Quick screening of priority beta-agonists in urine using automated TurboFlow™ - LC/Exactive mass spectrometry Thorsten Bernsmann, Peter Fuerst, Michal Godula To cite this version: Thorsten Bernsmann, Peter Fuerst, Michal Godula. Quick screening of priority beta-agonists in urine using automated TurboFlow™ - LC/Exactive mass spectrometry. Food Additives and Contaminants, 2011, 28 (10), pp.1352-1363. 10.1080/19440049.2011.619504. hal-00743048 HAL Id: hal-00743048 https://hal.archives-ouvertes.fr/hal-00743048 Submitted on 18 Oct 2012 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Food Additives and Contaminants This paper describes a method for the determination of priority β -agonists in urine based on a fully automated sample preparation procedure using the online TurboFlow™ chromatography clean -up step and determination on the Orbitrap™ mass analyzer technology. The principle of the method after enzymatic hydrolysis over night on a small column packed with a special stationary phase (TurboFlow™) while flushing away sample matrix and interfering compounds. Thereafter the analytes are transferred onto an analytical column and detected by id chromatography/high resolution mass spectrometry in full scan mode at a resolution of R=50,000 FWHM (full width at half maximum) and in HCD (Higher Energy Collisional Dissociation) scan mode at a resolving power of 10,000 FWHM. The optimization of each step of theFor Peer Review Only method, such as selection of the TurboFlowTM and analytical column as well as sample loading and elution parameters were performed using a standard solution containing salbutamol, clenbuterol and mabuterol at a veloped automated sample preparation significantly improved the throughput and efficiency of the previous used screening method and resulted in a considerable reduction in analysis time. Validation experiments including 24 β -agonists in urine gave 0.35 ug/L. The repeatability of analyses for urine samples spiked at 0.5 ug/L was within the range of 5 - 26% and recoveries for all compounds were found to be within 89 -107%. http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 1 of 26 Food Additives and Contaminants 1 2 3 4 5 6 7 8 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 2 of 26 1 2 Quick screening of priority beta-agonists in urine using automated 3 4 TurboFlow TM - LC/Exactive mass spectrometry 5 6 7 8 Thorsten Bernsmann a*, Peter Fürst a and Michal Godula b 9 10 11 a 12 Chemical and Veterinary Analytical Institute Münsterland-Emscher-Lippe, Joseph-König- 13 Straße 40, 48147 Münster, Germany 14 For Peer Review Only b 15 Thermo Fisher Scientific, Slunečná 27, 10000 Praha 10, Czech Republic 16 17 18 19 Abstract 20 21 22 This paper describes a method for the determination of priority β-agonists in urine based on a 23 24 fully automated sample preparation procedure using an online TurboFlow TM chromatography 25 TM 26 clean-up step and determination with Orbitrap mass analyzer technology. The principle of 27 28 the method was the enrichment of the β-agonists after enzymatic hydrolysis overnight on a 29 small column packed with a special stationary phase (TurboFlow TM ) while flushing away 30 31 sample matrix and interfering compounds. Thereafter the analytes were transferred onto an 32 33 analytical column and detected by liquid chromatography/high resolution mass spectrometry 34 35 in full scan mode at a resolution of R=50,000 FWHM (full width at half maximum) and in 36 HCD (Higher Energy Collisional Dissociation) scan mode at a resolving power of 10,000 37 38 FWHM. The optimization of each step of the method, such as selection of the TurboFlow TM 39 40 and analytical column as well as sample loading and elution parameters were performed using 41 42 a standard solution containing salbutamol, clenbuterol and mabuterol at a concentration of 100 43 44 µg/L. The developed automated sample preparation significantly improved the throughput and 45 efficiency of the previous used screening method and resulted in a considerable reduction in 46 47 analysis time. Validation experiments including 24 β-agonists in urine gave decision limits 48 49 (CCα) between 0.05-0.35 µg/L. The repeatability of analyses for urine samples spiked at 0.5 50 51 µg/L was within the range of 5-26% and recoveries for all compounds were found to be 52 within 89-107%. 53 54 55 Key Words: Beta-agonists, urine, TurboFlow TM , Orbitrap TM mass analyzer technology, 56 57 Exactive MS 58 59 60 * Corresponding author. Email: [email protected] http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 3 of 26 Food Additives and Contaminants 1 2 3 4 5 6 Introduction 7 8 9 Beta-agonists are synthetically produced compounds that, in addition to their bronchodilatory 10 and tocolytic effects, can promote live weight gain in food producing animals. There have 11 12 been documented cases when consumption of liver and meat from animals illegally treated 13 14 with clenbuterolFor has resulted Peer in serious humanReview intoxication (Botsoglou Only et al. 2001). Due to 15 16 their adverse effects, the use of clenbuterol and its analogues from the β-agonist group has 17 been banned by the European Union (EU 1996) and other regulatory agencies worldwide. 18 19 Monitoring programs have shown that β-agonists are still illegally used by food producers 20 21 (Fiori et al. 2002, Mazzanti et al. 2003). Moreover, newly developed analogues with modified 22 23 structures are obviously being continuously introduced in routine practice. Many papers have 24 25 been published in the past describing the analysis of β-agonists in various matrices using GC- 26 MS. Typically are those methods based on the determination of compounds after 27 28 derivatization by GC-MS (Montrade et al. 1993, Damasceno et al. 2000, Henze et al. 2001) or 29 30 GC-MS/MS (Biancotto et al. 1999, Amendola et al. 2002, Bocca et al. 2003). In most cases 31 32 the sample preparation steps include time-consuming evaporation of water based re-extracts 33 or solid phase extraction (SPE) clean-up steps. 34 35 36 37 In order to avoid derivatization steps the recently published methods use liquid 38 39 chromatography coupled to tandem mass spectrometry (MS/MS) instruments. In the case of 40 41 liver and kidney samples, the β-agonists are typically extracted from the tissue after enzymatic 42 digestion and the extracts are cleaned up using liquid-liquid extraction and SPE (Fesser et al. 43 44 2005). Beta-agonists from urine are generally alkaline extracted after enzymatic hydrolysis 45 46 and acidic re-extraction is then performed. After the evaporation of the acidic re-extract and 47 48 reconstitution in the mobile phase the analytes are determined using electrospray ionization 49 (ESI) LC-MS/MS (Thevis et al. 2003). Alternatively, the clean-up of the sample can be 50 51 improved using SPE and as an alternative to ESI LC-MS/MS atmospheric pressure chemical 52 53 ionization (APCI) is also used for LC-MS/MS determination (Dickson et al. 2005). The very 54 55 efficient alternative to the commonly used SPE approach is the use of molecularly imprinted 56 57 polymer (MIP) columns. Several papers have been published using MIP columns to 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 4 of 26 1 2 effectively remove sample matrix and to allow sub ng/mL determination of β-agonists in 3 4 various matrices (Widestrand et al. 2004, Fiori et al. 2005, Kootstra et al. 2005). Although 5 6 most of the above cited approaches make it possible to reach the required detection limits their 7 main limitation is the time-consuming and expensive column clean-up step. 8 9 10 11 Based on the annual national production figures, the EU Commission stipulates sampling 12 13 levels and frequencies for β-agonists and other veterinary drugs in animal products and 14 For Peer Review Only 15 matrices in order to check for illegally administered substances and in the case of authorized 16 compounds for compliance with prescribed withdrawal periods. Compared to time and effort 17 18 to fulfill these requirements, the number of positive findings is relatively low. This indicates 19 20 that there is a clear need for quick and simple screening methods to routinely and accurately 21 22 control levels of β-agonists in samples of animal origin, such as urine, plasma, and tissues. 23 24 25 The principle of TurboFlow TM chromatography is the separation of analytes from the matrix 26 27 using specific columns packed with large particles. In combination with high linear velocity of 28 29 the mobile phase the conditions are induced on the column that allow the retention of smaller 30 31 molecules (i.e. veterinary drugs) while the large molecules (such as proteins and lipids) are 32 passing through the column unretained (Quinn and Takarewski 1997).