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TITLE : MUSCLE, POULTRY LIVE R, WATER AND FISH - THE SCREENING, QUANTIFICATION AND CONFIRMATION OF A SELECTION OF GROWTH PROMOTORS - LC-MS/MS 1 OBJECTIVE AND SCOPE This SOP describes the analysis using LC-MS/MS of a selection of growth-promoting compounds in muscle, poultry liver, water and fish. These compounds are: 17 α–trenbolone, 17β–trenbolone, 17 α-nortestosterone, 17 β- nortestosterone, androsta-1,4-diene-3,17-dione (ADD), 17 α–boldenone, 17 β-boldenone, methylboldenone, 17 α- testosterone, 17 β-testosterone, 17 α-methyltestosterone, 16 β-OH-stanozolol, stanozolol, prednisolone, methylprednisolone, isoflupredone, flumethasone, dexamethasone, betamethasone, triamcinolon-acetonide, clobetasol, megestrol-acetate, melengestrol-acetate, medroxyprogesterone-acetate, chlormadinone-acetate, α- zeranol ( α-zearalanol), β-zeranol ( β-zearalanol), zearalanon, zearalenon, α-zearalenol, β-zearalenol. Bovine muscle is fully validated, other muscle, poultry liver, water and fish are partially validated. The method has a measuring range of 0.2 to 5.0 µg/kg. 2 DEFINITION MMS Matrix matched standards ES Electrospray MS Mass spectrometer UPLC Ultra Performance Liquid Chromatography ADD Androsta-1,4-diene-3,17-dione SPE Solid Phase Extraction ACN Acetonitrile MeOH Methanol FA Formic acid (HCOOH) Water MilliQ H2O DMSO Dimethyl sulfoxide HAc Acetic acid NH3 Ammonia 3 PRINCIPLE The method consists of the following steps: • Destruction of meat with a bead ruptor • Extraction of analytes from destructed meat • Sample clean-up with 96-wells SPE • Analysis with LC-MSMS 4 CHEMICALS AND REAGENTS All reagents and chemicals must be at least pro analysis quality. With ‘water’ is meant water, purified with a MilliQ® system with a minimum resistance of at least 18.2 M Ω.cm -1. Instead of listed below, chemical products from other suppliers may be used, as long as they are from comparable quality. 4.1 Standards 4.1.1 17 α–Trenbolone (NMI D708) 4.1.2 17 β–Trenbolone (Steraloids E3170-000) 4.1.3 17 β–Boldenone (Steraloids A200) 4.1.4 17 α–Boldenone (Rikilt, EU/CRL 53, this is an ampoule containing 0.1 mg 17 α–boldenon) 4.1.5 17 α-Testosterone (Steraloids, A6900) 4.1.6 17 β-Testosterone-d3 (NMI, D546) 4.1.7 Prednisolone (Sigma-Aldrich P6004) 4.1.8 Methylprednisolone (Sigma-Aldrich M0639) 4.1.9 Megestrol-acetate (Sigma-Aldrich M0513) 4.1.10 Melengestrol-acetate (Sigma-Aldrich 33998) 4.1.11 Chlormadinone-acetate (Sigma-Aldrich C5145) 4.1.12 Isoflupredone (Steraloids P0553-000) 4.1.13 Flumethasone (Sigma F9507) 4.1.14 Triamcinolone-acetonide (Sigma T6501) 4.1.15 Betamethasone (Steraloids P0520-000) 4.1.16 Dexamethasone (Sigma D1756) 4.1.17 Clobetasol (Steraloids P0086-000) 4.1.18 ADD (Androsta-1,4-diene-3,17-dione) (Steraloids A100) 4.1.19 α-Zeranol (Sigma Z0292) 4.1.20 β-Zeranol (Sigma Z0417) 4.1.21 Zearalanone (TRC Z270450 this is a vial containing 1 mg zearalanon) / Sigma Z0167 4.1.22 Zearalenone (Sigma Z2125) 4.1.23 α-Zearalenol (Sigma Z0166) 4.1.24 β-Zearalenol (Sigma Z2000)) 4.1.25 16 β-OH-Stanozolol (NMI D621, this is an ampul containing 1 mg 16 β-OH-stanozolol) 4.1.26 Isoflupredone-d3 (d2 major) (TRC I816602, this is an vial containing 1 mg Isoflupredone-d3 (d2 major). 4.1.27 17 β–Trenbolone-d3 (EURL 0102) (This is an ampoule containing 0.1 mg 17 β–trenbolone-d3) 4.1.28 17 β–Boldenone-d3 (EURL 0065) (This is an ampoule containing 0.1 mg 17 β–boldenone-d3) 4.1.29 α-Zeranol-d4/ β-zeranol-d4 (EURL 0006) (This is an ampoule containing 0.05 mg α-zeranol-d4 and 0.05 mg β-zeranol-d4) 4.1.30 α-Zearalenol-d4 (EURL 0048) (This is an ampoule containing 0,1 mg α-zearalenol-d4) 4.1.54 α-Zearalenol-d7 (EURL 0091) (This is an ampoule containing 0.05 mg α-zearalenol-d7) 4.1.31 β-Zearalenol-d4 (EURL 0049) (This is an ampoule containing 0,1 mg β-zearalenol-d4) 4.1.32 β-Zearalenol-d7 (EURL 0092) (This is an ampoule containing 0.05 mg β-zearalenol-d7) 4.1.33 16 β-OH-stanozolol-d3 (EURL 0064) (This is an ampoule containing 0.1 mg 16 β-OH-stanozolol-d3) 4.1.34 Triamcinolone-acetonide-d6 (EURL 0063) (This is an ampoule containing 0.1 mg triamcinolon-acetonide- d6) 4.1.35 Dexamethasone-d4 (Cachesyn, CSTD361) 4.1.36 Zearalenone-d6 (TRC Z270502) (This is an vial containing 1 mg zearalenone-d6) 4.1.37 Zearalanone-d6 (TRC 270442) (This is an vial containing 0.25 mg zearalanone-d6) 4.1.38 α-Zearalanol-d4/ β-zearalanol-d4 (EURL 0006) (This is an ampoule containing 0.05 mg α-zearalanol-d4 and 0.05 mg / β-zearalanol-d4) 4.1.39 17 β–Nortestosterone-d3 (EURL 0004) (This is an ampoule containing 0.1 mg nortestosterone-d3) 4.1.40 Melengestrol-acetate-d3 (TRC M215352) 4.1.41 Megestrol-acetate-d3 (EURL 0013) (This is an ampoule containing 0.1 mg megestrol-acetate-d3) 4.1.42 Prednisolone-d8 (major compound prednisolone-d7) (TRC P703742) 4.1.43 Medroxyprogesterone-acetate (Steraloids Q3021-000) 4.1.44 Medroxyprogesterone-acetate-d3 (EURL 011) (This is an ampoule containing 0.1 mg medroxyprogesterone-acetate-d3 4.1.45 Stanozolol (Cerilliant S-906) 4.1.46 Stanozolol-d3 (Cerilliant S-910) 4.1.47 Methylboldenone (Steraloids A0130-040) 4.1.48 Methylboldenone-d3 (EURL 52) (This is an ampoule containing 0.1 mg methylboldenone-d3) 4.1.49 17 β-Testosterone (Steraloids A6950-000) 4.1.50 17 α–Nortestosterone (Steraloids E4040-000) 4.1.51 17 β–Nortestosterone (Steraloids E4050-000) 4.1.52 17 α–Methyltestosterone (Steraloids A6280-000) 4.1.53 17 α–Methyltestosterone-d3 (EURL 05) (This is an ampoule containing 0.1 mg methyltestosterone-d3) 4.2 Chemicals 4.2.1 Ammonia 25% (Merck, 105432) 4.2.2 Acetic acid glacial 100% (Merck, 100063) 4.2.3 Formic acid 100% ULC grade (ActuAll, 806012817) 4.2.4 Methanol ULC grade (ActuAll, 813013802) 4.2.5 Acetonitrile ULC grade (ActuAll, 801022802) 4.2.6 Ethanol (100%) (Merck, 100983) 4.2.7 Water ULC grade (ActuAll, 823013802) 4.2.8 Ammonium formate (Sigma 156264) 4.2.9 Dimethylsulfoxide (DMSO) (Sigma-Aldrich D5879) 4.3 Standard solutions 4.3.1 Stock solutions 100 or 1000 mg/l Prepare individual stock solutions of each reference compound (see table below). Weigh reference compounds to 0,01 mg accurate in amber coloured vials with screw cap. Use an analytical balance. Add an amount of MeOH to obtain the concentration of 1000 mg/l. Take account of the test portion, purity and appearance of the standard substance. The amount of MeOH is added by weighing and converting the weighed amount to volume by using the density of ethanol at room temperature 0.79 g/cm ³. Internal standards expire after 5 years. Store in freezer. name stability (weeks) Concentration (mg/l) 17 α-testosterone 104 1000 17 β-testosterone 104 1000 17 α-trenbolone 4.5 1000 17 β-trenbolone 52 1000 17 α-methyltestosterone 104 1000 17 α-nortestosterone 104 1000 17 β nortestosterone 104 1000 17 α-boldenone 4.5 100 17 β-boldenone 52 1000 methylboldenone 4.5 1000 androstadienedione 52 1000 16 β-hydroxystanozolol 52 100 stanozolol 104 1000 α-zearalanol 104 1000 β-zearalanol 104 1000 α-zearalenol 13 1000 β-zearalenol 13 1000 zearalanone 70 1000 zearalenone 104 1000 chlormadinone acetate 52 1000 medroxyprogestrone-17-acetate 52 1000 megestrol acetate 52 1000 melengestrol acetate 52 1000 betamethasone 52 100 clobetasol 156 1000 dexamethasone 156 1000 flumethasone 156 1000 isoflupredone 156 1000 methylprednisolone 156 1000 prednisolone 156 1000 Triamcinolon-acetonide 156 1000 Internal standards 260 1000 4.3.2 Mix standard solution I 1 mg/l (MSS I) Pipette of each stock solution 10 µl (1000 mg/l) or 100 µl (100 mg/l) in an amber coloured vial with screw cap. Add an amount of MeOH till an exact total volume of 10 ml final volume. The amount of MeOH is added by weighing and converting the weighed amount to volume by using the density of MeOH at room temperature 0.79 g/cm³. Store in freezer. 4.3.3 Internal standard solution 1 mg/l (ISS I) Pipette of each internal stock solution 10 µl in an amber coloured vial with screw cap. Add an amount of MeOH till an exact total volume of 10 ml final volume. The amount of MeOH is added by weighing and converting the weighed amount to volume by using the density of MeOH at room temperature 0,79 g/cm ³. Expiration 3 years. Store in freezer. 4.3.4 MSS II 100 µg/l Pipette 500 µl of MSS I (4.3.2) in an amber coloured vial with screw cap. Add 4500 µl of MeOH. Expiration 1 month. Store in freezer. 4.3.5 ISS II 100 µg/l Pipette 500 µl of ISS I (4.3.3) in an amber coloured vial with screw cap. Add 4500 µl of MeOH. Expiration 1 month. Store in freezer. 4.3.6 MSS III 10 µg/l Pipette 500 µl of MSS II (4.3.4) in an amber coloured vial with screw cap. Add 4500 µl of MeOH. Expiration 1 month. Store in freezer. 4.4 Reagents 4.4.1 Ammonium formate 1M Dissolve 6.3 g ammonium formate in water and add water to a final volume of 100 ml. This solution is stable for 3 months at room temperature. 4.4.2 Wash solvent 1: 60% MeOH / 2% HAc (500 ml) Mix 190 ml water, 300 ml MeOH and 10 ml HAc.
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    Pharmaceuticals and Endocrine Active Chemicals in Minnesota Lakes May 2013 Authors Mark Ferrey Contributors/acknowledgements The MPCA is reducing printing and mailing costs This report contains the results of a study that by using the Internet to distribute reports and characterizes the presence of unregulated information to wider audience. Visit our website contaminants in Minnesota’s lakes. The study for more information. was made possible through funding by the MPCA reports are printed on 100 percent post- Minnesota Clean Water Fund and by funding by consumer recycled content paper manufactured the U.S. Environmental Protection Agency without chlorine or chlorine derivatives. (EPA), which facilitated the sampling of lakes for this study. The Minnesota Pollution Control Agency (MPCA) thanks the following for assistance and advice in designing and carrying out this study: Steve Heiskary, Pam Anderson, Dereck Richter, Lee Engel, Amy Garcia, Will Long, Jesse Anderson, Ben Larson, and Kelly O’Hara for the long hours of sampling for this study. Cynthia Tomey, Kirsten Anderson, and Richard Grace of Axys Analytical Labs for the expert help in developing the list of analytes for this study and logistics to make it a success. Minnesota Pollution Control Agency 520 Lafayette Road North | Saint Paul, MN 55155-4194 | www.pca.state.mn.us | 651-296-6300 Toll free 800-657-3864 | TTY 651-282-5332 This report is available in alternative formats upon request, and online at www.pca.state.mn.us. Document number: tdr-g1-16 Contents Contents ...........................................................................................................................................
  • Steroid Use in Prednisone Allergy Abby Shuck, Pharmd Candidate

    Steroid Use in Prednisone Allergy Abby Shuck, Pharmd Candidate

    Steroid Use in Prednisone Allergy Abby Shuck, PharmD candidate 2015 University of Findlay If a patient has an allergy to prednisone and methylprednisolone, what (if any) other corticosteroid can the patient use to avoid an allergic reaction? Corticosteroids very rarely cause allergic reactions in patients that receive them. Since corticosteroids are typically used to treat severe allergic reactions and anaphylaxis, it seems unlikely that these drugs could actually induce an allergic reaction of their own. However, between 0.5-5% of people have reported any sort of reaction to a corticosteroid that they have received.1 Corticosteroids can cause anything from minor skin irritations to full blown anaphylactic shock. Worsening of allergic symptoms during corticosteroid treatment may not always mean that the patient has failed treatment, although it may appear to be so.2,3 There are essentially four classes of corticosteroids: Class A, hydrocortisone-type, Class B, triamcinolone acetonide type, Class C, betamethasone type, and Class D, hydrocortisone-17-butyrate and clobetasone-17-butyrate type. Major* corticosteroids in Class A include cortisone, hydrocortisone, methylprednisolone, prednisolone, and prednisone. Major* corticosteroids in Class B include budesonide, fluocinolone, and triamcinolone. Major* corticosteroids in Class C include beclomethasone and dexamethasone. Finally, major* corticosteroids in Class D include betamethasone, fluticasone, and mometasone.4,5 Class D was later subdivided into Class D1 and D2 depending on the presence or 5,6 absence of a C16 methyl substitution and/or halogenation on C9 of the steroid B-ring. It is often hard to determine what exactly a patient is allergic to if they experience a reaction to a corticosteroid.