Sol–gel-immobilized Tris(2,2′-bipyridyl)ruthenium(II) electrogenerated chemiluminescence sensor for high-performance liquid chromatography

Analytica Chimica Acta 541 (2005) 49–56

Sol–gel-immobilized Tris(2,2
-bipyridyl)ruthenium(II) electrogenerated chemiluminescence sensor for high-performance liquid chromatography Han Nim Choi, Sung-Hee Cho, Yu-Jin Park, Dai Woon Lee, Won-Yong Lee∗ Department of Chemistry and Center for Bioactive Molecular Hybrids, Yonsei University, Seoul 120-749, Republic of Korea Received 22 April 2004; received in revised form 28 June 2004; accepted 28 June 2004 Available online 23 August 2004

Abstract The sol–gel-immobilized Tris(2,2
-bipyridyl)ruthenium(II) [Ru(bpy)3 2+ ] electrogenerated chemiluminescence (ECL) sensor was applied to the reversed-phase high-performance liquid chromatography (HPLC) determination of phenothiazine derivatives (promazine, chlorpromazine, triflupromazine, thioridazine, and trifluoperazine) and erythromycin in human urine samples. In this method, Ru(bpy)3 2+ was immobilized in sol–gel-derived titania (TiO2 )–Nafion nanocomposite films coated on a dual platinum electrode. This method eliminates an extra pump needed for the delivery of Ru(bpy)3 2+ reagent into a reaction/observation zone in front of photomultiplier tube because the immobilized-Ru(bpy)3 2+ is recycled on the electrode surface by an applied potential at +1.3 V versus Ag/AgCl (3 M NaCl) reference electrode. The resulting analytical performances such as detection limit, working range, sensitivity, and measurement precision were slightly worse than those obtained with the conventional post-column Ru(bpy)3 2+ addition approach. The lack of significant interferences and the low detection limits for phenothiazine derivatives and erythromycin indicate that the proposed HPLC-Ru(bpy)3 2+ ECL detection method is suitable for the determination of those compounds in biological fluids. © 2004 Elsevier B.V. All rights reserved. Keywords: Tris(2,2
-bipyridyl)ruthenium(II); Electrogenerated chemiluminescence; Sol–gel; Phenothiazines; Erythromycin; HPLC

1. Introduction In recent years, chemiluminescence (CL) has become an attractive detection method for high-performance liquid chromatography (HPLC) due to the very low detection limits and wide linear dynamic ranges which can be obtained with relatively simple instrumentation [1]. In particular, Tris(2,2
-bipyridyl)ruthenium(II) [Ru(bpy)3 2+ ] electrogenerated chemiluminescence (ECL) has been used for the HPLC determination of a wide range of compounds [2–4], such as aliphatic amines [5], antihistamines [6], amino acids [7,8], clindamicine antibiotics [9], dansyl derivatized amino acids [10,11], oxalate [12,13], erythromycin [14], tricyclic antidepressants [15], and ␤-blockers [16]. The ECL emission of the Ru(bpy)3 2+ /amine systems presumably arises from the ∗

Corresponding author. Tel.: +82 2 2123 2649; fax: +82 2 364 7050. E-mail address: [email protected] (W.-Y. Lee).

0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.06.076

energetic electron transfer reaction between the electrogenerated Ru(bpy)3 3+ and a strong reducing intermediate formed by the one-electron oxidation of amines [12,17,18]. 3+ − Ru(bpy)2+ 3 → Ru(bpy)3 + e ∗

2+ Ru(bpy)3+ 3 + reductant → [Ru(bpy)3 ] + product ∗

2+ [Ru(bpy)2+ 3 ] → Ru(bpy)3 + light (610 nm)

The reaction scheme for Ru(bpy)3 2+ ECL reveals that the starting material Ru(bpy)3 2+ is regenerated in situ when it is immobilized on an electrode surface. Therefore, the immobilization of Ru(bpy)3 2+ on the electrode surface can overcome several flaws in the conventional HPLC–ECL scheme based on the post-column Ru(bpy)3 2+ -addition approach, where Ru(bpy)3 2+ is delivered to a reaction/observation zone in front of photomultiplier tube by an extra pump, and re-

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acts with the HPLC-separated analyte to produce ECL emission. The use of the Ru(bpy)3 2+ -immobilized ECL sensor can eliminate the extra pump and also save the expensive Ru(bpy)3 2+ reagent. To date, several different approaches have been tried to immobilize Ru(bpy)3 2+ on a variety of different electrode surfaces. For example, Ru(bpy)3 2+ was immobilized in Nafion (perfluorosulfonated ionomer) film [19–21]. Since cationexchangable Nafion contains hydrophobic domain composed of fluorocarbon skeleton [22], a hydrophobic cation such as Ru(bpy)3 2+ can be easily incorporated into the films via both an ion-exchange process and hydrophobic interactions. However, the stability of this sensor is problematic because the immobilized Ru(bpy)3 2+ migrates into the electro-inactive hydrophobic region with time. In addition, the Nafion-based ECL sensor is rapidly destroyed upon exposure to mobile phases containing such high organic content [23]. Recently, a number of sol–gel-derived silicate/organic polymer composite films have been used to immobilize the Ru(bpy)3 2+ because the sol–gel-derived silica films possess physical rigidity, chemical inertness, negligible swelling, thermal stability, and optical transparency [24–26]. Ru(bpy)3 2+ -modified chitosan–silica composite films have been reported and the presence of silica gel in the ECL sensor improved the long-term stability of the sensor compared to that of the Nafion-based ECL sensor [27]. Khramov and Collinson have studied the ECL sensor consisting of Ru(bpy)3 2+ immobilized in Nafion/silica composite films [28]. The ECL sensor exhibited improved the ECL sensitivities for tripropylamime and oxalate compared to pure Nafion-based ECL sensor. Similarly, Dong and co-workers have reported the immobilization of Ru(bpy)3 2+ in poly(sodium 4-styrene sulfonate)/silica composite films and the ECL sensor showed very good storage stability during a period of six months [29,30]. Although, all reported ECL sensors based on the sol–gel-derived silicate films exhibited improved ECL characteristics compared to those based on pure Nafion films, the Ru(bpy)3 2+ ECL sensor was not applied as a detector for HPLC. Recently, we constructed the ECL sensor based on Ru(bpy)3 2+ immobilized in sol–gel-derived titania (TiO2 )–Nafion nanocomposite films coated on a glassy carbon electrode [31]. The ECL sensor exhibited improved ECL sensitivity and long-term stability compared to the ECL sensors based on pure Nafion films. Moreover, the sol–gelimmobilized ECL sensor is stable in a mobile phase containing organic solvent (30% acetonitrile, v/v). In the present work, the Ru(bpy)3 2+ ECL sensor based on titania–Nafion composite film was applied to the HPLC determination of model analytes such as phenothiazine derivatives (promazine, chlorpromazine, triflupromazine, thioridazine and trifluoperazine) and erythromycin in human urines. The resulting analytical performances such as detection limit, working range, sensitivity, and measurement precision were compared to those obtained with the conventional post-column Ru(bpy)3 2+ addition approach.

2. Experimental 2.1. Materials Ru(bpy)3 2+ chloride, titanium (IV) isopropoxide (Ti(OR)4 ) (R = CH(CH3 )2 , 99.99%), Nafion (perfluoinated ion-exchange resin, 5% (w/v) solution in a solution of 90% aliphatic alcohol/10% water mixture), and phenothiazine derivatives (hydrochlorides of promazine, chlorpromazine, triflupromazine, thioridazine, and trifluoperazine) were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Table 1 shows the structures of the phenothiazine derivatives examined. The buffers used in this work were sodium acetate trihydrate, glacial acetic acid, potassium dihydrogenphosphate, and potassium hydrogenphosphate from Aldrich Chemical Co. The pH of buffer solution was adjusted with glacial acetic acid and 1.0 M NaOH. Acetonitrile was J & T Baker (Phillipsburg, NJ, USA) HPLC grade solvent purchasing from commercial source. The urine sample was obtained from one healthy volunteer. Water for all solutions was purified using a Milli-Q water purification system (Millipore, Bedford, MA).

Table 1 Chemical structures and relative ECL intensities of five phenothiazine derivatives Compounda

Structure

Relative ECL intensityb

Promazine

1.00

Chlorpromazine

0.675

Triflupromazine

0.597

Thioridazine

0.785

Trifluoperazine

0.898

a All concentrations were 0.5 mM in 100 mM phosphate buffer at pH 6.0 in which 0.5 mM Ru(bpy)3 2+ was dissolved. b Intensity normalized to promazine = 1.00.

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Fig. 1. Schematic diagram of the HPLC-Ru(bpy)3 2+ ECL detection system. The Ru(bpy)3 2+ solution and extra HPLC pump were used only for the conventional post-column Ru(bpy)3 2+ -addition approach.

2.2. Instrumentation Cyclic voltammetric experiments were performed with an EG&G 273A potentiostat (Oak Ridge, TN, USA). Flow injection analysis and HPLC–ECL detection based on postcolumn Ru(bpy)3 2+ addition approach was performed with the ECL detection system as shown in Fig. 1. In the HPLCRu(bpy)3 2+ ECL sensor detection system, a flow cell was assembled from a conventional LC-EC dual platinum electrode (Bioanalytical Systems, West Lafayette, IN, USA) and placed against a transparent Plexiglass window for the detection of ECL emission as shown in Fig. 2. In addition to a dual platinum electrode, a stainless steel counter electrode at the cell outlet and silver wire quasi-reference electrode were used. The entire flow cell was directly placed in front of the photo multiplier tube (PMT) window. A Younglin M930 HPLC pump (Young-Lin Instrument, Seoul, Korea) was used to deliver a buffered mobile phase. Injection was made using a Rheodyne Model 9725 injector with 20 ␮L injection loop. A XTerra RP18 column (5 ␮m, 150 mm × 4.6 mm i.d.) from Waters (Milford, MA, USA) was used for reverse-phase separation of phenothiazine derivatives. The potential of the ECL detection system was held at 1.3 V versus silver quasireference electrode using a Won-A Tech potentiostat (Seoul, Korea). The photon multiplier tube used was a Hamamatsu

Photonics H5784 optical sensor module. The output of the PMT module was fed into Autochro Data Module (YoungLin Instrument). 2.3. Preparation of the Ru(bpy)3 2+ ECL sensor The Ru(bpy)3 2+ ECL sensor based on TiO2 –Nafion (50%) composite film was prepared as described in the previous paper [31]. 2.4. Experimental conditions In the pH study, solutions of each analyte were prepared at different pH values covering a range from 4 to 8. In order to cover wide range of pH values, it was necessary to use a couple of different buffer systems. Buffer solutions used in these studies were acetate buffer (pH 4.0–5.0) and phosphate buffer (pH 6.0–8.0). 1.0 mM Ru(bpy)3 2+ and each 1.0 mM phenothiazine solution were prepared in the 50 mM buffer solution. A pH study has been carried out with the sol–gelimmobilized ECL sensor in a single-channel flow injection system at a flow rate of 2.0 mL/min. In the HPLC–ECL experiments, an aliquot of a human urine sample was diluted four-fold with 0.05 M phosphate buffer solution at pH 7 and the mixture was spiked with a so-

Fig. 2. Schematic diagram of the flow cell placed in front of PMT.

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lution containing accurately measured phenothiazine derivatives or erythromycin. The urine samples were filtered using a syringe filter of 0.45 ␮m pore size before injection into HPLC column. The column was operated at an ambient temperature (25 ± 2 ◦ C). The mobile phase was also filtered using a vacuum filter system equipped with 0.45 ␮m nylon membrane filter (Millipore, Bedford, MA) and sonicated for 30 min. In the HPLC determination of phenothiazine derivatives, the mobile phase consisting of 50 mM phosphate buffer (pH 6.0)/acetonitrile (70:30, v/v) was carried at a flow rate of 2.0 mL/min. In the post-column Ru(bpy)3 2+ addition mode, the 0.5 mM Ru(bpy)3 2+ solution was delivered to a reaction/observation zone at a flow rate of 0.5 mL/min. In the HPLC determination of erythromycin, the mobile phase consisting of 50 mM phosphate buffer (pH 7.0)/acetonitrile (70:30, v/v) was carried at a flow rate of 1.0 mL/min. In the post-column Ru(bpy)3 2+ addition mode, the 0.5 mM Ru(bpy)3 2+ solution was delivered to a reaction/observation zone at a flow rate of 0.5 mL/min.

3. Results and discussion 3.1. Determination of phenothiazine derivatives Phenothiazine derivatives belong to a large group of tricyclic antidepressants which are commonly used for the treatment of psychiatric patients suffering depressions [32]. The monitoring of such compounds is important for quality assurance in pharmaceutical preparations and for obtaining optimum therapeutic concentrations in body fluids to minimize the risk of toxicity [33]. All phenothiazine derivatives contain aliphatic tertiary amine groups as seen in Table 1 so that they can be sensitively detected by the Ru(bpy)3 2+ ECL. Among them, promazine and chloropromazine have been previously detected with the solution-phase Ru(bpy)3 2+ ECL in static or flow injection analysis modes [33,34]. In our work, several phenothiazine compounds other than promazine and chloropromazine were examined in 50 mM phosphate buffer at pH 6.0 by flow injection analysis in order to see if they can be sensitively detected with Ru(bpy)3 2+ ECL. This pH was chosen because this condition is suitable for HPLC separation and Ru(bpy)3 2+ ECL detection of analytes. All the phenothiazine derivatives examined gave strong ECL emissions as we expected from their chemical structures containing tertiary amine groups. The results from this experiment are summarized in Table 1. The differences in ECL intensity between this series of compounds can be explained by considering their chemical structures. Previously, Danielson group and Bobbitt group have independently reported what effect the R group attached to the amine group of analytes has on the Ru(bpy)3 2+ ECL intensities [7,35]. In general, it has been found that electronwithdrawing groups tend to decrease ECL intensity while electron-donating groups increase ECL intensity. As shown in Table 1, promazine produced the largest ECL intensity

Fig. 3. The pH effect on ECL intensity of 0.5 mM phenothiazine derivatives with sol–gel-immobilized ECL sensor: chlorpromazine (
), trifluoperazine (䊉), triflipromazine ( ), promazine (), thioridazine ().

since it only contains two tertiary amine groups in comparison with other compounds. Chlorpromazine and triflupromazine containing electron withdrawing halogen atoms attached to one of the two aromatic rings produced smaller ECL intensities than that obtained with promazine. Thioridazine contains a sulfur group which is also electronegative, thus resulted in smaller ECL intensity compared to that of promazine. Although the general trend exists, it is still difficult to precisely predict which compounds will give more ECL emission. A pH study has been carried out with the sol–gelimmobilized ECL sensor in a flow injection system to determine the effect that pH has on the Ru(bpy)3 2+ ECL intensities of the five phenothiazine derivatives. In general, the ECL intensities for amine compounds are greatly affected by the pH of the buffer solution at the ECL detection. Fig. 3 illustrates the ECL intensity variation with pH for the compounds tested in acetate and phosphate buffer over a pH range of 4.0–8.0. The background corrected ECL signals for phenothiazine derivatives obtained at the ECL sensor based on TiO2 –Nafion (50%) composite-modified electrode increased significantly from pH 4.0 up to pH 7.0 and decreased at higher pH by around 40–75%. The ECL intensity for promazine obtained at pH 7 was 24-fold larger than that obtained at pH 4.0. This was similar to the result of the Ru(bpy)3 2+ -tripropylamine system [18]. As can be seen in the Ru(bpy)3 2+ ECL mechanism, all phenothiazine compounds should be first oxidized before they react with the oxidized Ru(bpy)3 3+ species. Therefore, ECL intensities for all phenothiazine compounds are dependent upon the degree of oxidation, thus, anodic current of phenothiazine compounds in cyclic voltammograms. The effect of pH on the anodic peak current of phenothiazine compounds was quite similar to the pH effect on the ECL intensity. The anodic peak current increased significantly from pH 4.0 up to pH 7.0 and slightly decreased at higher pH. In addition, Ru(bpy)3 3+ species is most stable at pH 6–7 as reported previously by the Bard and his co-workers [36]. Therefore, as the pH increases, some decomposition of Ru(bpy)3 3+ species

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would be expected, leading to a diminished ECL reagent available for ECL reaction, thus decreased ECL intensity because the ECL intensity is strongly dependent upon the amount of Ru(bpy)3 3+ concentration. The ECL signals for the erythromycin obtained at the solution-phase Ru(bpy)3 2+ addition approach also exhibited the same trend as in the ECL sensor. Although, the highest ECL intensity for promazine was observed at pH 7.0, phosphate buffer at pH 6.0 was used in the following HPLC–ECL detection experiments because all the phenothiazine compounds are well separated and detected with similar Ru(bpy)3 2+ ECL sensitivities at that pH. Since the phenothiazine compounds are among the most widely used drugs in medical practice, the present sol–gelimmobilized Ru(bpy)3 2+ ECL sensor was applied to the HPLC determination of the phenothiazine derivatives in human urine samples. An aliquot of a human urine sample was diluted four-fold with 50 mM phosphate buffer solution at pH 6.0 and the mixture was spiked with a solution containing an accurately measured amount of phenothiazine compounds. The mobile phase consisting of 50 mM phosphate buffer (pH 6.0) and acetonitrile (70:30, v/v) was delivered at a flow rate of 2.0 mL/min. Fig. 4a shows the chromatogram obtained with isocratic separation of human urine sample, spiked with a mixture of five 25 ␮M phenothiazine compounds. A large ECL signal resulting from amino acids is observed near the solvent front in the chromatogram because the reversed-phase HPLC column very weakly retains charged amino acids. The five phenothiazine compounds were well resolved from each other. Thus, those phenothiazine compounds can be easily determined from the resulting simple chromatogram. In the HPLC–ECL sensor detection system, the linear range for phenothiazine compounds extended from 5.0 ␮M to 1.0 mM with a detection limit of 0.5–3.0 ␮M (S/N = 3). The precision was examined by repeated determinations (n = 10) of phenothiazines concentrations in urine spiked with each drug (25 ␮M). The relative standard deviations were below 5% for phenothiazine compounds. The present ECL sensor was quite stable over experimental time scales under the exposure to the mobile phase containing 30% acetonitrile organic solvent. The ECL response decreased less than 10% over 5 h of operation period. In contrast, pure Nafion-based ECL sensor is rapidly destroyed upon exposure to mobile phases containing such high organic content as previously reported so that the ECL response decreased 50% in 1 h operation. The longterm stability of the ECL sensor was studied by measuring the ECL signal of 25 ␮M promazine solution over time. The ECL sensor retained 50% of its initial ECL signal after oneweek period of its storage in the mobile phase having 30% acetonitrile. Therefore, further efforts should be directed to the development of the ECL sensor having superior long-term stability. The conventional post-column Ru(bpy)3 2+ -addition approach was also examined to compare the analytical characteristics of the two approaches. Fig. 4b shows the chromatogram obtained with isocratic separation of human

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Fig. 4. Chromatograms obtained by two modes of isocratic HPLC separation and Ru(bpy)3 2+ ECL detection for human urine samples spiked with phenothiazine derivatives (a) sol–gel-immobilized Ru(bpy)3 2+ ECL sensor mode for 25 ␮M phenothiazine derivatives; (b) post-column Ru(bpy)3 2+ addition mode for 10 ␮M phenothiazines. A: promazine; B: chlorpromazine; C: triffupromazine; D: thioridazine; E: trifluoperazine.

urine sample, spiked with a mixture of five 10 ␮M phenothiazine compounds. In the post-column Ru(bpy)3 2+ -addition approach, a flow of 0.5 mM Ru(bpy)3 2+ in 50 mM phosphate buffer at pH 6.0 was delivered to the detection flow cell by an extra peristaltic pump at a flow rate of 0.5 mL/min. In the post-column Ru(bpy)3 2+ -addition approach, the linear range for phenothiazine compounds extended from 1.0 ␮M to 1.0 mM with a detection limit of 0.3–2.1 ␮M (S/N = 3) with relative standard deviations below 4% for 10 replicate injected samples. The analytical figures of merits in the two approaches were summarized in Table 2. As can be seen in those figures, the conventional post-column Ru(bpy)3 2+ addition approach produced higher ECL intensities, thus higher sensitivity and lower LOD compared to those obtained with sol–gel-immobilized Ru(bpy)3 2+ ECL sensor. This result is reasonable. In the sol–gel-immobilized ECL Ru(bpy)3 2+ ECL sensor, the ECL emission occurs from the reaction of the oxidized Ru(bpy)3 3+ reagent and the oxidized

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Table 2 Calibration data obtained with sol–gel-immobilized ECL sensor and conventional solution-phase post-column Ru(bpy)3 2+ -addition ECL method R

Compound

Detection method

Slopea

Promazine

ECL sensor Solution-phase

170 505

0.998 0.999

0.529 0.328

Chlorpromazine

ECL sensor Solution-phase

108 223

0.998 0.999

0.833 0.846

Triflupromazine

ECL sensor Solution-phase

55.0 77.0

0.997 0.999

1.64 2.127

Thioridazine

ECL sensor Solution-phase

52.6 192

0.997 0.998

1.71 0.857

Trifluoperazine

ECL sensor Solution-phase

30.6 84.7

0.996 0.997

2.94 1.998

Erythromycin

ECL sensor Solution-phase

81 95

0.998 0.999

1.11 0.95

LOD (␮M)b

a

Slope (a.u./␮M). b The detection limit was calculated as three times the signal from the standard deviation of the background noise.

phenothiazine compounds in the diffusion layer near the electrode surface. Since the diffusion coefficient of Ru(bpy)3 2+ in the sol–gel-derived titania–Nafion composite film (Dapp = 4 × 10−9 cm2 /s) is much smaller compared to that in buffer solution (Dapp = ∼1 ×10−5 cm2 /s, in the case of the post-column addition approach) [31], the reaction rate of the oxidized Ru(bpy)3 3+ reagent and the oxidized phenothiazine compounds in the diffusion layer on the ECL sensor is much smaller than that in the solution-phase Ru(bpy)3 2+ ECL. Therefore, ECL intensities in the sol–gel-immobilized ECL sensor are smaller than those obtained with the post-column addition approach. Although the smaller ECL intensities are obtained, the present ECL sensor in HPLC detection provides distinctive advantages such as cost-effectiveness and simple experimental set-up. The detection limits for phenothiazines in urine obtained with the present ECL sensor are almost equal to those obtained with the HPLC–UV absorbance detection method, but slightly higher than those obtained with a fluorescence detection method [37]. The detection limits for phenothiazines in urine obtained with the present ECL sensor were sufficiently low as to be valuable for detecting these compounds at therapeutic concentration range. 3.2. Determination of erythromycin Erythromycin is an important macrolide antibiotic that is used for treatment of bacterial infections in humans and animals [38]. The determination of erythromycin in urine and plasma using microbore HPLC with Ru(bpy)3 2+ in mobile phase has been reported by Nieman group [14]. In the present study, the erythromycin in human urine sample was determined by the HPLC with the Ru(bpy)3 2+ ECL sensor based on the TiO2 –Nafion composite films and the analytical performance was compared to that obtained with the solution-phase Ru(bpy)3 2+ addition approach.

Fig. 5. The pH effect on ECL intensity of 0.5 mM erythromycin with sol–gelimmobilized ECL sensor.

First, a pH study has been carried out in a flow injection system to determine the effect that pH has on the Ru(bpy)3 2+ ECL intensities of the erythromycin. Fig. 5 shows the ECL intensity change with pH for the compounds tested in acetate and phosphate buffer over a pH range of 4.0–8.0. The background corrected ECL signals for the erythromycin obtained at the ECL sensor increased slightly from pH 4.0 up to pH 7.0 and then decreased at higher pH. The ECL intensity for the erythromycin obtained at pH 7 was almost 1.4-fold larger than that obtained at pH 4.0. Therefore, phosphate buffer at pH 7.0 was used in all HPLC–ECL detection experiments. An aliquot of a human urine sample was diluted fourfold with 50 mM phosphate buffer solution at pH 7.0 and the mixture was spiked with 25 ␮M erythromycin solution. The mobile phase consisting of 0.05 M phosphate buffer (pH 7.0) and acetonitrile (70:30, v/v) was delivered at a flow rate of 1.0 mL/min. Fig. 6a shows the chromatogram obtained with isocratic separation. A large ECL signal resulting from amino acids was also observed near the solvent front in the chromatogram because of charged amino acids. Since the ECL signal of erythromycin is not interfered with various amino acids, the erythromycin can be easily determined from the resulting simple chromatogram. In the HPLC–ECL detection system, the linear range for erythromycin extended from 5.0 ␮M to 1.0 mM (R2 = 0.998) at the TiO2 –Nafion (50%) composite-modified electrode with a detection limit of 1.1 ␮M (S/N = 3). The precision was examined by repeated determinations (n = 10) of erythromycin concentration in urine spiked with erythromycin (25 ␮M). The relative standard deviation was 3.0%. The conventional post-column Ru(bpy)3 2+ -addition approach was also examined to compare the analytical characteristics of the two approaches. Fig. 6b shows the chromatogram obtained with isocratic separation of human urine sample spiked with 25 ␮M erythromycin. In the post-column Ru(bpy)3 2+ -addition approach, a flow of 0.5 mM Ru(bpy)3 2+ in 50 mM phosphate buffer at pH 7.0 was delivered to the detection flow cell by an extra peristaltic pump at a flow rate of 0.5 mL/min. In the

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in human urine samples. The resulting analytical performances such as detection limit, working range, sensitivity, and measurement precision were slightly worse than those obtained with the conventional post-column Ru(bpy)3 2+ addition approach. The lack of significant interferences and the achieved detection limit for phenothiazine compounds and erythromycin indicate that the proposed HPLC-Ru(bpy)3 2+ ECL detection method is suitable for the determination of amine-containing compounds in pharmaceutical preparations and biological fluids such as urine and maybe plasma. Since the present method eliminates the need for a second pump delivering the Ru(bpy)3 2+ solution, the use of the sol–gel-immobilized Ru(bpy)3 2+ ECL sensor in HPLC allows the HPLC–ECL instrumentation to be greatly simplified compared to the conventional post-column Ru(bpy)3 2+ addition approach. Furthermore, this sol–gel-immobilized Ru(bpy)3 2+ ECL sensor could be very valuable for the miniaturization of detection systems in microseparation techniques such as microbore HPLC and capillary electrophoresis, in which extra band broadening caused by post-column reagent mixing is an important concern due to small peak volume.

Acknowledgements This work was supported by Korea Research Foundation Grant (KRF-2000-015-DP0239).

Fig. 6. Chromatograms obtained by two modes of isocratic HPLC separation and Ru(bpy)3 2+ ECL detection for human urine samples spiked with 25 ␮M erythromycin (a) sol–gel-immobilized Ru(bpy)3 2+ ECL sensor mode; (b) post-column Ru(bpy)3 2+ addition mode.

post-column Ru(bpy)3 2+ -addition approach, the linear range for erythromycin extended from 5.0 ␮M to 1.0 mM with a detection limit of 0.9 ␮M (S/N = 3). The relative standard deviation was below 2.9% for 10 replicate injected samples. The analytical figures of merits in the two approaches were also summarized in Table 2. The detection limit of 1.11 ␮M for erythromycin in urine obtained with the present ECL sensor is slightly better than 1.36 ␮M obtained with the HPLC–UV absorbance detection method [38], but one order higher than that obtained with a post-column ion-pair extraction along with the strongly fluorescent 9,10-dimethoxy-anthracene-2sulfonate [39].

4. Conclusions Because of good ECL sensitivity and long-term stability of the sol–gel-immobilized Ru(bpy)3 2+ ECL sensor based on titania/Nafion composite films, the present ECL sensor can be used as an HPLC detector for the determination of alkylamine drugs such as phenothiazine derivatives and erythromycin

References [1] T.A. Nieman, Chemiluminescence and Photochemical Reaction Detection in Chromatography, VCH Publishers, New York, 1989, p. 99. [2] W.-Y. Lee, Mikrochim. Acta 127 (1997) 19. [3] A.W. Knight, Trend Anal. Chem. 18 (1999) 47. [4] R.D. Gerardi, N.W. Barnett, S.W. Lewis, Anal. Chim. Acta 378 (1999) 1. [5] J.B. Noffsinger, N.D. Danielson, J. Chromatogr. 387 (1987) 520. [6] J.A. Holeman, N.D. Danielson, J. Chromatogr. A. 679 (1994) 277. [7] S.N. Brune, D.R. Bobbitt, Anal. Chem. 64 (1992) 166. [8] W.A. Jackson, D.R. Bobbitt, Anal. Chim. Acta 285 (1994) 309. [9] M.A. Targove, N.D. Danielson, J. Chromatogr. Sci. 28 (1990) 505. [10] W.-Y. Lee, T.A. Nieman, J. Chromatogr. A 659 (1994) 111. [11] D.R. Skotty, W.-Y. Lee, T.A. Nieman, Anal. Chem. 68 (1996) 1530. [12] J.B. Noffsinger, N.D. Danielson, Anal. Chem. 59 (1987) 865. [13] D.R. Skotty, T.A. Nieman, J. Chromatogr. B 665 (1995) 27. [14] J.S. Ridlen, D.R. Skotty, P.T. Kissinger, T.A. Nieman, J. Chromatogr. B 694 (1997) 393. [15] H. Yoshida, K. Hidaka, J. Ishida, K. Yoshikuni, H. Nohta, M. Yamaguchi, Anal. Chim. Acta 378 (2000) 137. [16] Y.-J. Park, D.W. Lee, W.-Y. Lee, Anal. Chim. Acta 471 (2002) 51. [17] I. Rubinstein, A.J. Bard, J. Am. Chem. Soc. 103 (1981) 512. [18] J.K. Leland, M.J. Powell, J. Electrochem. Soc. 137 (1990) 3127. [19] I. Rubinstein, A.J. Bard, J. Am. Chem. Soc. 102 (1980) 6642. [20] T.M. Downey, T.A. Nieman, Anal. Chem. 64 (1992) 261. [21] W.-Y. Lee, T.A. Nieman, Anal. Chem. 67 (1995) 1789. [22] P.C. Lee, D. Meisel, J. Am. Chem. Soc. 102 (1980) 5477. [23] A.J. Tudos, J.J. Ozinga, H. Poppe, W.Th. Kok, Anal. Chem. 62 (1990) 367.

56

H.N. Choi et al. / Analytica Chimica Acta 541 (2005) 49–56

[24] B.C. Dave, B. Dunn, J.S. Valentine, J.I. Zink, Anal. Chem. 66 (1994) 1120A. [25] O. Lev, M. Tsionsky, L. Rabinovich, V. Glezer, S. Sampath, I. Pankratov, J. Jun, Anal. Chem. 67 (1995) 22A. [26] J. Lin, C.W. Brown, Trends Anal. Chem. 16 (1997) 200. [27] C.-Z. Zhao, N. Egashira, Y. Kurauchi, K. Ohga, Anal. Sci. 14 (1998) 439. [28] A.N. Khramov, M.M. Collinson, Anal. Chem. 72 (2000) 2943. [29] H. Wang, G. Xu, S. Dong, Analyst 126 (2001) 1095. [30] H. Wang, G. Xu, S. Dong, Electroanalysis 14 (2002) 853. [31] H.N. Choi, S.-H. Cho, W.-Y. Lee, Anal. Chem. 75 (2003) 4250.

[32] J. Karpimska, B. Starczewska, H. Puzanowska-Tarasiewicz, Anal. Sci. 12 (1996) 161. [33] G.M. Greenway, S.J.L. Dolman, Analyst 124 (1999) 759. [34] G. Xu, S. Dong, Anal. Chem. 72 (2000) 5308. [35] J.B. Noffsinger, N.D. Danielson, Anal. Chem. 59 (1987) 865. [36] D. Ege, W.G. Becker, A.J. Bard, Anal. Chem. 56 (1984) 2413. [37] M.C. Quintana, M.H. Blanco, J. Lacal, L. Hern´andez, Talanta 59 (2003) 417. [38] C. Stubbs, J.M. Haigh, I. Kanfer, J. Pharm. Sci. 74 (1985) 1126. [39] K. Kahn, J. Paesen, E. Roets, J. Hoogmartens, J. Liq. Chromatogr. 17 (1994) 4195.