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Confirmatory method for the determination of various acetylgestagens in animal kidney fat using liquid chromatography tandem mass spectrometry Edward Malone, Geraldine Dowling, Chris Elliott, David Glenn Kennedy, Liam Regan

To cite this version:

Edward Malone, Geraldine Dowling, Chris Elliott, David Glenn Kennedy, Liam Regan. Confirmatory method for the determination of various acetylgestagens in animal kidney fat using liquid chromatog- raphy tandem mass spectrometry. Food Additives and Contaminants, 2009, 26 (05), pp.672-682. ￿10.1080/02652030802642110￿. ￿hal-00577354￿

HAL Id: hal-00577354 https://hal.archives-ouvertes.fr/hal-00577354 Submitted on 17 Mar 2011

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 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 method for the determination of various acetylgestagens in animal kidney fat using liquid chromatography tandem mass spectrometry

Journal: Food Additives and Contaminants

Manuscript ID: TFAC-2008-294.R1

Manuscript Type: Original Research Paper

Date Submitted by the 19-Nov-2008 Author:

Complete List of Authors: Malone, Edward; The State Laboratory, Veterinary Toxicology Dowling, Geraldine; The State Laboratory, Veterinary Toxicology Elliott, Chris; Queens University, Belfast, School of Biological Sciences Kennedy, David; Chemical Surveillance Branch, VSD, AFBI Regan, Liam; The State Laboratory, Veterinary Toxicology

Methods/Techniques: Extraction, LC/MS, Method validation

Drug residues - , Veterinary residues - anabolic Additives/Contaminants: , Veterinary

Food Types: Animal, Animal products – meat, Oils and fats

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1 2 3 4 5 6 Confirmatory method for the determination of various acetylgestagens in 7 8 9 animal kidney fat using liquid chromatography tandem mass 10 11 12 spectrometry 13 14 15 16 For Peer Review Only a,b a b c 17 Edward Malone , Geraldine Dowling Chris Elliott , Glenn Kennedy , and Liam 18 19 Regan a 20 21 22 23 a 24 The State Laboratory, Backweston, Celbridge, Co. Kildare, Ireland 25 26 b School of Biological Sciences, Queen’s University, Belfast, Northern Ireland 27 28 c 29 Agri-Food and Bioscience Institute, Belfast, Northern Ireland 30 31 Abstract 32 33 34 A confirmatory method has been developed and validated that allows for the 35 36 37 simultaneous detection of (MPA), acetate 38 39 (MGA), acetate (MLA), acetate (CMA) and 40 41 42 acetate (DMA) in animal kidney fat using liquid chromatography 43 44 tandem mass spectrometry (LC-MS/MS). The compounds were extracted from kidney 45 46 fat using acetonitrile, defatted using a hexane wash and subsequent saponification. 47 48 TM 49 Extracts were then purified on Isolute CN solid phase extraction cartridges and 50 51 analysed by LC-MS/MS. The method was validated in animal kidney fat in 52 53 accordance with the criteria defined in Commission Decision 2002/657/EC. The 54 55 -1 56 decision limit (CC α) was calculated to be 0.12, 0.48, 0.40, 0.63 and 0.54 µg kg , 57 58 respectively, for MPA, MGA, MLA, DMA and CMA detection capability 59 60 (CC β) values of 0.20, 0.81, 0.68, 1.07 and 0.92 µg kg -1 respectively, were obtained.

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1 2 3 The measurement uncertainty of the method was estimated at 16, 16, 19, 27 and 26 % 4 5 6 for MPA, MGA, MLA, DMA and CMA respectively. Fortifying kidney fat samples 7 8 (n = 18) in three separate assays, show the accuracy of the method to be between 98 9 10 11 and 100 %. The precision of the method, expressed as % RSD values for the within- 12 13 lab reproducibility at the three levels of fortification (1, 1.5 and 2 µg kg -1 for MPA, 5, 14 15 7.5 and 10 µg kg -1 for MGA, MLA, DMA and CMA) was less than 5 % for all 16 For Peer Review Only 17 18 analytes. 19 20 21 22 : 23 Keywords Acetylgestagens; Kidney Fat; Mass Spectrometry; Decision Limit; 24 25 Detection Capability 26 27 28 29 30 Introduction 31 32 Under European Union (EU) Council Directive 96/22/EC (Council Directive EC 33 34 96/22 1996), any substances exhibiting, thyreostatic, androgenic, oestrogenic or 35 36 37 gestagenic effects are prohibited for use in food producing animals. The synthetic 38 39 progestagens, medroxyprogesterone acetate (MPA), (MGA), 40 41 42 (MLA), (CMA) and 43 44 (DMA) exhibit gestagenic effects when animals are treated with these substances and 45 46 are prohibited for use in animals reared for human consumption. Incidents of the 47 48 49 illegal administration of gestagens have been previously reported in several European 50 51 countries (Rapp et al. 1989; Vanoosthuyze et al. 1994). 52 53 54 55 56 The acetylgestagen group of compounds exhibit lipophilic properties and hence are 57 58 more likely to bio-accumulate in fatty, adipose tissues (Krzeminski et al. 1981]. 59 60

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1 2 3 Therefore the preferred matrix when testing for these compounds is usually kidney fat 4 5 6 as it offers the greatest chance of detecting residues after illegal administration. 7 8 9 10 11 In order to maintain a zero tolerance policy with regards to these compounds suitably 12 13 sensitive analytical methods are required. Low limits of detection are required 14 15 especially for MPA as it has a minimum required performance level (MRPL) set by 16 For Peer Review Only 17 -1 18 the EU at 1 µg kg in porcine kidney fat (Commission Decision 2003/181 2003). 19 20 There is no MRPL set for MGA, MLA, CMA and DMA. However laboratories 21 22 -1 23 should be capable of monitoring at levels of 5 g kg which is the required 24 25 performance level (RPL) set by the community reference laboratory (CRL Guidance 26 27 Paper 2007). 28 29 30 31 32 Various analytical methods exist that describe the analysis of gestagens in kidney fat, 33 34 a wide array of extraction techniques have been applied for this purpose. They include 35 36 37 solid phase extraction (SPE) (Lohmus et al 2007, Impens et al 2002, Impens et al 38 39 2003, Hageleit et al 2001); supercritical fluid extraction (SFE) (Stolker et al 2002) , 40 41 42 accelerated solvent extraction (ASE) (Hooijerink et al 2003) and matrix solid phase 43 44 dispersion (MSPD) (Rosen et al 1994). A rigorous clean-up is required when 45 46 analysing for these compounds. Hexane wash steps as well as saponification have 47 48 49 been described previously in the literature in order to remove some of the lipophilic 50 51 interferences (Impens et al 2003, Lohmus et al 2007). Other purification techniques 52 53 used include SPE, which often occurs after hexane washing and/or saponification 54 55 56 (Impens et al 2003, Lohmus et al 2007), various types of cartridge chemistry are 57 58 utilised to achieve. These include C 18 (Daeseleire et al 1998, Hooijerink et al 2003, 59 60 Hageleit et al 2001), Silica+NH 2 (Impens et al 2003) and CN cartridges (Impens et al

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1 2 3 2003). A number of analytical techniques have been published for the determination 4 5 6 of acetylgestagens compounds; these include enzyme linked immunoassay (ELISA) 7 8 (Daxenberger et al 1999), gas chromatography-mass spectrometry (GC-MS) (Impens 9 10 11 et al 2002; Impens et al 2003; Stolker et al 1998; Daeseleire et al 1998, Daeseleire et 12 13 al 1994) and liquid chromatography-mass spectrometry (LC-MS) (Stolker et al 2002, 14 15 Hooijerink et al 2003, Lohmus et al 2007, Mortensen et al 2007). Other matrices have 16 For Peer Review Only 17 18 also been investigated and there is published work on a variety of these. These include 19 20 the analysis of MPA in bovine hair (Rambaud et al. 2005). Methods have been 21 22 described to detect gestagens in sites that utilise high performance thin layer 23 24 25 chromatography (HPTLC) and detection under UV light at 366 nm and GC-MS 26 27 (Daeseleire et al 1994). Work on the analysis of gestagens in bovine muscle using 28 29 high performance liquid chromatography (HPLC) clean-up and fractionation followed 30 31 32 by GC-MS analysis has also been published (Daeseleire et al 1998). Methods have 33 34 been described that analyse gestagens from and muscle using GC-MS (Marchand 35 36 37 et al 2000) and from bovine and porcine plasma using LC-MS/MS (Mortensen et al 38 39 2007) and from skin and meat by GC-MS (Stolker et al 1998). 40 41 42 43 44 For Group A substances, mass spectrometry is one of the techniques deemed to 45 46 provide sufficient confirmatory data under Commission Decision 2002/657/EC 47 48 (Commission Decision 2002/657/EC 2002). LC-MS has an advantage over GC-MS 49 50 51 analysis in the analysis of acetylgestagens as this mode of detection avoids the time 52 53 consuming and often non robust step of having to hydrolyze and derivatise samples 54 55 prior to analysis. 56 57 58 59 60

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1 2 3 The first aim of this work was to develop a multi-residue LC-MS/MS confirmatory 4 5 6 method. This method should be capable of simultaneously identifying and quantifying 7 8 five acetylgestagen compounds in kidney fat of bovine, ovine, porcine and equine 9 10 11 species. The method should be validated according to Commission Decision 12 13 2002/657/EC. and establish a measurement of uncertainty for the analytical method 14 15 developed. The second aim of the work was to accredit the method according to the 16 For Peer Review Only 17 18 IS0 17025 standard (ISO/IEC 17025 2005). A third aim was to implement the method 19 20 into the National Monitoring Plan in Ireland to monitor for misuse of these substances 21 22 under the requirements of Council Directive 96/23/EC (Council Directive EC 96/23 23 24 25 1996). 26 27 28 29 The developed method was based on previously published work (Impens et al 2003). 30 31 32 It was modified to reduce the analytical sample size required, to include the analysis 33 34 of delmadinone acetate, to extend the range of species it is applicable to and replace 35 36 37 detection using GC-MS/MS with LC-MS/MS. 38 39 40 41 Materials and methods 42 43 44 Materials and reagents 45 46 Water, methanol, ethyl acetate, acetonitrile and n-hexane, all (HPLC grade) were 47 48 obtained from BDH (Merck, UK). chloride, sodium hydroxide and 49 50 51 acetate, all Analar grade were obtained from BDH (Merck, UK). MPA, 52 53 MGA, MLA, CMA (Vetranal analytical standards) were obtained from Riedel de 54 55 Haen (Sigma Aldrich, Ireland). DMA, MPA-d3, MGA-d3, MLA-d3 were obtained 56 57 58 from RIVM (Bilthoven, The ). Primary stock standard solutions of MPA, 59 60 MGA, MLA and CMA were prepared in methanol at a concentration of 1 mg mL -1.

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1 2 3 Primary stock standard solutions of the deuterated internal standards as well as DMA 4 5 6 were prepared by reconstitution of ampoules containing 0.1 mg with 1 mL of 7 8 methanol yielding a concentration of 0.1 mg mL -1. Stock standard solutions with a 9 10 -1 11 concentration of 1 mg mL were stored in the dark at less than -20°C until use, up to 12 13 a period of 12 months. A final working solution containing all the analytes, except 14 15 internal standards, was prepared in methanol at a concentration of 300 ng mL -1 (for 16 For Peer Review Only 17 -1 18 MGA, MLA, CMA and DMA) and 60 ng mL (MPA) solutions of all analytes (stable 19 20 for 6 months). A final internal standard working solution was prepared in methanol at 21 22 a concentration of 600 ng mL -1(stable for 6 months). All standards were stored at - 23 24 TM 25 20°C in the dark. Isolute CN solid phase extraction cartridges (6 mL, 500 mg) 26 27 were obtained from Biotage (Biotage, UK). 28 29 30 31 32 LC conditions 33 34 The LC system consisted of a model Finnigan TSQ Quantum Ultra (MS) coupled to a 35 36 37 Finnigan Surveyor LC Pump and a Finnigan Surveyor Autosampler (Thermo Electron 38 39 Corporation, CA, USA). A gradient LC system (Table 1) using 0.5 mM ammonium 40 41 acetate:methanol (70:30, v/v Mobile phase A) and 0.5 mM ammonium 42 43 -1 44 acetate:methanol (5:95, v/v Mobile phase B) at a flow of 0.300 mL min , was used to 45 46 separate the analytes on a Phenomenex Luna 3 m C 18 (100 x 2.0 mm) (Phenomenex, 47 48 49 UK) column equipped with a Phenomenex Luna 3 m C 18 guard column (10.0 × 2.0 50 51 mm I.D.). The column temperature was maintained at 45 °C. Data acquisition and 52 53 54 integration were performed using XCalibur version 1.4 chromatographic management 55 56 software (Thermo Electron Corporation, CA, USA). A linear gradient (Table 1) was 57 58 used, the total runtime was 28 min. 59 60

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1 2 3 MS/MS parameters 4 5 6 The analysis was performed using electrospray ionization operated in positive polarity 7 8 and data was gathered using multiple reaction monitoring (MRM) mode. Two 9 10 11 transitions per compound were monitored (one transition for the internal standards) 12 13 and the collision voltages were optimised as shown (Table 2). The MS/MS detector 14 15 conditions were as follows: mode ESI+; sheath gas pressure 60 psi; auxiliary gas 16 For Peer Review Only 17 o 18 pressure 30 psi; capillary temperature 350 C; source CID -10 V; collision pressure 19 20 1.5 torr; collision gas argon. 21 22 23 24 25 Kidney fat samples 26 27 Bovine, porcine, ovine and equine kidney fat samples were received at our laboratory 28 29 within 24 h of animal slaughter for testing as part of the National Residue Control 30 31 32 Plan. The samples are stored at -20 °C until analysis. Kidney fat ran in previous 33 34 batches and found to contain no detectable residues of MPA, MGA, MLA, DMA or 35 36 37 CMA was used as negative control material. 38 39 40 41 Sample extraction and clean-up 42 43 44 A fat tissue sample (approximately 10 g) sliced into very fine pieces was transferred 45 46 into a small glass funnel previously packed with glass wool; this funnel was then 47 48 49 placed into a conical flask. This was then placed for at least three hours into an oven 50 51 (65°C). A portion (3 g) of the molten fat is then weighed into a polypropylene 52 53 centrifuge tube (15 mL), 50 µL of the mixed internal standard is added at this time, 54 55 -1 56 corresponding to a concentration of 10 µg kg . Samples are fortified at levels 57 58 corresponding to 5, 7.5 and 10 µg kg -1 for MGA, MLA, CMA, DMA and 1, 1.5 and 2 59 60 µg kg -1 for MPA by adding 50, 75 and 100 L portions of a 300 ng mL -1 solution of

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1 2 3 MGA, MLA, CMA, DMA and 60 ng mL -1 solution of MPA. After fortification, 4 5 6 samples are held for 15 min prior to extraction. Acetonitrile (8 mL) is added and the 7 8 samples are placed into the oven to allow the fat to become molten again and hence 9 10 11 mix the acetonitrile. After 15 minutes the samples are removed from the oven and 12 o 13 shaken vigorously (30 seconds). They are then centrifuged (3500 rpm, 10 min, 4 C) 14 15 and the supernatant is transferred to a clean polypropylene tube. The sample pellet is 16 For Peer Review Only 17 18 re-extracted with acetonitrile (8 mL) using the same procedure as above and the 19 20 supernatants are combined and dried under nitrogen with heat (55 oC) to an 21 22 approximate volume of 7 mL. N-hexane (5 mL) is then added to the samples and they 23 24 o 25 are shaken (1 min) and centrifuged (3500 rpm, 10 min, 4 C). The upper hexane layer 26 27 is then removed and discarded and the acetonitrile extracts are evaporated to dryness 28 29 under nitrogen with heat (55 oC). The extract is then reconstituted in n-hexane (5 mL) 30 31 32 and vortexed (1 min), the saponification step is then carried out by the addition of 0.1 33 34 M sodium hydroxide (NaOH) (1.4 mL) followed by of 1 M magnesium chloride 35 36 37 (MgCl 2) (850 µL) This is then shaken gently (30 seconds) and placed in an oven (60 38 o 39 C) to aid saponification. The samples are then removed and centrifuged (3500 rpm, 40 41 10 min, 4 oC), the upper hexane layer is then removed and transferred to a clean 15 ml 42 43 44 glass test tube. 45 46 47 48 Solid Phase Extraction 49 50 51 The sample extracts are purified by SPE using Isolute CN SPE cartridges. Sample 52 53 extracts are loaded under gravity onto the cartridges (preconditioned with ethyl 54 55 acetate (3 mL) and n-hexane (5 mL). The cartridges are then eluted (2 x 2.5 mL) with 56 57 58 ethyl acetate :n-hexane (90:10, v/v). The eluates are then evaporated to dryness under 59 60

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1 2 3 nitrogen with heat (55 oC) and reconstituted in Mobile Phase A (200 µL) (70:30 0.5 4 5 6 mM ammonium acetate: methanol), vortexed (1 min) and transferred to LC vials. 7 8 9 10 11 Matrix-Matched Calibration 12 13 Matrix matched calibration curves were prepared and used for quantification. Control 14 15 kidney fat previously tested and shown to contain no residues was prepared as above 16 For Peer Review Only 17 18 (2.4). One kidney fat sample is used for each calibration standard level. Samples were 19 20 fortified at levels corresponding to 0, 0.5, 1, 2, 3, 4 and 8 µg kg -1 MPA and 0, 2.5, 5, 21 22 -1 23 10, 15, 20 and 40 µg kg MGA, MLA, DMA, CMA by adding 0, 25, 50, 100, 150, 24 25 200 and 400 L portions of a 300 ng mL -1 standard solution of MGA, MLA, DMA, 26 27 -1 28 CMA and 60 ng mL of MPA. After fortification, samples were held for 15 min prior 29 30 to the extraction procedure as described above (2.5). Calibration curves were prepared 31 32 by plotting the response factor (area of the analyte/area of internal standard) as a 33 34 -1 -1 35 function of analyte concentration (0 to 8 µg kg for MPA) and (0 to 40 µg kg for 36 37 MGA, MLA, DMA and CMA) to quantify samples. 38 39 40 41 42 Method validation 43 44 For estimation of accuracy, blank kidney fat samples were fortified with MGA, MLA, 45 46 -1 47 DMA and CMA at 5, 7.5 and 10 µg kg which is 1, 1.5 and 2 times the (RPL) of 48 49 5 µg kg -1 set for these compounds by the Community Reference Laboratory and 50 51 -1 52 fortified with MPA at 1, 1.5 and 2 µg kg which is 1, 1.5 and 2 times the (MRPL) set 53 54 by the EU. Six replicate test portions, at each of the three fortification levels, were 55 56 analysed. Analysis of the 18 test portions was carried out on three separate occasions. 57 58 59 For the estimation of the precision of the method, repeatability and within-laboratory 60 reproducibility was calculated. The decision limit (CC α) of the method was calculated

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1 2 3 according to the calibration curve procedure using the intercept (value of the response 4 5 6 factor, y, where the concentration, x is equal to zero) and 2.33 times the standard error 7 8 of the intercept for a set of data with 6 replicates at 3 levels. The detection capability 9 10 11 (CC β) was calculated by adding 1.64 times the standard error to the CC α. 12 13 14 15 Results and Discussion 16 For Peer Review Only 17 18 Preliminary experiments:- 19 20 The LC-MS/MS method was developed to provide confirmatory data for the analysis 21 22 23 of animal kidney fat for MPA, MGA, MLA, DMA and CMA. An excellent response 24 25 was noted for each of the analytes using electrospray ionisation in positive polarity 26 27 and this was the chosen ionization mode for this work. The MS/MS fragmentation 28 29 30 conditions were investigated and collision energies were optimised, the fragmentation 31 32 patterns observed were similar to those found in previously published work 33 34 (Hooijerink et al. 2003; Mortensen et al. 2007). For a method to be deemed 35 36 37 confirmatory in accordance with Commission Decision 2002/657/EC four 38 39 identification points are required. These identification points are obtained by 40 41 monitoring one precursor ion and two transition products (corresponding to strong and 42 43 44 weak ion transitions). The precursor and product transitions for each of the 45 46 acetylgestagen compounds assayed for are listed in Table 2 as well as their associated 47 48 49 collision energy. 50 51 52 53 The extraction and sample purification steps in the method described in this paper are 54 55 56 based on those described in a previously published work by (Impens et al 2003). 57 58 However in this paper we have downscaled the sample size from 5 grams to 3 grams, 59 60 included an extra analyte, delmadinone acetate as well as extended the method to also

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1 2 3 include ovine and equine species. The determination step described in this work is 4 5 6 carried out by LC-MS/MS as opposed to GC-MS/MS (Impens et al 2003) and yields 7 8 lower CC α and CC β values for all of the analytes examined. 9 10 11 12 13 MPA, MGA, MLA, DMA and CMA were chromatographed on a Phenomenex Luna 14 15 C18 , 100 x 3mm, 3 µm column, with retention times of 18.5, 18.2, 18.6, 17.7 and 18.3 16 For Peer Review Only 17 18 min respectively. Deuterated analogues of MPA, MLA and MGA were used as 19 20 internal standards for these compounds. It was found also that MLA-d3 corrected best 21 22 for CMA and that MGA-d3 was best at correcting for DMA so these were chosen as 23 24 37 25 their corresponding internal standards. chlormadinone Cl acetate was obtained, in 26 27 order to be used as an I.S. for chlormadinone acetate. However during development it 28 29 was noted that in samples only fortified with the various I.S. compounds that quite a 30 31 32 large response was found in the chlormadinone acetate trace that originated from 33 34 chlormadinone 37 Cl acetate, hence it was decided to omit using this as an internal 35 36 37 standard. 38 39 40 41 Validation study 42 43 44 Validation of the method was according to procedures described in Commission 45 46 Decision 2002/657/EC covering specificity, calibration curve linearity, recovery 47 48 (accuracy), precision, decision limit (CC α) and detection capability (CC β). 49 50 51 52 53 Specificity 54 55 56 The technique of LC-MS/MS itself offers a high degree of selectivity and specificity. 57 58 To establish the selectivity/specificity of the method, a variety of fat samples from 59 60 different species (bovine, ovine, porcine and equine) were fortified with MPA, MGA,

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1 2 3 MLA, DMA and CMA (to check if any of these compounds give rise to peaks for 4 5 6 transitions for any of the other analytes, also the data obtained was used in calculating 7 8 the measurement of uncertainty) and their non-fortified equivalent samples were also 9 10 11 analysed. No interfering peaks were observed at the retention time for any of the 12 13 analytes (Figure 1). Additionally samples were fortified with a variety of steroidal 14 15 compounds including α and β , , , , 16 For Peer Review Only 17 18 , , flumethasone, methylboldenone, 19 20 and . No interferences were observed in the retention windows of 21 22 MPA, MGA, MLA, DMA and CMA in chromatograms when fortified with these 23 24 25 substances. 26 27 28 29 Linearity of the response 30 31 32 The linearity of the chromatographic response was tested with matrix matched curves 33 34 using 7 calibration points in the concentration range of 0 to 40 µg kg -1 for MGA, 35 36 -1 2 37 MLA, DMA and CMA and 0 to 8 µg kg for MPA. The regression coefficients ( r ) 38 39 for all the calibration curves used in this study were ≥≥≥ 0.997. A plot of residuals was 40 41 42 also carried out to further demonstrate the linearity of the response. For each observed 43 44 value of x (concentration), the residual is the observed y value (response factor) minus 45 46 the corresponding fitted Y. The residual was then plotted against the concentration; 47 48 49 this is shown in Figure 3 for each of the five analytes. The residuals were acceptable, 50 51 they have a random scatter rather than any systematic pattern and also they are 52 53 54 normally distributed with an approximate mean of zero. 55 56 57 58 Accuracy 59 60

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1 2 3 The accuracy of the method was determined using kidney fat samples fortified at 5.0, 4 5 -1 -1 6 7.5 and 10.0 µg kg for MGA, MLA, DMA and CMA and 1, 1.5 and 2 µg kg for 7 8 MPA. The resulting values were calculated from matrix matched calibration curves. 9 10 11 Mean corrected recovery (n = 18) of the analyte, determined in three separate assays 12 13 (Table 3) was between 98 and 100 % for MPA, MGA, MLA, DMA and CMA. 14 15 16 For Peer Review Only 17 18 Precision 19 20 The usefulness of suitable isotopically labelled internal standards is demonstrated in 21 22 23 the excellent repeatability and within-laboratory reproducibility obtained for MPA, 24 25 MGA and MLA (Table 3). No deuterated analogue was available for CMA or DMA, 26 27 however these compounds are structurally very similar to the other gestagen 28 29 30 compounds which had deuterated analogues, so these internal standards also worked 31 32 very well for CMA and DMA. In the case of CMA, MLA-d3 was used as the 33 34 corresponding internal standard and in the case of DMA, MGA-d3 was used as the 35 36 37 corresponding internal standard. The precision of the method, expressed as RSD 38 39 values for the within-lab reproducibility at the three levels of fortification (5.0, 7.5 40 41 -1 -1 42 and 10.0 µg kg for MGA, MLA, DMA and CMA and 1, 1.5 and 2 µg kg for MPA) 43 44 was less than 6 %. 45 46 47 48 49 CC α and CC β 50 51 The decision limit is defined as the limit above which it can be concluded with an 52 53 54 error probability of α, that a sample contains the analyte. In general, for prohibited 55 56 substances an α value equal to 1 % is applied. The detection capability is the smallest 57 58 59 content of the substance that may be detected, identified and quantified in a sample, 60 with a statistical certainty of 1-β, were β = 5 %. CC α and CC β were calculated using

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1 2 3 the intercept (value of the signal, y, were the concentration, x is equal to zero) and the 4 5 6 standard error of the intercept for a set of data with 6 replicates at 3 levels on three 7 8 separate days (5.0, 7.5 and 10.0 µg kg -1 for MGA, MLA, DMA and CMA and 1, 1.5 9 10 -1 11 and 2 µg kg ). Blank kidney fat was fortified at 1, 1.5 and 2 times the minimum 12 13 required performance level of 1 µg kg -1 for MPA and 5 µg kg -1 for MGA, MLA, 14 15 16 DMA and CMA.For CC αPeer is the concentration Review corresponding Onlyto the intercept + 2.33 times 17 18 the standard error of the intercept. CC α values of 0.12, 0.48, 0.40, 0.54 and 0.63 µg 19 20 -1 21 kg were achieved for MPA, MGA, MLA, CMA and DMA respectively. CCβ is the 22 23 concentration corresponding to the signal at CC α + 1.64 times the standard error of 24 25 26 the intercept (i.e the intercept + 3.97 times that standard error of the intercept). CC β 27 28 values of 0.20, 0.81, 0.68, 0.92 and 1.07 µg kg -1 were achieved for MPA, MGA, 29 30 31 MLA, CMA and DMA respectively. 32 33 34 35 36 Measurement Uncertainty 37 38 The measurement of uncertainty was estimated by taking into account the within 39 40 laboratory reproducibility over days 1, 2 and 3 as well as considering the repeatability 41 42 43 on day 4 due to matrix effects caused by different species (bovine, ovine, porcine and 44 45 equine) and different animals. These two variability’s were combined and multiplied 46 47 by a coverage factor of three to give an overall figure for the uncertainty of the 48 49 50 measurement. This approach of using the within laboratory reproducibility as a good 51 52 estimator of measurement of uncertainty is taken from the SANCO/2004/2726rev1 53 54 document (SANCO 2004/2726rev1 2004). It recommends using the within laboratory 55 56 57 reproducibility and using a coverage factor of 2.33 to estimate expanded uncertainty, 58 59 however as it was felt that not all the environmental and other factors that could be 60 varied over the course of the validation were examined, it was felt that a coverage

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1 2 3 factor of 2.33 may underestimate the true uncertainty of the method. So a value of 3 4 5 6 was chosen instead to give a more realistic value for the true uncertainty, this 7 8 approach was acceptable to the ISO17025 (ISO/IEC 17025 2005) auditors as well. 9 10 11 12 13 Evaluation 14 15 The method developed in this study has been used to monitor for the presence of 16 For Peer Review Only 17 18 MPA, MGA, MLA, CMA and DMA residues in kidney fat of bovine, porcine, ovine 19 20 and equine kidney fat as part of the National Monitoring Plan in Ireland in some of, 21 22 2007 and all of 2008. The method has been carried out by different analysts over a 23 24 25 period of two years under varying environmental conditions and the method was 26 27 shown to be robust. The method has been accredited by the Irish National 28 29 Accreditation Board in accordance with ISO17025 standard which is the main 30 31 32 internationally recognised standard used by testing and calibration laboratories. 33 34 Samples of incurred porcine kidney fat received from the Community Reference 35 36 37 Laboratory (CRL), RIVM, Bilthoven, The Netherlands as part of a proficiency 38 39 scheme. Figure 4 shows a chromatogram with both strong and weak transitions for 40 41 MPA for one of these incurred kidney fat samples of at a measured concentration of 42 43 -1 44 1.2 µg kg . These samples were tested using the method described here and were 45 46 found to yield satisfactory results. In two samples containing incurred MPA, the 47 48 described method was able to detect and confirm its presence and also the calculated 49 50 51 concentrations were close to the CRL’s assigned values. In fact when the method’s 52 53 measurement of uncertainty is applied, the assigned values falls within the possible 54 55 range of concentrations given by this method, no z-scores for this proficiency scheme 56 57 58 have been released by the CRL yet. Also the described method found no detectable 59 60 peaks in the negative control sample sent by the CRL.

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1 2 3 4 5 Conclusions 6 7 8 The aim of this work was to develop, validate and accredit a multi-residue LC- 9 10 MS/MS confirmatory method that can simultaneously identify and quantify five 11 12 acetylgestagen compounds in kidney fat of bovine, ovine, porcine and equine species. 13 14 15 This goal was successfully met and the developed method has been successfully 16 For Peer Review Only 17 utilised as part of the National Residue Control Plan in the Republic of Ireland. It also 18 19 20 has been accredited by the Irish National Accreditation Board to ISO17025 standard 21 22 and been successfully used in analysing proficiency test samples. 23 24 25 26 27 The obtained data fulfils the requirements laid down in Commission Decision 28 29 2002/657/EC and allows the calculation of all relevant performance characteristics. 30 31 This study shows that the developed method meets the required sensitivity of 1 g kg - 32 33 1 34 which is the MRPL set by the EU for MPA, and also meets the recommended 35 36 concentration level of 5 g kg -1set by the CRL for the other acetylgestagen 37 38 39 compounds. The CC α and CC β values determined for each analyte are considerably 40 41 lower than these levels. The method performs very well in terms of accuracy and 42 43 44 repeatability for each of the analytes due to the utilisation of different deuterated 45 46 internal standards. The values achieved for accuracy (%), RSD (%) and measurement 47 48 of uncertainty (%) all fall within acceptable ranges. The applicability of the method 49 50 51 for use on different species (bovine, ovine, porcine and equine) was demonstrated by 52 53 the satisfactory results obtained from day 4 and also ongoing data gathered as part of 54 55 56 National Residue Monitoring Plan in Ireland for all the different species mentioned 57 58 previously. 59 60

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1 2 3 4 5 6 Acknowledgements 7 8 This work was funded by The State Laboratory, Co. Kildare in Ireland. The authors 9 10 11 would like to thank the staff at The State Laboratory, Co. Kildare for their practical 12 13 assistance. 14 15 16 For Peer Review Only 17 References 18 19 20 Commision Decision 2002/657/EC of 12 August 2002 implementing Council 21 22 Directive 96/23/EC concerning the performance of analytical methods and the 23 24 interpretation of results. Off. J. Eur. Commun. L2218. 25 26 27 Commission Decision 2003/181/EC of 13 March 2003 amending Decision 28 29 2002/657/EC as regards the setting of minimum required performance limits 30 31 (MRPLs) for certain residues in food of animal origin. Off. J. Eur. Commun. L7117. 32 33 34 Council Directive 96/22/EC. 1996. Concerning the prohibition on the use in 35 36 stockfarming of certain substances having a hormonal or thyrostatic action and of ß- 37 38 . Off. J Eur Commun. L 125. 39 40 41 Council Directive 96/23/EC. 1996. On measures to monitor certain substances and 42 43 residues thereof in live animals and animal products. Off. J. Eur. Commun. L221. 44 45 46 47 CRL Guidance Paper (7 December 2007) CRLs view on state of the art analytical 48 methods for national residue control plans. 49 50 51 52 Daeseleire E, Vandeputte R, Van Peteghem C. 1998. Validation of multi-residue 53 54 methods for the detection of anabolic steroids by GC-MS in muscle tissues and 55 samples from cattle. Analyst (123) 2595-2598. 56 57 58 59 Daeseleire E, Vanoosthuyze K, Van Peteghem C. 1994 Application of high- 60 performance thin-layer chromatography and gas chromatography-mass spectrometry

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1 2 3 to the detection of new anabolic steroids used as growth promotors in cattle fattening. 4 5 J. of Chromatogr. A (674) 247-253. 6 7 8 9 Daxenberger A, Meyer K, Hageleit M, Meyer HHD. 1999. Detection of melengestrol 10 11 acetate residues in plasma and edible tissues of heifers. Vet. Quart. (21) 154-157. 12 13 14 General requirements for the competence of testing and calibration laboratories 2005. 15 16 ISO/IEC 17025:2005For (E)Peer Review Only 17 18 19 Guidelines for the implementation of Commission Decision.. 2002/657/EC. 2004. 20 21 SANCO/2004/2726rev1. 22 23 24 25 Hageleit M, Daxenberger A, Meyer HHD. 2001. A sensitive enzyme immunoassay 26 (EIA) for the determination of melengestrol acetate (MGA) in adipose and muscle 27 28 tissues. Food Addit. Contam. (18) 285-291. 29 30 31 32 Hooijerink H, van Bennekom EO, Nielen MWF. 2003 Screening for gestagens in 33 34 kidney fat using accelerated solvent extraction and liquid chromatography 35 electrospray tandem mass spectrometry. Anal. Chim. Acta (483) 51-59. 36 37 38 39 Impens S, Courtheyn D, De Wasch K. 2003 Faster analysis of anabolic steroids in 40 41 kidney fat by downscaling the sample size and using gas chromatography-tandem 42 mass spectrometry. Anal. Chim. Acta (483) 269-280. 43 44 45 46 Impens S, De Wasch K, Cornelius M, De Brabander HF. 2002. Analysis on residues 47 48 of , gestagens and in kidney fat and meat with gas 49 50 chromatography-tandem mass spectrometry. J. Chromatogr A (970) 235-247. 51 52 53 Joos PE, Van Ryckeghem M. 1999. Liquid chromatography-tandem mass 54 55 spectrometry of some anabolic steroids. Anal. Chem. (71) 4701-4710. 56 57 58 Krzeminski LF, Cox BL, Gosline RE. 1981. Fate of radioactive melengestrol acetate 59 60 in the bovine. J. Agric. Food Chem. (29) 387-391.

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1 2 3 Lohmus M, Kender T. 2007. Determination of gestagens in kidney fat by liquid 4 5 chromatography tandem mass spectrometry. Anal. Chim. Acta (586) 233-238. 6 7 8 9 Marchand P, Le Bizec B, Gade C, Monteau F, Andre F. 2000. Ultra trace detection of 10 11 a wide range of anabolic steroids in meat by gas chromatography coupled to mass 12 spectrometry. J. Chromatogr A, (867) 219-233. 13 14 15 16 Mortensen For SK and PedersenPeer M. 2007Review Confirmatory analysis Only of acetylgestagens in 17 18 plasma using liquid chromatography-tandem mass spectrometry. Anal. Chim. Acta 19 (586) 217-222. 20 21 22 23 Rambaud L, Bichon E, Cesbron N, Andre F, Le Bizec B. 2005 Study of 17-- 24 25 3-benzoate, 17-methyltestosterone and medroxyprogesterone acetate fixation in 26 bovine hair. Anal. Chim. Acta (532) 165-176. 27 28 29 30 Rapp M, Meyer HHD. 1989. Control of illegal Medroxyprogesterone acetate- 31 32 application in veal calves by residue analysis in adipose tissues using HPLC/RIA 33 34 method. Food Addit. Contam. (6) 59-70. 35 36 37 Rosen J, Hellenas KE, Tornqvist P, Shearan P. 1994 Automated extraction of 38 39 acetylgestagens from kidney fat by matrix solid phase dispersion. Analyst (119) 2635- 40 41 2638. 42 43 44 Stolker AAM, Zoontjes PW, Schwillens PLWJ, Kootstra PR,van Ginkel LA, 45 46 Stephany RW, Brinkman UATh. 2002. Determination of acetyl gestagenic steroids in 47 48 kidney fat by automated supercritical fluid extraction and liquid chromatography ion- 49 50 trap mass spectrometry. Analyst (127) 748-754. 51 52 53 Stolker AAM, Zoontjes PW, van Ginkel LA. 1998. The use of supercritical fluid 54 55 extraction for the determination of steroids in animal tissues. Analyst (123) 2671- 56 57 2676. 58 59 60

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1 2 3 Vanoosthuyze K, Daeseleire E, Van Overbeke A, Van Peteghem C. 1994. Survey of 4 5 the hormones used in cattle fattening based on the analysis of Belgian injection sites. 6 7 Analyst. (119) 2655-2658. 8

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11 12 13 14 15 16 For Peer Review Only 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

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1 2 3 4 5 6 7 (a) 8 9 10 11 12 13 14 For Peer Review Only 15 16 (b) 17 MPA 18 19 20 21 22 23 24 25 (a) 26 27 28 29 30 31

32 33 (b) 34 MGA 35 36 37 38 39 40 41 42 43

44 (a) 45 46 47 48 49 50 51 52 53 (b) MLA 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 22 of 28

1 2 3 4 5 6 7 (a) 8 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 (b) 19 CLA 20 21 22 23 24 25 26 (a) 27 28 29 30 31 32 33 34 35 36 (b) 37 DLA 38 39 40 41 42 Figure 1. (a) Negative control kidney fat (b) Kidney fat fortified with 0.5 µg kg -1 43 -1 44 MPA and 2.5 µg kg of MGA, MLA, CLA and DLA respectively. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 23 of 28 Food Additives and Contaminants

1 2 3 4

5 MGA MLA 6 7 8 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 19 20 21 MPA 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 CLA DLA 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 Figure 2. Structures of the various acetylgestagen compounds 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 24 of 28

1 2 3 4 5 MGA

6 0.12 0.1 7 0.08 8 0.06 0.04 9 0.02 0

10 Residual -0.02 0 10 20 30 40 50 11 -0.04 -0.06 12 -0.08 13 Concentration 14 For Peer Review Only 15 MPA

16 0.015 17 0.01 18 19 0.005 0

20 Residual 0 2 4 6 8 10 21 -0.005 22 -0.01 23 Concentration 24 25 MLA

26 0.08 27 0.06 28 0.04 29 0.02 0

30 Residual -0.02 0 10 20 30 40 50 31 -0.04 32 -0.06 33 Concentration 34 35 CLA

36 0.01 37 0.008 0.006 38 0.004 0.002 39 0 -0.002 0 10 20 30 40 50

40 Residual -0.004 41 -0.006 -0.008 42 -0.01 43 Concentration 44 45 DLA

46 0.02 47 0.015 48 0.01 0.005 49 0 -0.005 0 10 20 30 40 50

50 Residual 51 -0.01 -0.015 52 -0.02 53 Concentration 54 55 56 Figure 3. Plot of residuals for each acetylgestagen compound, concentration is µg kg -1 in 57 each case. 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 25 of 28 Food Additives and Contaminants

1 2 3 4 5 6 7 8 (a) 9 10 11 12 13 14 For Peer Review Only 15 16 17 18 19 20 21 22 23 24 25 26 27 (b) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Figure 4. Incurred sample of kidney fat at a concentration of 1.2 µg kg -1. (a) Strong 48 Transition (m/z) 387.0>327.2 and (b) Weak Transition (m/z) 387.0>285.2 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 26 of 28

1 2 3 Table 1: LC gradient profile for determination of MPA, MGA, MLA, DLA and CMA. 4 5 6 Time Component A Component B 7 8 (min) (%) (%) 9 10 0 90 10 11 12 6.5 90 10 13 14 10.0 For Peer 35 Review 65 Only 15 16 20.0 35 65 17 18 19 22.5 90 10 20 21 28.0 90 10 22 23 24 Component A: 0.5 mM ammonium acetate buffer + methanol (70:30, v/v). 25 26 Component B: 0.5 mM ammonium acetate buffer + methanol (5:95, v/v). 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] Page 27 of 28 Food Additives and Contaminants

1 2 3 4 5 6 Table 2: MS/MS parameters for determination of each of the investigated analytes and 7 their associated deuterated analogues. 8 9 10 11 Compound Transition Polarity Collision Retention 12 (m/z) Energy (eV) Time (min) 13 Medroxyprogesterone 387.0>327.2 * 15 14 Positive 18.5 AcetateFor Peer Review Only 15 387.0>285.2 18 16 Megestrol 385.3>325.1 * 19 17 Positive 18.2 Acetate 18 385.3>267.3 15 19 Melengestrol 397.3>337.3 * 20 20 Positive 18.6 21 Acetate 397.3>279.2 15 22 Delmadinone 403.2>205.1 * 19 23 Positive 17.7 24 Acetate 403.2>181.1 21 25 Chlormadinone 405.0>308.9 * 17 26 Positive 18.3 Acetate 27 405.0>344.9 13 28 Medroxyprogesterone 390.0>330.2 Positive 15 18.4 29 Acetate-d3 30 31 Megestrol 388.3>328.2 Positive 15 18.2 32 Acetate-d3 33 34 Melengestrol 400.2>340.2 Positive 15 18.6 35 Acetate-d3 36 37 * Denotes strong transition 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 28 of 28

1 2 3 4 5 6 Table 3: CC α, CC β, Within-laboratory Reproducibility RSD (%), Accuracy (%) and 7 Measurement of Uncertainty (%) for each analyte. 8 9 10 Compound CC α CC β RSD Measurement Accuracy 11 -1 -1 12 (µµµg kg ) (µµµg kg ) (%) of Uncertainty (%) 13 (%) 14 MedroxyprogesteroneFor Peer Review Only 0.12 0.20 4.4 16 99.8 15 Acetate 16 17 Megestrol 0.48 0.81 5.2 16 99.1 18 Acetate 19 20 Melengestrol 21 0.40 0.68 4.2 18 98.4 Acetate 22 23 Delmadinone 24 0.63 1.07 4.7 26 98.4 Acetate 25 26 Chlormadinone 27 0.54 0.92 4.4 26 98.4 28 Acetate 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]