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Clinical Chemistry 48: 1049-1058, 2002;
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(Clinical Chemistry. 2002;48:1049-1058.)
© 2002 American Association for Clinical Chemistry, Inc.

Determination of Sulpiride by Capillary Electrophoresis with End-Column Electrogenerated Chemiluminescence Detection

Jifeng Liu1, Weidong Cao1, Haibo Qiu1, Xiuhua Sun1, Xiurong Yang1 and Erkang Wanga1

1 State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, People’s Republic of China.

aAuthor for correspondence. Fax 86-431-5689711; e-mail ekwang{at}ns.ciac.jl.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Capillary electrophoresis (CE) with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)32+]-electrogenerated chemiluminescence (ECL) detection is a promising method for clinical analysis. In this study, a method combining CE with Ru(bpy)32+ ECL (CE-ECL) detection that can be applied to amine-containing clinical species was developed, and the performance of CE-ECL as a quantitative method for determination of sulpiride in human plasma or urine was evaluated.

Methods: Sulpiride was separated by capillary zone electrophoresis in uncoated fused-silica capillaries [50 cm x 25 µm (i.d.)] filled with phosphate buffer (pH 8.0) and a driving voltage of +15 kV, with end-column Ru(bpy)32+ ECL detection. A platinum disc electrode was used as working electrode. Sulpiride in human plasma or urine samples (100 µL) was extracted by a double-step liquid-liquid extraction procedure, dried under nitrogen at 35 °C in a water bath, and reconstituted with 100 µL of filtered water. The extraction solvent was ethyl acetate–dichloromethane (5:1 by volume).

Results: Under optimum conditions (pH 8.0 phosphate buffer, injection for 6 s at 10 kV, and +1.2 V as detection potential), separation of sulpiride was accomplished within 4 min. The calibration curve was linear over a concentration range of 0.05–25.0 µmol/L, and the limit of detection was 2.9 x 10-8 mol/L for sulpiride. Intra- and interday CVs for ECL intensities were <6%. Extraction recoveries of sulpiride were 95.6–101% with CVs of 2.9–6.0%. The method was clinically validated for patient plasma and urine samples.

Conclusions: CE combined with Ru(bpy)32+ ECL is reproducible, precise, selective, and enables the analysis of sulpiride in human plasma and urine. It thus is of value for rapid and efficient analysis of amine-containing analytes of clinical interest.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Capillary electrophoresis (CE)1 is an exceptional tool for performing rapid and highly efficient biochemical separations (1)(2). CE uses only picoliter to nanoliter sample volumes, and these small sample volumes require a very sensitive detection method. It is well known that laser-induced fluorescence detection, radiometric detection, and electrochemical detection are the most sensitive detection techniques available for CE, providing low detection limits. Although impressive analyses have been performed with available CE detection methods, there is still a need for new, highly sensitive CE detection methods. In addition to providing sensitivity, it would be desirable for new detection methods to be simpler and able to detect new types of analytes at trace concentrations. Chemiluminescence (CL) detection has been shown to be ideally suited for the challenging volume and detection limit requirements characteristic of CE separations. Various CL reagents and schemes have been reported for CE, using luminol (3)(4), peroxyoxalate (5)(6), and acridinium-based CL (7)(8). CL detection offers excellent detection limits because of the extremely low background noise. In addition, the instrumentation necessary to collect a CL signal is simple, consisting of a photomultiplier tube, a light-tight box, and minimal optics (9).

Electrogenerated CL (ECL) is the production of light by an oxidation or reduction reaction at an electrode surface. Compared with CL, ECL offers the ability to generate luminescence in a defined location on the surface of the electrode. Detection of photons emitted from an ECL reaction should provide an extremely sensitive means of detection for CE. Furthermore, the use of ECL should reduce interference from solution impurities and eliminate the need for complicated construction of post-column reactors for CL detection because the electrode replaces the catalyst added for CL (9).

One of the most efficient and thoroughly examined CL compounds is tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)32+], which can emit light in both a CL and an ECL reaction. Ru(bpy)32+ ECL is observed when Ru(bpy)33+ reacts with Ru(bpy)3+ and yields an excited-state molecule, Ru(bpy)32+ (10). ECL emission can also be obtained when various oxidants and reductants react with the reduced or oxidized forms of Ru(bpy)32+; therefore, either the reductant or the oxidant can be treated as an analyte.

Ru(bpy)32+ ECL has been widely used as a detection method for various amine-containing analytes in flowing streams, such as HPLC and flow injection analysis (11)(12). Comparatively, the application of Ru(bpy)32+ ECL detection in CE is very limited. To our knowledge, no more than 10 reports about CE-ECL have been published (13)(14)(15)(16)(17)(18)(19), and some of them suffer from poor separation efficiency, low sensitivity, and complicated instrumentation. In these reports, Ru(bpy)32+ was added to the CE running buffer and converted to Ru(bpy)33+ at the exit end of the capillary by use of a separate working electrode (13) or was added in a small reservoir at the interface between the separation capillary and the working electrode, which was then used to oxidize Ru(bpy)32+ to Ru(bpy)33+ (14)(16)(17). Additionally, Ru(bpy)33+ has been generated in situ at the interface. In this approach, Ru(bpy)32+ was transported to the interface by a syringe pump through a reaction tube, and the separation capillary was inserted into the reaction tube (15). Beta blockers, amino acids, and tripropylamine were used as analytes to characterize the CE-ECL systems. However, none of the above-mentioned studies explored the performance of CE-ECL for application to clinical analyses.

In our laboratory, CE-ECL detection has been applied to the analysis of biochemicals and pharmaceuticals. A Ru(bpy)32+ ECL detection cell with end-column detection has been designed. In a separate experiment, we found that the detection limit for tripropylamine was 8 x 10-9 mol/L, which is four orders lower than that of the reported CE-ECL system for this compound (14). From these data, we concluded that the detector was very sensitive. In addition to sensitivity, the analysis system was compact and convenient. The inner diameter of separation capillary we used was 25 µm, thus eliminating the need for on-column fractionation, which was used in the reported systems for separating CE current from the ECL detector (13)(14)(15)(16)(17). The diameter of the working electrode was 300 µm, which was comparable to the outer diameter of the separation capillary; thus, alignment of the working electrode with the separation capillary outlet could be achieved easily under a microscope (x72 magnification).

Sulpiride {5-(aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxybenzamide}, a selective dopamine D2 antagonist with antipsychotic and antidepressant activity, is effective in the treatment of mental disorders, e.g., as a behavior regulator in the psychopathology of senescence, depression, and schizophrenia, and the frequency of extrapyramidal side effects is low (20)(21). The recommended oral dose of sulpiride in the treatment of schizophrenia is 200–300 mg three times a day with a gradual increase to a maximum of 1200 mg daily (20). Various methods have been reported for the determination of sulpiride; most of them were HPLC coupled to ultraviolet (22)(23)(24)(25) or fluorescence detection (21)(26)(27). Gas chromatography with mass spectrometric detection (22)(28) and oscillopolarographic detection (29) methods have also been reported.

The goal of this work was to develop a CE separation method with end-column Ru(bpy)32+ ECL detection for clinical analysis and to evaluate the performance of CE-ECL as a quantitative assay for determining sulpiride concentrations in human plasma and urine. For this purpose, we used the newly designed Ru(bpy)32+ ECL detection cell to detect sulpiride after CE separation. The intra- and interday precision and accuracy of the assay were evaluated. This method was applied to the quantitative detection of sulpiride in human body fluids, and seven patients were investigated for sulpiride concentrations in plasma and urine 10 h after oral intake of 200 mg of sulpiride.

In this experiment, the sulpiride in the human plasma and urine samples was extracted by a double-step liquid–liquid extraction procedure using ethyl acetate–dichloromethane (5:1 by volume) as the extraction solvent.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
reagents and chemicals
All reagents and chemicals used were at least analytical reagent grade. Tris(2,2'-bipyridyl)ruthenium (II) chloride hexahydrate was purchased from Aldrich Chemical Co. Sulpiride was purchased from Sigma Chemical Co. and was dissolved in 0.6 mol/L acetic acid at a concentration of 10 mmol/L for use as a stock solution. Before use, the stock solution was diluted to the desired concentrations. Sulpiride tablets were obtained from Jinan Pharmaceutical Factory; each tablet contains 100 mg of sulpiride. The buffers used in this experiment were sodium dihydrogen phosphate and disodium hydrogen phosphate. The buffer pH was adjusted with sodium hydroxide (98% purity; Sigma). D-Arginine, L-aspartic acid, L-glutamic acid, L-glycine, L-tyrosine, L-threonine, and L-valine were obtained from Shanghai Biochemical Co. Perphenazine, lidocaine hydrochloride, and megimide were purchased from Shanghai Zhaohui Pharmaceutical Factory. Haloperidol and phentolamine methanesulfonate were obtained from Shanghai Haipu Pharmaceutical Co. Promethazine hydrochloride was obtained from Shanghai Hefeng Pharmaceutical Co. Anisodamine hydrochloride and glyphylline were obtained from Shanghai No. 1 and Shanghai Xinyi Pharmaceutical Factory, respectively, and procaine hydrochloride was purchased from Beijing Yongkang Pharmaceutical Factory. All solutions were prepared with water purified by a Milli-Q system (Millipore) and stored at 4 °C in a refrigerator. Before use, all samples and buffer solutions were filtered through 0.22 µm cellulose acetate filters (Shanghai Xinya Purification Material Factory). Drug-free human plasma and urine were obtained from Changchun blood bank (Changchun, China) and a healthy male volunteer, respectively, for use in constructing the calibration curve and preparing the samples for recovery studies.

patient protocol
To determine whether CE-ECL could give reliable results for identification and quantification of sulpiride, plasma and urine samples were obtained from three male and four female patients newly admitted to Shandong Mental Health Center (Jinan, China), who were not on sulpiride treatment until this study began. The diagnosis, according to the Chinese Classification and Diagnostic Criteria of Mental Disorders, 3rd edition (30), was schizophrenia in six patients and mental retardation in one patient. The patients’ ages ranged from 19 to 43 years, and body weights were 45–68 kg. This study received approval from the Department of Medicine and Research Administration of Shangdong Mental Health Center, and patients enrolled in the study provided informed consent. The patients received 200 mg of sulpiride (two 100-mg tablets) orally at night. Baseline blood and urine (~2 mL) samples were collected 0.5 h before oral intake of the drug, and blood and urine samples (~2 mL) were collected the next morning, 10 h after the dosing. Blood samples were collected in test tubes without anticoagulants and centrifuged at 1020g for 10 min; the plasma was then transferred to suitably labeled tubes. Urine samples were also collected in labeled tubes, and all the plasma and urine samples were stored at -20 °C until analysis.

capillary zone electrophoresis and detection system
Electrophoresis in the capillary was driven by a high-voltage power supply (CZE 1000R; Spellman). Uncoated fused-silica capillaries (25-µm i.d.; 360-µm o.d.) were purchased from Hebei Yongnian Optical Conductive Fiber Plant. A Model 800 Electrochemical Analyzer (CH Instruments) was used to provide potential for the oxidation of Ru(bpy)32+ to Ru(bpy)33+. CL emission was detected by a Model BPCL Ultra-weak Luminescent Analyzer (Institute of Biophysics, Chinese Academy of Sciences, Beijing, China); the photomultiplier tube was operated in pulse mode and set at 900 V for detection.

A piece of capillary was cut to 50 cm in length and was used as separation capillary. An ~5-mm section of the polyimide coating was burned off at the distal end to allow light to pass. The capillary was placed between two buffer reservoirs. A high voltage was applied at the injection end, with the reservoir in the ECL detection cell held at ground potential.

ECL detection was carried out using a three-electrode system with a platinum disk working electrode, a 300-µm diameter Ag/AgCl reference electrode (in saturated KCl solution), and a 1-mm diameter platinum wire auxiliary electrode. The working electrode was placed at the outlet of the separation capillary.

The detection cell and the detector (detailed below) were located in the light-tight box of the luminescent analyzer to prevent stray room light from contributing to background noise.

ecl detection cell
ECL detection at a constant potential with CE was performed using the end-column approach; a schematic diagram of the detection cell and light detection apparatus is given in Fig. 1 . Axial alignment was achieved by adjusting the three nylon screws around the electrode. Once the electrode and capillary were axially aligned, the distance between the capillary and the electrode could be adjusted by adjusting the capillary holder under a microscope (x72 magnification). A distance of 70 ± 5 µm between the capillary and electrode was found to be optimal. The reservoir (300 µL) was refilled with Ru(bpy)32+ solution and 50 mmol/L phosphate supporting electrolyte before each analysis. A piece of 1-mm thickness optical glass was mounted at the bottom of the reservoir; photons produced during the Ru(bpy)32+ ECL reaction passed through the glass window and were detected by a photomultiplier tube.



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  		Figure 1. Schematic diagram of the Ru(bpy)32+ ECL detection cell.

1, Plexiglass cell body; 2, separation capillary; 3, separation capillary holder; 4, platinum disc working electrode; 5, working electrode holder; 6, platinum wire auxiliary electrode; 7, Ag/AgCl (in saturated KCl) reference electrode; 8, three electrode alignment nylon screws; 9, optic glass window; 10, photomultiplier tube (PMT); 11, reservoir (300 µL) for Ru(bpy)32+ and buffer.

platinum disk working electrode
The working electrode was constructed with a 300-µm diameter platinum wire. Approximately 2.5 cm was cut and carefully soldered to a copper wire with soldering tin at the end of the electrode; it was then inserted into a polypropylene tube (1-mm i.d.; 3 mm-o.d.) until ~100 µm of the platinum wire protruded from the tip of the tube. The tube was heated with continuous rolling above a micro flame alcohol burner until the plastic melted around and adhered tightly to the wire. Leakage of the electrolyte could be avoided by this technique. The electrode protruding from the plastic tube was cut and carefully polished with 1.0- and 0.3-µm {alpha}-Al2O3 on a polishing cloth until a mirror-smooth surface was obtained. Before use, the platinum disk electrode was sonicated and rinsed with doubly distilled water for ~3 min.

After each analysis, the electrode was treated by cyclic voltammetry at a potential of -0.5 to 0 V and a scanning rate of 100 mV/s for 2 min. This treatment allowed the electrode to be used at least 1 week without any loss in activity for oxidizing Ru(bpy)32+.

cyclic voltammetry
Cyclic voltammetry of 1 mmol/L Ru(bpy)32+ and 5 mmol/L sulpiride plus 50 mmol/L phosphate buffer (pH 8.0) was carried out in the same ECL detection cell with the same three-electrode system and electrochemical analyzer that were used in ECL detection.

electrophoresis procedure
A new fused-silica capillary was filled with 0.1 mol/L NaOH solution for 24 h before use and then flushed with filtered water and the corresponding running buffer for 15 min by means of a syringe. The running buffer in the experiment was 10 mmol/L phosphate solution. During the experiment, +15 kV (6 µA) separation voltage was applied across the capillary, and the detection potential was applied at the working electrode. After the baseline CL signal reached a constant value (~10 min), electromigration injection was used for sample introduction, and the electropherogram was recorded. Between two electrophoretic runs, the capillary was rinsed with filtered water and running buffer for ~5 min, respectively. The number of theoretical plates (N), was calculated according to the equation:

(1)
Where tm is the migration time, and W1/2 is the width at half height of the electrophoretic peak.

liquid–liquid sample extraction
We pipetted 100 µL of human plasma or urine samples containing sulpiride, enriched standards, and blank plasma or urine into 1.5-mL centrifuge tubes. The sample was alkalinized by adding 10 µL of 1 mol/L NaOH solution; 1 mL of ethyl acetate–dichloromethane (5:1 by volume) was then added to the tube, and the tube was capped. The samples were vortex-mixed for 10 s in a Model G-560E vortex-mixer (Scientific Industries, INC.) with a 25 Hz frequency (5 mm amplitude), mixed at 120 rpm (20 mm amplitude) on a tube shaker (Shenzhen Guohua Co.) for 10 min, cooled to -20 °C for 10 min to break the emulsion formed during mixing, and then centrifuged at 1700g for 5 min. The top organic layer was transferred to a clean tube, and the process was repeated. The organic layer obtained this time was transferred to the same tube, and the combined organic layers were evaporated to dryness under a gentle stream of nitrogen in a water bath at 35 °C. Filtered water (100 µL) was added to dissolve the residue, and the tube was vortex-mixed for 5 min at the same frequency as above. The sample was then injected into the CE-ECL system by electromigration.

calibration curves and extraction recovery
Calibrators for sulpiride were prepared by adding appropriate amounts of sulpiride to a series of 100-µL aliquots of drug-free human plasma or urine to achieve concentrations of 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, and 25.0 µmol/L, respectively; the samples were then extracted and analyzed using CE-ECL as described above. A calibration curve was obtained from the least-squares linear regression of the ECL peak intensities vs the concentrations of sulpiride added. This curve was used to calculate sulpiride concentrations in the unknown human plasma or urine samples based on the ECL peak intensities.

Two sets of samples, a control group and a recovery group, were prepared to determine the extraction recovery. The control samples were prepared as follows: 100-µL aliquots of drug-free human plasma or urine were extracted by the liquid–liquid sample extraction procedure described above, and the residues were reconstituted with 100 µL of 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, or 25.0 µmol/L sulpiride solution, respectively. The recovery samples were prepared according to the procedure for preparing calibrators; the control group and recovery group samples were then analyzed by CE-ECL.

The absolute recovery was calculated as the ratio of measured ECL peak intensities for the recovery samples to that of the corresponding control samples at each sulpiride concentration.

validation of the assay
Several enriched human plasma or urine samples were tested for the presence of interfering compounds. The intra- and interday CVs and relative error of the mean were used to validate the precision and recovery of the assay, based on sulpiride calibrators in plasma or urine. To assess intraday precision and recovery, we assayed five sets of controls at nine concentrations (0.05–25.0 µmol/L) with one calibration curve in the same run. To assess interday precision and recovery, we evaluated five sets of control samples at nine different concentrations on 5 different days in 1 week (five calibration curves were prepared).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ELECTROCHEMISTRY OF Ru(bpy)32+ AND SULPIRIDE
In the cyclic voltammograms, the Ru(bpy)32+/Ru(bpy)33+ couple displayed reversible electrochemistry ({Delta}Ep = 59 mV) on the platinum disc electrode (Fig. 2 , voltammogram III), whereas sulpiride did not show an obvious electrochemical response in the potential range from 0 to +1.4 V (Fig. 2 , voltammogram II). When cyclic voltammetry of Ru(bpy)32+ was repeated in the presence of sulpiride, the Ru(bpy)33+ reduction disappeared and an electrochemical catalytic wave, produced by the reduction of Ru(bpy)33+ by sulpiride, was observed (Fig. 2 , voltammogram IV).



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  		Figure 2. Cyclic voltammograms of Ru(bpy)32+ and sulpiride on a platinum disc electrode (300-µm diameter).

Voltammograms: I, phosphate buffer only; II, 5 mmol/L sulpiride; III, 1 mmol/L Ru(bpy)32+; IV, 1 mmol/L Ru(bpy)32+ plus 5 mmol/L sulpiride. Potential sweep rate was 100 mV/s. Supporting electrolyte was 50 mmol/L phosphate buffer, pH 8.0.

optimum conditions of ce with end-column ecl detection
The intensity of the emitted light is dependent on the rate of the light-emitting chemical reaction, and this reaction rate is dependent on the potential applied to the electrode (31). We evaluated the potential at which a maximum ECL signal was observed. Applied potentials of 1.0–1.3 V were explored, with 1.2 V producing the maximum ECL response. The ECL increased when the electrode potential was changed from 1.0 to 1.2 V and then decreased slightly after 1.2 V. We therefore set 1.2 V as the detection potential in our experiments.

As discussed above, there were roughly three modes for introducing Ru(bpy)32+ at the electrode–capillary interface. In the end-column detection mode presented here, the reservoir is filled with Ru(bpy)32+, and Ru(bpy)33+ is generated on the surface of the working electrode. However, there is a strong flow of effluent from the electrophoresis capillary over the electrode, which may reduce the concentration of Ru(bpy)33+, reducing the efficiency of light-producing reaction (9). In addition, only a fraction of the total amount of Ru(bpy)32+ is converted to Ru(bpy)33+ on the electrode surface. Thus, the electrogenerated Ru(bpy)33+ is the limiting reagent for the ECL reaction (15)(32). The amount of Ru(bpy)33+ at the capillary–electrode interface could also limit the detection reaction. Thus, increasing the concentration of Ru(bpy)32+ increases the concentration of Ru(bpy)33+ and, thus, the ECL intensity (32). We found that the ECL intensity increased with increasing Ru(bpy)32+ concentration from 0.2 to 5 mmol/L; we therefore selected a Ru(bpy)32+ concentration of 5 mmol/L to achieve a high ECL signal.

Because the injection volume is one of the most important factors in the separation and detection, we observed the effect of injection voltage and time on the ECL intensity of sulpiride to optimize the value of the injection voltage.

The ECL intensity increased, whereas N decreased, with increasing injection voltage over a range from 5 to 17.5 kV (Fig. 3A ). The CL reaction occurs in the diffusion layer near the electrode when Ru(bpy)32+ is oxidized to Ru(bpy)33+ at the electrode surface and the oxidized Ru(bpy)33+ contacts the analyte (32). Because the CL reaction rate is a function of the concentrations of species involved in the reaction and because emission intensity is dependent on the rate of the reaction (11), when more analyte in the effluent is present in the diffusion layer of the working electrode, a higher ECL signal can be obtained. On the other hand, when too much analyte gets into the diffusion layer, it frequently causes peak broadening. As a compromise, to obtain a high ECL signal and large N, we selected 10 kV as the optimal injection voltage. As illustrated in Fig. 3B , ECL intensity increases with increasing injection time, whereas N decreases. The reason for the change is similar to that for the effect of injection voltage (described above), and a 6-s injection time was chosen.



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  		Figure 3. Effect of injection voltage (A) and injection time (B) on the ECL intensity ({square}) of Ru(bpy)32+ and the number of theoretical plates (N; •).

Sulpiride solution (1 µmol/L) was injected in electromigration mode. The injection time was 3 s in A, and the injection voltage was 10 kV in B. Separation was carried in 10 mmol/L phosphate buffer, pH 8.0. The fused-silica capillary [50 cm x 25 µm (i.d.)] was uncoated. Separation voltage was 15 kV (~6 µA). The reservoir contained 300 µL of 5 mmol/L Ru(bpy)32+ plus 50 mmol/L phosphate buffer, pH 8.0. A working potential of +1.2 V (vs Ag/AgCl) was applied to the platinum disc working electrode.

The effect of pH on the ECL intensity was investigated in a pH range from 4.5 to 10.0 in 0.5 pH units. With various amines, optimum pH values have been reported at pH 4–6 (33), and values remain essentially constant from pH 3 to 9 (34). In our present experiment, we observed that the ECL intensity for sulpiride was dependent on pH. As shown in Fig. 4 , at a pH range of 4.5–7.5, ECL intensity increased steadily and reached a maximum value of ~1500 at pH 8.0. At pH values >8.5, the ECL intensity decreased rapidly. A pH of 8.0 was chosen to obtain high ECL signals.



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  		Figure 4. Effect of buffer pH on the ECL intensity of Ru(bpy)32+.

Sulpiride solution (1 µmol/L) was injected in electromigration mode at 10 kV for 6 s (~3 nL). Separation was carried in 10 mmol/L phosphate buffer. A driving voltage of 15 kV was applied to the fused-silica capillary [50 cm x 25 µm (i.d.)]. The reservoir contained 300 µL of 5 mmol/L Ru(bpy)32+ plus 50 mmol/L phosphate buffer. The potential of platinum disc working electrode was +1.2 V (vs Ag/AgCl). The pH of phosphate buffer was adjusted to the corresponding values.

extraction recovery
For human specimens, it has been reported that ethyl acetate–dichloromethane (5:1 by volume) is suitable as the extraction solvent for plasma because it produces minimal interference (27). In our experiments, we found that extraction with this solvent mixture provided maximum recoveries (95.6–101%) and minimum CVs for recovery (2.9–6.0%) and also produced clean extracts devoid of CE and ECL interference from the human plasma or urine. We therefore used ethyl acetate–dichloromethane (5:1 by volume) as the extraction solvent in this study.

interference study
Shown in Fig. 5 are typical electropherograms of the extracts from plasma and urine containing 10 µmol/L sulpiride under optimum experimental conditions: pH 8.0 phosphate buffer, injection for 6 s at 10 kV, and a detection potential of +1.2 V. Drug-free human plasma and urine showed no interference for sulpiride.



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  		Figure 5. Electropherograms of extracts from human plasma (A) and human urine (B).

Electropherograms: I, extract of plasma containing 10 µmol/L sulpiride; II, drug-free human plasma; III, urine containing 10 µmol/L sulpiride; IV, drug-free human urine. For each condition, 100 µL of plasma or urine was extracted. The extracts were reconstituted with 100 µL of H2O, and ~3 nL was electromigrated into the capillary [50 cm x 25 µm (i.d.)]. Separation conditions were as follows: buffer, 10 mmol/L phosphate, pH 8.0; separation voltage, 15 kV. The reservoir contained 300 µL of 5 mmol/L Ru(bpy)32+ plus 50 mmol/L phosphate buffer, pH 8.0. The working electrode potential was +1.2 V (vs Ag/AgCl).

To examine other potential interferences, we added the following substances and drugs to plasma or urine samples containing 10 µmol/L sulpiride: D-arginine (100 µmol/L), L-aspartic acid (100 µmol/L), L-glutamic acid (100 µmol/L), L-glycine (100 µmol/L), L-tyrosine (100 µmol/L), L-threonine (100 µmol/L), L-valine (100 µmol/L), perphenazine (14 µmol/L), lidocaine hydrochloride (10 µmol/L), megimide (16 µmol/L), haloperidol (13 µmol/L), phentolamine methanesulfonate (26 µmol/L), promethazine hydrochloride (10 µmol/L), anisodamine hydrochloride (30 µmol/L), glyphylline (40 µmol/L), and procaine hydrochloride (10 µmol/L). Approximately five additional peaks were observed within the 5-min migration time compared with the sample without these substances and drugs (Fig. 6 ). The sulpiride peak was well resolved, and the recovery and migration time of sulpiride were not affected.



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  		Figure 6. Electropherogram of extracted human plasma containing 10 µmol/L sulpiride to which seven amino acids and nine other drugs were added (concentrations are given in the text).

Plasma (100 µL) containing sulpiride, seven amino acids, and nine other drugs was extracted. The dried extract was reconstituted with 100 µL of H2O and injected for 6 s at 10 kV. Separation was carried in 10 mmol/L phosphate buffer, pH 8.0. A separation voltage of 15 kV was applied to the fused-silica capillary [50 cm x 25 µm (i.d.)]. The reservoir contained 300 µL of 5 mmol/L Ru(bpy)32+ plus 50 mmol/L phosphate buffer, pH 8.0. The working electrode potential was +1.2 V (vs Ag/AgCl).

assay validation
Assay validation involved the estimation of intra- and interday imprecision and relative error in human plasma and urine. The results for plasma are presented in Table 1 . The imprecision and relative error data for urine were similar and are not presented. The intra- and interday CVs were 2.9–5.3% and 4.3–6.0%, respectively. The intra- and interday relative errors of the mean were -4.38% to 1.78% and -3.38 to 1.61%, respectively.


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  		Table 1. Intra- and interday precision of the sulpiride assay.1

The mean intraday migration time was 3.76 ± 0.02 min with a CV of 0.48%, and the mean interday migration time was 3.81 ± 0.03 min with a CV of 0.86%. All these data depicted the high precision of the analysis.

linearity and detection limit
The sulpiride calibration curve (Fig. 7 ) was linear (r >0.998) at 0.05–25.0 µmol/L. The experimental detection limit, defined as the lowest concentration of sulpiride that gave a signal-to-noise ratio 3, was 2.9 x 10-8 mol/L (9.9 µg/L, or 0.029 µmol/L). Thus, the assay is sufficiently sensitive and reliable for monitoring sulpiride in human plasma or urine.



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  		Figure 7. Typical calibration curves for the assay of sulpiride in human plasma or urine.

Regression lines obtained by least-squares regression analysis for sulpiride. The regression equations were calculated by plotting the ECL intensities vs the plasma or urine concentrations of sulpiride. The equations for the regression lines are: intraday (top), y = 940.97x + 457.02 (r = 0.9989); interday (bottom), y = 897.88x + 469.89 (r = 0.9987).

application to study of sulpiride in patient plasma and urine
The method described here was applied to the determination of sulpiride in plasma and urine from seven patients treated with sulpiride. Sulpiride was clearly detected in all samples examined, and no interfering peaks were observed from endogenous blank plasma or urine matrices (Fig. 8 ).



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  		Figure 8. Electropherograms of extracted patient plasma (left) and urine (right) samples.

The seven patients received oral doses of 200 mg (two 100-mg tablets) sulpiride at night. Blank blood and urine samples (~2 mL) were collected at 0.5 h before oral intake of the drug and 10 h after dosing (the next morning). Plasma sample was obtained by centrifugation at 1020g for 10 min. The plasma and urine samples were extracted by double-step liquid–liquid extraction, as described in the text. The extracts were reconstituted with 100 µL of H2O, and ~3 nL was electromigrated into the capillary [50 cm x 25 µm (i.d.)]. Separation buffer was 10 mmol/L phosphate, pH 8.0. Separation voltage was 15 kV. The reservoir contained 300 µL of 5 mmol/L Ru(bpy)32+ plus 50 mmol/L phosphate buffer, pH 8.0. The working electrode potential was +1.2 V (vs Ag/AgCl). I–VII refer to the seven patients. The calculated plasma sulpiride concentrations were 0.56, 1.00, 0.38, 0.43, 0.65, 0.44, and 0.58 µmol/L, and the calculated urinary concentrations were 6.71, 6.16, 8.98, 10.35, 8.76, 14.22, and 11.61 µmol/L for patients I–VII, respectively.

The regression equation for sulpiride concentrations in patient plasma and urine samples, as measured by CE-ECL, was: y = 940.97x + 457.02, where x is the sulpiride concentration (µmol/L) and y is the ECL intensity (counts; see top panel in Fig. 7Up ). The sulpiride concentrations in the plasma samples from patients I–VII were 0.56, 1.00, 0.38, 0.43, 0.65, 0.44, and 0.58 µmol/L, respectively, which were comparable to those obtained by HPLC [0.29–0.58 µmol/L (100–200 µg/L)] for the plasma samples collected at the same time (10 h) after the patients received the same oral dose (200 mg) (26). The discrepancy may be attributable to the variability of the bioavailability of sulpiride among the patients. The urinary sulpiride concentrations measured by CE-ECL were 6.71, 6.16, 8.98, 10.35, 8.76, 14.22, and 11.61 µmol/L for patients I–VII, respectively.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CE is available as a clinical tool for measurement of serum protein and quantification of hemoglobin variants; for drug, vitamin, and isoenzyme analysis; and for analysis of metabolic disorders, porphyria, and DNA (35). The Ru(bpy)32+ ECL is a well-established, sensitive detection technique for the determination of various amine-containing analytes without derivatization in flowing effluent streams, and the application of this method will be continue to grow in the biological and clinical sciences.

Sulpiride possesses a chromophore and a fluorophore in its structure, which enable ultraviolet or fluorescent detection. Sulpiride also has a tertiary amine group; it thus can be treated as an analyte when Ru(bpy)32+ ECL detection is adopted. The electrochemical and ECL mechanisms for the reaction of Ru(bpy)32+ with amines on different electrodes have been reported previously by other investigators (11)(36)(37)(38). In this study, when a platinum electrode was used and the amine was sulpiride, we think that the mechanism was identical, based on the cyclic voltammograms (Fig. 2Up ). The protonated sulpiride radical can be formed by direct electrochemical oxidation of sulpiride or by oxidation of Ru(bpy)33+ (Fig. 2Up , voltammogram IV). The protonated sulpiride radical then spontaneously loses a proton, forming a neutral sulpiride radical. The main route for light emission is reduction of Ru(bpy)33+ by the neutral sulpiride radical to electronically excited Ru(bpy)32+, which emits light when it relaxes to the ground state. According to this mechanism, our CE-ECL method can also be used to detect other amine-containing substances.

Sulpiride is slowly and poorly absorbed from the gastrointestinal tract, with peak serum concentrations occurring within 2–6 h and 30% of an oral dose excreted unchanged in the urine in 48 h (20). Sulpiride in human plasma or urine thus can be detected at an appropriate time after the administration of the drug. HPLC methods are routinely used for sulpiride assays.

In this report we have presented, for the first time, the performance characteristics of CE-ECL as a quantitative method for the determination of sulpiride in human plasma and urine and as a clinical assay for analyzing sulpiride in patient plasma or urine samples. This CE-ECL method seems to offer several advantages compared with HPLC: it is rapid (run time <4 min), inexpensive (small buffer and reagent volumes; less-expensive fused-silica capillary and instrumentation), relatively robust, reproducible, and selective. We have provided optimal analytical conditions and data on reproducibility, linearity, and sensitivity. Using capillary zone electrophoresis separation with end-column Ru(bpy)32+ ECL detection, we achieved good precision and high recoveries. Sulpiride concentrations in patient plasma samples measured by CE-ECL correlated well with those determined by HPLC (26). The calibration curve was linear at 0.05–25.0 µmol/L (r >0.998). This linear range was wider than those reported for HPLC methods: 14.7–293 µmol/L (23), 0.029–4.4 µmol/L (21), 0.029–2.9 µmol/L (26), and 0.058–4.4 µmol/L (27).

The detection limit for the CE-ECL method presented here is comparable to the detection limits reported for two fluorescence methods (2.9 x 10-8 mol/L) (25)(26), but lower than the detection limits reported for another fluorescence method (5.8 x 10-8 mol/L) (27), for a gas chromatographic–mass spectrometric method (7.31 x 10-8 mol/L) (22), for HPLC with ultraviolet detection [2.92 x 10-6 (23) and 3.5 x 10-7 mol/L (24)], and for a oscillopolarographic detection method (1 x 10-7 mol/L) (29) and is sensitive enough for clinical analysis. It should be noted that only 100 µL of plasma or urine is necessary in the extraction procedure to achieve this low limit of detection and that this sample volume is much smaller than the volume needed for a HPLC method (>1 mL). Taking into consideration the low sample injection volume (3 nL), the mass limit of detection obtained by this CE-ECL method was only 8.7 x 10-17 mol, which is several orders of magnitude lower than the detection limit for the HPLC method, for which the sample injection volumes are ~100 µL (21)(27).

In conclusion, we have developed and validated a new method combining CE with end-column Ru(bpy)32+ ECL detection that is reproducible, reliable, and precise for the analysis of sulpiride in human plasma or urine. In the clinical laboratory, this CE-ECL method may be of great value for drug monitoring, pharmacokinetic or bioavailability studies, and other clinical analysis.


   Acknowledgments
 
We thank the staff of the Shandong Mental Health Center for obtaining patient samples. This project was supported by the National Natural Science Foundation of China.


   Footnotes
 
1 Nonstandard abbreviations: CE, capillary electrophoresis; CL, chemiluminescence; ECL, electrogenerated CL; and Ru(bpy)32+, tris(2,2'-bipyridyl)ruthenium(II).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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