Tandem Mass Spectrometry Mikael Pedersen, Jens Hinge Andersen
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Confirmatory analysis of steroids in muscle using liquid chromatography - tandem mass spectrometry Mikael Pedersen, Jens Hinge Andersen To cite this version: Mikael Pedersen, Jens Hinge Andersen. Confirmatory analysis of steroids in muscle using liq- uid chromatography - tandem mass spectrometry. Food Additives and Contaminants, 2011, pp.1. 10.1080/19440049.2010.549153. hal-00671233 HAL Id: hal-00671233 https://hal.archives-ouvertes.fr/hal-00671233 Submitted on 17 Feb 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 For Peer Review Only Confirmatory analysis of steroids in muscle using liquid chromatography – tandem mass spectrometry Journal: Food Additives and Contaminants Manuscript ID: TFAC-2010-345.R1 Manuscript Type: Original Research Paper Date Submitted by the 09-Dec-2010 Author: Complete List of Authors: Pedersen, Mikael; DTU Food, Food Chemistry Andersen, Jens; DTU Food, Food Chemistry Methods/Techniques: Chromatography - LC/MS, Method validation Additives/Contaminants: Veterinary drug residues - anabolic steroids Food Types: Animal products – meat http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 1 of 23 Food Additives and Contaminants 1 2 3 4 5 1 Confirmatory analysis of steroids in muscle using liquid chromatography – 6 7 2 8 tandem mass spectrometry 9 10 3 11 12 4 Mikael Pedersen and Jens Hinge Andersen 13 14 15 5 16 For Peer Review Only 17 6 National Food Institute (DTU-FOOD), Mørkhøj Bygade 19, DK-2860 Søborg, Denmark 18 19 20 7 21 22 8 Abstract: 23 24 9 A method is described for screening and confirmation of synthetic and endogenous steroids in 25 26 27 10 muscle tissue. The method is sensitive, selective, rapid and the consumption of organic solvents is 28 29 11 low, compared to previously published methods. The procedure involves hydrolysis, defattening 30 31 12 with heptane and final clean up with SPE using C18 cartridge. After filtration the analytes are 32 33 34 13 analysed by LC/MS/MS, and quantification is performed using deuterated internal standards. 35 36 14 Decision limits (CC α) varied from 0.02 to 0.33 µg kg -1 and the detection capabilities (CC β) were < 37 38 -1 39 15 0.50 µg kg . The mean within-laboratory reproducibility ranged from 5 – 22% (%RSD IR ). 40 41 16 Endogenous steroids (ex. testosterone, epitestosterone and androstenedione), have been included in 42 43 17 the method, to provide insights into the levels of these steroids as findings of these endogenous 44 45 46 18 steroids have been made several times during the period where analysis has been made of imported 47 48 19 meat. 49 50 51 20 52 21 Keywords: Steroids, LC-MS/MS, Muscle, Method validation, LC-TOFMS, Import control 53 22 54 55 23 Introduction 56 57 58 24 The use of androgen steroids is forbidden for fattening purposes in the European Community [1]. 59 60 25 The most suitable matrices for control of abuse is often urine, faeces, plasma and kidney fat, but for 1 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 2 of 23 1 2 3 4 5 26 control of abuse of these compounds in import from third countries to the EU, these matrices are not 6 7 27 available. Therefore a sensitive and specific method for screening and confirmation of these 8 9 28 substances in muscle, has been developed. In 2007 Denmark imported 6300 tons of beef directly 10 11 12 29 from third countries (primarily South America and Australia) corresponding to app. 5% of beef 13 14 30 consumed in Denmark. 15 16 31 For Peer Review Only 17 18 -1 19 32 Since the European Commission has set 1 µg kg as the recommended concentrations for most 20 21 33 steroids in muscle tissue [2] a very sensitive method has to be used and it must be in compliance 22 23 24 34 and validated in accordance with the criteria of the Commission Decision 2002/657/EC [3]. 25 26 35 A confirmatory method has been developed for the determination of chlormadinone acetate, 27 28 36 megestrol acetate, melengestrol acetate, medroxyprogesterone acetate, methyltestosterone, α/β- 29 30 31 37 boldenone, methylboldenone, α/β -trenbolone, α/β-nortestosterone, altrenogest, testosterone, 32 33 38 epitestosterone, androstenedione and clostebolacetate (screening only) in muscle from bovine. The 34 35 36 39 steroids represent both gestagens and androgens and both synthetic and endogenous steroids have 37 38 40 been included. The method has the advantages that the amount of organic solvents consumed is 39 40 41 very low, no evaporation steps are needed and because of appropriate cleanup and reduced amount 41 42 43 42 of sample material, the matrix effect is low. Since 2007, analysis for steroids in meat from third 44 45 43 countries has been included in the Danish residue plan and the developed method is part of the 46 47 48 44 flexible accreditation our laboratory has been assigned by the Danish Accreditation and Metrology 49 50 45 Fund - DANAK [4]. As a consequence of the survey on the use of veterinary medicinal products in 51 52 46 third countries prepared in 2009 [5], the substances altrenogest and clostebolacetat were included in 53 54 55 47 the method. Clostebolacetate, though, was not included in the accreditation because some criteria 56 57 48 regarding confirmation was not fulfilled. 58 59 49 60 2 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 3 of 23 Food Additives and Contaminants 1 2 3 4 5 50 The procedure involves hydrolysis, defattening with heptane, clean up with SPE using C18 6 7 51 cartridges and final filtration. Analytes were analysed using liquid chromatography (LC) coupled 8 9 52 with tandem mass spectrometry (MS 2), and quantification was performed using spiked standards in 10 11 12 53 combination with deuterated internal standards. Two ion transitions were monitored for each 13 14 54 analyte. The decision limits (CC α) and detection capabilities (CC β) were well below the 15 16 For Peer Review-1 Only 17 55 recommended concentration which is set at 1 µg kg . The mean within-laboratory reproducibility 18 19 56 ranged from 5 to 22% (%RSD IR ). The method is sensitive and reliable and has been used for import 20 21 57 control during the last two years. In addition to the detection and quantification by LC-MS/MS, 22 23 24 58 detection by exact mass measurement using time-of-flight mass spectrometry (LC-TOFMS) has 25 26 59 been demonstrated. The three endogenous steroids testosterone, epitestosterone and 27 28 29 60 androstenedione, have been included in the method to get knowledge about the levels of these 30 31 61 steroids and findings of these steroids have been made several times. A fractional factorial design 32 33 62 was used to evaluate the performance of the method when introducing changes in possible critical 34 35 36 63 factors. 37 38 39 64 Materials and methods 40 41 42 65 Materials and reagents 43 66 All reagents were of analytical or HPLC grade and supplied by Merck (Darmstadt, Germany) 44 45 67 and Rathburn Chemicals (Walkerburn, Scotland). Water was ultra-purified using a Maxima 46 47 48 68 purification system from USF Elga (Bucks, UK). Protease Type VIII from B. Licheniformis was 49 50 69 from Sigma-Aldrich (St. Louis, MO, USA). Medroxyprogesteronacetate (MPA), Megestrolacetate 51 52 70 (MGA), Chlormadinonacetate (CMA), Methyltestosterone (MT), β-Nortestosterone (BNO), 53 54 55 71 Testosterone (TES), epitestosterone (ETE), Androstenedione (ASD), Clostebolacetate (CLAc) and 56 57 72 Altrenogest (ALTR) were purchased from Sigma-Aldrich, Melengestrolacetate (MLA) was 58 59 60 73 purchased from Steraloids Inc. (Newport, USA), α-Trenbolon (ATR) was bought from LGC 3 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 4 of 23 1 2 3 4 5 74 PromoChem (National Analytical Reference Laboratory, Australia), Methylboldenon (MBO), α- 6 7 75 Boldenon (ABO), β-Boldenon (BBO), β-Trenbolon (BTR) and α-Nortestosteron (ANO), 8 9 10 76 Medroxyprogesteronacetate-d3 (MPA-d3), Megestrolacetate-d3 (MGA-d3), Melengestrolacetate-d3 11 12 77 (MLA-d3), Testosteron-d2 (TES-d2), Methyltestosteron-d3 (MT-d3), β-Boldenone-d3 (BBO-d3), 13 14 15 78 Methylboldenone-d3 (MBO-d3), β-Nortestosteron-d3 (BNO-d3) and β-Trenbolon-d2 (BTR-d2) were 16 For Peer Review Only 17 79 purchased from RIVM (Bilthoven, The Netherlands). Stock solutions of each compound were 18 19 80 prepared separately in ethanol at concentrations of 1 mg mL -1. Stock solutions of internal standards, 20 21 22 81 however, were prepared by reconstitution of ampoules containing a fixed amount of material with 23 24 82 ethanol at a concentration of 0.1 mg mL -1. Stock solutions were stored at –18 °C until use for up to 25 26 -1 27 83 3 year. A final working solution containing all the compounds at 0.1 µg mL , except internal 28 29 84 standards, was prepared by diluting stock solutions with ethanol. A final working solution of 30 31 -1 32 85 internal standards containing all nine internal standards at 0.1 µg mL was also prepared by diluting 33 34 86 stock solutions with ethanol.