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Analyst, November 1994, Vol. 119 2431

Rapid Detection of Paracetamol Using a

Disposable, Surface-modif ied Screen-printed
Carbon Electrode

Markas A. T. Gilmartin and John P. Hart*

Faculty of Applied Sciences, University of the West of England, Bristol,
Coldharbour Lane, Frenchay, Bristol BS16 1QY, U K
Published on 01 January 1994 on http://pubs.rsc.org | doi:10.1039/AN9941902431

A method for the rapid and simple determination of
paracetamol in urine is described. The transducer is a
reagentless, amperometric sensor that was developed through
the combined use of screen-printing and permselective
membrane technologies. The sensor operates selectively by
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(N-AC) supplies endogenous thiols that intervene with this
reaction and, consequently, constitutes the most successful
antidotal therapy for paracetamol intoxification.9-11
It is exigent that N-AC is administered within 8 h of
poisoning, treatment after 15 h is ineffective and hence an

means of an anti-interference barrier, cellulose acetate, which early diagnosis is a prerequisite for the efficient management
is drop-coated directly on to the screen-printed carbon of the condition.12,13This, coupled with the fact that certain
electrodes. The disposable, amperometric sensor require no individuals evince N-AC-sensitive reactions, predicates the
further modification procedures to enhance selectivity, i.e., no need for a rapid and straightforward means of selectively
enzymes are involved, and hence their fabrication is simple and determining paracetamol, i. e . , for use in an emergency room.
extremely economical. Given the rapid response times, 13 X Techniques typically employed in clinical laboratories
10-6 mol dm-3 limit of detection and wide functional range, encompass titrimetry,*4 chromatography,15 spectropho-
linear up to 2 x 10-3 mol dm-3, they represent an attractive tometry16 and immunoassays. 17 Generally, the analysis times
alternative for paracetamol monitoring. The surface-modified are lengthy and the suitability for routine bedside measure-
strips were applied to the detection of paracetamol in urine, ments is compromised by the necessity for dedicated and
correlating well with a standard enzymwolour reagent kit. cumbersome analytical equipment. Amperometry is an elec-
troanalytical interfacial technique, which, when used in
Keywords: Amperometry; paracetamol; screen printing;
conjunction with disposable sensors, has many inherent
permselectivity; urine analysis
advantages that address these limitations. The most striking
features of amperometric sensors are their simplicity, low cost
and amenability to miniaturization. The drawback associated
Introduction with this approach is that any compound that rapidly
exchanges electrons with the electrode will, theoretically, be
Paracetamol (N-acetyl-p-aminophenol or acetaminophen) is
detected. Specificity may be attained by deliberate tailoring of
an acylated aromatic amide (see Fig. 1 for structure) first
the sensors' response characteristics with a host of modifying
introduced into medicine as an antipyretic/analgesic by Von
Mering in 1893 (as stated in ref. 1). It attracted little clinical
attention, however, until recognized as the chief metabolite of
(a) NHCOCH, NCOCH,
acetanilide and phenacetin in 1949.2 Paracetamol usage has
subsequently burgeoned in many countries as an alternative to
aspirin and phenacetin.
Paracetamol's popularity stems from the innocuous nature
of the drug when administered in therapeutic doses.3 There
have, however, been numerous reports on the incidence of
OH 0
paracetamol hepatotoxicity in animals. EdeI-4 described hep-
atic necrosis in cats (1964), Boyd and Bereczky5 observed Paracetamol N-acetyl-pquinoneimine
extensive centriolobular liver necrosis in rats (1966) and (N-acetyl-paminophenol) (NAPQI)
severe and fatal liver damage was independently first recorded
in humans in 1966.6.7 Cases of self-poisoning have risen in
recent years, concurrent with the availability of the non-
prescription drug. Today, paracetamol intoxification rep-
resents one of the most commonly used overdose agents in (b) NHCOCH, NCOCH,
suicide attempts.8
During overdosage, the predominant biotransformation of
paracetamol in man is oxidative metabolism to produce
mainly cysteine and mercaptouric acid conjugates. The initial
step in the formation of these compounds is conjugation with
glutathione (GSH); thus when stores of hepatic GSH are 0- 0
depleted, as in cases of poisoning, the intermediate is free to
react with vital cellular macromolecules.9~~~)N-Acetylcysteine Phenoxide of paracetamol N-acetyl-pquinoneimine
(N-acetyl-pamino phenoloxide) (NAPQI)

Fig. 1 pH-induced interconversions and subsequent solution electro-

* To whom correspondence should be addressed.
chemistry of paracetamol ( a ) below and (h) above pH 9.0.

2432 Analyst, November 1994, Vol. 119
agents. Indeed, amperometric, disposable sensors have been
fabricated for glucose,l8 salicylate,19 3-hydroxybutyrate,20
urate,21 NADH22 and thiocholine23 on this basis.
Amperometry has been used for determining paracetamol
in liquid chromatographic eluates24 and, more recently,
directly in whole bIood.11.25 The latter two reports are
particularly germane in this context as single-use sensors were
used as transducers. The principle of both methods involved
the measurement of the product of the enzymatic hydrolysis of
paracetamol catalysed by aryl acylamidase. The product,
p-aminophenol, has a lower oxidation potential than the
parent compound and hence lowers the susceptibility of the
system to interfering compounds that would elicit a response
Published on 01 January 1994 on http://pubs.rsc.org | doi:10.1039/AN9941902431
at the sensor surface if higher voltages were applied. Vaughan
et al.25 achieved additional specificity by the juxtaposition of a
GSH-impregnated paper over the paracetamol-sensing
enzyme strip. The GSH layer serves to sequester endogenous
thiols that would otherwise hamper analyses by their 1,4-
Michael reaction with the benzene ring in the drug, generating
new electroactive compounds. Although the device exhibited
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several desirable performance characteristics, such as a
functional range up to 3 x 10-3 rnol dm-3 and a relatively fast
response time (30 s), it was not entirely specific for par-
acetamol. Indeed, a significant background current was
obtained for the oxidation of coexisting species (e.g., ascorbic
and uric acids) present in the samples tested.
Recently, we have developed epoxy-resin and screen-
printed amperometric urate sensors whose selectivity accrued
from the production of a cellulose acetate (CA) membrane
over the sensing assembly.26 This functions as an anti-interfer-
ence barrier, permitting determinations in complex matrices.
Subsequently, we have fine-tuned the solvent-casting proce-
dure to permit the direct deposition of the permselective
membrane on to the electrode surface.27The surface-modified
electrodes confer favourable interferent rejection properties
that may be chemically manipulated to allow the dedicated
measurement of the biomolecule of interest.
In this paper, the development of a disposable, ampero-
metric sensor for the rapid testing of urine paracetamol, based
on surface-modified, screen-printed carbon electrodes
(SPCEs), is described. The benefits of the present CA-SPCE
ensemble lie in their simplicity, low cost and selectivity.
Additionally, as labile biocatalytic recognition components
have been omitted in the sensor design, the operational
lifetime and storage properties are likely to be advantageous.
Experimental
Chemicals and Reagents
All chemicals were of analytical-reagent grade and obtained
from BDH (now Merck, Poole, Dorset, UK), unless stated
otherwise. The ink (R14) and apposite solvent system used for
screen-printing electrodes were kindly donated by Gwent
Electronic Materials (GEM, Newport, UK). Uric acid,
L-methionine, gentisic acid, 5-sulfosalicylic acid and reduced
glutathione (GSH) were obtained from Sigma (St.Louis, MO,
USA). Ascorbic acid and cellulose acetate were obtained from
Aldrich (Poole, Dorset, UK). L-Cysteine, malic and oxalic
acid were purchased from BDH, now Merck. The PVC
support (Pentawhite) was obtained from ADP (Bristol, UK)
and salicylic acid from BDH (Romford, Essex, UK).
Standard urate, ascorbate and glutathione solutions were
prepared daily and immediately wrapped in aluminium foil to
prevent photo- and thermal biomolecule degradation. Uric
acid was dissolved in 50 cm3 of 0.05 mol dm-3 sodium
hydroxide by sonication for 20 min with a Decon FSlOO
sonicator (Ultrasonics, Hove, Sussex, UK). Stock solutions of
paracetamol were prepared in 5% v/v ethanol-water. Par-
View Online
acetamol-fortified formulations were acquired under the guise
of Anadin Extra (Whitehall Laboratories, Taplo, Berkshire,
UK) and LemSip (Reckitt & Colman Products, Hull, UK).
An in vitro paracetamol assay kit was used, courtesy of the
clinical biochemistry department at Frenchay hospital (Bris-
tol, UK). The enzyme-colour diagnostic test was originally
purchased from Cambridge Life Sciences (CLS, Cambridge,
Ely, UK).
The supporting electrolyte used throughout this study was a
0.05 rnol dm-3 phosphate buffer, which was prepared by
mixing 0.5 rnol dm-3 solutions of disodium hydrogenphos-
phate, sodium dihydrogen phosphate, orthophosphoric acid
and sodium hydroxide to give the desired pH. These were then
diluted with de-ionized water (if necessary) to yield the
desired concentrations for ionic strength experiments. All
solutions were prepared using water that had been de-ionized
with an R0200-Stillplus HP system (Purite, Thame, Oxford-
shire, UK).
Apparatus
Cyclic voltammetry (CV), amperometry and chronoamper-
ometry were performed using an E612 VA scanner (Metrohm,
Herisau, Switzerland) in conjunction with an E611 VA
detector, connected to a Linseis LY18100 x-y plotter
(Recorder Laboratory Services, Colworth, Bedfordshire,
UK) to record the voltammograms. A three-electrode cell was
employed incorporating an SPCE as the working electrode, a
saturated calomel reference electrode (SCE) (Russel Elec-
trodes, Autermuchty, Fife, UK) and a laboratory-constructed
platinum wire counter electrode. A small circular stirring disc
(14 mm diameter; BDH) was placed in the bottom of the cell
and rotated by a Mini-MR stirrer (Whatman, Maidstone,
Kent, UK) for amperometry in stirred solutions. The temper-
ature of the amperometric cell was controlled with a ther-
mostated water-jacket.
Fabrication and Surface Modifwations of SPCEs
The base unmodified SPCEs were prepared using a previously
reported method.26 Once dry, the connecting strip of each
electrode was trimmed to 15 mm and covered with insulating
tape (RS Components). This left a 3 mm square-ended
working area exposed, in addition to a 6 mm length permitting
electrical contact with the spade connector in the electrode
holder. The SPCEs were surface modified by drop-coating 20
p1 of a 2% m/v CA solution directly on to the square-ended
electrode bulb (geometrical area 9.0 mm2).
Results and Discussion
In the proposed method, a disposable, SPCE was used as a
base transducer for the amperometric determination of
paracetamol. Preliminary studies were therefore performed to
examine its electrochemical behaviour at SPCEs under a
variety of solution and instrumental conditions.
Paracetamol Solution Electrochemistry: Voltammetric
Behaviour and Optimization
Cyclic voltammetry
CV was initially employed to probe the redox properties of
paracetamol. Fig. 2 displays representative voltammograms of
7.5 x 10-5 rnol dm-3 paracetamol obtained at an SPCE in 0.05
mol dm-3 phosphate buffers (pH 2.0-11.0). A quasi-revers-
ible couple is produced in Fig. 2(b)-(f), denoted peak I, for
the oxidation and I, for the reduction process. The voltammet-
ric data are given in Table 1. Peak I, represents the
electrochemical oxidation of paracetamol to N-acetyl-p-
 View Online

Analyst, November 1994, Vol. 119 2433

quinoneimine (NAQI) and I, the electrochemical reduction of

NAQI. Peak I, is the analytically useful signal used for the
development of the amperometric sensor.
Comparisons of Fig. 2(b) and (f) and Table 1 show that the
oxidation becomes kinetically less favourable with increasing
pH. This is highlighted in the anodic peak I, broadening and a
decrease in the values for the electron transfer coefficient
((xn,)28 from 1.6 to 0.78 (Table 1). Such ill-defined peaks
obtained at high pHs may be attributable to adsorption of
paracetamol at the sensory interface followed by a diffusion-
controlled oxidation. Alternatively, Hart et a f . 2 9 reported on a
similar phenomenon occurring at carbon electrodes with other
aromatic compounds and postulated that a progressively
I L
Published on 01 January 1994 on http://pubs.rsc.org | doi:10.1039/AN9941902431

’a
slower electron transfer occurs with increasing deprotonation
of the drug in the bulk solution. Investigations performed to
ascertain the nature of the electrode process are described in
the adsorption study section.
The redox behaviour of paracetamol with pH provides a
valuable insight into the mechanisms by which this compound
is electrolysed at SPCEs. The negative shift in anodic peak
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potential (Epa)with pH incrementation (2-9) is described by

E,, (V versus SCE) = + 0.72 - 0.0465 {pH} (1)
The theoretical gradient for a classical Nernstian two-
electron, two-proton process is 59 mV pH-1. In this study,
however, a slope of 46.5 mV pH-1 was obtained, indicating
that a more complex oxidation mechanism proceeds at the
SPCE. On the basis of these results a 2e-, 2H+ electrochem-
ical oxidation is proposed. This is consistent with the findings
of Miner et af.,”) using coulometry and a carbon electrode,
calculated the number of electrons involved to be 2.1 k 0.1.
Also in accordance with our results, the same group”)
reported a slope of 37 mV pH-1 for an E, versus pH plot over
a similar range to that studied here.
It is noteworthy that the cathodic wave is lost below pH 3
[Fig. 2 ( a ) ] ;Miner et al.30 published similar findings, conclud-
ing that hydration of the NAQI molecule accounted for its
disappearance from the voltammogram.
Between pH 9 and 10, the reported pK, value of the
phenolic group,3* there is a more marked dependence on
solution pH. This may be explained by the partial formation of
the phenoxide, which, being negatively charged, is preferen-
tially attracted to the positively polarized electrode surface.
E,, is a linear function between pH 9 and 10 according to the
following relationship:
E,, (V versus SCE) = + 0.98 - 0.0652 {pH} (2)

Table 1 Voltammetric data for the solution electrochemistry of

paracetamol at SPCEs. E, and i, signify the peak potentials and
currents and subscripts a and c, refer to oxidation (anodic) and
reduction (cathodic) processes, respectively

Ep,I Ep,I iPal ipJ AE,I Srrp.,* Sr, *

pH v v PA PA v ah,>, a4up, (W (G
2 0.63 - 2.16 - - 1.6 - 5.9 -
3.2 0.57 0.47 1.53 0.24 0.1 1.6 0.96 3.2 6.4
6.1 0.43 0.2 1.52 0.66 0.23 1.4 0.55 4.8 4.3
7 0.39 0.12 1.56 0.88 0.27 0.96 0.58 2.2 1.3
7.7 0.37 0.11 1.31 0.75 0.26 0.96 0.53 5.5 1.5
9 0.3 0.08 1.25 0.7 0.22 0.96 0.65 6 5.7
9.4 0.35 0.07 1.35 0.84 0.28 0.96 0.53 4.2 6
EN--, 9.5 0.27 0.09 1.38 0.98 0.18 0.96 0.87 5.8 6.6
Fig. 2 Typical cyclic voltammograms recorded using SPCEs for 7.5 10 0.23 0.07 1.14 0.98 0.16 0.96 0.87 6 5.3
X mol dm-3 paracetamol solutions in 0.05 mol dm-3 phosphate 10.5 0.2 0.06 1.3 1 0.14 0.8 1.1 5.6 5.7
buffers adjusted to pH ( a ) 2.0; ( b ) 6.1; ( c ) 9.0; ( d ) 9.5; ( e ) 10.5; and 10.8 0.2 0.05 0.99 0.75 0.15 0.78 1 6 4.3
cf) 1 I .O. Instrumental variables: initial potential, 0 V; scan rate, 20 12.1 0.15 0.08 0.8 0.3 0.07 - - 5.4 3.1
mV s - l , current settings. switching potentials, direction of scans and * Relative standard deviations determined from the anodic and
of value zero. @ (solid line), as indicated. Peak I,, electrochemical cathodic peak currents (ips and ip,) obtained for three separate
oxidation of paracetamol; and peak I,. reduction of N-acetyl-p- voltammograms using fresh SPCEs in each instance.
quinone imine.

2434 Analyst, November 1994, Vol. 119
The Epashift is drastically reduced between pH 10 and 11,
attributable to the total conversion of the undissociated form
of paracetamol into the phenoxide monoanion. The oxidation
of this anionic species involves one less proton [according to
Fig. l ( b ) ]and therefore exhibits a diminished dependence on
the solution pH (30 mV pH-1). Schemes for the proposed
pH-induced interconversions and subsequent electro-
chemistry are provided in Fig. l ( a ) and ( b ) .
Adsorption studies
The symmetrical appearance of the peaks below pH 8 coupled
with the broad anodic peaks obtained above pH 11 suggested
that adsorption studies would be worthy of investigation. If
Published on 01 January 1994 on http://pubs.rsc.org | doi:10.1039/AN9941902431
accumulation of the drug did occur, then this could be
exploited for quantitative purposes.
Chronoamperometry and cyclic voltammetry were per-
formed on a 0.4 x 10-3 mol dm-3 paracetamol solution at low
( 3 ) , neutral (7.5) and high (12) pH with SPCEs. The
subsequent analysis of current-time transients and the effect
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of scan rate were used to elucidate the nature of the electrode
processes.
The current (i,,)-time ( t ) chronoamperograms, or more
specifically the Cottrel equation-derived relationship between
i,, and l/&,32 was used as a mechanistic tool. The diagnostic
test for a diffusion-controlled reaction is that i,, versus l/d is a
linear function, this law is clearly obeyed in Fig. 3(a). Fig. 3(b)
shows the relationship between current function (ipa/cvh)and
the square root of sweep rate (Y;). These parameters are
components of the Randles-Sevcick equation, which states
that a reversible, diffusion-controlled process should be
independent of scan rate.32 Inspection of Fig. 3 ( b ) shows that
this rule is followed; however, some variation in current
function predicates some degree of irreversibility. This
assumption is further substantiated by the shift in E, values
from 0.32 to 0.39 V when using scan rates of 20 and 200
mV s - l , respectively (not shown), as such a peak shift is
indicative of a progressively more irreversible oxidation.
These results combined show that the oxidation is a diffusion-
controlled process and not governed by paracetamol adsorp-
tion at the sensor interface. To our knowledge, this appears to
be the first record documenting adsorption studies with
paracetamol.
Analytical variables
The solution conditions were optimized to ensure that the
maximum current densities were obtained for the oxidation of
.
i A
I I I I I I
0.1 0.2 0.3 0.4 0.5 0.6
1/t+
0.4 I I I I I I
0 2 4 6 8 10
Vf
Fig. 3 Paracetamol adsorption tests using SPCEs. ( a ) current
function, ip,/cvr versus Y+. A, pH 3; B, pH 12; and C, pH 7.5. ( b ) ip,
versus I/tt, A, pH 3; B , pH 12; C, pH 7.5.
View Online
paracetamol at SPCEs. Selection of a suitable pH environ-
ment involves a compromise between sensitivity and compati-
bility in biomatrices, and given the electrochemical behaviour
of paracetamol in acidic and alkaline media (Fig. 1 and Table
1) and the typical pH profile of urine, it was clear that pH 7
was preferable. This pH was subsequently selected in order to
optimize the ionic strength and temperature of the supporting
electrolyte, which were found to be 0.05 mol dm-3 and 25 "C,
respectively (data not shown).
The potential of SPCEs as detection systems for par-
acetamol has been highlighted by the voltammetric informa-
tion described herein. For routine bedside measurements
however, the instrumentation must be hand-held and simple,
affording decentralized testing.33 Amperometry satisfies these
criteria and was therefore pursued in the ensuing studies.
In order to maximize the selectivity and sensitivity of the
amperometric sensors, a number of parameters had to be
optimized. Hydrodynamic voltammetry (HDV) was used to
select a convenient potential for the oxidation of paracetamol.
All potentials are quoted with reference to an SCE.
Hydrodynamic voltammetry
An SPCE was introduced into a plain, contacting buffer
solution and polarized with an applied voltage of 0.0 V; the
resulting current was allowed to reach the steady state before
paracetamol was introduced into the cell. Any currents
derived from the electro-oxidation of the drug were noted and
then the operating potential was incremented manually in
small steps. Again, observed currents were calculated and the
resulting anodic currents were plotted against applied voltage
to construct an HDV (Fig. 4). The above procedure was
repeated using a CA-coated SPCE (required to enhance
selectivity in complex biomatrices, see later) to establish
whether or not the film juxtaposed over the electrode surface
had altered their electrochemical characteristics. An operat-
ing potential of +0.4 V was selected from the HDV,
representing a trade-off between the sensitivity and selectiv-
ity. This voltage was subsequently used in the remaining
experiments focused on fixed-potential amperometry .
Amperometry
A bare SPCE was polarized at +0.4 V and its analytical utility
as a base paracetamol-sensing transducer was evaluated.
Amperometric calibrations were performed to ascertain the
functional range, precision and sensitivity of the devices. The
procedure was repeated for SPCEs surface modified with CA.
Linearity, calibration and precision
Paracetamol solutions were introduced into the amperometric
cell over the range 0-3.0 x 10-3 mol dm-3 and their
relationship with the steady-state current (iSJexamined. The
0.8 1 1
0.7 <
3 0.7
5 0.6
2 0.5
5 0.4
0.3
g 0.2
2 0.1
I I
0 0.2 0.4 0.6 0.8
12 14
Applied voRageN
Fig. 4 Hydrodynamic voltammetric behaviour of a 0.1 X
rnol dm-3 paracetamol solution (pH 7) at (A) CA-coated and (B) bare
SPCEs.

functional range was linear up to 2.0 x 10-3 rnol dm-3 with a
sensitivity factor of 9.98 PA mmol-1 ( r = 0.999, n = 3). The
intra-batch precision (relative standard deviation, s,) was
calculated by measuring the slopes of individual amperometric
calibration graphs and was found to be 6.2% ( n = 5 ) .
Selectivity
When subjected to a range of potentially interfering com-
pounds, the selectivity of the sensor was severely compro-
mised, attributable to the relatively high operating voltage
employed. This undesirable outcome was exacerbated in more
complex physiological biomatrices, such as urine.
In the light of this drawback, a strategy was pursued to
Published on 01 January 1994 on http://pubs.rsc.org | doi:10.1039/AN9941902431
enhance the selectivity of the devices. In previous papers26727
we described a surface-modification technique to effectively
isolate the indicator electrode from readily electro-oxidizable
biomolecules. CA membranes cast directly on the surface of
SPCEs (hereafter referred to as CA-SPCE) have proved to be
suitable for the elimination of interferents. We therefore
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sought to exploit such permselective technology to tailor the
sensors' response to the dedicated measurement of par-
acetamol.
Fig. 4 shows that a suitable signal for the oxidation of the
drug is apparent from the HDV at +0.4 V. Amperometric
calibrations [Fig. 5(A)-(D)] were subsequently conducted
with the CA-SPCEs and these were found to be linear over a
similar dynamic range (2.0 x 10-3 rnol dm-3, r = 0.999; n = 3)
as that of the bare SPCEs. Although a 60% diminution in
current was obtained with the coated electrodes, it was clear
that the signal was still significant. The slopes, and hence
sensitivities, of these calibrations were accordingly reduced
(0.503 pA x 10-3 rnol dm-3, s, = 7.8%), when compared with
experiments performed with their bare counterparts (slope =
9.98 FA x 10-3 rnol dm-3, s, = 6.2%). This observation is
consistent with other analyte retardation findings in cellulosic
matrices.32 It was also noteworthy that the time taken to reach
95% of the steady-state currents [iss(TgS)]increased from 25 to
Time -
Fig. 5 Ampcrometric paracetamol calibrations using screen-printed
strips coated with A, 1.0%; B, 1.5% ; C, 1.8%;and D, 2.0% CA. The
arrows indicate the point of addition of 0.4 x 10-3 mol dm--7
concentrations of the drug. Analytical and instrumental parameters:
0.05 mol dm--7 phosphate buffer (pH 7.0); temperature, 25 "C;
applied voltage, +0.4 V; current ranges and time base are given on the
amperogram.
View Online
Analyst, November 1994, Vol. 11 9 2435
75 s with increasing CA concentration [Fig. 5(A)-(D)]. The
fact that the uncoated SPCE produces a iss(Tg5)of 15 s (not
shown) strongly suggests that paracetamol is partially retarded
in the CA membrane.34
Analytical performance of CA-SPCEs
The selectivity of the CA-SPCEs was assessed by exposing
them to a number of substances that may interfere with
analyses. Fig. 6(A)-(C) show the results of studies directed
towards evaluating the permselective characteristics of CA
films of various compositions. The sensors were tested for
their amperometric response to a 1.0 x 10-3 rnol dm-3
paracetamol solution using SPCEs coated with 1-2% CA.
Clearly, the lower concentrations of CA fail to provide the
required selectivity, predicated by the signals obtained for the
non-target species [Fig. 6(A)]. As thiols are not only
indigenous compounds but are also present in the circulatory
system as a result of chemotherapy, it must be ascertained that
they, themselves, do not elicit a signal. Using a 1.5%
CA-coated SPCE [Fig. 6(B)] a response is evident for
cysteine; however, when the composition of the CA matrix is
altered to 1.8% [Fig. 6(C)], the thiol is preferentially excluded
from the electrode surface. SPCEs coated with a 1.8% CA
film afford effective interferent screening, whilst still permit-
ting the detection of paracetamol [Fig. 6(C)]. These results
highlight the analytical utility of amperometric sensors for the
discriminative measurement of paracetamol in complex bio-
logical fluids. The optimized 1.8% CA-SPCEs were sub-
sequently used for the remaining studies.
Analytical Recovery
First , the endogenous urine paracetamol concentration fol-
lowing oral administration was determined by the method of
multiple standard additions. An aliquot of voided urine was
collected following 45 min ingestion of 2 x 500 mg par-
acetamol tables (Anadin) and the mean paracetamol concen-
tration was found to be 0.38 x 10-3 rnol dm-3 ( n = 3, s, =
4.1%). Next, increasing concentrations of paracetamol (up to
3.0 x 10-3 rnol dm-3) were added to 10 cm3 aliquots of urine
and the resulting levels of paracetamol were calculated by
back-extrapolation of a graph of the amount of paracetamol
versus the amount found. The resulting values were added to
(b) 1 E
1 c
G
4. D,
1\ c
- 4
1-;1
0.075 pA
50 s
G
8.02 pA
I
50 s
Time -+
Fig. 6 Amperometric responses of surface-modified SPCEs [ ( u ) ,
1.0; ( 6 ) . 1.5; ( c ) , 1.8% CAI to a selection of interferents. The arrows
indicate the point of addition of A, paracetamol; B, gentisic acid. C,
uric acid; D. ascorbic acid; E, glutathione; F, salicylic acid; and G,
cysteine at final concentrations of 0.3 x 10-3 rnol dm-3. Analytical
and instrumental parameters: 0.05 rnol dm-3 posphate buffer (pH
7.0); temperature, 25 "C; applied voltage, +0.4 V; current ranges and
time base are given on the amperogram.
 View Online

2436 Analyst, November 1994, Vol. 119

the endogenous concentration of paracetamol (0.38x 10-3
mol dm-3) to yield the total concentration of the drug found.
The percentage recoveries were derived by calculating the
slope of this plot. Table 3 shows the data obtained from
duplicate amperometric recovery assays. Evidently, the pro-
posed method yields recoveries of 90-loo%, further substan-
tiating that the sensors withstand critical analytical evaluation.
Pharmacokinetic Study
Following the oral ingestion of a therapeutic dose of par-
acetamol (1 sachet of LemSip, containing 650 mg of par-
acetamol), periodic urine samples were collected and tested
Published on 01 January 1994 on http://pubs.rsc.org | doi:10.1039/AN9941902431
resulting paracetamol concentrations. A graph was sub-
sequently constructed of drug concentraton versus time of
appearance in the urine. Table 4 shows the efficiency of
paracetamol elimination from the circulation in that it is
detected in the urine after as short a time as 14 min. This
finding is expected as it is known that paracetamol does not
impart its therapeutic effect prior to its excretion in the
urine.3.30 The apogee at 60 min represents the optimum renal
clearance time; this decays as expected to yield a low
paracetamol concentration after 24 h, which is also consistent
with other workers’ results.30
Interlaboratory Correlation

for the levels of excreted drug. For this time course study,
10 cm3 of urine were introduced into the cell along with 10 (33173
of the optimized phosphate buffer. Again, the method of
multiple standard additions was employed to establish the
Table 2 Optimization of the cellulose acetate composition for surface
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A range of paracetamol-fortified urine samples were assayed
using the amperometric strips described in this paper and an
enzyme-colour reagent kit. Plots of the paracetamol concen-
trations measured using the two methods (x and y , respect-
ively) gave a linear relationship with the equation y = 1.103x
- 0.016( n = 2;r = 0.977).As two of the measurements were

modification of the sensor interface using potential interferents as

probes. Interferents include organic acids, thiol-containing com-
taken near to the detection limits (around 10-5 mol dm-3) for
pounds and selected drugs and metabolites. Responses: f, negligible; the described methods, it was thought that these may be
+, significant; -, no signal adversely affecting the results. When such readings were
omitted from the graphs, the equation for the resulting
[CAI (Yo) straight line ( y = 1.003 x - 0.003)exhibited a much closer
Interferent 1.0 1.5 1.8 relationship ( r = 0.995,n = 2). Given the clinical significance
Salicylic acid - - - of elevated and not trace urine paracetamol concentrations,
5-Sulfosalicylic acid - - - the results obtained at the higher levels are particularly
Gentisic acid + * - germane.
Ascorbic acid + k -
Uric acid + - -
Oxalic acid - - - Conclusions
Malic acid - - -
Cysteine + - - Emergency paracetamol assays demand an analytical tech-
Methionine - - - nique that is simple, rapid and specific for the parent drug. By
Glutathione + - - exploiting screen-printing and permselective membrane tech-
nologies, a disposable, amperometric strip has been success-
fully developed that satisfies these criteria. Additionally,
these devices are inexpensive to fabricate and, in conjunction
Table 3 Analytical paracetamol recoveries in urine using screen- with amperometry, may be readily translated into hand-held
printed, surface-modified, amperometric strips systems, clearly propitious for diagnostic tests. Another
beneficial feature inherent in the sensor design is the absence
Theoretical
Paracetamol Paracetamol paracetamol
of a biorecognition component, such as an enzyme. Apart
added/ found/ S, concentration/ Recovery from somewhat complicating matters, these biospecificity
mmol dm-3 mmol dm-3 (%) mmol dm-3 (%) agents are generally labile and thus compromise the longevity
0.08 0.43 9.2 0.46 93.5 of the sensors. Moreover, advantages also accrue when
0.16 0.52 7.9 0.54 96.3 physiological fluids are taken from diseased individuals. In
0.20 0.54 6.7 0.58 93.1 particular, renal failure patients excrete elevated levels of
0.32 0.65 4.1 0.70 92.9 phenolic compounds which are known to inhibit certain
0.80 1.1 5.6 1.18 93.2 enzymes. The presence of such foreign substances may be
0.95 1.36 5.8 1.33 102.3 detrimental to the efficacy of enzyme-based measuring
regimes.
The disposable, screen-printed strip described here may be
produced reliably and on a large-scale basis. The sensor
Table 4 Paracetamol pharmacokinetic and interlaboratory correlation
study
permitted the selective determination of paracetamol in
laboratory-contrived and real samples over a wide functional
Paracetamol concentration range.
mmol dm-3
The authors thank U W E and Gwent Electronic Materials for
CLS financial and material support. In addition, colleagues at
Time/ Amperometric enzyme-colour UWE, particularly, M. Choi, Dr. R . Osborne, S. Sprules and
min assay assay* I. Hartley, are thanked for helpful discussions. We thank D.
0 - - Fletcher and the clinical chemists at Frenchay Hospital,
14 0.033 (+ 0.001) 0.009 Bristol for their assistance.
28 0.070 (k 0.004) 0.060
65 0.11 (k0.005) 0.11 References
90 0.11 (zk0.005) 0.11
1440 0.043 (+ 0.002) 0.013 1 Smith, J. M., Roberts, W. O., Hall, S. M . , White, T. A.. and
* Results for the enzyme-colour assay varied by k0.007 mmol dm-3 Gibertson, A. A . , Br. Med. 1..1978, 1, 331.
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Paper 4l03083D
Received May 23, 1994
Accepted June 3, I994