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Determination of clenbuterol in pig liver by high-performance liquid chromatography with a coulometric electrode array system
Analytica Chimica Acta 489 (2003) 95–101
Determination of clenbuterol in pig liver by high-performance liquid chromatography with a coulometric electrode array system X.Z. Zhang, Y.R. Gan∗ , F.N. Zhao Department of Biochemical Engineering, School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, China Received 17 March 2003; received in revised form 26 May 2003; accepted 5 June 2003
Abstract A method has been developed to determine clenbuterol in pig liver using HPLC with coulometric electrode array system, for this compound can be irreversibly oxidized at high potentials by ordinary methods. Investigation into the effect of the pH of mobile phase on the retention factor and peak height of clenbuterol was made. The electrochemical behavior of clenbuterol at graphite electrodes was taken into account. Optimization of different extract conditions was also performed. The samples were pretreated using liquid–liquid extraction based on diethyl ether and the organic layer was evaporated to dryness. The residue was dissolved in mobile phase and monitored by an ESA electrochemical detector. Four electrodes in series were used for quantitation and the potentials of electrodes were set at 450, 600, 650 and 680 mV, respectively. Calibration curve showed good linearity and the detection limit of clenbuterol was 1.2 ng/g. This method developed using HPLC–ECD is reproducible, and sensitive enough for the determination of clenbuterol in pig liver. It is easy to perform. © 2003 Elsevier B.V. All rights reserved. Keywords: HPLC; Electrochemical method; Clenbuterol
1. Introduction Clenbuterol is a selective 2 -adrenoceptor agonist which is widely used orally in the treatment of asthma [1]. Some studies have been reported that the 2 -agonist including clenbuterol can promote muscle growth and reduce body fat [2]. Recently, the misuse of the 2 -agonist in animal feed and the residues of these compounds in animal tissue have drawn substantial attention. When animals are treated with 2 -agonist, residues can accumulate in their meat ∗ Corresponding author. Tel.: +86-22-27409598; fax: +86-22-27409598. E-mail address: [email protected] (Y.R. Gan).
and liver which may have a pharmacological effect in human [3,4]. Therefore, the use of 2 -agonists in meat-producing animals is now banned [5]. Clenbuterol residues have been shown to persist in the liver for 25–30 days after withdrawal. Repartitioning effects have been shown to be present for up to 70 days after withdrawal [6]. So, liver was usually used to analyze its clenbuterol residues. A number of methods for the analysis of clenbuterol in biological matrices have been reported, including radioimmunoassay [7], enzyme immunoassay [8], GC–MS [9], HPLC with UV detection [2,10], mass spectrometric detection [11,12] and electrochemical detection [13–17], HPTLC [18]. All these methods have their own disadvantages. The first two methods
0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0003-2670(03)00707-4
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can provide very low limits of detection for clenbuterol but they had to couple with HPLC as detection technique sometimes and the time of sample preparation is too long. The GC–MS method needs a derivatization procedure due to the polar nature of clenbuterol. The amperometric detection has been used in all HPLC–ECD methods reported to determine clenbuterol in biological matrices. Koole et al. [13] reported a method for clenbuterol in human and calf urine that used an amperometric detector with glassy carbon electrode and Ag/AgCl reference electrode. A solid-phase extraction clean up procedure was used and the ion-pair reagent was added to mobile phase in the method. The extracts were cleaned and the detection limit was 4 ng/ml. Unfortunately, the extract procedure was time-consuming. Lin et al. [15] developed a method for clenbuterol in bovine retinal tissue that involved an amperometric detector with glassy carbon electrode and Ag/AgCl reference electrode. The limit of detection for this method was 10 ng/g and the spiked recovery was 75%. But in some instances, interferences were co-eluted with clenbuterol resulted in an overestimation of its concentration. Ali Qureshi et al. [16] described an ion-pair liquid chromatography method for determination of clenbuterol in equine plasma using an amperometric detector with glassy carbon electrode and saturated calomel reference electrode. However, the working electrode should be polished daily and the final sample solution was stored at 4 ◦ C overnight to reduced baseline disturbances and the number of interfering peaks from the plasma. In order to prevent exploitation of the growth promotion effects, a much more sensitive and simple method is required to determine the very low concentration of clenbuterol in liver sample. As clenbuterol contains an electroactive aromatic amino group, electrochemical detection is an ideal choice. The coulometric electrode array detection consists of 16 electrochemical cells arranged in series and the potential of each cell can be set independently. This enables the concurrent detection of different chemical classes, each at their optimal potential settings. The porous graphite working electrode, palladium reference electrode and platinum counter electrode were used in each cell. A piece of chromatogram collected from the system contains a number of curves, which allows for the identification of the compound of in-
terest based on the retention time and its oxidation (reduction) characteristic on several traces. The detection system can offer superior sensitivity over other amperometric detectors commonly used with HPLC and co-eluting analytes can be resolved by making use of differences in their electrochemical behavior. So, the coulometric electrode array detection was chosen to analyse the concentration of clenbuterol. In this paper, we describe a simple, sensitive and reliable HPLC method for the determination of clenbuterol in pig liver using the coulometric electrode array detector. A 1.2 ng/g detection limit was obtained for clenbuterol using this method. We have studied the influence of the pH of mobile phase on the retention factor and peak height of clenbuterol. The current–potential curve was made according to the electrochemical behavior of clenbuterol at graphite electrodes and the optimized potentials of cells were selected based on it. The recoveries that obtained from different pH of extract were also compared. 2. Experimental 2.1. Chemicals and reagents Clenbuterol was obtained from Sigma. Hydrochloric acid, sodium hydroxide, phosphoric acid, triethylamine and diethyl ether were analytical grade from Sigma. Methanol and acetonitrile were HPLC purity reagents from Fisher Scientific (New Jersey, USA). The water used in all experiments had a resistivity of 18.2 M cm obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). The primary stock standard solution of clenbuterol in concentration 0.2 mg/ml was made by dissolving 20 mg of clenbuterol in 100 ml of methanol and was stable at 4 ◦ C for several months. The spiking standard solution was made by diluting the primary solution with methanol in order to give a concentration of 1 g/ml. 2.2. Apparatus The HPLC system used was an ESA chromatographic system (ESA, Chelmsford, MA, USA) equipped with two 582 pumps, an organizer chamber, a PEEK pulse damper, a manual injector fitted with a
X.Z. Zhang et al. / Analytica Chimica Acta 489 (2003) 95–101
20 l loop (Rheodyne 7725i, CA, USA) and a 5600A 16 channels CoulArray detector. In this method, clenbuterol was detected on four channels attached in series after the HPLC column. ESA software was used for data acquisition and processing. In addition, Sartorius BS 110S electronic balance (Beijing, China), WTW inolab pH level 1 pH meter (Weiheim, Germany) and Dupont Sorvall RC 5C plus centrifuge (Newtown, CT, USA) were used in the study. 2.3. Chromatographic conditions Clenbuterol was analyzed by reversed-phase HPLC analysis using ODS Hypersil (150 mm×4 mm i.d. and 5 m particle size, Hewlett-Packard, USA) column. A guard column (Hypersil, 5 m, Alltech Associates Inc., USA) was used to protect the analytical column. The mobile phase component A was 50 mM phosphoric acid–30 mM triethylamine and the pH was adjusted to 4.0 with 2 M sodium hydroxide solution. The mobile phase component B was acetonitrile and methanol in the proportion of 45:30 (v/v). An 80:20 (v/v) mixture of mobile phase components A and B was used in the method and the flow rate was kept constantly at 0.8 ml/min. The injection volume was 20 l. Mobile phase was always freshly prepared, filtered through a 0.22 m membrane and sonicated before use. The column effluent was monitored using CoulArray electrochemical detector with porous graphite electrodes operated in the oxidative screen mode. The electrodes potential were set at 450, 600, 650 and 680 mV, respectively. Typical retention time obtained from the final system is 8.1 min for clenbuterol. 2.4. Liquid–liquid extraction procedure The pig liver sample was homogenized and stored at −20 ◦ C. The frozen pig liver samples were thawed at 4 ◦ C when analysis. Ten milliliters of 1 M hydrochloric acid was added to 10 g pig liver sample. The mixture was shaken vigorously for 10 min and centrifuged for 10 min at 4000 × g. The supernatant was decanted and placed at another container. The container was placed in 60–70 ◦ C water bath for 30 min, and then was taken out and cooled down. After that, 10 ml diethyl ether was added to the container. The mixture
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was vortex mixed for 5 min and centrifuged for 10 min at 4000 × g. The organic layer was discarded. Another 10 ml diethyl ether was added to the aqueous layer and the process was repeated. The pH of the remaining aqueous layer was adjusted to 11.6 by adding 2 M sodium hydroxide solution. Five milliliters diethyl ether was added and vortex mixed vigorously for 10 min, and then the sample was centrifuged for 10 min at 4000 × g. The diethyl ether layer was removed, placed in a glass tube and evaporated at 60 ◦ C under a continuous flow of nitrogen. To the remaining aqueous phase, another 5 ml diethyl ether was added and the sample was vortex mixed and centrifuged. The diethyl ether layer was removed in the same glass tube. The diethyl ether layer was evaporated to dryness under the nitrogen at 60 ◦ C. The residue was redissolved in 1 ml mobile phase, passed through a 0.22 m filter and 20 l was injected in the HPLC–ECD system. 2.5. Method validation The method was validated by determining of the following operational characteristics: linearity range, selectivity, limit of detection, precision and accuracy. The standard curve was constructed by extracting blank pig liver samples spicked with different concentrations of clenbuterol ranging from 1.88 to 60.16 ng/g. The samples were treated under the experiment conditions described above and analyzed in triplicate. The sum of peak height of clenbuterol at all sensitive channels was measured and plotted against concentration. The selectivity of the method was studied by analyzing of blank pig liver samples and checking any interfering substances which presented at the retention time of clenbuterol at the same potential. The detection limit of the assay was estimated using a signal to noise ratio of 3:1. Precision and accuracy were assessed at performing replicate analysis of spiked samples. Three different concentration samples within calibration range were prepared and analyzed with calibration curve to determine intra-day (six replicates per concentration) and inter-day (six replicates per concentration over 1–3 days) variability. The intra-day and inter-day precisions were determined as the relative standard deviation (%RSD) and accuracy was determined as percentage relative error (%RE).
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3. Results and discussion
1400
Proper selection of applied electrode potentials is critical for accurate, interference-free measurement. The working potentials of the detector cells were optimized by experimentation under the analytical conditions being used. A 1 g/ml solution of clenbuterol was prepared by diluting 0.5 ml of the stock standard solution (0.2 mg/ml) to 100 ml with mobile phase. Two electrodes were selected. The potential of electrode 1 (upstream electrode) was set at 200 mV and the potential of electrode 2 (downstream electrode) at 800 mV initially. A 20 l of the 1 g/ml standard solution was injected in the system and the peak height of clenbuterol was recorded at electrode 1. The potential of the electrode 1 was increased by 50 mV. The procedure above was repeated until increases of potential did not result in significant changes in peak height. The relationship between the peak height (current) and potential (voltage) was shown in Fig. 1. From Fig. 1, we can see clenbuterol could be oxidized at 500 mV and reached a plateau at 700 mV where no significant increase in peak height occured when the potential was increased. Based on the curve, the following potentials were selected for electrodes: 450, 600, 650 and 680 mV. Although greater response could be obtained with higher potential than 700 mV, the background current or noise also increased. Therefore, the potentials of 600, 650 and 680 mV were selected to provide adequate sensitivity for clenbuterol determination. The first electrode was set to remove
Peak Height/nA
1200
3.1. Voltammetric behavior of clenbuterol
1000 800 600 400 200 0 0
1
2
3
4
5
6
7
pH Fig. 2. Peak height–pH curve of clenbuterol.
those interfering substances that may co-elute with clenbuterol and can be oxidized at low potential. Another character is obtained when using the array of electrodes at potentials along with the oxidative curve of each analyte, i.e. the peak height response ratios between adjacent channels are descriptive of the voltammetric behavior of each analyte. Comparison of these ratios between authentic standard and sample provides an estimate of the purity of each analyte peak in the sample. The peak height response ratio obtained for clenbuterol in the external standard was automatically calculated to be 1.26 for E3/E4. The ratio obtained from the sample height identified as clenbuterol was 1.41 for E3/E4. The E3/E4 ratio in the standard was divided by the E3/E4 ratio in the unknown equals 1.12. Since the sample ratio accuracy is within 20% of 1.26, the sample was deemed as the same compound as standard. 100
extraction efficiency
Peak Height/nA
1200 1000 800 600 400 200 0
80 60 40 20 0
0
200
400
600
800
Applied Potential/mV Fig. 1. C–V curve for the oxidation of clenbuterol.
3
5
7
9
11
13
15
pH Fig. 3. The extraction efficiency–pH curve of clenbuterol.
X.Z. Zhang et al. / Analytica Chimica Acta 489 (2003) 95–101
3.2. Optimization of the pH of the mobile phase The pH of mobile phase was found have effect on both the retention factor and the peak height of clenbuterol. The retention factor of clenbuterol increases with the increase in pH. But the peak height of clenbuterol climbs continuously at first and reaches its highest point when the pH equals to 4.0. After that, the peak height decreases sharply (Fig. 2). Therefore,
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to maximize the sensitivity of the system, the following experiments were carried out at pH 4.0. 3.3. Optimization of the pH of extract process The results of initial experiments showed that the extraction efficiency varied with the pH of extract process (Fig. 3). From Fig. 3, we can see the extraction efficiency was larger than 70% when the pH ranged
Fig. 4. HPLC chromatograms obtained for (a) blank pig liver sample and (b) liver sample collected from a pig administered with clenbuterol.
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from 11.1 to 11.8 and reached its highest point at the pH of 11.6. When the pH was adjusted to 10.8, the efficiency dropped to 53%. And when the pH was modified to 13.5, it was difficult to find clenbuterol. So, the pH of 11.6 ± 2 was chosen as the optimum value.
organic layer. As a result, the chromatogram will contain endogenous contaminants. Because clenbuterol in acidic media can not dissolve in diethyl ether and diethyl ether is a good solvent to fat, diethyl ether was used to remove the particulates of fat existing in pig liver tissue homogenate. The function of heating procedure in course of sample treatment is to oxidize those interfering substances which can be oxidized easily and extracted by diethyl ether. The concentrations of the incurred samples were determined by calibration curve and the spiked standard sample was included with every 10 samples. The concentrations of these incurred samples were 7.52, 7.31, 4.23, 7.16 and 5.60 ng/g, respectively. One of the chromatograms was shown in Fig. 4. The sample was tested positive for the presence of clenbuterol, containing 7.52 ng/g clenbuterol in pig liver.
3.4. Extraction efficiency The extraction efficiency of clenbuterol was assessed at three concentration levels (3.76, 15.04 and 56.40 ng/g) from blank pig liver sample and water. To simulate the incurred sample, clenbuterol was added to the blank pig liver sample homogenate, mixed and the mixtures were kept in the refrigerator for 24 h. The spiked pig liver samples were analyzed along with the method described above, while the aqueous standards were dried by rotary-evaporator and the residues were dissolved in 1 ml mobile phase. The spiked pig liver samples were run in triplicate while the spiked water solutions were run in duplicate in three separate runs. The mean percentage recovery of the extraction procedure was 77% for pig liver sample with a standard deviation of 6.2%.
3.6. Calibration range, linearity, limit of detection, precision and accuracy The calibration curve was obtained by adding clenbuterol to blank pig liver samples to get concentrations of 1.88, 3.76, 7.52, 15.04, 30.08 and 60.16 ng/g. These standards were treated under the experimental condition described above and analyzed in triplicate. Calibration curve show good linearity within the selected concentration range. The correlation coefficient was 99.53% for pig liver sample. A series of blank pig liver samples were checked using the method developed, no interfering substances presented at the retention time of clenbuterol at the same potential. Under the experimental condition described above, the minimum detectable concentration was 1.2 ng/g for pig liver sample.
3.5. Sample analysis Pig liver samples collected from pigs that were administered with clenbuterol were tested. Many kinds of fat and lipoid exist in pig liver tissue. When the pig liver samples were smashed into homogenate and centrifuged, the particulates of fat and lipoid would disperse in 1 M hydrochloric acid solution but not deposit with dregs. When the solution was treated with organic solvents, the fat particulates would be extracted into
Table 1 Precision and accuracy for clenbuterol from the spiked pig liver sample (n = 6) Sample
Theoretical concentration (ng/g)
Concentration found (ng/g)
Recovery (%)
%RE
Mean
S.D.
%RSD
Intra-day
3.76 15.04 56.40
3.70 14.68 57.19
0.32 0.75 2.79
8.65 5.11 4.88
98.41 97.60 101.4
1.59 2.40 1.40
Inter-day
3.76 15.04 56.40
3.66 14.91 55.68
0.42 1.08 4.35
11.48 7.24 7.81
97.35 99.13 98.72
2.65 0.87 1.28
X.Z. Zhang et al. / Analytica Chimica Acta 489 (2003) 95–101
Precision and accuracy results were shown in Table 1. This table demonstrated good precision and accuracy within the concentration range selected. 3.7. Electrode maintenance Clenbuterol is known to foul carbon electrodes. Although the coulometric electrode remains unaffected by poisoning until more than 90% of the electrode surface is fouled, the electrodes are cleaned at the end of each working day in order to prolong the using time of electrodes and get the reproducible results. All the electrodes were kept at 900 mV for 1 or 2 min in order to restore them. Those components that have fouled the electrodes will be oxidized thoroughly and come out of them. 4. Conclusions An extraction method and an HPLC method with coulometric electrode array system have been developed for determining clenbuterol in pig liver sample. The method is simple, precise and accurate for the determination of clenbuterol in pig liver. Electrochemical detection is chosen to enhance selectivity as well as sensitivity for low level analytes. References [1] A. Baronti, A. Grieco, C. Vibelli, Int. J. Clin. Pharmacol. 18 (1980) 21.
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[2] H. Hooijerink, R. Schilt, W. Haasnoot, D. Courtheijn, J. Pharm. Biomed. Anal. 9 (1991) 485. [3] L. Salleras, A. Donguez, E. Mata, J.L. Taberrer, I. Moro, P. Salva, Public Health Rep. 110 (1995) 338. [4] J.F. Navarro-Martinez, Lancet 336 (1990) 1311. [5] Council directive 96/22/EU, Off. J. Eur. Commun. L125 (1996) 3. [6] C.T. Elliott, S.R.H. Crooks, J.G.D. MeEvoy, W.J. MeCaughey, S.A. Hewitt, D. Patterson, D. Kilpatrick, Vet. Res. Commun. 17 (1993) 459. [7] D. Boyd, P. Shearan, J.P. Hopkins, M. O’Keefe, M.R. Smyth, Anal. Chim. Acta 275 (1993) 221. [8] H.H.D. Meyer, L. Rinke, I. Dursch, J. Chromatogr. 564 (1991) 551. [9] W.J. Blanchflower, S.A. Hewitt, A. Cannavan, C.T. Elliott, D.G. Kennedy, Biol. Mass Spectrom. 22 (1993) 326. [10] C. Eddins, J.A. Hamann, K. Johnson, J. Chromatogr. Sci. 23 (1985) 308. [11] L. Debrauwen, G. Bories, Anal. Chim. Acta 275 (1993) 231. [12] D.R. Doerge, S. Bajic, S. Lowes, Rapid Commun. Mass Spetrom. 7 (1993) 462. [13] A. Koole, J. Bosman, J.P. Franke, R.A. de Zeeuw, J. Chromatogr. B 726 (1999) 149. [14] G.J. McGrath, E. O’Kane, W.F. Smyth, F. Tagliaro, Anal. Chim. Acta 322 (1996) 159. [15] L.A. Lin, J.A. Tomlinson, R. Duane Satzger, J. Chromatogr. A 762 (1997) 275. [16] G. Ali Qureshi, A. Eriksson, J. Chromatogr. 441 (1988) 197. [17] B. Diquet, L. Doare, P. Simon, J. Chromatogr. 336 (1984) 415. [18] J. Henion, G.A. Maylin, B.A. Thomson, J. Chromatogr. 271 (1983) 107.
Determination of clenbuterol in pig liver by high-performance liquid chromatography with a coulometric electrode array system X.Z. Zhang, Y.R. Gan∗ , F.N. Zhao Department of Biochemical Engineering, School of Chemical Engineering & Technology, Tianjin University, Tianjin 300072, China Received 17 March 2003; received in revised form 26 May 2003; accepted 5 June 2003
Abstract A method has been developed to determine clenbuterol in pig liver using HPLC with coulometric electrode array system, for this compound can be irreversibly oxidized at high potentials by ordinary methods. Investigation into the effect of the pH of mobile phase on the retention factor and peak height of clenbuterol was made. The electrochemical behavior of clenbuterol at graphite electrodes was taken into account. Optimization of different extract conditions was also performed. The samples were pretreated using liquid–liquid extraction based on diethyl ether and the organic layer was evaporated to dryness. The residue was dissolved in mobile phase and monitored by an ESA electrochemical detector. Four electrodes in series were used for quantitation and the potentials of electrodes were set at 450, 600, 650 and 680 mV, respectively. Calibration curve showed good linearity and the detection limit of clenbuterol was 1.2 ng/g. This method developed using HPLC–ECD is reproducible, and sensitive enough for the determination of clenbuterol in pig liver. It is easy to perform. © 2003 Elsevier B.V. All rights reserved. Keywords: HPLC; Electrochemical method; Clenbuterol
1. Introduction Clenbuterol is a selective 2 -adrenoceptor agonist which is widely used orally in the treatment of asthma [1]. Some studies have been reported that the 2 -agonist including clenbuterol can promote muscle growth and reduce body fat [2]. Recently, the misuse of the 2 -agonist in animal feed and the residues of these compounds in animal tissue have drawn substantial attention. When animals are treated with 2 -agonist, residues can accumulate in their meat ∗ Corresponding author. Tel.: +86-22-27409598; fax: +86-22-27409598. E-mail address: [email protected] (Y.R. Gan).
and liver which may have a pharmacological effect in human [3,4]. Therefore, the use of 2 -agonists in meat-producing animals is now banned [5]. Clenbuterol residues have been shown to persist in the liver for 25–30 days after withdrawal. Repartitioning effects have been shown to be present for up to 70 days after withdrawal [6]. So, liver was usually used to analyze its clenbuterol residues. A number of methods for the analysis of clenbuterol in biological matrices have been reported, including radioimmunoassay [7], enzyme immunoassay [8], GC–MS [9], HPLC with UV detection [2,10], mass spectrometric detection [11,12] and electrochemical detection [13–17], HPTLC [18]. All these methods have their own disadvantages. The first two methods
0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0003-2670(03)00707-4
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X.Z. Zhang et al. / Analytica Chimica Acta 489 (2003) 95–101
can provide very low limits of detection for clenbuterol but they had to couple with HPLC as detection technique sometimes and the time of sample preparation is too long. The GC–MS method needs a derivatization procedure due to the polar nature of clenbuterol. The amperometric detection has been used in all HPLC–ECD methods reported to determine clenbuterol in biological matrices. Koole et al. [13] reported a method for clenbuterol in human and calf urine that used an amperometric detector with glassy carbon electrode and Ag/AgCl reference electrode. A solid-phase extraction clean up procedure was used and the ion-pair reagent was added to mobile phase in the method. The extracts were cleaned and the detection limit was 4 ng/ml. Unfortunately, the extract procedure was time-consuming. Lin et al. [15] developed a method for clenbuterol in bovine retinal tissue that involved an amperometric detector with glassy carbon electrode and Ag/AgCl reference electrode. The limit of detection for this method was 10 ng/g and the spiked recovery was 75%. But in some instances, interferences were co-eluted with clenbuterol resulted in an overestimation of its concentration. Ali Qureshi et al. [16] described an ion-pair liquid chromatography method for determination of clenbuterol in equine plasma using an amperometric detector with glassy carbon electrode and saturated calomel reference electrode. However, the working electrode should be polished daily and the final sample solution was stored at 4 ◦ C overnight to reduced baseline disturbances and the number of interfering peaks from the plasma. In order to prevent exploitation of the growth promotion effects, a much more sensitive and simple method is required to determine the very low concentration of clenbuterol in liver sample. As clenbuterol contains an electroactive aromatic amino group, electrochemical detection is an ideal choice. The coulometric electrode array detection consists of 16 electrochemical cells arranged in series and the potential of each cell can be set independently. This enables the concurrent detection of different chemical classes, each at their optimal potential settings. The porous graphite working electrode, palladium reference electrode and platinum counter electrode were used in each cell. A piece of chromatogram collected from the system contains a number of curves, which allows for the identification of the compound of in-
terest based on the retention time and its oxidation (reduction) characteristic on several traces. The detection system can offer superior sensitivity over other amperometric detectors commonly used with HPLC and co-eluting analytes can be resolved by making use of differences in their electrochemical behavior. So, the coulometric electrode array detection was chosen to analyse the concentration of clenbuterol. In this paper, we describe a simple, sensitive and reliable HPLC method for the determination of clenbuterol in pig liver using the coulometric electrode array detector. A 1.2 ng/g detection limit was obtained for clenbuterol using this method. We have studied the influence of the pH of mobile phase on the retention factor and peak height of clenbuterol. The current–potential curve was made according to the electrochemical behavior of clenbuterol at graphite electrodes and the optimized potentials of cells were selected based on it. The recoveries that obtained from different pH of extract were also compared. 2. Experimental 2.1. Chemicals and reagents Clenbuterol was obtained from Sigma. Hydrochloric acid, sodium hydroxide, phosphoric acid, triethylamine and diethyl ether were analytical grade from Sigma. Methanol and acetonitrile were HPLC purity reagents from Fisher Scientific (New Jersey, USA). The water used in all experiments had a resistivity of 18.2 M cm obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). The primary stock standard solution of clenbuterol in concentration 0.2 mg/ml was made by dissolving 20 mg of clenbuterol in 100 ml of methanol and was stable at 4 ◦ C for several months. The spiking standard solution was made by diluting the primary solution with methanol in order to give a concentration of 1 g/ml. 2.2. Apparatus The HPLC system used was an ESA chromatographic system (ESA, Chelmsford, MA, USA) equipped with two 582 pumps, an organizer chamber, a PEEK pulse damper, a manual injector fitted with a
X.Z. Zhang et al. / Analytica Chimica Acta 489 (2003) 95–101
20 l loop (Rheodyne 7725i, CA, USA) and a 5600A 16 channels CoulArray detector. In this method, clenbuterol was detected on four channels attached in series after the HPLC column. ESA software was used for data acquisition and processing. In addition, Sartorius BS 110S electronic balance (Beijing, China), WTW inolab pH level 1 pH meter (Weiheim, Germany) and Dupont Sorvall RC 5C plus centrifuge (Newtown, CT, USA) were used in the study. 2.3. Chromatographic conditions Clenbuterol was analyzed by reversed-phase HPLC analysis using ODS Hypersil (150 mm×4 mm i.d. and 5 m particle size, Hewlett-Packard, USA) column. A guard column (Hypersil, 5 m, Alltech Associates Inc., USA) was used to protect the analytical column. The mobile phase component A was 50 mM phosphoric acid–30 mM triethylamine and the pH was adjusted to 4.0 with 2 M sodium hydroxide solution. The mobile phase component B was acetonitrile and methanol in the proportion of 45:30 (v/v). An 80:20 (v/v) mixture of mobile phase components A and B was used in the method and the flow rate was kept constantly at 0.8 ml/min. The injection volume was 20 l. Mobile phase was always freshly prepared, filtered through a 0.22 m membrane and sonicated before use. The column effluent was monitored using CoulArray electrochemical detector with porous graphite electrodes operated in the oxidative screen mode. The electrodes potential were set at 450, 600, 650 and 680 mV, respectively. Typical retention time obtained from the final system is 8.1 min for clenbuterol. 2.4. Liquid–liquid extraction procedure The pig liver sample was homogenized and stored at −20 ◦ C. The frozen pig liver samples were thawed at 4 ◦ C when analysis. Ten milliliters of 1 M hydrochloric acid was added to 10 g pig liver sample. The mixture was shaken vigorously for 10 min and centrifuged for 10 min at 4000 × g. The supernatant was decanted and placed at another container. The container was placed in 60–70 ◦ C water bath for 30 min, and then was taken out and cooled down. After that, 10 ml diethyl ether was added to the container. The mixture
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was vortex mixed for 5 min and centrifuged for 10 min at 4000 × g. The organic layer was discarded. Another 10 ml diethyl ether was added to the aqueous layer and the process was repeated. The pH of the remaining aqueous layer was adjusted to 11.6 by adding 2 M sodium hydroxide solution. Five milliliters diethyl ether was added and vortex mixed vigorously for 10 min, and then the sample was centrifuged for 10 min at 4000 × g. The diethyl ether layer was removed, placed in a glass tube and evaporated at 60 ◦ C under a continuous flow of nitrogen. To the remaining aqueous phase, another 5 ml diethyl ether was added and the sample was vortex mixed and centrifuged. The diethyl ether layer was removed in the same glass tube. The diethyl ether layer was evaporated to dryness under the nitrogen at 60 ◦ C. The residue was redissolved in 1 ml mobile phase, passed through a 0.22 m filter and 20 l was injected in the HPLC–ECD system. 2.5. Method validation The method was validated by determining of the following operational characteristics: linearity range, selectivity, limit of detection, precision and accuracy. The standard curve was constructed by extracting blank pig liver samples spicked with different concentrations of clenbuterol ranging from 1.88 to 60.16 ng/g. The samples were treated under the experiment conditions described above and analyzed in triplicate. The sum of peak height of clenbuterol at all sensitive channels was measured and plotted against concentration. The selectivity of the method was studied by analyzing of blank pig liver samples and checking any interfering substances which presented at the retention time of clenbuterol at the same potential. The detection limit of the assay was estimated using a signal to noise ratio of 3:1. Precision and accuracy were assessed at performing replicate analysis of spiked samples. Three different concentration samples within calibration range were prepared and analyzed with calibration curve to determine intra-day (six replicates per concentration) and inter-day (six replicates per concentration over 1–3 days) variability. The intra-day and inter-day precisions were determined as the relative standard deviation (%RSD) and accuracy was determined as percentage relative error (%RE).
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3. Results and discussion
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Proper selection of applied electrode potentials is critical for accurate, interference-free measurement. The working potentials of the detector cells were optimized by experimentation under the analytical conditions being used. A 1 g/ml solution of clenbuterol was prepared by diluting 0.5 ml of the stock standard solution (0.2 mg/ml) to 100 ml with mobile phase. Two electrodes were selected. The potential of electrode 1 (upstream electrode) was set at 200 mV and the potential of electrode 2 (downstream electrode) at 800 mV initially. A 20 l of the 1 g/ml standard solution was injected in the system and the peak height of clenbuterol was recorded at electrode 1. The potential of the electrode 1 was increased by 50 mV. The procedure above was repeated until increases of potential did not result in significant changes in peak height. The relationship between the peak height (current) and potential (voltage) was shown in Fig. 1. From Fig. 1, we can see clenbuterol could be oxidized at 500 mV and reached a plateau at 700 mV where no significant increase in peak height occured when the potential was increased. Based on the curve, the following potentials were selected for electrodes: 450, 600, 650 and 680 mV. Although greater response could be obtained with higher potential than 700 mV, the background current or noise also increased. Therefore, the potentials of 600, 650 and 680 mV were selected to provide adequate sensitivity for clenbuterol determination. The first electrode was set to remove
Peak Height/nA
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3.1. Voltammetric behavior of clenbuterol
1000 800 600 400 200 0 0
1
2
3
4
5
6
7
pH Fig. 2. Peak height–pH curve of clenbuterol.
those interfering substances that may co-elute with clenbuterol and can be oxidized at low potential. Another character is obtained when using the array of electrodes at potentials along with the oxidative curve of each analyte, i.e. the peak height response ratios between adjacent channels are descriptive of the voltammetric behavior of each analyte. Comparison of these ratios between authentic standard and sample provides an estimate of the purity of each analyte peak in the sample. The peak height response ratio obtained for clenbuterol in the external standard was automatically calculated to be 1.26 for E3/E4. The ratio obtained from the sample height identified as clenbuterol was 1.41 for E3/E4. The E3/E4 ratio in the standard was divided by the E3/E4 ratio in the unknown equals 1.12. Since the sample ratio accuracy is within 20% of 1.26, the sample was deemed as the same compound as standard. 100
extraction efficiency
Peak Height/nA
1200 1000 800 600 400 200 0
80 60 40 20 0
0
200
400
600
800
Applied Potential/mV Fig. 1. C–V curve for the oxidation of clenbuterol.
3
5
7
9
11
13
15
pH Fig. 3. The extraction efficiency–pH curve of clenbuterol.
X.Z. Zhang et al. / Analytica Chimica Acta 489 (2003) 95–101
3.2. Optimization of the pH of the mobile phase The pH of mobile phase was found have effect on both the retention factor and the peak height of clenbuterol. The retention factor of clenbuterol increases with the increase in pH. But the peak height of clenbuterol climbs continuously at first and reaches its highest point when the pH equals to 4.0. After that, the peak height decreases sharply (Fig. 2). Therefore,
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to maximize the sensitivity of the system, the following experiments were carried out at pH 4.0. 3.3. Optimization of the pH of extract process The results of initial experiments showed that the extraction efficiency varied with the pH of extract process (Fig. 3). From Fig. 3, we can see the extraction efficiency was larger than 70% when the pH ranged
Fig. 4. HPLC chromatograms obtained for (a) blank pig liver sample and (b) liver sample collected from a pig administered with clenbuterol.
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from 11.1 to 11.8 and reached its highest point at the pH of 11.6. When the pH was adjusted to 10.8, the efficiency dropped to 53%. And when the pH was modified to 13.5, it was difficult to find clenbuterol. So, the pH of 11.6 ± 2 was chosen as the optimum value.
organic layer. As a result, the chromatogram will contain endogenous contaminants. Because clenbuterol in acidic media can not dissolve in diethyl ether and diethyl ether is a good solvent to fat, diethyl ether was used to remove the particulates of fat existing in pig liver tissue homogenate. The function of heating procedure in course of sample treatment is to oxidize those interfering substances which can be oxidized easily and extracted by diethyl ether. The concentrations of the incurred samples were determined by calibration curve and the spiked standard sample was included with every 10 samples. The concentrations of these incurred samples were 7.52, 7.31, 4.23, 7.16 and 5.60 ng/g, respectively. One of the chromatograms was shown in Fig. 4. The sample was tested positive for the presence of clenbuterol, containing 7.52 ng/g clenbuterol in pig liver.
3.4. Extraction efficiency The extraction efficiency of clenbuterol was assessed at three concentration levels (3.76, 15.04 and 56.40 ng/g) from blank pig liver sample and water. To simulate the incurred sample, clenbuterol was added to the blank pig liver sample homogenate, mixed and the mixtures were kept in the refrigerator for 24 h. The spiked pig liver samples were analyzed along with the method described above, while the aqueous standards were dried by rotary-evaporator and the residues were dissolved in 1 ml mobile phase. The spiked pig liver samples were run in triplicate while the spiked water solutions were run in duplicate in three separate runs. The mean percentage recovery of the extraction procedure was 77% for pig liver sample with a standard deviation of 6.2%.
3.6. Calibration range, linearity, limit of detection, precision and accuracy The calibration curve was obtained by adding clenbuterol to blank pig liver samples to get concentrations of 1.88, 3.76, 7.52, 15.04, 30.08 and 60.16 ng/g. These standards were treated under the experimental condition described above and analyzed in triplicate. Calibration curve show good linearity within the selected concentration range. The correlation coefficient was 99.53% for pig liver sample. A series of blank pig liver samples were checked using the method developed, no interfering substances presented at the retention time of clenbuterol at the same potential. Under the experimental condition described above, the minimum detectable concentration was 1.2 ng/g for pig liver sample.
3.5. Sample analysis Pig liver samples collected from pigs that were administered with clenbuterol were tested. Many kinds of fat and lipoid exist in pig liver tissue. When the pig liver samples were smashed into homogenate and centrifuged, the particulates of fat and lipoid would disperse in 1 M hydrochloric acid solution but not deposit with dregs. When the solution was treated with organic solvents, the fat particulates would be extracted into
Table 1 Precision and accuracy for clenbuterol from the spiked pig liver sample (n = 6) Sample
Theoretical concentration (ng/g)
Concentration found (ng/g)
Recovery (%)
%RE
Mean
S.D.
%RSD
Intra-day
3.76 15.04 56.40
3.70 14.68 57.19
0.32 0.75 2.79
8.65 5.11 4.88
98.41 97.60 101.4
1.59 2.40 1.40
Inter-day
3.76 15.04 56.40
3.66 14.91 55.68
0.42 1.08 4.35
11.48 7.24 7.81
97.35 99.13 98.72
2.65 0.87 1.28
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Precision and accuracy results were shown in Table 1. This table demonstrated good precision and accuracy within the concentration range selected. 3.7. Electrode maintenance Clenbuterol is known to foul carbon electrodes. Although the coulometric electrode remains unaffected by poisoning until more than 90% of the electrode surface is fouled, the electrodes are cleaned at the end of each working day in order to prolong the using time of electrodes and get the reproducible results. All the electrodes were kept at 900 mV for 1 or 2 min in order to restore them. Those components that have fouled the electrodes will be oxidized thoroughly and come out of them. 4. Conclusions An extraction method and an HPLC method with coulometric electrode array system have been developed for determining clenbuterol in pig liver sample. The method is simple, precise and accurate for the determination of clenbuterol in pig liver. Electrochemical detection is chosen to enhance selectivity as well as sensitivity for low level analytes. References [1] A. Baronti, A. Grieco, C. Vibelli, Int. J. Clin. Pharmacol. 18 (1980) 21.
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