.5 g ml
-1
; 4, 3 g ml
-1
].

Table 1Application of signal increment version of HPSAM
in a mixture of paracetamol (1.5 g ml
-1
) and
caffeine (1.0 g ml
-1
)

Time interval (s)
10-150 10-200 15-180 20-180 30-200 50-200
Paracetam
ol
(g ml
-1
)
1.52 1.49 1.48 1.48 1.47 1.46
Recovery
(%)
101.33 99.33 98.66 98.66 98.00 97.33
RSD (%)
(n = 6)
1.6 1.5 1.7 1.6 1.7 1.8
Table 2Analysis of paracetamol and caffeine present in
different concentration ratios

A-C equation R
2

Amt taken(found)
(g ml
-1
)
Paracetamol Caffeine

A
200
= 0.1219C + 0.3905
A
10
= 0.0158C + 0.2325
0.9999
0.9999
1.50
(1.49)
0.80
(0.80)

A
200
= 0.1989C + 0.6901
A
10
= 0.024C + 0.4292
0.9998
0.9992
1.50
(1.49)
3.00
(3.03)

A
200
= 0.189C + 0.7253
A
10
= 0.0399C + 0.4306
0.9999
0.9999
2.00
(1.98)
2.50
(2.52)

A
200
= 0.1869C + 0.8759
A
10
= 0.026C + 0.3862
0.9998
0.9992
3.00
(3.04)
2.00
(1.99)

INDIAN J CHEM, SEC A, JUNE 2009


816
Remidon, containing 500.0 mg of paracetamol and
65.0 mg of caffeine, capsule Novafen containing
325 mg of paracetamol, 40 mg of caffeine and 200 mg
of ibuprofen per capsule and Pirosal tablet containing
160.0 mg of paracetamol, 30.0 mg of caffeine and
220.0 mg metamizol.
The quantitative results of this analysis are shown
in Table 3. Good agreement between the obtained
results and certified amounts indicates the successful
applicability of the HPSAM for simultaneous
determination of paracetamol and caffeine.

Acknowledgement
The authors gratefully acknowledge the support of
this work by Payame Noor University (PNU)
Research Council of Iran.

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Table 3Simultaneous determination of paracetamol and caffeine in pharmaceutical samples

Sample Cert amt. (g ml
-1
) Found
a
(g ml
-1
) (Recovery, %)
Paracetamol Caffeine Paracetamol Caffeine
Remidon tablet,
Deva Pharm.Ind., Turkey,
Batch no. 706-1770

500.0 65.0 504.0(100.8) 65.7(101.1)

Novafen capsule,
Brown & Burk Ind., UK,
Batch no. NVF34E3

325.0 40.0 327.0(100.6) 40.50(101.2)

Pirosal

tablet,
Saba Pharm., Turkey,
Batch no. 48

160.0 30.0 159.0(99.4) 30.80(102.7)

a
Mean of five replicate determinations after dilution and determination by the proposed method.