536

Determination of Codeine in Human Plasma and Drug Formulation

Using a Chemically Modified Electrode
Jyh-Myng Zen,*þ Ming-Ren Chang,þ Hsieh-Hsun Chung,þ and Ying Shihþþ
þ
Department of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan
þþ
Department of Applied Cosmetology, Hung-Kuang Institute of Technology, Taichung 433, Taiwan

Received: January 28, 1998

Final version: March 24, 1998

Abstract
Both flow injection methodology and square-wave voltammetry were developed and evaluated for determining codeine in human plasma and
pharmaceutical formulations using a Nafion/ruthenium oxide pyrochlore chemically modified electrode. Combining the electrocatalytic function of
the ruthenium-oxide pyrochlore with charge-exclusion and the preconcentration features of Nafion perform well in codeine detection. Compared to
a bare glassy carbon electrode, the chemically modified electrode exhibits a shift of the oxidation potential in cathodic direction and a marked
enhancement of the current response. A linear calibration plot is obtained over the 0–32 mM range in 0.05 M HClO4 solution with a detection limit
(3j) of 10 nM in the square-wave voltammetric method. While, in flow-injection analysis, a linear calibration plot is obtained over the 0.5–40 mM
range with a detection limit of 0.86 ng. Quantitative analysis was performed by the standard addition method for codeine content in human plasma
and a commercially available drug.

Keywords: Codeine, Flow injection analysis, Human plasma, Square-wave voltammetry, Chemically modified electrode

1. Introduction without further purification. Aqueous solutions were prepared with

double distilled deionized water.
Electrochemistry was performed on a BAS-50W electrochemical
Codeine has long been used as an effective analgesic and
analyzer. A BAS VC-2 electrochemical cell was employed in
antitussive agent in pharmaceutical preparations. A sensitive
these experiments. The three-electrode system consisted of either
and specific bioanalytical method is essential for studying the
a glassy carbon electrode (GCE) or a Nafion/ruthenium oxide
bioavailability of codeine from oral formulations. Previous
pyrochlore CME working electrode, a Ag/AgCl reference electrode
approaches, including GC, RIA, and HPLC, were reported for
(Model RE-5, BAS), and a platinum wire auxiliary electrode. Since
codeine determination [1–6]. However, these methods either
dissolved oxygen did not interfere with the anodic voltammetry, no
involve a tedious extraction process prior to the determination
deaeration was performed.
or are time consuming. A relatively simple and rapid electro-
The flow injection system consisted of a carrier reservoir, a BAS
chemical method was recently reported, which uses plastic
PM-80 solvent delivery system, a Rheodyne Model 7125 sample
membrane electrodes for the potentiometric determination of
injection valve (20 mL loop), interconnecting Teflon tubing, and
codeine in pharmaceutical preparation [7]. Nevertheless, till date,
a BAS CC-5 thin-layer electrochemical detector with a BAS
more sensitive and selective methods for the determination of
MR-3068 dual GCE. A CH-660 electrochemical workstation was
codeine in human plasma or pharmaceutical formulations are
connected in the FIA experiments.
still needed.
The preparation of the Nafion/ruthenium oxide pyrochlore CME
We report here both a square-wave voltammetric (SWV) method
was described elsewhere [8]. Electrodes were prepared with the
and flow injection analysis (FIA) for the determination of
optimum coating solution of 1.25 wt. % Nafion at 3000 rpm spin-
codeine using a Nafion/ruthenium oxide pyrochlore chemically
coating rate. The Nafion/ruthenium oxide pyrochlore CME was
modified electrode (CME). This CME has been used for several
equilibrated in the test solution containing codeine before measure-
determinations with excellent sensitivity and selectivity in our
ment. SW voltammograms were obtained by scanning the potential
previous studies [8–12]. Significant advantages have been achieved
from þ0.6 to þ1.4 V at a SW frequency of 15 Hz and SW amplitude
by combining the electrocatalytic function of the ruthenium oxide
of 50 mV. At a step height of 4 mV, the effective scan rate is 60 mV/s.
pyrochlore with charge-exclusion and preconcentration features
The codeine quantitation was achieved by measuring the current
of Nafion. In this article, the CME is further applied to the
of the oxidation peak after background subtraction. For most of
determination of codeine in human plasma and pharmaceutical
the experiments, a 0.05 M HClO4 solution was used as supporting
formulations. The optimal experimental conditions were thor-
electrolyte. In order to fit into the linear range, all samples used
oughly investigated. Practical analytical utility was illustrated
for detection were suitably diluted with the supporting electrolyte.
by selective measurements of codeine in human plasma and a
A stock solution prepared by dissolving 194 mg of codeine
commercially available drug.
phosphate in 100 mL 0.05 M HClO4 solution. An aliquot was
diluted to the appropriate concentrations with 0.05 M HClO4 before
actual analysis. The standard addition method was used to evaluate
the content of codeine in real samples. Human plasma was obtained
2. Experimental from healthy volunteers and was mixed with 5 M HClO4 (3:1) to
cause deproteination. After gentle agitation, the mixture was
Nafion perfluorinated ion-exchange powder, 5 wt. % solution in centrifuged at 1000 g for 15 min at 4 8C. The upper layer of solution
a mixture of lower aliphatic alcohols and 10 % water, was obtained was collected and then spiked with exogenous codeine. In order to
from Aldrich Chemical Co. (Milwaukee, WI). Codeine and all fit into the linear range, the drug sample used for detection was
the other compounds (ACS-certified reagent grade) were used suitably diluted.

Electroanalysis 1998, 10, No. 8 q WILEY-VCH Verlag GmbH, D-69469 Weinheim, 1998 1040-0397/98/0807-0536 $ 17.50þ.50/0
Determination of Codeine in Human Plasma and Drug Formulation 537

Fig. 2. Dependence of the anodic peak current on [HClO4 ] in SWV for 10 mM

codeine at the CME. SWV parameters are as in Figure 1.

Fig. 1. SW voltammograms for 10 mM codeine in 0.05 M HClO4 solution at the CME as indicated in our previous studies, so that the effect
a bare GCE (a), the Nafion/GCE (b), and the Nafion/ruthenium oxide of the concentration of HClO4 on the voltammetric oxidation of
pyrochlore CME (c). SWV amplitude: 50 mV; SW frequency: 15 Hz; step
codeine was first studied. The results obtained are shown in
height: 4 mV.
Figure 2. As can be seen, the current response starts to increase
rapidly in more acidic environment and the optimal condition was
3. Results and Discussion found to be around 0.05 M HClO4. The above supporting electrolyte
was thus used in the subsequent caffeine detection work. Note
that the optimum concentration of HClO4 supporting electrolyte
3.1. Voltammetric Behavior is the same as that used in the detection of caffeine at the CME [11].
Apparently, the same catalytic mechanism of the ruthenium
Figure 1 demonstrates the catalytic function of the Nafion/
oxide pyrochlore is operated in both cases.
ruthenium oxide pyrochlore CME in the determination of codeine
The effects of preconcentration potential and preconcentration
by SWV. On scanning from þ0.6 V toward a positive potential at
time on the SW response for codeine were studied next and the
a bare GCE, only a much smaller anodic peak around þ1.2 V was
results obtained are shown in Figures 3A and B, respectively. As
observed for 10 mM codeine (Fig. 1, curve a). A clear increase in
anodic peak was observed when a Nafion-coated GCE was used
(Fig. 1, curve b). The increase in codeine response for the Nafion-
coated GCE is an indication of the ion-exchange process between
Nafion and codeine. Whereas, a large increase in the peak current
at þ1.07 V was observed when the CME was used (Fig. 1, curve c).
The enhancement in current response and the shift in oxidation
potential are clear evidences of the catalytic effect of the CME
toward codeine oxidation.
Further investigation was made to the transport characteristics
of codeine at the CME. The linear scan voltammetric current
response obtained at the CME was found to be linearly proportional
to the square root of the scan rate, which illustrated that the process
was diffusion-controlled. Further evidence for the nonadsorptive
behavior of codeine was demonstrated by the following experiment.
When the CME was switched to a medium containing only
supporting electrolyte after being used in measuring a codeine
solution, no voltammetric peak signal was observed at all. Note
that, since the process is diffusion-controlled, the response time
for the CME is very fast. All the measurements can be done
immediately after the CME is immersed into the test samples.

3.2. Analytical Characterization

Both the electrode and the detection aspects should be considered
to arrive at the optimum conditions for codeine determination. As Fig. 3. The effects of preconcentration potential (A) and preconcentration
to the electrode aspect, the optimum conditions generally follow time (B) on the SWV response for codeine at the CME. SWV parameters are
those used in previous studies [7–11]. The pH range is critical for as in Figure 1.

Electroanalysis 1998, 10, No. 8

538
Fig. 4. Hydrodynamic voltammograms at a bare GCE (a) and the CME (b) for
codeine obtained under flow injection conditions with 0.05 M HClO4
solution as a carrier at 100 mV intervals between þ0:8 and þ1:6 V.
can be seen in Figure 3A, the peak current increases, as the
potential of the electrode becomes more negative between þ0.6
and 0.0 V. This behavior is explained by the fact that codeine bears
a positive charge in strong acidic environment; as a result, the
accumulation of codeine is favored at more negative potentials.
However, the peak current drops rapidly if the potential is more
negative than ¹0.2 V. A preconcentration potential of 0.0 V was
therefore chosen in all subsequent work. As to the effect of
preconcentration time, for 10 mM of codeine, the peak current
increases as the preconcentration time increases and starts to level
off around 15 s as shown in Figure 3B. Note that it takes longer time
for the peak current to level off for a lower concentration of codeine
(not shown). This phenomenon is as expected and further confirms
the ion-exchange process between Nafion and codeine. Therefore,
to increase the sensitivity of detection, a longer preconcentration
time is needed for the lower concentration of codeine.
The peak current obtained in SWV is dependent on various
instrumental parameters such as SW amplitude, SW frequency, and
step height. These parameters are interrelated and have a combined
effect on the response, but here only the general trends will be
examined. It was found that these parameters had little effect on the
peak potential. When the SW amplitude was varied in the range
of 100–100 mV, the peak currents increased with increasing
amplitude until 50 mV. However, the peak width increased at the
same time, in particular when the amplitude was greater than
50 mV. Hence, 50 mV was chosen as the SW amplitude. The step
height together with the frequency defines the effective scan rate.
Hence an increase with either the frequency or the step height
results in an increase in the effective scan rate. The response for
codeine increases with SW frequency, however, above 15 Hz the
peak current was unstable and obscured by a large residual current.
By maintaining the frequency as 15 Hz, the effect of step height was
studied. At step height of 4 mV the response was more accurately
recorded. Overall, the optimized parameters can be summarized as
follows: SW frequency, 15 Hz; SW amplitude, 50 mV; step height,
4 mV. The effective scan rate was 60 mV/s. Under optimum
conditions, the SWV current response was linearly dependent on
the concentration of codeine between 0 and 32 mM in 0.05 M HClO4
solution with slope ( mA/mM), intercept ( mA), and correlation
Electroanalysis 1998, 10, No. 8
J.-M. Zen et al.
Fig. 5. FIA responses with increasing codeine concentrations of 5 mM to
25 mM at 5 mM intervals using the CME.
coefficient of 0.796, 0.589, and 0.998, respectively. The detection
limit (3j) was 10 nM.
To characterize the reproducibility of the CME, repetitive
measurement-regeneration cycles were carried out. The results of
15 successive measurements showed a relative standard deviation
of 2.7 % for 10 mM codeine. Thus, the electrode renewal gives a
good reproducible surface. The stability of the CME was also
evaluated and the results showed an excellent long-term stability.
No decrease in codeine response with regard to either peak potential
or peak current was observed after the electrode was repeatedly
used and stored in 1 M NaOH solution for more than 2 months.
3.3. FIA Amperometric Detection
Parameters affecting the amperometric response to codeine such
as the applied potential, the concentration of carrier, and the flow
rate were optimized. Figure 4 shows the hydrodynamic voltammo-
grams for codeine obtained under flow injection conditions with
0.05 M HClO4 solution as a carrier, at 100 mV intervals between
þ0.8 and þ1.6 V. As can be seen, only a much smaller anodic peak
current was observed for 10 mM codeine at the bare GCE ( Fig. 4,
curve a). Whereas, a large increase in the peak current was observed
when the CME was used (Fig. 4, curve b). The enhancement in
current response of codeine in FIA is once again clear evidence
of the catalytic effect of the CME toward codeine oxidation. The
potential to obtain the maximum response for codeine was at about
þ1.5 V, which is an agreement with the SW voltammetric data. An
applied potential of þ1.5 V was selected for subsequent work to
maintain a low background current and to increase the electrode
stability.
The current increased with flow rate from 0.3 to 1.0 mL/min
and then decreased rapidly. The increase of the response arises from
the reaction being able to go to completion in a shorter time.
Beyond 0.6 mL/min the kinetics are limiting leading to lower
currents as the reaction fails to keep up with the passing analyte.
A flow rate of 0.6 mL/min was chosen as the optimum flow rate
in FIA.
A typical FIA response for codeine using the CME is shown
in Figure 5. A good reproducible flow injection response was
obtained at þ1.5 V applied potential with 0.05 M HClO4 carrier at
a flow rate of 0.6 mL/min. The calibration plot was linear between
0.5 and 40 mM with a detection limit (S/N ¼ 3) of 0.14 mM (i.e.,
Determination of Codeine in Human Plasma and Drug Formulation
Fig. 6. Typical SWV and FIA responses for the determination of codeine in human plasma with increasing codeine concentrations of 10 mM to 50 mM at 10 mM
intervals (A) and commercially available drug with increasing codeine concentrations of 10 mM to 50 mM at 10 mM intervals (B) and 5 mM to 25 mM at 5 mM
intervals (C).
0.86 ng). The repeatability of the response of the CME to codeine
was determined by ten replicate injections of 20 mL of codeine
and the relative standard deviations were 2.86 % and 2.53 % for
5 mM and 50 mM codeine, respectively. Again, the electrode
renewal gives a good reproducible surface.
3.4. Sample Analysis
The CME was applied to the measurement of codeine in human
plasma and a commercially available drug. Typical SWV and FIA
responses for the determination of codeine with and without
the spike of codeine are shown in Figure 6. Since no codeine is
supposed to be found in healthy human plasma, when spiked with
exogenous codeine from 10 mM to 50 mM at 10 mM intervals, a
comprehensive linearity (Y ¼ 0:15 þ 0:11X ) in plasma codeine
level was observed with correlation coefficients of 0.998 by SWV
(Fig. 6A). Note that the codeine level in human plasma can not
be determined by FIA due to the existence of other oxidizable
interferants. In a commercially available drug, good linearity of
539
Y ¼ 1:47 þ 0:51X and Y ¼ 3:64 þ 0:12X with correlation coeffi-
cients of 0.999 and 0.998 were observed with SWV (Fig. 6B) and
FIA (Fig. 6C), respectively. The signals were confirmed as being
due to codeine by enhancement of the peak when standard codeine
solution was added to the sample solutions. The recovery was found
to be 99 % for both cases.
4. Conclusions
This study has demonstrated that the Nafion/ruthenium oxide
pyrochlore CME can be applied to the detection of codeine in
human plasma and pharmaceutical formulations with excellent
sensitivity and selectivity by both SWV and FIA. Significant
advantages have been achieved by combining the electrocatalytic
function of the ruthenium oxide pyrochlore with charge-
exclusion and preconcentration features of Nafion. The reliability
and stability of the CME offer a good possibility for extending the
technique in routine analysis of codeine.
Electroanalysis 1998, 10, No. 8
540 J.-M. Zen et al.

5. Acknowledgement
The authors gratefully acknowledge financial support from the
National Science Council of the Republic of China under Grant
NSC 87-2113-M-005-021.
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[4] K.R. Bedford, P.C. White, J. Chromatogr. 1985, 347, 398.
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