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Simple and rapid voltammetric determination of morphine at electrochemically pretreated glassy carbon electrodes
Talanta 79 (2009) 845–850
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Simple and rapid voltammetric determination of morphine at electrochemically pretreated glassy carbon electrodes Fei Li, Jixia Song, Dongmei Gao, Qixian Zhang, Dongxue Han, Li Niu ∗ State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, and Graduate University of the Chinese Academy of Sciences, Chinese Academy of Sciences, Changchun 130022, PR China
a r t i c l e
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Article history: Received 18 January 2009 Received in revised form 9 May 2009 Accepted 11 May 2009 Available online 20 May 2009 Keywords: Morphine Electrochemical pretreatment Voltammetric detection Glassy carbon electrode
a b s t r a c t A simple and rapid method for morphine detection has been described based on electrochemical pretreatment of glassy carbon electrode (GCE) which was treated by anodic oxidation at 1.75 V, following potential cycling in the potential range from 0 V to 1.0 V vs. Ag|AgCl reference electrode. The sensitivity for morphine detection was improved greatly and the detection limit was 0.2 M. The reproducibility of the voltammetric measurements was usually less than 3% RSD for six replicate measurements. Moreover, this method could readily discriminate morphine from codeine. And an electrochemical detection of morphine in spiked urine sample was succeeded with satisfactory results. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Morphine as a major component in opium is frequently used to relieve severe pain in patients, especially those undergoing a surgical procedure. However, it is toxic in excess and when abused. To prevent overdose-induced toxication, it is necessary to sensitively monitor the concentrations of morphine in a patient’s blood or urine. Various analytical methods have been developed for the determination of morphine and its major metabolites. The most common analytical techniques currently used include gas chromatography [1,2], high-performance liquid chromatography (HPLC) [3–8] and their combination with other detection methods. Benefited from high efficient separation, they have the advantage of high sensitivity, but suffer from time-consuming and expensive equipment. Use of capillary electrophoresis and microchip capillary electrophoresis [9–11] have partially solved this problem. However, the difficulty in preparing capillary column and its short life restrict its development. Immunoassay, such as surface plasmon resonance (SPR) based immunosensors [12,13] and radioimmunoassay (RIA) [14] are also reported for morphine detection, although they are very sensitive but antibody cross-reactivity may exist. Research in more depth has been devoted to develop a simple, economical, fast and efficient method for detecting morphine. The electrochemical method is widely used because it is highly sensitive, cheap, simple, easily miniaturized and conve-
∗ Corresponding author. Fax: +86 431 8526 2800. E-mail address: [email protected] (L. Niu). 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.05.011
nient. The use of bare electrodes such as glassy carbon electrode [15], platinum electrode and graphite electrode [16] are proved to be straightforward and simple on detecting morphine. However, there are a number of limitations, such as slow electron transfer, low sensitivity and electrode contamination, etc. To avoid those shortcomings, combination of HPLC and electrochemical detection has been designed [17]. Recently, some new electrochemical detection methods have been proposed for morphine detection. For example, an adsorptive differential pulse stripping method [18] and its conjugation with least-squares support vector machines [19] have been developed for trace morphine detection. Fast Fourier transformation with continuous cyclic voltammetry at Au microelectrode [20,21] has been devised for morphine detection in a flow injection system. Furthermore, different modified electrodes have been developed for morphine detection. For example, Jin and co-workers prepared a kind of cobalt hexacyanoferrate modified carbon paste electrode, combined it with HPLC and successfully detected morphine in vivo [22]. Ho et al. devised a Prussian blue-modified indium tin oxide (ITO) electrode [23] and molecularly imprinted electrodes for morphine determination [24,25]. Multiwalled carbon nanotubes modified preheated glassy carbon electrode has also been used for the morphine detection [26]. Prussian blue film modified-palladized aluminum electrode [27] has recently been used for morphine detection. It is also well known that electrochemical pretreatment of glassy carbon electrode (PGCE) are usually prepared by potentiostatic polarization at oxidizing potentials or by potential cycling in a wide potential range. The pretreatment introduces oxygen-containing
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functional groups on the electrode surface. These functional groups exhibit certain interaction, such as electron donor–acceptor, hydrogen bonding, electrostatic, dispersive and solvophobic interactions [28,29], with various species. For example, PGCE has been applied in the preconcentration and electroanalysis of copper [28] and manganese species [30]. A number of organic molecules such as erythromycin [31], vitamin B2 [29] have also been adsorbed strongly at such activated glassy carbon electrodes. Besides, PGCE has also been used to determine biomolecules, such as DNA [32,33]. However, to our best knowledge, the report concerning the detection of morphine at PGCE is quite few. In this work, it was found that morphine could also be effectively adsorbed and accumulated on the electrochemically pretreated glassy carbon electrode. So a simple, rapid and sensitive voltammetric method for morphine detection was explored. The method could readily discriminate morphine even from coexist codeine. As a potential application, the electrochemical detection of morphine in spiked urine sample was performed too.
2.4. Procedure for determination of morphine The electrochemical experiments were performed in 0.05 M PBS (pH 7.4) with different concentrations of morphine. The adsorptive accumulation of morphine at the working electrode was done in a stirred solution at open circuit for 90 s. After a 2 s quiet time, the cyclic voltammograms were recorded from 0 V to 0.7 V at 0.1 V/s. The electrode can be used for next measurement after continuous sweep for four cycles at potential range from 0 V to 1.0 V in 0.05 M PBS (pH 7.4). 2.5. Sample preparation Urine samples were obtained from healthy laboratory volunteers without any pretreatment, except that they were diluted by 100 times with 0.05 M PBS (pH 7.4). A quantity of the morphine was added to the diluted urine and the resulting drug urine solutions were used for voltammetric analysis. The content of the morphine was determined by the standard addition method [31].
2. Experimental 3. Results and discussion 2.1. Reagents 3.1. Morphology and surface composition after pretreatment Morphine hydrochloride and codeine phosphate were supplied by the Institute of Forensic Science Ministry of Public Security (China). Unless otherwise stated, other reagents were of analytical grade and used as received. Phosphate buffer solution (0.05 M PBS), which was prepared by mixing different volume of Na2 HPO4 solution (0.05 M) and KH2 PO4 solution (0.05 M), was employed as supporting electrolyte. Britton–Robinson (B–R) buffer was prepared by dissolving the same concentrations (0.04 M) of orthophosphoric acid, boric acid and glacial acetic acid in water and adjusting to the desired pH value with sodium hydroxide (0.2 M). All aqueous solutions were prepared with high pure water from a Millipore system (>18 M cm). 2.2. Apparatus Scanning electron microscopy (SEM) pictures were imaged by an XL30 ESEM FEG field emission scanning electron microscope. X-ray photoelectron spectroscopy (XPS) was conducted with a VG ESCALAB MKII spectrometer (VG Scientific, UK) employing a monochromatic Mg-K␣ source (h = 1253.6 eV) and the elemental content ratio was evaluated by using the software installed with the instrument. Cyclic voltammetric (CV) measurements were performed in a conventional three-electrode cell with a platinum wire as the auxiliary electrode and an Ag|AgCl (saturated KCl) as the reference electrode with a CHI 660 Electrochemical Workstation (CHI, USA). The working electrode was bare or pretreated glassy carbon electrode (GCE, d = 3 mm). All potentials reported here refer to the Ag|AgCl (sat. KCl) reference electrode.
Through the electrochemical pretreatment a thin layer was found on the electrode surface, which was in agreement with the previous report [34,35]. This layer is composed of carbon oxidation products of the electrode itself and has been termed electrochemical graphite oxide. The morphology of the glassy carbon electrode before and after the anodic oxidation pretreatment was examined by SEM (data not shown here). Both of them were flat and uniform in the submicrometer scale, indicating that the pretreatment was mild and did not cause significant changes in GCE surface morphology. The surface of the glassy carbon electrode was further examined by XPS measurement. As shown in Fig. 1, the O1s spectra show an increase in amplitude after the pretreatment (dotted), although the peak shape changed little. The major peak in the C1s spectrum (284.6 eV) changed in shape as a result of pretreatment. The width at half-maximum height went from 0.86 eV at the polished electrode (solid) to 1.42 eV at the pretreatment electrode. New C1s peaks were also found at the binding energy centered at ca. 286.9 eV, 288.6 eV, 292.9 eV and 295.8 eV, indicating the presence of different forms of carbon having higher binding energy, such as carbonyl, carboxyl and quinone [36,37]. Calculations indicated that the oxygen-to-carbon ratio of the electrode surface were 0.12 and 0.33 for the freshly polished and pretreated electrode, respectively. The increase in the O/C ratio and the O1s amplitude showed
2.3. Pretreatment of the glassy carbon electrode The glassy carbon electrode was polished carefully with 1.0 m, 0.3 m, and 0.05 m alumina slurry to a mirror finish, respectively. And it was cleaned in an ultrasonicating bath for 2 min before use. The general procedure for electrochemical pretreatment of glassy carbon electrode was modified from previously published procedures [32]. The preparation of PGCE was performed by anodic oxidation at 1.75 V for 200 s in 0.05 M PBS at pH 5. The electrode was then cycled between 0 and 1.0 V at a scan rate of 0.1 V/s until a stable current–voltage curve was obtained.
Fig. 1. XPS spectra of freshly polished GCE (solid) and pretreated GCE (dotted).
F. Li et al. / Talanta 79 (2009) 845–850
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Fig. 2. (A) Cyclic voltammograms of 25 M morphine in 0.05 M PBS (pH 7.4) (A) at (a) unpretreated GCE (dotted) and (b and c) pretreated GCE without (dash) and with accumulation (solid) at 0.1 V/s. Accumulation was performed in open circuit for 90 s. (B) Influence of accumulation time on the peak current of morphine at different concentrations. (a) 5 M; (b) 10 M; (c) 25 M. Scan rate: 0.1 V/s.
that the surface became oxygenated during pretreatment, which was consistent with previous reports, i.e. oxygen-containing groups could be created on the surface of glassy carbon electrodes through pretreatment [34,36,37]. Thus, the environment of surface carbon became more complicated upon pretreatment [36]. According to previous report [34], the pretreatment of GCE involved the formation of a new phase, which contained a significant amount of microcrystallinity and graphite oxide. 3.2. Voltammetric behaviour of morphine at pretreated glassy electrode Morphine is a kind of phenolic compounds, which can transfer electrons to electrodes at a certain potential. Fig. 2A shows the cyclic voltammograms of 25 M morphine at the unpretreated GCE (dotted) and pretreated GCE without (dash) and with accumulation (solid) in 0.05 M PBS (pH 7.4). At this pretreated GCE, the morphine gave a well-defined oxidation peak at ca. 0.43 V. The oxidation reaction of the phenolic group at the 3-position involving one-electron transferring is responsible for the major peak. The oxidation of the phenolic group leads to the formation of pseudomorphine as the main product. The oxidation of morphine can be described by the following mechanism (Scheme 1). The peak potential of the oxidation of morphine at the pretreated GCE was slightly more negative than that obtained at unpretreated GCE. The peak current of pretreated GCE was higher than that of the unpretreated GCE, although the background current of the pretreated GCE was somewhat higher than the unpretreated GCE. From curve b, it could be seen that morphine was quickly oxidized at the pretreated GCE without accumulation, and the peak current was 4-fold larger than that at the unpretreated GCE. And after a 90 s accumulation (curve c), an approximately twice enhancement of the peak current was observed over that obtained without accumulation. Therefore a considerable enhancement in sensitivity could be obtained by applying adsorption step.
Moreover, it was also found that the adsorption efficiency of morphine was affected strikingly by preanodization time, accumulation time and stirring. It is generally accepted that the number of active adsorption sites increases with the preanodization time [28,29]. We have investigated the influence of the activation time. Longer activation time usually resulted in larger number of adsorption sites. However, the thickness of the activated film became larger and the adsorption ability of morphine at the electrode decreased. Meanwhile, thicker film might result in memory effects of the electrode and thus affected the reproducibility too. We found that the adsorptive ability of morphine at the electrode decrease when the time duration for electrode pretreatment exceeds over 200 s (data not shown here). So a preanodization time of 200 s was selected. The influence of accumulation time of morphine at different concentrations (i.e. 2 M, 10 M and 25 M) to the peak current was employed. When the accumulation time was less than 90 s, the peak currents increased with the time, and after that it reached a platform. It indicated that the equilibrium between the concentration of morphine in solution and that on the surface of the electrode occurred at ca. 90 s. Thus, the accumulation of 90 s was selected as the optimum condition at open circuit (Fig. 2B). The peak current obtained under constant stirring also increased with stirring assistance. The results also showed that the peak currents decreased to a constant with succeeding potential scan, as previously reported [33], which was attributed to adsorption of oxidation product at this PGCE. So it is necessary to renew the electrode surface by potential cycling before each measurement. As shown in Fig. 3, the peak currents (ip ) at the pretreated electrode in 25 M morphine solution (pH 7.4) varied with change of scan rate (). In the range of 10–150 mV/s, the relations obey the following question (R = 0.9964, where is in mV/s and ip is in A), log ip = −1.342 + 0.888 log
(1)
This indicated that the electrode process was controlled simultaneously both by diffusion and adsorption [31]. 3.3. Effects of pH
Scheme 1. The reaction scheme of morphine.
Effect of pH on the peak current and peak potential (as shown in Fig. 4) for the morphine oxidation was also investigated. The peak potential shifted negatively with increase of pH. It could be explained by the consequence of deprotonation involved in the oxidation process which was facilitated at higher pH values [26]. A plot of peak potentials vs. pH value was found to be linear over the pH range of 3–9, corresponding to a mechanism involving the same number of electrons and protons (as shown in Fig. 4B). It
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Fig. 3. Cyclic voltammograms of 25 M morphine in 0.05 M PBS (pH 7.4) at pretreated GCE at various scan rates: 10 mV/s, 25 mV/s, 50 mV/s, 75 mV/s, 100 mV/s, 125 mV/s and 150 mV/s from inner to outer, respectively. Inset is the calibrated plot of log ip vs. log .
was found in Fig. 4A that the current increased with the decrease of the pH. Our results showed that morphine adsorbed readily on pretreatment glassy carbon electrode in acidic medium, while morphine was usually easily adsorbed on bare electrode under alkaline medium. Firstly, this might be related to the differences in the surface properties of the electrodes and the adsorption interactions between morphine and the electrode surfaces. According to previous report [34], the pretreatment of GCE involved the formation of a new phase, which contained a significant amount of microcrystallinity and graphite oxide. This graphite oxide film probably acts as a reservoir for morphine and causes the observed adsorption. But this graphite oxide film would be partially dissolved in alkaline medium [35] and resulted in the decrease of morphine adsorption. Secondly, the variation of electrostatic interaction and hydrogen bonding between morphine and the electrode surface at different pH could also be responsible for this phenomenon. Thirdly,
Fig. 5. Calibration curves of current response vs. morphine concentration. Inset is the calibration curve corresponding (a) to 4–18 M morphine and (b) to 18–100 M morphine. Accumulation was performed in open circuit for 90 s. Scan rate: 0.1 V/s.
the decrease of the peak current might be resulted from the decomposition of morphine in alkaline medium. Based on above reasons, the current decreased with the increase of the pH. 3.4. Calibration curve Fig. 5 displays the calibration curve for morphine determination at PGCE. The peak current increased with the morphine concentration up to 100 M. There were two linear relations with different slopes in the range of 4–100 M. In the low concentration, the linear range (4–18 M) gave a large slope of 0.0715 A L mol−1 , and a correlation coefficient of 0.997. When the concentration exceeded 18 M, it deviated from the linear relationship (4–18 M) and followed another linear relationship (18–100 M) which gave a relative small slope of 0.0353 A L mol−1 and a correlation coefficient of 0.998. Due to the limited adsorption sites, the saturated coverage of the electrode surface was reached and peak current tended to immovability when the morphine concentration is more than 400 M. The detection limit was estimated to be ca. 0.2 M based on the signal corresponding to three times the noise of the response following the accumulation of 90 s. Reproducibility of the PGCE for determination of morphine was also investigated. It was performed in the solution of 10 M morphine in 0.05 M PBS (pH 7.4). A relative standard deviation of 2.77% was obtained for six replicate measurements. 3.5. Discrimination of morphine from codeine
Fig. 4. (A) Influences of pH values on the peak current () and peak potential () of 25 M morphine in B–R buffers at 0.1 V/s. (B) Calibration plot of pH vs. potential.
Voltammetric response at the PGCE in morphine solution containing codeine was also examined further. Codeine, with the methyl ether group (–OCH3 ) substituted for the phenolic group (–OH) in the 3-position, is very similar to morphine in structure (as shown in Fig. 6 inset). It usually interferes with morphine analysis in urine or blood. The oxidation peak of codeine was found at 0.96 V (data not shown here) due to the absence of phenolic group at the 3-position. Therefore, the pretreated electrode operating at 0.43 V (as shown in Fig. 2A) can easily discriminate morphine from codeine. The adsorption of morphine by the PGCE involved interactions between morphine molecules and the activated surfaces. The presence of codeine may affect such detection of morphine due to its
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Table 2 Comparison the proposed method with other reported methods. Detection method
Limit of detection
Sample
Recovery (%)
Reference
Microchip CEa LPMEb –HPLCc Amperometry Amperometry-MIPd SIAe DPVf Voltammetry
0.2 mol L−1 0.05 mg L−1 0.2 mmol L−1 0.3 mmol L−1 0.076 g mL−1 0.01 mol L−1 0.2 mol L−1
Urine Urine Not applied Not applied Urine Plasma Urine
96.2 92.4–106.8 – – 96.3 98.5–102.5 95.1–106.6
[11] [39] [24] [25] [40] [19] This work
a b c d e f
Fig. 6. Cyclic voltammograms of 10 M morphine in the presence of various concentrations of codeine: 0 M, 5 M, 10 M and 20 M at the pretreated GCE, respectively. Accumulation was performed in open circuit for 90 s. Scan rate: 0.1 V/s. Inset: the molecular structure of morphine and codeine.
competitive adsorption. The degree of interference was explored at various codeine concentrations. The results, as shown in Fig. 6, exhibited that codeine did not show significant interference while its concentration was lower than 2-fold concentration of morphine. It was well known that it was important criteria for judging recent heroin use while the morphine-to-codeine ratio was higher than 2 [38]. And morphine was the major component in opium poppy. Therefore, the PGCE may be used for the quantitative and qualitative determination of morphine in opium poppy and for judging heroin abusers. When the concentration of codeine was 100 times larger than that of morphine, the PGCE was still available to detect morphine qualitatively. 3.6. Determination of morphine in urine The proposed method in real sample analysis was also examined in human urine samples. The recovery was evaluated by comparing the analytic signals of morphine obtained from the spiked urine with those of the same concentration standard solution (n = 5) (Table 1). The recovery of the spiked samples ranged between 95.1% and 106.6%. The R.S.D. (n = 5) was less than 6.0%. In Table 2, response characteristics of the proposed method are compared with those obtained by some reported methods. In comparison with some other voltammetric methods of morphine determination, our method showed advantages in several aspects. For example, Prussian blue film modified-palladized aluminum electrode [27] has been recently used for morphine detection. Here the PGCE has lower detection limit. Moreover, the preparation process of PGCE was simpler and the reproducibility was also good. Meanwhile, it could be reused through the simple electrochemical treatment and also could be even used in real sample. Niazi et al. [19] reported using of adsorptive differential pulse stripping voltammetric method on a hanging mercury drop electrode for determination of morphine with excellent detection limit (0.01 M). But the mercury is very poisonous and it was only used Table 1 Results of determination of morphine in urine sample. Urine sample
Spike (mol L−1 )
Found (mol L−1 )
Recovery (%)
R.S.D. (%)a
1 2 3
5.0 10.0 15.0
4.76 10.65 14.62
95.1 106.6 97.5
3.4 4.8 5.3
a
Average of five replicate measurements.
Capillary electrophoresis. Liquid phase microextraction. High-performance liquid chromatography. Molecularly imprinted polymer. Sequential injection analysis. Differential pulse voltammetry.
in the pretreated plasma sample. Compared with other methods to determine morphine in urine, such as microchip electrophoresis [11], liquid phase microextraction-HPLC [39] and sequential injection analysis [40], this method had the similar detection limit and the accuracy. At the same time, it also show extra advantages: simple preparation process of PGCE; short time and low cost for the analysis; and no pretreatment needed before the measurement. 4. Conclusion Voltammetric determination of morphine has been performed at the electrochemically pretreated GCE. The main advantage of the proposed method is simple and fast compared to other determination methods. Furthermore, a low detection limit and a wide range of concentrations are enough for usual analytical purpose. The method has been demonstrated that it easily discriminate morphine from codeine. Moreover, the determination morphine in real human urine without any sample pretreatment has been succeeded with consistent result with those of HPLC and microchip electrophoresis. The proposed method provides the basis for designing portable morphine sensor due to its easy and fast preparation, and low cost. Acknowledgement The authors are most grateful to the NSFC, China (No. 20827004) and Ministry of Science and Technology (Nos. 2006BAKB05 and 2007BAK26B06) for their financial support. References [1] B. Fryirs, M. Dawson, L.E. Mather, J. Chromatogr. B 693 (1997) 51. [2] U. Hofmann, S. Seefried, E. Schweizer, T. Ebner, G. Mikus, M. Eichelbaum, J. Chromatogr. B 727 (1999) 81. [3] P.P. Rop, F. Grimaldi, J. Burle, M.N. Desaintleger, A. Viala, J. Chromatogr. B 661 (1994) 245. [4] S.R. Edwards, M.T. Smith, J. Chromatogr. B 814 (2005) 241. [5] M. Mabuchi, S. Takatsuka, M. Matsuoka, K. Tagawa, J. Pharm. Biomed. Anal. 35 (2004) 563. [6] K.L. Crump, I.M. McIntyre, O.H. Drummer, J. Anal. Toxicol. 18 (1994) 208. [7] R. Aderjan, S. Hofmann, G. Schmitt, G. Skopp, J. Anal. Toxicol. 19 (1995) 163. [8] S.O. Mashayekhi, M. Ghandforoush-Sattari, R.D.W. Hain, J. Clin. Pharm. Ther. 33 (2008) 419. [9] J.Q. Mi, X.X. Zhang, W.B. Chang, J. Immunoass. Immunochem. 25 (2004) 57. [10] J.L. Tsai, W.S. Wu, H.H. Lee, Electrophoresis 21 (2000) 1580. [11] Q.L. Zhang, J.J. Xu, X.Y. Li, H.Z. Lian, H.Y. Chen, J. Pharm. Biomed. Anal. 43 (2007) 237. [12] G. Sakai, K. Ogata, T. Uda, N. Miura, N. Yamazoe, Actuators Sens. B 49 (1998) 5. [13] N. Miura, K. Ogata, G. Sakai, T. Uda, N. Yamazoe, Chem. Lett. (1997) 713. [14] D.J. Chapman, S.P. Joel, G.W. Aherne, J. Pharm. Biomed. Anal. 12 (1994) 353. [15] R.S. Schwartz, C.R. Benjamin, Anal. Chim. Acta 141 (1982) 365. [16] B. Proksa, L. Molnár, Anal. Chim. Acta 97 (1978) 149. [17] P.H. Jordan, J.P. Hart, Analyst 116 (1991) 991. [18] A. Niazi, A. Yazdanipour, Chin. Chem. Lett. 19 (2008) 465.
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Simple and rapid voltammetric determination of morphine at electrochemically pretreated glassy carbon electrodes Fei Li, Jixia Song, Dongmei Gao, Qixian Zhang, Dongxue Han, Li Niu ∗ State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, and Graduate University of the Chinese Academy of Sciences, Chinese Academy of Sciences, Changchun 130022, PR China
a r t i c l e
i n f o
Article history: Received 18 January 2009 Received in revised form 9 May 2009 Accepted 11 May 2009 Available online 20 May 2009 Keywords: Morphine Electrochemical pretreatment Voltammetric detection Glassy carbon electrode
a b s t r a c t A simple and rapid method for morphine detection has been described based on electrochemical pretreatment of glassy carbon electrode (GCE) which was treated by anodic oxidation at 1.75 V, following potential cycling in the potential range from 0 V to 1.0 V vs. Ag|AgCl reference electrode. The sensitivity for morphine detection was improved greatly and the detection limit was 0.2 M. The reproducibility of the voltammetric measurements was usually less than 3% RSD for six replicate measurements. Moreover, this method could readily discriminate morphine from codeine. And an electrochemical detection of morphine in spiked urine sample was succeeded with satisfactory results. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Morphine as a major component in opium is frequently used to relieve severe pain in patients, especially those undergoing a surgical procedure. However, it is toxic in excess and when abused. To prevent overdose-induced toxication, it is necessary to sensitively monitor the concentrations of morphine in a patient’s blood or urine. Various analytical methods have been developed for the determination of morphine and its major metabolites. The most common analytical techniques currently used include gas chromatography [1,2], high-performance liquid chromatography (HPLC) [3–8] and their combination with other detection methods. Benefited from high efficient separation, they have the advantage of high sensitivity, but suffer from time-consuming and expensive equipment. Use of capillary electrophoresis and microchip capillary electrophoresis [9–11] have partially solved this problem. However, the difficulty in preparing capillary column and its short life restrict its development. Immunoassay, such as surface plasmon resonance (SPR) based immunosensors [12,13] and radioimmunoassay (RIA) [14] are also reported for morphine detection, although they are very sensitive but antibody cross-reactivity may exist. Research in more depth has been devoted to develop a simple, economical, fast and efficient method for detecting morphine. The electrochemical method is widely used because it is highly sensitive, cheap, simple, easily miniaturized and conve-
∗ Corresponding author. Fax: +86 431 8526 2800. E-mail address: [email protected] (L. Niu). 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.05.011
nient. The use of bare electrodes such as glassy carbon electrode [15], platinum electrode and graphite electrode [16] are proved to be straightforward and simple on detecting morphine. However, there are a number of limitations, such as slow electron transfer, low sensitivity and electrode contamination, etc. To avoid those shortcomings, combination of HPLC and electrochemical detection has been designed [17]. Recently, some new electrochemical detection methods have been proposed for morphine detection. For example, an adsorptive differential pulse stripping method [18] and its conjugation with least-squares support vector machines [19] have been developed for trace morphine detection. Fast Fourier transformation with continuous cyclic voltammetry at Au microelectrode [20,21] has been devised for morphine detection in a flow injection system. Furthermore, different modified electrodes have been developed for morphine detection. For example, Jin and co-workers prepared a kind of cobalt hexacyanoferrate modified carbon paste electrode, combined it with HPLC and successfully detected morphine in vivo [22]. Ho et al. devised a Prussian blue-modified indium tin oxide (ITO) electrode [23] and molecularly imprinted electrodes for morphine determination [24,25]. Multiwalled carbon nanotubes modified preheated glassy carbon electrode has also been used for the morphine detection [26]. Prussian blue film modified-palladized aluminum electrode [27] has recently been used for morphine detection. It is also well known that electrochemical pretreatment of glassy carbon electrode (PGCE) are usually prepared by potentiostatic polarization at oxidizing potentials or by potential cycling in a wide potential range. The pretreatment introduces oxygen-containing
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functional groups on the electrode surface. These functional groups exhibit certain interaction, such as electron donor–acceptor, hydrogen bonding, electrostatic, dispersive and solvophobic interactions [28,29], with various species. For example, PGCE has been applied in the preconcentration and electroanalysis of copper [28] and manganese species [30]. A number of organic molecules such as erythromycin [31], vitamin B2 [29] have also been adsorbed strongly at such activated glassy carbon electrodes. Besides, PGCE has also been used to determine biomolecules, such as DNA [32,33]. However, to our best knowledge, the report concerning the detection of morphine at PGCE is quite few. In this work, it was found that morphine could also be effectively adsorbed and accumulated on the electrochemically pretreated glassy carbon electrode. So a simple, rapid and sensitive voltammetric method for morphine detection was explored. The method could readily discriminate morphine even from coexist codeine. As a potential application, the electrochemical detection of morphine in spiked urine sample was performed too.
2.4. Procedure for determination of morphine The electrochemical experiments were performed in 0.05 M PBS (pH 7.4) with different concentrations of morphine. The adsorptive accumulation of morphine at the working electrode was done in a stirred solution at open circuit for 90 s. After a 2 s quiet time, the cyclic voltammograms were recorded from 0 V to 0.7 V at 0.1 V/s. The electrode can be used for next measurement after continuous sweep for four cycles at potential range from 0 V to 1.0 V in 0.05 M PBS (pH 7.4). 2.5. Sample preparation Urine samples were obtained from healthy laboratory volunteers without any pretreatment, except that they were diluted by 100 times with 0.05 M PBS (pH 7.4). A quantity of the morphine was added to the diluted urine and the resulting drug urine solutions were used for voltammetric analysis. The content of the morphine was determined by the standard addition method [31].
2. Experimental 3. Results and discussion 2.1. Reagents 3.1. Morphology and surface composition after pretreatment Morphine hydrochloride and codeine phosphate were supplied by the Institute of Forensic Science Ministry of Public Security (China). Unless otherwise stated, other reagents were of analytical grade and used as received. Phosphate buffer solution (0.05 M PBS), which was prepared by mixing different volume of Na2 HPO4 solution (0.05 M) and KH2 PO4 solution (0.05 M), was employed as supporting electrolyte. Britton–Robinson (B–R) buffer was prepared by dissolving the same concentrations (0.04 M) of orthophosphoric acid, boric acid and glacial acetic acid in water and adjusting to the desired pH value with sodium hydroxide (0.2 M). All aqueous solutions were prepared with high pure water from a Millipore system (>18 M cm). 2.2. Apparatus Scanning electron microscopy (SEM) pictures were imaged by an XL30 ESEM FEG field emission scanning electron microscope. X-ray photoelectron spectroscopy (XPS) was conducted with a VG ESCALAB MKII spectrometer (VG Scientific, UK) employing a monochromatic Mg-K␣ source (h = 1253.6 eV) and the elemental content ratio was evaluated by using the software installed with the instrument. Cyclic voltammetric (CV) measurements were performed in a conventional three-electrode cell with a platinum wire as the auxiliary electrode and an Ag|AgCl (saturated KCl) as the reference electrode with a CHI 660 Electrochemical Workstation (CHI, USA). The working electrode was bare or pretreated glassy carbon electrode (GCE, d = 3 mm). All potentials reported here refer to the Ag|AgCl (sat. KCl) reference electrode.
Through the electrochemical pretreatment a thin layer was found on the electrode surface, which was in agreement with the previous report [34,35]. This layer is composed of carbon oxidation products of the electrode itself and has been termed electrochemical graphite oxide. The morphology of the glassy carbon electrode before and after the anodic oxidation pretreatment was examined by SEM (data not shown here). Both of them were flat and uniform in the submicrometer scale, indicating that the pretreatment was mild and did not cause significant changes in GCE surface morphology. The surface of the glassy carbon electrode was further examined by XPS measurement. As shown in Fig. 1, the O1s spectra show an increase in amplitude after the pretreatment (dotted), although the peak shape changed little. The major peak in the C1s spectrum (284.6 eV) changed in shape as a result of pretreatment. The width at half-maximum height went from 0.86 eV at the polished electrode (solid) to 1.42 eV at the pretreatment electrode. New C1s peaks were also found at the binding energy centered at ca. 286.9 eV, 288.6 eV, 292.9 eV and 295.8 eV, indicating the presence of different forms of carbon having higher binding energy, such as carbonyl, carboxyl and quinone [36,37]. Calculations indicated that the oxygen-to-carbon ratio of the electrode surface were 0.12 and 0.33 for the freshly polished and pretreated electrode, respectively. The increase in the O/C ratio and the O1s amplitude showed
2.3. Pretreatment of the glassy carbon electrode The glassy carbon electrode was polished carefully with 1.0 m, 0.3 m, and 0.05 m alumina slurry to a mirror finish, respectively. And it was cleaned in an ultrasonicating bath for 2 min before use. The general procedure for electrochemical pretreatment of glassy carbon electrode was modified from previously published procedures [32]. The preparation of PGCE was performed by anodic oxidation at 1.75 V for 200 s in 0.05 M PBS at pH 5. The electrode was then cycled between 0 and 1.0 V at a scan rate of 0.1 V/s until a stable current–voltage curve was obtained.
Fig. 1. XPS spectra of freshly polished GCE (solid) and pretreated GCE (dotted).
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Fig. 2. (A) Cyclic voltammograms of 25 M morphine in 0.05 M PBS (pH 7.4) (A) at (a) unpretreated GCE (dotted) and (b and c) pretreated GCE without (dash) and with accumulation (solid) at 0.1 V/s. Accumulation was performed in open circuit for 90 s. (B) Influence of accumulation time on the peak current of morphine at different concentrations. (a) 5 M; (b) 10 M; (c) 25 M. Scan rate: 0.1 V/s.
that the surface became oxygenated during pretreatment, which was consistent with previous reports, i.e. oxygen-containing groups could be created on the surface of glassy carbon electrodes through pretreatment [34,36,37]. Thus, the environment of surface carbon became more complicated upon pretreatment [36]. According to previous report [34], the pretreatment of GCE involved the formation of a new phase, which contained a significant amount of microcrystallinity and graphite oxide. 3.2. Voltammetric behaviour of morphine at pretreated glassy electrode Morphine is a kind of phenolic compounds, which can transfer electrons to electrodes at a certain potential. Fig. 2A shows the cyclic voltammograms of 25 M morphine at the unpretreated GCE (dotted) and pretreated GCE without (dash) and with accumulation (solid) in 0.05 M PBS (pH 7.4). At this pretreated GCE, the morphine gave a well-defined oxidation peak at ca. 0.43 V. The oxidation reaction of the phenolic group at the 3-position involving one-electron transferring is responsible for the major peak. The oxidation of the phenolic group leads to the formation of pseudomorphine as the main product. The oxidation of morphine can be described by the following mechanism (Scheme 1). The peak potential of the oxidation of morphine at the pretreated GCE was slightly more negative than that obtained at unpretreated GCE. The peak current of pretreated GCE was higher than that of the unpretreated GCE, although the background current of the pretreated GCE was somewhat higher than the unpretreated GCE. From curve b, it could be seen that morphine was quickly oxidized at the pretreated GCE without accumulation, and the peak current was 4-fold larger than that at the unpretreated GCE. And after a 90 s accumulation (curve c), an approximately twice enhancement of the peak current was observed over that obtained without accumulation. Therefore a considerable enhancement in sensitivity could be obtained by applying adsorption step.
Moreover, it was also found that the adsorption efficiency of morphine was affected strikingly by preanodization time, accumulation time and stirring. It is generally accepted that the number of active adsorption sites increases with the preanodization time [28,29]. We have investigated the influence of the activation time. Longer activation time usually resulted in larger number of adsorption sites. However, the thickness of the activated film became larger and the adsorption ability of morphine at the electrode decreased. Meanwhile, thicker film might result in memory effects of the electrode and thus affected the reproducibility too. We found that the adsorptive ability of morphine at the electrode decrease when the time duration for electrode pretreatment exceeds over 200 s (data not shown here). So a preanodization time of 200 s was selected. The influence of accumulation time of morphine at different concentrations (i.e. 2 M, 10 M and 25 M) to the peak current was employed. When the accumulation time was less than 90 s, the peak currents increased with the time, and after that it reached a platform. It indicated that the equilibrium between the concentration of morphine in solution and that on the surface of the electrode occurred at ca. 90 s. Thus, the accumulation of 90 s was selected as the optimum condition at open circuit (Fig. 2B). The peak current obtained under constant stirring also increased with stirring assistance. The results also showed that the peak currents decreased to a constant with succeeding potential scan, as previously reported [33], which was attributed to adsorption of oxidation product at this PGCE. So it is necessary to renew the electrode surface by potential cycling before each measurement. As shown in Fig. 3, the peak currents (ip ) at the pretreated electrode in 25 M morphine solution (pH 7.4) varied with change of scan rate (). In the range of 10–150 mV/s, the relations obey the following question (R = 0.9964, where is in mV/s and ip is in A), log ip = −1.342 + 0.888 log
(1)
This indicated that the electrode process was controlled simultaneously both by diffusion and adsorption [31]. 3.3. Effects of pH
Scheme 1. The reaction scheme of morphine.
Effect of pH on the peak current and peak potential (as shown in Fig. 4) for the morphine oxidation was also investigated. The peak potential shifted negatively with increase of pH. It could be explained by the consequence of deprotonation involved in the oxidation process which was facilitated at higher pH values [26]. A plot of peak potentials vs. pH value was found to be linear over the pH range of 3–9, corresponding to a mechanism involving the same number of electrons and protons (as shown in Fig. 4B). It
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Fig. 3. Cyclic voltammograms of 25 M morphine in 0.05 M PBS (pH 7.4) at pretreated GCE at various scan rates: 10 mV/s, 25 mV/s, 50 mV/s, 75 mV/s, 100 mV/s, 125 mV/s and 150 mV/s from inner to outer, respectively. Inset is the calibrated plot of log ip vs. log .
was found in Fig. 4A that the current increased with the decrease of the pH. Our results showed that morphine adsorbed readily on pretreatment glassy carbon electrode in acidic medium, while morphine was usually easily adsorbed on bare electrode under alkaline medium. Firstly, this might be related to the differences in the surface properties of the electrodes and the adsorption interactions between morphine and the electrode surfaces. According to previous report [34], the pretreatment of GCE involved the formation of a new phase, which contained a significant amount of microcrystallinity and graphite oxide. This graphite oxide film probably acts as a reservoir for morphine and causes the observed adsorption. But this graphite oxide film would be partially dissolved in alkaline medium [35] and resulted in the decrease of morphine adsorption. Secondly, the variation of electrostatic interaction and hydrogen bonding between morphine and the electrode surface at different pH could also be responsible for this phenomenon. Thirdly,
Fig. 5. Calibration curves of current response vs. morphine concentration. Inset is the calibration curve corresponding (a) to 4–18 M morphine and (b) to 18–100 M morphine. Accumulation was performed in open circuit for 90 s. Scan rate: 0.1 V/s.
the decrease of the peak current might be resulted from the decomposition of morphine in alkaline medium. Based on above reasons, the current decreased with the increase of the pH. 3.4. Calibration curve Fig. 5 displays the calibration curve for morphine determination at PGCE. The peak current increased with the morphine concentration up to 100 M. There were two linear relations with different slopes in the range of 4–100 M. In the low concentration, the linear range (4–18 M) gave a large slope of 0.0715 A L mol−1 , and a correlation coefficient of 0.997. When the concentration exceeded 18 M, it deviated from the linear relationship (4–18 M) and followed another linear relationship (18–100 M) which gave a relative small slope of 0.0353 A L mol−1 and a correlation coefficient of 0.998. Due to the limited adsorption sites, the saturated coverage of the electrode surface was reached and peak current tended to immovability when the morphine concentration is more than 400 M. The detection limit was estimated to be ca. 0.2 M based on the signal corresponding to three times the noise of the response following the accumulation of 90 s. Reproducibility of the PGCE for determination of morphine was also investigated. It was performed in the solution of 10 M morphine in 0.05 M PBS (pH 7.4). A relative standard deviation of 2.77% was obtained for six replicate measurements. 3.5. Discrimination of morphine from codeine
Fig. 4. (A) Influences of pH values on the peak current () and peak potential () of 25 M morphine in B–R buffers at 0.1 V/s. (B) Calibration plot of pH vs. potential.
Voltammetric response at the PGCE in morphine solution containing codeine was also examined further. Codeine, with the methyl ether group (–OCH3 ) substituted for the phenolic group (–OH) in the 3-position, is very similar to morphine in structure (as shown in Fig. 6 inset). It usually interferes with morphine analysis in urine or blood. The oxidation peak of codeine was found at 0.96 V (data not shown here) due to the absence of phenolic group at the 3-position. Therefore, the pretreated electrode operating at 0.43 V (as shown in Fig. 2A) can easily discriminate morphine from codeine. The adsorption of morphine by the PGCE involved interactions between morphine molecules and the activated surfaces. The presence of codeine may affect such detection of morphine due to its
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Table 2 Comparison the proposed method with other reported methods. Detection method
Limit of detection
Sample
Recovery (%)
Reference
Microchip CEa LPMEb –HPLCc Amperometry Amperometry-MIPd SIAe DPVf Voltammetry
0.2 mol L−1 0.05 mg L−1 0.2 mmol L−1 0.3 mmol L−1 0.076 g mL−1 0.01 mol L−1 0.2 mol L−1
Urine Urine Not applied Not applied Urine Plasma Urine
96.2 92.4–106.8 – – 96.3 98.5–102.5 95.1–106.6
[11] [39] [24] [25] [40] [19] This work
a b c d e f
Fig. 6. Cyclic voltammograms of 10 M morphine in the presence of various concentrations of codeine: 0 M, 5 M, 10 M and 20 M at the pretreated GCE, respectively. Accumulation was performed in open circuit for 90 s. Scan rate: 0.1 V/s. Inset: the molecular structure of morphine and codeine.
competitive adsorption. The degree of interference was explored at various codeine concentrations. The results, as shown in Fig. 6, exhibited that codeine did not show significant interference while its concentration was lower than 2-fold concentration of morphine. It was well known that it was important criteria for judging recent heroin use while the morphine-to-codeine ratio was higher than 2 [38]. And morphine was the major component in opium poppy. Therefore, the PGCE may be used for the quantitative and qualitative determination of morphine in opium poppy and for judging heroin abusers. When the concentration of codeine was 100 times larger than that of morphine, the PGCE was still available to detect morphine qualitatively. 3.6. Determination of morphine in urine The proposed method in real sample analysis was also examined in human urine samples. The recovery was evaluated by comparing the analytic signals of morphine obtained from the spiked urine with those of the same concentration standard solution (n = 5) (Table 1). The recovery of the spiked samples ranged between 95.1% and 106.6%. The R.S.D. (n = 5) was less than 6.0%. In Table 2, response characteristics of the proposed method are compared with those obtained by some reported methods. In comparison with some other voltammetric methods of morphine determination, our method showed advantages in several aspects. For example, Prussian blue film modified-palladized aluminum electrode [27] has been recently used for morphine detection. Here the PGCE has lower detection limit. Moreover, the preparation process of PGCE was simpler and the reproducibility was also good. Meanwhile, it could be reused through the simple electrochemical treatment and also could be even used in real sample. Niazi et al. [19] reported using of adsorptive differential pulse stripping voltammetric method on a hanging mercury drop electrode for determination of morphine with excellent detection limit (0.01 M). But the mercury is very poisonous and it was only used Table 1 Results of determination of morphine in urine sample. Urine sample
Spike (mol L−1 )
Found (mol L−1 )
Recovery (%)
R.S.D. (%)a
1 2 3
5.0 10.0 15.0
4.76 10.65 14.62
95.1 106.6 97.5
3.4 4.8 5.3
a
Average of five replicate measurements.
Capillary electrophoresis. Liquid phase microextraction. High-performance liquid chromatography. Molecularly imprinted polymer. Sequential injection analysis. Differential pulse voltammetry.
in the pretreated plasma sample. Compared with other methods to determine morphine in urine, such as microchip electrophoresis [11], liquid phase microextraction-HPLC [39] and sequential injection analysis [40], this method had the similar detection limit and the accuracy. At the same time, it also show extra advantages: simple preparation process of PGCE; short time and low cost for the analysis; and no pretreatment needed before the measurement. 4. Conclusion Voltammetric determination of morphine has been performed at the electrochemically pretreated GCE. The main advantage of the proposed method is simple and fast compared to other determination methods. Furthermore, a low detection limit and a wide range of concentrations are enough for usual analytical purpose. The method has been demonstrated that it easily discriminate morphine from codeine. Moreover, the determination morphine in real human urine without any sample pretreatment has been succeeded with consistent result with those of HPLC and microchip electrophoresis. The proposed method provides the basis for designing portable morphine sensor due to its easy and fast preparation, and low cost. Acknowledgement The authors are most grateful to the NSFC, China (No. 20827004) and Ministry of Science and Technology (Nos. 2006BAKB05 and 2007BAK26B06) for their financial support. References [1] B. Fryirs, M. Dawson, L.E. Mather, J. Chromatogr. B 693 (1997) 51. [2] U. Hofmann, S. Seefried, E. Schweizer, T. Ebner, G. Mikus, M. Eichelbaum, J. Chromatogr. B 727 (1999) 81. [3] P.P. Rop, F. Grimaldi, J. Burle, M.N. Desaintleger, A. Viala, J. Chromatogr. B 661 (1994) 245. [4] S.R. Edwards, M.T. Smith, J. Chromatogr. B 814 (2005) 241. [5] M. Mabuchi, S. Takatsuka, M. Matsuoka, K. Tagawa, J. Pharm. Biomed. Anal. 35 (2004) 563. [6] K.L. Crump, I.M. McIntyre, O.H. Drummer, J. Anal. Toxicol. 18 (1994) 208. [7] R. Aderjan, S. Hofmann, G. Schmitt, G. Skopp, J. Anal. Toxicol. 19 (1995) 163. [8] S.O. Mashayekhi, M. Ghandforoush-Sattari, R.D.W. Hain, J. Clin. Pharm. Ther. 33 (2008) 419. [9] J.Q. Mi, X.X. Zhang, W.B. Chang, J. Immunoass. Immunochem. 25 (2004) 57. [10] J.L. Tsai, W.S. Wu, H.H. Lee, Electrophoresis 21 (2000) 1580. [11] Q.L. Zhang, J.J. Xu, X.Y. Li, H.Z. Lian, H.Y. Chen, J. Pharm. Biomed. Anal. 43 (2007) 237. [12] G. Sakai, K. Ogata, T. Uda, N. Miura, N. Yamazoe, Actuators Sens. B 49 (1998) 5. [13] N. Miura, K. Ogata, G. Sakai, T. Uda, N. Yamazoe, Chem. Lett. (1997) 713. [14] D.J. Chapman, S.P. Joel, G.W. Aherne, J. Pharm. Biomed. Anal. 12 (1994) 353. [15] R.S. Schwartz, C.R. Benjamin, Anal. Chim. Acta 141 (1982) 365. [16] B. Proksa, L. Molnár, Anal. Chim. Acta 97 (1978) 149. [17] P.H. Jordan, J.P. Hart, Analyst 116 (1991) 991. [18] A. Niazi, A. Yazdanipour, Chin. Chem. Lett. 19 (2008) 465.
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