Analytica Chimica Acta 539 (2005) 257–261
Direct determination of paracetamol in powdered pharmaceutical
samples by fluorescence spectroscopy
Altair B. Moreira a , Hueder P.M. Oliveira a , Teresa D.Z. Atvars a , Iara L.T. Dias b ,
Graciliano O. Neto a , Elias A.G. Zagatto c , Lauro T. Kubota a,∗
a
Instituto de Quı́mica, Universidade Estadual de Campinas, Unicamp P.O. Box 6154, CEP 13083-970, Campinas SP, Brazil
b Universidade São Francisco, Bragança Paulista SP, Brazil
c CENA, Universidade de São Paulo, Piracicaba SP, Brazil
Received 8 November 2004; received in revised form 22 February 2005; accepted 7 March 2005
Available online 31 March 2005
Abstract
The native fluorescence of paracetamol (PA) in the solid state is demonstrated, allowing the development of a rapid, simple and rugged
method for direct analysis of pharmaceutical formulations. It is easily adaptable to any spectrofluorimeter, and no chemical treatment of
the sample is needed. The fluorescence measurements (λex = 333 nm; λem = 382 nm) are performed directly on the powdered sample, the
active substance being diluted in lactose, maize starch, poly(vinylpyrrolidone), talc and stearic acid. The influence of the ingredients of PA
formulations is discussed. Fluorescence intensity is linearly dependent on PA concentration within the 100–400 mg g−1 range. The analytical
frequency is 200 h−1 . Detection and quantification limits were estimated within the 13.0–16.7 and 43.1–55.7 mg g−1 ranges for samples with
different ingredient proportions. The method was applied to pharmaceutical formulations and the relative standard deviation of results was
<2.7% (n = 20) for all tested ingredient proportions. Results were compared with those obtained by a method recommended by the British
Pharmacopoeia and no statistical difference between methods was found at the 95% confidence level.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Solid-phase analysis; Fluorescence spectroscopy; Paracetamol; Acetaminophen
1. Introduction
Paracetamol (acetaminophen or N-acetyl-4-aminophenol), herein referred to as PA (Fig. 1), is a popular antipyretic
and analgesic agent. In several countries, it is one of the most
used medicines as an alternative to aspirin (acetylsalicylic
acid).
For PA determination in drug formulations, different
methods exploiting, e.g. titrimetric [1,2], electrochemical
[3–5], spectrofluorimetric [6–8], chromatographic [9–11]
and spectrophotometric [12–19] techniques among others
have been proposed. Most of the reported procedures require a previous oxidation of PA [7,9,12,13,16,17,19], which
may increase the reagent consumption, time required for
∗
Corresponding author. Tel.: +55 37883091; fax: +55 37882987.
E-mail address: kubota@iqm.unicamp.br (L.T. Kubota).
0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2005.03.012
analysis and possibility of sample contamination. A sample preparation step prior to the determination makes the
above-mentioned procedures somewhat laborious and less
attractive for muti-sample analyses. This is often a limiting
factor in the chemical and pharmaceutical industries, where a
large number of samples must be assayed for quality control
purposes. Simpler procedures performed in a shorter time are
preferred and analytical procedures that do not involve use of
chemicals and/or sample pre-treatment steps are then a good
alternative.
In this context, near-infrared (NIR) [20,21] and FT-Raman
[22] spectroscopy have been used for PA determination in
the solid phase. However, quantitative determinations with
these techniques are only feasible by applying chemometric
algorithms, especially in relation to NIR, where the spectra
do not have high resolution, and complex-calibrating models
must be constructed.
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A.B. Moreira et al. / Analytica Chimica Acta 539 (2005) 257–261
Fig. 1. Molecular formula of paracetamol.
Spectrofluorimetry in the UV–vis region can be used
to perform the measurements directly in the solid matrix
[23,24], leading to favorable characteristics of simplicity,
sensitivity, ruggedness, selectivity, rapidity, etc. Nondestructive analyses are carried out that follow the present
tendency towards Clean Chemistry. Moreover, the possibility
of using optical-fiber accessories for on-line and/or in situ
analysis [25] becomes feasible. To date, this strategy has
scarcely been exploited in relation to solid samples of
pharmaceutical interest. Regarding PA determination, a
literature survey reveals that it usually requires the use of
derivative reactions [6–8] because PA is not intrinsically
fluorescent in aqueous solutions. In this regard, preliminary
experiments demonstrated that PA is fluorescent in the solid
phase.
The aim of this work was the development of a simple, rugged and reliable procedure for PA determination in
pharmaceutical preparations exploiting its inherent fluorescence in the solid phase. The measurements are performed
directly on the solid sample without any prior chemical
treatment.
2. Experimental
2.1. Chemicals and samples
PA was of analytical reagent grade (Synth) and lactose,
maize starch, poly(vinylpyrrolidone), talc and stearic acid
used for the tablet preparation were of pharmaceutical grade
(Galena). These ingredients were chosen because they are
usually employed in pharmaceutical formulations. The
proportions used were those indicated in a pharmaceutical
handbook [26] and used to prepare dilutions of the active
ingredient in accordance with the desired concentrations.
The samples were prepared according to the recommendations of the British Pharmacopoeia [27]. The sample dilutions to reach the required concentrations were carried out
after powdering the samples until a homogeneous particle
size was attained; the powder was further mixed with the
solid ingredients. The average particle sizes were smaller
than 100 ␮m and in this size range no significant changes
in the fluorescence signal were observed.
2.2. Apparatus
Steady-state fluorescence measurements were carried out
with a model LS-55 Perkin-Elmer spectrofluorimeter, fur-
nished with a xenon discharge lamp (20 kW, 8 ␮s), two MonkGillieson monochromators, a Hamamatsu photomultiplier,
an optical-fiber accessory and an ELISA plate, where the
powdered samples were placed. Slits for the excitation and
emission beams were set at 13 and 15 nm, the photomultiplier voltage was adjusted to 800 mV and the monochromator scan rates were 700 nm min−1 . For data acquisition and
treatment, a PC microcomputer running FT Winlab software
was used.
Fluorescence decays were obtained with a pulsed FL 900
spectrofluorometer (Edinburgh Analytical Instruments, Edinburgh, UK) using the time correlated single photon counting
(TCSPC) method. Excitation of the samples was carried out
by a pulsed hydrogen flash-lamp controlled by a Thyratron
tube operating with a repetition frequency rate of 40 kHz.
Small disks prepared by compressing solid PA (41 MPa)
were placed inside 1-cm quartz cuvettes. Measurements
were performed at room temperature (λex = 333 nm; λem
= 382 nm).
The observed decays of fluorescence intensity R(t) are
given by the convolution integral [28]:
t
R(t) =
F (t − t )L(t )dt
(1)
0
where L(t ) = time distribution of the lamp pulse; F(t ) =
(theoretical) response function corresponding to an infinitely
short excitation lamp pulse; t = sample decay time and t =
standard decay time.
L(t ) was measured with a solution of LUDOX (DuPont)
used to determine instrument response function closely
spaced in time to specific fluorescence decay experiments.
Data were analyzed by the exponential series method
(ESM) that assumes that the fluorescence decay can be
analyzed as a multiple exponential function where the
best fit uses the “Marquardt algorithm” to minimize the
χ2 (summation of the square of the differences between
experimental data and modeling function multiplied by
the weighting factor) [28]. A good fit is obtained when
χ2 approaches 1.0 and a random residual distribution is
observed.
2.3. Procedure
Synthetic samples containing lactose, maize starch,
talc, poly(vinylpyrrolidone) and stearic acid in the 70:14:
9:4:3 w/w (#1), 75:12:8:3:2 w/w (#2) or 80:10:7:2:1 w/w
(#3) proportions were prepared in order to match the
paracetamol tablets that generally present these proportions
[26]. The ingredients were mixed with PA and powdered in
an agata mortar for 0.5 min. The tablets underwent dilutions
in order to get PA concentrations within the 100–400 mg g−1
range. Twenty-five milligram samples were placed into the
96-well plate, where the fluorescence measurements were
carried out directly on the powdered samples. Spectra were
obtained at 25 ± 1 ◦ C. For wavelength selection, excitation
A.B. Moreira et al. / Analytica Chimica Acta 539 (2005) 257–261
259
and emission radiation were scanned from 305 to 355 nm
and from 360 to 475 nm, respectively.
3. Results and discussion
3.1. Solid-phase PA fluorescence
For the fluorimetric determination of PA in aqueous
medium, the analyte is usually oxidized under slightly alkaline conditions by hexacyanoferrate(III), yielding a fluorescent substance [29]. Fluorescence emission of native PA in
aqueous medium seems to be not yet observed and, in agreement with the literature, the authors did not observe emission
in aqueous phase. To the best of our knowledge, PA native
fluorescent in the solid phase has not yet been reported. Surprisingly, some emission was observed in the solid phase,
probably due to the rigidity of the PA molecule in the solid
phase. In fact, it is well known that increasing structural rigidity can improve the fluorescent quantum efficiency [30]. In
view of this pioneering aspect, it is important to demonstrate
experimentally the PA fluorescence.
PA fluorescence in the solid phase was investigated under
different excitation wavelengths, and excitation with lower
absorption radiation produces emission spectrum with lower
intensity (Fig. 2, inset). In addition, no spectral shifts in the
emission spectra were observed by varying the excitation
wavelength, which is often observed for light scattering phenomena [31].
In order to get some additional insights into this emission,
the emission decay (Fig. 3) of PA in the solid state was studied using the single photon counting time resolved (SPCTR)
fluorescence technique. A mono-exponential decay was observed, with lifetime τ F = 1.77 ± 0.01 ns, χ2 = 1.093. Both the
steady-state emission and the dynamic spectra indicate that
solid PA does exhibit fluorescence emission. This emission
Fig. 2. Fluorescence spectra (2) of pure PA related to excitation wavelength
(1) between 325 and 349 nm with increments of 1 nm. Inset: dependence of
the emission intensity with the excitation wavelength.
Fig. 3. Fluorescence decays for PA collected at λem = 382 nm and
λex = 333 nm; spectrum 1 is the instrument response function of the LUDOX solution and spectrum 2 is the sample decay, both obtained at 298 K
in the solid state.
signal can thus be exploited for quantitative PA determination.
3.2. Analytical studies
The method developed in the present work involved fluorescence measurements directly on the solid sample. Wavelengths for maximum absorption and emission were selected
as 333 and 382 nm, respectively (Fig. 4).
3.2.1. Distance between optical fiber and sample
The angle of incident light for total internal reflectance,
as well as the acceptable angle, is an important aspect to
be considered when using optical fibers. As the capacity of
an optical fiber to catch light is linked to this angle, it is
necessary to optimize the distance between the optical fiber
Fig. 4. Excitation (1) and emission (2) spectra of PA and their corresponding blank spectra (3 and 4) in the solid phase. Ingredient
proportion = 70:14:9:4:3 w/w; scan rate = 700 nm min−1 ; PA concentration = 250 mg g−1 .
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A.B. Moreira et al. / Analytica Chimica Acta 539 (2005) 257–261
Table 1
Effect of some ingredients
Ingredient
C
I
Lactose
Maize starch
Talc
Poly(vinylpyrrolidone)
Stearic acid
133
147
189
186
196
166
62
149
35
353
I/C
±
±
±
±
±
4
2
3
1
8
1.3
0.4
0.8
0.2
1.8
I = fluorescence intensity with estimated standard deviation for n = 3, in
arbitrary units; C = paracetamol concentration, in mg g−1 .
in the formulations have fluorescence and any of them was
observed.
Fig. 5. Effects of the sample amount (A, dashed line) and the distance between optical fiber and sample (B, solid line). Figure refers to 250 mg g−1
PA.
and the sample. In the present procedure, best sensitivity was
attained with a distance of 0.7–0.9 cm (Fig. 5).
3.2.2. Sample amount
The sample amount added to each well of the 96-well plate
was also investigated. Depending on this amount, a variation
in the fluorescence intensity might occur due to the eventual
radiation scattering provoked by the radiation crossing the
sample. Moreover, the number of molecules might not be
enough for a proper interaction with the incident radiation.
Thus, the diameter of the well is an important parameter to
be controlled as well as the diameter of the radiation light
beam. Increasing the sample amount increased the analytical signal, and maximum analytical signal was attained
for an amount of 17-mg sample. Increasing the sample
amount beyond this value did not improve the fluorescence
intensity.
3.2.3. Presence of ingredients
Influence of ingredients such as lactose, maize starch,
talc, poly(vinylpyrrolidone), and stearic acid on the PA fluorescence intensity was evaluated individually. For this task,
different mixtures of each ingredient with PA were prepared.
It was found that lower signal/concentration (I/C) relationship was observed for the PA plus poly(vinylpyrrolidone)
mixtures (Table 1). These data reveal that the proposed
method is dependent on the quantity and kind of the
ingredients used as constituents of the medicine. It is
important to have previous knowledge of the sample bulk
composition before applying the method, in order to prepare
standards matching the sample matrices. Hopefully, the
ingredients used in the production process are always
known; therefore, their effect can be easily circumvented. It
is also very important to know if any of the ingredients used
3.2.4. Analytical curve
For a 25-mg sample and the proportion #1 (70:14:
9:4:3 w/w), a linear response was noted within 100 and
400 mg g−1 PA. The analytical equation was:
y = 31(±7) + 0.97(±0.03)x
(r = 0. 9990, n = 7)
where x = PA concentration, expressed as mg g−1 ; y = fluorescence intensity, in arbitrary units. For the other investigated
proportions (#2 and #3, 75:12:8:3:2 and 80:10:7:2:1 w/w, respectively) the analytical equations were:
#2 :
y = 46(±9) + 0.90(±0.03)x
#3 :
y = 41(±10) + 0.97(±0.04)x
(r = 0.9981, n = 7)
(r = 0.9985, n = 7)
The different equations related to the different ingredient
proportions suggest that it is necessary to calibrate the method
for each production process, using the proportion actually involved. This is a limitation of the method that the previous
knowledge of the ingredients and proportions is needed. Even
though the PA contents in these samples were higher than the
optimized linear range, the proposed method can be applied
without diluting the sample. In fact, modifying the distance
between optical fiber and sample, reducing the intensity of the
radiation source, and/or the excitation slit width can be performed in order to match the dynamic instrument range with
the assayed samples. Moreover, smaller amounts of sample
can be used. This potentiality of the proposed method minimizes sample handling, allowing the direct determination in
the production line. However, for much more concentrated
samples, self-absorption eventually decreasing the quantic
yield cannot be disregarded.
3.2.5. Figures of merit
Repeatability of the analytical results was evaluated by
performing 10 determinations on different samples (ca.
250 mg g−1 PA). The R.S.D. was lower than 2.7% for all
ingredient proportions, highlighting the good repeatability
inherent in the proposed method.
Detection and quantification limits were estimated in accordance with the 3σ and the 10σ criteria [32], respectively,
where σ is the standard deviation of three measurements of
A.B. Moreira et al. / Analytica Chimica Acta 539 (2005) 257–261
Table 2
Paracetamol contents in tablets (mg per tablet) as determined by the proposed
and reference [27] (British Pharmacopoeia) methods
Sample
Nominal
Proposed method
1
2
3
4
5
6
750a
750a
750b
750b
750c
750c
763
759
761
745
768
742
±
±
±
±
±
±
14
13
10
11
10
8
British Pharmacopoeia
759
746
753
756
755
754
±
±
±
±
±
±
6
7
6
4
5
7
Experimental data (with uncertainties) based on three replicates.
a Proportion 1.
b Proportion 2.
c Proportion 3.
the blank. The values for the three ingredient proportions
studied were 13.0 and 43.6 mg g−1 (proportion #1), 14.2
and 47.4 mg g−1 (proportion #2), and 16.7 and 55.7 mg g−1
(proportion #3). Even though the detection and quantification
limits obtained for this method are somewhat high, a great
advance is reached as it is not necessary to use chemometric
methods, normally employed in NIR and Ramam techniques
for PA determination [20–22], and in which there is the need
to build a model for each sample.
The fluorescence spectrum was recorded in 18 s, leading
to a possible analytical frequency of 200 samples per hour,
considering an automated system or that the samples were
ready to be assayed.
3.2.6. Applications
The applicability of this new method was demonstrated by
applying it to six samples containing lactose, maize starch,
talc, poly(vinylpyrrolidone) and stearic acid as ingredients
in three different proportions, and the results are shown in
Table 2 together with results obtained with the UV spectrophotometric method recommended by the British Pharmacopoeia [27]. After applying the Student’s t-test, no difference
between these methods was found at 95% of confidence level.
4. Conclusions
The results presented in this work demonstrate the potentiality of the proposed method for PA determination in solid
matrices. Precise results, in agreement with those obtained by
the British Pharmacopoeia reference method, were obtained.
The procedure is simple, rapid and non-destructive. No chemicals are required; therefore, hazardous residues are not generated, thus contributing to Clean Chemistry. The method,
however, is dependent on the ingredients and powdering, being necessary a previous knowledge of the ingredients of
the sample, and a good powdering system. Circumvented
these points, a simple reading of the fluorescence intensity
in the solid sample makes an easy and fast determination
possible.
261
Acknowledgments
Partial support from the Fundação de Amparo à Pesquisa
do Estado de São Paulo (FAPESP) and the Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq)
is greatly appreciated.
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