This article was downloaded by: [Northwest Teacher Training University] On: 15 April 2014, At: 19:32 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Simultaneous and confirmative detection of multiresidues of β2-agonists and β-blockers in urine using LC-MS/MS/MS coupled with β-receptor molecular imprinted polymer SPE clean-up a
b
c
c
c
a
S. Fan , J.H. Zou , H. Miao , Y.F. Zhao , H.J. Chen , R. Zhao & Y.N. Wu
ac
a
Institute of Nutrition and Food Hygiene, Beijing Centre for Disease Control and Prevention, Beijing, China b
General Hospital of the Second Artillery, Chinese People’s Liberation Army, Beijing, China
c
Key Laboratory of Food Safety Risk Assessment, Ministry of Health, China Center for Food Safety Risk Assessment, Beijing, China Accepted author version posted online: 10 Sep 2013.Published online: 21 Oct 2013.
To cite this article: S. Fan, J.H. Zou, H. Miao, Y.F. Zhao, H.J. Chen, R. Zhao & Y.N. Wu (2013) Simultaneous and confirmative detection of multi-residues of β2-agonists and β-blockers in urine using LC-MS/MS/MS coupled with βreceptor molecular imprinted polymer SPE clean-up, Food Additives & Contaminants: Part A, 30:12, 2093-2101, DOI: 10.1080/19440049.2013.840929 To link to this article: http://dx.doi.org/10.1080/19440049.2013.840929
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part A, 2013 Vol. 30, No. 12, 2093–2101, http://dx.doi.org/10.1080/19440049.2013.840929
Simultaneous and confirmative detection of multi-residues of β2-agonists and β-blockers in urine using LC-MS/MS/MS coupled with β-receptor molecular imprinted polymer SPE clean-up S. Fana, J.H. Zoub, H. Miaoc*, Y.F. Zhaoc, H.J. Chenc, R. Zhaoa and Y.N. Wua,c a
Institute of Nutrition and Food Hygiene, Beijing Centre for Disease Control and Prevention, Beijing, China; bGeneral Hospital of the Second Artillery, Chinese People’s Liberation Army, Beijing, China; cKey Laboratory of Food Safety Risk Assessment, Ministry of Health, China Center for Food Safety Risk Assessment, Beijing, China
Downloaded by [Northwest Teacher Training University] at 19:32 15 April 2014
(Received 22 April 2013; accepted 1 September 2013) A liquid chromatography–linear ion-trap spectrometry (LC-MS3) method using β-receptor molecular-imprinted polymer (MIP) solid-phase extraction (SPE) as clean-up was developed to determine simultaneously and confirmatively residues of 25 β2-agonists and 21 β-blockers in urine samples. Urine samples were subjected to enzymatic hydrolysis by β-glucoronidase/arylsulphatase, and then extracted with perchloric acid. Sample clean-up was performed using β-receptor MIP SPE. A Supelco Ascentis® express Rp-Amide column was used to separate the analytes, and MS3 detection used an electrospray ionisation source in positive-ion mode. Recovery studies were carried out using blank urine samples fortified with the 46 analytes at the levels of 0.5, 1.0 and 2.0 μg l–1. Recoveries were obtained ranging from 60.1% to 109.9% with relative standard deviations (RSDs, n = 7) from 0.5% to 19.4%. The limits of detection (LODs) and limits of quantitation (LOQs) of the 46 analytes in urine were 0.02–0.18 and 0.05–0.60 μg l–1, respectively. As a result of the selective clean-up by MIP SPE and MS3 detection of the target drugs, the sensitivity and accuracy of the present method was high enough for monitoring β2-agonist and β-blocker residues in urine samples. Satisfactory results were obtained in the process of the determination of positive urine samples. Keywords: β2-agonists; β-blockers; MIP SPE; LC-MS; urine
Introduction Nowadays, β2-agonists are sometimes misused in livestock production since they can improve the fat-to-muscle ratio in animals and promote the growth of animal muscle tissues. β-blockers are often used to reduce the stress of animals during transport to the slaughterhouse. Such stress usually results in a loss of meat quality and even in premature death (Balizs & Hewitt 2003; Zhang et al. 2009). Sedatives are also illicitly used in animal husbandry to enhance the feed conversion ratio by reducing animal activity. However, the misuse of these substances may lead to drug residues in meat or offal of the animals that poses a potential health hazard to humans. The use of some or all of these substances in animals raised for human consumption has been banned in a number of countries, such as in the European Union, China and others. Therefore, sensitive analytical methods for quantification and confirmation of residues in tissues and urine are required for regulatory enforcement. When controlling misuse in livestock production, urine is the most readily available non-invasive sample. The detection of β2-agonists (Damasceno et al. 2002; Blanca et al. 2005; Gallo et al. 2007; Paik et al. 2007; Liu et al. 2009; Moragues & Igualada 2009) or β-blockers (Delamoye et al. 2004; Liu et al. 2009; Murray & Danaceau 2009) in urine samples has *Corresponding author. Email:
[email protected] © 2013 Taylor & Francis
been reported. However, few references have focused on simultaneous detection of the two kinds of drugs. GC/MS methods were widely used for analysis of β2-agonist residues in various biological samples (Damasceno et al. 2000, 2002; Gallo et al. 2007; Lee et al. 2007; Paik et al. 2007; Liu et al. 2009) years ago. Due to the high polarity and low volatility of β2-agonists and β-blockers, derivatisation was needed before analysis by GC/MS, which is time-consuming and laborious. Compared with GC/MS, LC-MS has the advantage of not needing derivatisation for β2-agonists and β-blockers, and more and more LC-MS references (Shishani et al. 2003; Blanca et al. 2005; Huang et al. 2007; Siluk et al. 2008; Croes et al. 2009; Moragues & Igualada 2009; Murray & Danaceau 2009; Shao et al. 2009) focusing on the determination of β2-agonists and β-blockers have been published in recent years. However, ion suppression remains a problem in LC-MS analysis and needs be overcome. Usually, two techniques are used to circumvent this problem: stable isotope dilution and matrix-matched calibration. In addition, in the present method MS3 detection using an electrospray ionisation source in a positive mode was used to minimise ion suppression. The improvement in sample preparation and clean-up is considered to be the most laborious but effective process to overcome ion suppression. For β2-agonists and β-blockers the purification techniques were mainly based on mixed-
Downloaded by [Northwest Teacher Training University] at 19:32 15 April 2014
2094
S. Fan et al.
phase SPE such as strong cation exchange combined with C8 or C18 cartridges (Damasceno et al. 2000, 2002; Hernandez-Carrasquilla 2000; Blanca et al. 2005; Van Hoof et al. 2005; Lee et al. 2007; Siluk et al. 2008; Shao et al. 2009). However, these SPE procedures have proven to be selective not only for β2-agonists and β-blockers, but also for other basic drugs. Therefore, molecular imprinted polymers (MIPs) designed for β2-agonists or β-blockers, with the advantage of high selectivity and specificity, were used in the clean-up procedure of different sample matrices (Widstrand et al. 2004; Fiori et al. 2005; Kootstra et al. 2005; Van Hoof et al. 2005; Gros et al. 2008; Hu et al. 2009). Nathalie Van Hoof et al. (2005) compared the effect of clean-up by mixed-phase SPE and MIP SPE for 14 β2agonists in a urine sample and found that the MIP technique was better for clean-up. However, the use of MIP SPE for simultaneous purification of β2-agonists and β-blockers has not been reported. In this paper, MIP for β-receptors was used for simultaneous clean-up of β2-agonists and β-blockers for the first time. Based on the β-receptor MIP SPE, a simple, rapid, accurate and reliable confirmative method for simultaneously confirmative detecting 46 β2-agonists and β-blockers in urine by LC-MS3 was established. The method was successfully applied to the analysis of human and pig urine samples. Analyte identification and confirmation was performed using an LC-MS3 in compliance with European Union regulations (EU Commission Decision 2002/657/EC), which was set for the determination of residues in food samples. The development of this method can contribute to the monitoring of the illegal use of β2agonists and β-blockers in livestock and poultry breeding.
cimaterol were from Boehringer Ingekheim (Ingekheim, Germany). Salmeterol was obtained from Toronto Research Chemical, Inc. (Toronto, ON, Canada). Clenproperol, clenpenterol, d7-cimaterol, d6-salbutamol, d9-cimbuterol, d7-clenproperol, d5-ractopamine, d9-mabuterol, d11-mapenterol and d6-clenbuterol were purchased from the EU Reference Laboratory (Freiburg, Germany). Metaproterenol, terbutaline, salbutamol, procaterol, fenotero, clenbuterol, ractopamine, tulobuterol, formoterol, fumarate, bambuterol hydrochloride, ritodrine hydrochloride, metoprolol, labetalol hydrochloride, propranolol hydrochloride, betaxolol, penbutolol sulfate, sotalol hydrochloride and esmolol hydrochloride were obtained from Sigma-Aldrich (St. Louis, MO, USA). Clorprenaline hydrochloride was from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). NA-1141 was a gift from the EU Reference Laboratory in Germany. Nadolol, timolol, oxprenolol, alprenolol, bunolol, carazolol, acebutolol, celiprolol, atenolol and carvedilol were purchased from Dr Ehrenstorfer GmbH (Augsburg, Germany). Metipranolol were from the British Pharmacopoeia Commission Laboratory (London, UK). d3-Salmeterol was purchased from Cambridge Isotope Laboratories Inc. (Tewksbury, MA, USA). The purity of all these standards was not less than 96%. Individual stock standard solution (1000 mg l–1) of the standard was prepared in methanol and stored in the dark at –18°C. Serial mixed working standard solutions were obtained by pooling aliquots of individual standard stock solution together and followed by subsequent dilution with methanol. Sample collection
Materials and methods Reagents and materials Methanol and acetonitrile of HPLC grade were supplied by Fisher Scientific (Fair Lawn, NJ, USA). Formic acid (99%) was from Acros Organics (Morris Plains, NJ, USA). Distilled/deionised water was obtained using a Milli-Q Ultrapure system (Millipore, Bedford, MA, USA). βGlucuronidase/arylsulfatase (116 400 unit ml–1) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Perchloric acid, trichloracetic acid and sodium hydroxide of analytical grade were purchased from Beijing Chemical Reagents Co. (Beijing, China). SupelMIPTM beta-receptor SPE cartridges (25 mg, 10 ml) were purchased from Sigma-Aldrich (Bellefonte, PA, USA). Standards Brombuterol hydrochloride, clenisopenterol hydrochloride, clencyclohexerol, clenhexerol, cimbuterol and mapenterol hydrochloride were purchased from WITEGA Laboratorien Berlin-Adlersh GmbH (Berlin, Germany). Mabuterol and
Pig urine samples were provided by farmers of one pig farm located in the suburb of Beijing. However, the positive pig urine samples were not available, and the preparation of positive pig urine would be not only labour demanding and time-consuming, but also contrary to animal welfare. Pigs and humans are similar in their drug metabolism, so positive urine from a human was used to demonstrate that the method could be used for the detection of drugs in urine samples. The positive urine samples were donated by patients with hypertension and asthma who had been treated with multiple β2-agonist and β-blocker drugs in the General Hospital of the Second Artillery of the Chinese People’s Liberation Army. The samples were kept at –20°C before extraction. Sample pre-treatment Urine samples quots of 5 ml polypropylene acetate buffer
were centrifuged at 8000g for 10 min; aliurine samples were transferred into 15 ml centrifuge tubes and 5 ml of 0.2 mol l–1 (pH 5.2) and 100 μl β-glucuronidase/
Downloaded by [Northwest Teacher Training University] at 19:32 15 April 2014
Food Additives & Contaminants: Part A arylsulfatase were added. A total of 10 μl of the internal standard (1 mg l–1) was spiked into the solution. The mixture was vortexed for 30 s and incubated at 55°C for 2 h. After cooling to RT, the solution was adjusted to pH 1.0 with 1.0 mol l–1 perchloric acid, and then ultrasonicated for 30 min. The mixture was centrifuged at 10 000 rpm for 10 min at 4°C. The supernatant was adjusted to pH 7.0 with 1.0 mol l–1 NaOH and then centrifuged at 8000g for 10 min at 4°C. The supernatant was subjected to clean-up using SupelMIP SPE cartridges. SupelMIPTM beta-receptor SPE cartridges (25 mg, 10 ml) were preconditioned with 1 ml acetonitrile and 1 ml water. The extracts were applied to the cartridges at a speed of 1 ml min–1. The cartridge was washed with 3 ml water and vacuum dried, and then washed with 1 ml acetonitrile and 1 ml acetonitrile + water (60:40, v/v) and vacuum dried. The targeted compounds were eluted with 2 ml acetonitrile containing 1% formic acid. The effluent was condensed to dryness under a gentle nitrogen stream. The residue was reconstituted into 200 μl solution of methanol + 0.1% formic acid (2:8, v/v) and then filtered through a 0.22-μm nylon filter for LC-MS3 analysis. Chromatographic system LC was performed by a Thermo Fisher HPLC system (Thermo Fisher, Waltham, MA, USA) with a Supelco Ascentis® express Rp-Amide column (150 mm × 2.1 mm, 2.7 μm). The column oven temperature was set at 30°C. The flow rate of the mobile phase was 100 μl min–1, and the injection volume was 10 μl. Optimal separation of the target compounds was achieved by gradient elution using methanol (A) and 0.1% formic acid in water (B). Mobile phase gradient used (A: B, v/v) began with 20:80, and held for 5 min, and then changed linearly to 25:75 at 20 min, 40:60 at 35 min, 60:40 at 45 min, 100:0 at 50 min, and then held for 1 min to elute impurities from the column, 20:80 at 52 min and equilibrated for 3 min before the next injection. Mass spectrometry MS analysis was carried out on a linear ion-trap mass spectrometry (LTQ) (Thermo Fisher) using the positive electrospray ionisation mode (ESI+). The consecutive reaction monitoring (CRM, MS3) scan mode was used. The mass fragments including the parent ions, daughter ions and the granddaughter ions used for the method are listed in Table 1. The voltage of the Ispray was set at 4.5 kV. The flow rates of the sheath gas and the auxiliary gas were 35 and 15 arb, respectively. The temperature of the capillary was 325°C; the capillary voltage was set at 35 V. Helium gas was used as the collision gas in the linear ion trap. The parameters of the linear ion trap were set as follows: full automatic gain control (AGC) target
2095
was 10 000.0, SIM AGC target was 5000.0, MSn AGC target was 5000.0, and Zoom AGC target was 3000.0. Calibration and quantification For the quantification of the 46 β2-agonists and β-blockers, nine deuterium isotope standards were used as internal standards. The peak areas of the analytes (A) and that of the internal standards (Ai) were recorded. To compensate for the possible influence from matrix, linear matrixmatched calibration curves were established for 46 analytes on the basis of the ratio of A:Ai versus the corresponding concentration. The calibration standard series (1.0, 5.0, 10.0, 25.0, 50.0, 100, 200 μg l–1) were prepared using blank urine extracts. The concentration of the samples was calculated using the matrix-matched calibration curves. Samples should be reanalysed in case the concentration of the injected extracts exceeded the calibration range. Results and discussion LC-ESI-MS/MS optimisation LC separation was performed on Supelco Ascentis® Express RP-Amide column (150 mm × 2.1 mm, 2.7 μm). During the process of LC separation optimisation, the initial ratio of eluent A was set at 5%. The six compounds eluting first included metaproterenol, cimaterol, sotalol, salbutamol, terbutaline and cimbuterol, and showed tailing chromatographic peaks. The shape of the six peaks improved by increasing the ratio of eluent A to 20%, as shown in Figure 1. Therefore the initial ratio of eluent A was set at 20%. The MS parameters, such as the flow rates of the sheath gas and the auxiliary gas, the voltage of the Ispray and the capillary, and the collision energy were optimised by flowinjecting the individual standard solutions (200 μg l–1) into the mobile phase of solution of methanol and 0.1% formic acid (50:50, v/v) eluting through the LC system. The base peak in the mass spectrum of each analyte was [M + H]+, which was subsequently used as the precursor ion for the resulting MS2 (SRM). Analytes, such as cimaterol, clencyclohexerol, clenproperol, clenisopenterol and clenhexerol, have only one MS2 product ion, which was 220 > 202, 319 > 301, 263 > 245, 291 > 273 and 305 > 287, respectively. It is different from the results of triple-quadrupole MS (Shao et al. 2009), in which all the five analytes contributed two daughter ions under MS/MS scan mode. In the point view of MS2 transitions, the identification points (IPs) of the five analytes were less than four according to European Union Directive 2002/657/EC, which did not meet the requirement for confirmative determination. Therefore the scan mode of MS3, that is CRM, was applied to the 46 target analytes to provide more detailed mass information.
2096
S. Fan et al.
Table 1. Mass spectrum parameters for β2-agonists and β-blockers.
Downloaded by [Northwest Teacher Training University] at 19:32 15 April 2014
Analyte Metaproterenol d7-Cimaterol Cimaterol Atenolol Sotalol d6-Salbutamol Salbutamol Terbutaline d9-Cimbuterol Cimbuterol Procaterol Carteolol Clencyclohexerol Fenoterol Nadolol NA-1141 d7-Clenproperol Clenproperol Ritodrine Clorpenaline d6-Clenbuterol Clenbuterol Metoprolol Timolol Tulobuterol d5-Ractopamine Ractopamine Bromchlorbuterol Acebutolol Brombuterol Formoterol d9-Mabuterol Bunolol Esmolol Levobunolol Mabuterol Clenpenterol Oxprenolol Bambuterol Celiprolol Bisoprolol Clenisopenterol d11-Mapenterol Mapenterol Labetalol Metipranolol Alprenolol Propranolol Betaxolol Carazolol Clenhexerol Carvedilol Penbutolol d3-Salmeterol Salmeterol
Retention time (min) 3.31 3.56 3.58 3.67 3.69 3.70 3.72 3.76 4.12 4.14 5.04 6.28 6.42 6.49 6.52 6.98 7.23 7.25 7.26 8.17 10.50 10.69 11.23 11.24 12.05 12.19 12.23 12.86 14.21 15.56 15.95 16.15 16.16 16.21 16.22 16.54 17.32 18.62 19.49 20.07 22.73 23.60 23.76 24.19 26.12 26.77 27.43 27.57 28.08 28.22 31.92 35.89 37.42 38.13 38.14
Parent ion Q (m/z) value 212 227 220 267 273 246 240 226 243 234 291 293 319 304 310 293 270 263 288 214 283 277 268 317 228 307 302 323 337 367 345 320 292 296 292 311 291 266 368 380 326 291 336 325 329 310 250 260 308 299 305 407 292 419 416
0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25
Isolation width (m/z)
Collision energy (%)
Daughter ion (m/z)
Collision energy (%)
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
27 19 25 30 20 20 21 30 20 22 22 22 20 24 24 18 19 24 25 23 18 24 34 24 28 18 20 20 25 18 20 20 25 26 22 20 20 30 20 24 27 18 20 21 20 29 32 30 32 29 20 24 25 20 20
194*, 152 209* 202* 225*, 190 255*, 213 228*, 167 222*, 166 152*, 167 225*, 161 216*, 160 273*, 232 237* 301* 286*, 135 254*, 236 275* 252* 245* 270*, 150 196* 265*, 204 259*, 203 191*, 116 261*, 244 154*, 172 289*, 167 284*, 164 305*, 249 319*, 260 349*, 293 327*, 149 302*, 238 236*, 201 219*, 254 236* 293*, 237 273*, 203 248*, 225 312*, 294 307*, 306 116*, 222 273* 318*, 238 307*, 237 311*, 207 233*, 191 116*, 173 183*, 116 116*, 177 222*, 116 287* 283*, 224 236*, 201 401*, 383 398*, 380
34 25 30 27 28 25 26 35 20 25 30 22 24 25 25 22 25 30 30 30 21 20 30 23 40 20 20 20 25 20 25 18 26 28 22 22 22 30 20 25 42 27 18 21 25 25 41 36 42 36 30 25 29 20 20
Note: *Quantitatve ion; the collision energy (%) is the normalisation energy.
Granddaughter ions (m/z) 152*, 161*, 160*, 208*, 213*, 148*, 166*, 125*,
177 146 145 190 176 149 148 135 161* 160* 231*, 214 202*, 164 203*, 188 135*, 107 236*, 201 203* 204*, 189 203*, 188 150*, 121 154* 204*, 203 203* 159*, 131 244*, 188 118*, 119 167*, 121 164*, 121 249*, 207 244*, 260 293* 149*, 121 238* 201*, 189 145*, 187 201*, 189 237* 203* 206*, 189 294* 251*, 233 72*, 74 217*, 188 238* 237* 207*, 294 191*, 233 74*, 56 155*, 165 74*, 72 194*, 180 188*, 217 210*, 212 201*, 168 383*, 382 380*, 232
Downloaded by [Northwest Teacher Training University] at 19:32 15 April 2014
Food Additives & Contaminants: Part A
2097
Figure 1. Optimisation of the initial mobile phase using blank urine matrix-matched blended stands: (a) the gradient elution began with a 95% solution (B); (b) the gradient elution began with a 90% solution (B); (c) the gradient elution began with an 85% solution (B); and (d) the gradient elution began with an 80% solution (B). These chromatograms are the CRM chromatograms of the mentioned analytes, and from the top to the bottom of the figure the compounds are metaproterenol, cimaterol, sotalol, salbutamol, terbutaline and cimbuterol.
Optimisation hydrolysis of urine sample Most of the β2-agonist and β-blocker drugs are known to form glucuronate and sulfate conjugates in vivo and excreta. Therefore, it is necessary to release the drugs from conjugates by hydrolysis in order to ensure that the drugs are completely extracted from the matrix. Enzymatic hydrolysis (Fiori et al. 2005; Croes et al. 2009; Shao et al. 2009) and acid hydrolysis (Guy et al. 1999) are the commonly used techniques. However, due to the large number of compounds involved in this study, it is difficult to collect samples containing all 46 compounds. In the present study, two positive urine samples were selected to investigate the efficiency of enzymatic hydrolysis and acid hydrolysis. One contains salbutamol (excreted in the form of a sulfate conjugate and a glucuronic acid conjugate), procaterol (excreted in the form of a glucuronic acid conjugate) and bisoprolol (excreted as original drug); the other contains terbutaline (excreted in the form of a sulfate conjugate) and bisoprolol. The hydrolysis effect study on these four drugs can represent most of the β2-agonist and β-blocker drugs. The two positive urine samples were mixed and then divided into nine portions, each of 5 ml. Three portions were acid hydrolysed using 0.1 mol ml–1 perchloric acid; the other three were acid hydrolysed by 5% trichloroacetic acid; and the remaining three were enzymatic hydrolysed by 100 μl β-glucuronidase/arylsulfatase at 55°C for 2 h. The results are shown in Figure 2. A higher response was observed from enzymatic hydrolysis. The lowest response
was obtained by 5% trichloracetic acid hydrolysis. It was inferred that the lowest response was not caused by trichloroacetic acid hydrolysis, but by the following MIP clean-up. Trichloracetic acid in the extracts, because of its strong oxidisation, destroyed the space structure of MIP materials in the cartridges, which resulted in bad purification. The same hydrolysis experiment was conducted by using Oasis MCX cartridges as clean-up, and good results were obtained by trichloroacetic acid hydrolysis. Therefore, trichloroacetic acid hydrolysis is not suitable for the present sample pre-treatment, and finally enzymatic hydrolysis by β-glucuronidase/arylsulfatase was used. Sample purification Interfering substances such as proteins are introduced into the extracts during the process of enzymatic hydrolysis. To precipitate those interferences, 1.0 mol l–1 perchloric acid was added into the extracts until the pH reached 1.0, and the precipitate was separated by centrifugation. MIPs have the ability to identify specific target compounds from a complex sample matrix (Widstrand et al. 2004; Fiori et al. 2005; Kootstra et al. 2005; Van Hoof et al. 2005; Gros et al. 2008; Hu et al. 2009). In the current purification procedure, SupelMIPTM beta-receptor SPE cartridges were used. During the washing step, a multi-step clean-up operation was used. Water was used for removing matrix and interference by salts, and acetonitrile was used
2098
S. Fan et al. 800
Enzymolysis Perchloric acid acidolysis Trichloroacetic acid acidacidolysis
700 600 500 400 300 200
Downloaded by [Northwest Teacher Training University] at 19:32 15 April 2014
100 35 30 25 20 15 10 5 0
Figure 2.
Terbutaline
Salbutamol
Bisoprolol
Comparison of hydrolysis.
for removing the interference from compounds with hydrophobic groups, and 60% acetonitrile in water was used for removing the interference from compounds with hydrophilic groups. Most of the interferences in the extracts were effectively removed by the above multi-step washing procedure. In fact, the new method MIP SPE used in the present case is more time-consuming; however, it is more selective than the normal SPE procedure (Widstrand et al. 2004). Therefore, the MIP SPE procedure can get a better
Figure 3.
Procaterol
purification effect, which can reduce contamination of the instruments. On the other hand, compared with the previous MIP, which was just designed for β2-agonists or β-blockers (Widstrand et al. 2004; Fiori et al. 2005; Kootstra et al. 2005; Van Hoof et al. 2005; Gros et al. 2008; Hu et al. 2009), the MIP SPE used in this case was revealed to be selective not only for β2-agonists, but also for β-blockers. In this paper, the MIP SPE was used firstly for the simultaneous clean-up of β2-agonists and β-blockers.
Chromatograms of the quantification transition of MS3 of blank urine matrix-matched standard (0.080 mg l–1).
Food Additives & Contaminants: Part A
2099
Table 2. Recoveries, LOD and LOQ of β2-agonists and β-blockers in urine samples (n = 7). Spike level (0.5 μg l–1)
Downloaded by [Northwest Teacher Training University] at 19:32 15 April 2014
Compounds Metaproterenol Salbutamol Sotalol Cimaterol Cimbuterol Terbutaline Atenolol Procaterol Clencyclohexerol Carteolol Nadolol Fenoterol NA-1141 Ritodrine Clenproperol Clorpenaline Clenbuterol Timolol Metoprolol Tulobuterol Ractopamine Bromchlorbuterol Acebutolol Brombuterol Formoterol Levobunolol Esmolol Mabuterol Bunolol Clenpenterol Oxprenolol Bambuterol Celiprolol Clenisopenterol Bisoprolol Mapenterol Labetalol Metipranolol Alprenolol Betaxlol Carazolol Propranolol Clenhexerol Carvedilol Penbutolol Salmeterol
Spike level (1 μg l–1)
Spike level (2 μg l–1)
Recovery (%)
RSD (%)
Recovery (%)
RSD (%)
Recovery (%)
RSD (%)
LOD (μg kg–1)
LOQ (μg kg–1)
62.7 67.1 64.9 90.8 60.7 80.7 90.9 60.1 61.0 104.5 102.2 105.6 81.1 70.6 68.6 102.5 100.4 105.3 71.3 91.1 62.7 66.5 104.7 68.1 65.4 73.4 64.3 66.2 86.1 65.2 90.9 100.6 92.5 60.7 104.5 89.1 70.7 94.5 88.7 91.9 69.3 79.7 71.8 73.3 73.1 68.8
4.4 4.5 8.5 1.8 4.0 4.3 5.4 1.4 1.1 4.2 1.2 1.1 2.4 4.3 6.7 1.2 11.3 2.6 9.0 1.7 11.7 2.8 4.8 6.4 4.5 12.4 4.1 4.2 7.4 3.9 0.7 3.7 3.1 3.2 17.1 1.1 5.3 1.1 1.6 5.2 7.3 6.2 2.2 12.0 0.6 8.6
74.9 79.7 98.5 73.3 72.3 100.5 100.3 76.1 73.3 107.3 101.3 100.1 88.5 85.7 70.1 92.6 72.7 104.4 102.2 105.6 81.7 93.8 104.1 76.8 75.6 101.0 74.0 77.5 107.9 77.7 100.6 89.2 108.7 70.1 92.5 101.9 72.6 105.9 105.3 104.0 107.7 108.4 77.3 73.9 79.2 77.0
2.5 2.5 4.9 1.2 1.5 1.4 5.0 1.6 1.6 2.0 3.7 2.8 3.3 1.0 0.6 0.9 1.2 1.8 1.6 1.2 0.6 0.9 3.6 0.6 5.2 1.5 0.9 0.8 1.1 0.8 1.4 6.7 1.6 1.1 3.5 1.1 9.2 1.0 2.2 3.7 6.7 6.9 4.1 6.2 5.4 0.5
82.6 83.3 103.4 85.1 86.0 109.9 101.4 81.9 82.8 109.0 97.5 82.7 96.2 93.3 83.9 88.8 83.2 107.3 104.2 106.0 88.7 95.3 102.9 84.1 86.1 101.9 82.6 83.6 109.7 82.3 102.3 87.5 108.3 87.1 94.9 97.7 82.2 107.8 98.9 102.9 100.7 106.8 88.2 86.2 83.1 84.0
2.5 4.1 4.2 3.9 2.6 1.7 2.8 4.5 3.1 2.5 3.5 3.6 0.8 4.5 2.8 8.2 3.3 1.8 4.0 9.3 3.3 3.9 4.5 2.6 1.8 6.1 2.0 4.1 5.7 3.4 4.9 8.7 2.4 7.1 9.2 6.2 10.9 4.4 5.7 7.1 11.6 10.6 3.7 7.4 8.9 4.7
0.08 0.14 0.09 0.09 0.08 0.05 0.09 0.05 0.05 0.09 0.05 0.09 0.05 0.09 0.05 0.05 0.05 0.02 0.02 0.05 0.02 0.02 0.03 0.02 0.05 0.03 0.02 0.05 0.02 0.02 0.05 0.09 0.02 0.02 0.05 0.02 0.02 0.02 0.02 0.09 0.09 0.09 0.09 0.05 0.18 0.05
0.23 0.45 0.30 0.30 0.23 0.15 0.30 0.15 0.15 0.30 0.15 0.30 0.15 0.30 0.15 0.15 0.15 0.05 0.06 0.15 0.06 0.06 0.08 0.06 0.15 0.08 0.05 0.15 0.05 0.06 0.15 0.30 0.06 0.06 0.15 0.06 0.06 0.06 0.06 0.30 0.30 0.30 0.30 0.15 0.60 0.15
Calibration curves The total ion chromatogram and the chromatograms of quantitative transition of CRM of the matrix-matched standard solution are shown in Figure 3. No interference with any of the analytes was observed and the well-shaped peak for each analyte was obtained. All the analytes showed good linearity in the range of 1–200 μg l–1 with the correlation coefficient (r) values not less than 0.995.
Recovery experiments The recovery experiment was carried out with the spiked blank urine samples. The results are shown in Table 2. The recoveries of the 46 compounds were 60.1–105.6%, 70.1–108.7% and 81.9–109.9% at the spiked levels of 0.5, 1.0 and 2.0 μg l–1, and the RSD were 0.6–17.1%, 0.5–9.2% and 0.8–11.6%, respectively. The results were satisfied for the simultaneous and
2100
S. Fan et al. Table 3.
Concentrations of the four drugs in human urine samples (μg l–1).
Downloaded by [Northwest Teacher Training University] at 19:32 15 April 2014
Patient 1 Patient 2
Figure 4.
Terbutaline
Salbutamol
Procaterol
Bisoprolol
– 1439.5
57.0 –
22.0 –
259.2 230.2
MS3 chromatograms and MS3 spectrum of a positive urine sample.
confirmative determination of 46 analytes in urine samples. LOD and LOQ Before the extraction procedure, blank urine samples were fortified with working standard solutions of 46 β2-agonists and β-blockers at the levels of 0.05, 0.1, 0.2, 0.5 and 1.0 μg l–1. The LOD and LOQ were expressed as the concentration in the blank spiked samples with ratios of signal-tonoise (S/N) of 3 and 10, respectively. The results of LOD and LOQ in urine are listed in Table 3. All the LODs were in the range of 0.02–0.18 μg l–1; LOQs were in the range of 0.05–0.60 μg l–1.
Conclusion This work presented an LC-MS3 method for the simultaneous confirmatory determination of β2-agonists and β-blockers in urine. The sample preparation procedure comprised a two-step hydrolysis, followed by beta-receptor MIP SPE clean-up, which ensured a better clean-up than the normal SPE. Compared with LC-MS2, LC-MS3 showed a better ability on analyte identification and confirmation. The method was demonstrated to have good linearity, accuracy and precision, and is now used in our laboratory for monitoring the illegal use of β2-agonists and β-blockers in livestock. Acknowledgements
Application to real samples The method was successfully applied to the analysis of human and pig urine samples. By using this method, terbutaline, salbutamol, procaterol and bisoprolol were detected in the positive human urine samples. The concentrations of the four drugs are listed in Table 3. The chromatograms of the positive human urine samples are shown in Figure 4. No target β2-agonists and β-blockers were detected in the pig urine samples.
The authors would like to thank Michael Ye of Supelco, Division of Sigma-Aldrich, for his help in the preparation of this manuscript.
Funding This study was supported by the National Natural Science Founding of China [grant number 20837003]; and by the Science and Technology Projects of the Ministry of Health [grant number 200902009].
Food Additives & Contaminants: Part A
Downloaded by [Northwest Teacher Training University] at 19:32 15 April 2014
References Balizs G, Hewitt A. 2003. Determination of veterinary drug residues by liquid chromatography and tandem mass spectrometry. Anal Chim Acta. 492:105–131. Blanca J, Munoz P, Morgado M, Mendez N, Aranda A, Reuvers T, Hooghuis H. 2005. Determination of clenbuterol, ractopamine and zilpatero]in liver and urine by liquid chromatography tandem mass spectrometry. Anal Chim Acta. 529:199–205. Croes K, Goeyens L, Baeyens W, Van Loco J, Impens S. 2009. Optimization and validation of a liquid chromatography tandem mass spectrometry (LC/MSn) method for analysis of corticosteroids in bovine liver: evaluation of Keyhole Limpet beta-glucuronidase/sulfatase enzyme extract. J Chromatogr B Anal Technol Biomed Life Sci. 877:635–644. Damasceno L, Ventura R, Cardoso J, Segura J. 2002. Diagnostic evidence for the presence of beta-agonists using two consecutive derivatization procedures and gas chromatographymass spectrometric analysis. J Chromatogr B Anal Technol Biomed Life Sci. 780:61–71. Damasceno L, Ventura R, Ortuño J, Segura J. 2000. Derivatization procedures for the detection of beta(2)-agonists by gas chromatographic/mass spectrometric analysis. J Mass Spectrom. 35:1285–1294. Delamoye M, Duverneuil C, Paraire F, de Mazancourt P, Alvarez JC. 2004. Simultaneous determination of thirteen beta-blockers and one metabolite by gradient high-performance liquid chromatography with photodiode-array UV detection. Alvarez Forensic Sci Int. 141:23–31. European Union 2002. Commission decision of 12 August 2002 implementing council directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. (2002/657/EC). Off J Eur Union. L 221:8–36. Fiori M, Civitareale C, Mirante S, Magaro E, Brambilla G. 2005. Evaluation of two different clean-up steps, to minimize ion suppression phenomena in ion trap liquid chromatographytandem mass spectrometry for the multi-residue analysis of beta agonists in calves urine. Anal Chim Acta. 529:207–210. Gallo P, Brambilla G, Neri B, Fiori M, Testa C, Serpe L. 2007. Purification of clenbuterol-like beta2-agonist drugs of new generation from bovine urine and hair by alpha1-acid glycoprotein affinity chromatography and determination by gas chromatography-mass spectrometry. Anal Chim Acta. 587:67–74. Gros M, Pizzolato TM, Petrović M, de Alda MJ, Barceló D. 2008. Trace level determination of beta-blockers in waste waters by highly selective molecularly imprinted polymers extraction followed by liquid chromatography-quadrupole-linear ion trap mass spectrometry. J Chromatogr A. 1189:374–384. Guy PA, Savoy MC, Stadler RH. 1999. Quantitative analysis of clenbuterol in meat products using liquid chromatographyelectrospray ionisation tandem mass spectrometry. J Chromatogr B Biomed Sci Appl. 736:209–219. Hernandez-Carrasquilla M. 2000. Gas chromatography-mass spectrometry analysis of β2-agonists in bovine retina. Anal Chim Acta. 408:285–290. Hu X, Pan J, Hu Y, Li G. 2009. Preparation and evaluation of propranolol molecularly imprinted solid-phase micro
2101
extraction fiber for trace analysis of beta-blockers in urine and plasma samples. J Chromatogr A. 1216:190–197. Huang JF, Zhang HJ, Lin B, Yu QW, Feng YQ. 2007. Multiresidue analysis of beta-agonists in pork by coupling polymer monolith microextraction to electrospray quadrupole time-of-flight mass spectrometry. RCM. 21:2895–2904. Kootstra PR, Kuijpers CJPF, Wubs KL, Van Doorn D, Sterk SS, Van Ginkel LA, Stephany RW. 2005. The analysis of betaagonists in bovine muscle using molecular imprinted polymers with ion trap LCMS screening. Anal Chim Acta. 529:75–81. Lee HB, Peart TE, Svoboda ML. 2007. Determination of betablockers and beta2-agonists in sewage by solid-phase extraction and liquid chromatography-tandem mass spectrometry. J Chromatogr A. 1148:158–167. Liu W, Zhang L, Wei Z, Chen S, Chen G. 2009. Analysis of beta-agonists and beta-blockers in urine using hollow fibreprotected liquid-phase microextraction with in situ derivatization followed by gas chromatography/mass spectrometry. J Chromatogr A. 1216:5340–5346. Moragues F, Igualada C. 2009. How to decrease ion suppression in a multiresidue determination of β2-agonists in animal liver and urine by liquid chromatography-mass spectrometry with ion-trap detector. Anal Chim Acta. 637:193–195. Murray GJ, Danaceau JP. 2009. Simultaneous extraction and screening of diuretics, beta-blockers, selected stimulants and steroids in human urine by HPLC-MS/MS and UPLCMS/MS. J Chromatogr B Anal Technol Biomed Life Sci. 877:3857–3864. Paik MJ, Lee J, Kim KR. 2007. Simultaneous screening analysis of multiple beta-blockers in urine by gas chromatographymass spectrometry in selected ion monitoring mode. Anal Chim Acta. 601:230–233. Shao B, Jia XF, Zhang J, Meng J, Wu YN, Duan HJ, Tu XM. 2009. Multi-residual analysis of 16 β-agonists in pig liver, kidney and muscle by ultra performance liquid chromatography tandem mass spectrometry. Food Chem. 114:1115–1121. Shishani E, Chai SC, Jamokha S, Aznar G, Hoffman MK. 2003. Determination of ractopamine in animal tissues by liquid chromatography-fluorescence and liquid chromatography/tandem mass spectrometry. Anal Chim Acta. 483:137– 145. Siluk D, Kim HS, Cole T, Wainer IW. 2008. HPLC-electrospray mass spectrometric assay for the determination of (R,R)fenoterol in rat plasma. J Pharm Biomed Anal. 48:960–964. Van Hoof N, Courtheyn D, Antignac JP, Van de Wiele M, Poelmans S, Noppe H, De Brabander H. 2005. Multi-residue liquid chromatography/tandem mass spectrometric analysis of beta-agonists in urine using molecular imprinted polymers. RCM. 19:2801–2808. Widstrand C, Larsson F, Fiori M, Civitareale C, Mirante S, Brambilla G. 2004. Evaluation of MISPE for the multiresidue extraction of beta-agonists from calves urine. J Chromatogr B Anal Technol Biomed Life Sci. 804:85–91. Zhang J, Shao B, Yin J, Wu YN, Duan HJ. 2009. Simultaneous detection of residues of β-adrenergic receptor blockers and sedatives in animal tissues by high-performance liquid chromatography/tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci. 877:1915–1922.
