Bilag 3 Appendix B: Function tests

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1 Bilag 3 Appendix B: Function tests Purchase of two (2) UPLC/UHPLC-MS/MS systems 2017 B1 B2 B3 B4 Function test for Nitrofuran metabolites in muscle Function test for Antibiotics in milk Function test for Mycotoxins in flour Function test for Ergot alkaloids in flour Sagsnr.: Bilag 3, Appendix B, Function tests Page 1 of 11

2 Function tests The instrument must demonstrate its ability to analyze veterinary drug residues and biotoxins. The Function Test is divided into four parts (B1 B4): B1 Nitrofuran metabolites in muscle B2 Antibiotics in milk B3 Mycotoxins in flour B4 Ergot alkaloids in flour The methods will be used for routine analysis, which requires robust methods. The robustness of these methods should be described, including, for example: Frequency for cleaning the ion source and column rinse, ion suppression and the possibility of a "switch to waste". An important parameter is the time of analysis per sample. Runtimes less than 10 minutes for each part of the function test (B1 B4) is preferred. It is a prerequisite for submission of tender that the functional test (B1 B4) is complete. The result of the function test will be included in the evaluation of the tender. If two suppliers are equal in relation to sub-criteria sensitivity is an essential parameter in the choice of instrument. The test solutions must be obtained by sending an with postal information to the following e- mail addresses: Gitte Geertsen and Udo Jensen All test solutions must be stored at -20 C or lower. Bilag 3, Appendix B, Function tests Page 2 of 11

3 B1 Function test for Nitrofuran metabolites in muscle. ATTENTION: As nitrophenyl derivatives are very light-sensitive. All work takes place in rooms with lamps which do not emit UV light. The method use standards with matrix. Pig muscles. The samples are extracted with ethyl acetate and end up in 20% acetonitril in water, 0.1% acetic acid. Two vials with standard without matrix are also provided (B1- NP tune and B1- DNP tune) the solvent is methanol. The concentration is 10 µg/ml. UPLC/UHPLC-MS/MS method An important parameter for the choice of instrument is the runtime of analysis. Basically it is required that analysis of the compounds listed in Table B1-a (Nitrofuran metabolites) can be done in one run of less than 10 minutes. Table B1-a: Compounds and proposals for transitions and polarity Compound Polarity MRM m/z MRM m/z NP-AMOZ (nitrophenyl derivative of Furaltadone) + 335> >262 NP-AOZ (nitrophenyl derivative of Furazolidone) + 236> >104 NP-AHD (nitrophenyl derivative of Nitrofurantoin) + 249> >178 NP-SEM (nitrophenyl derivative of Nitrofurazone) + 209> >166 NP-AMOZ-d 5 (Nitrophenyl derivative of AMOZ-d 5 ) + 340>296 NP-AOZd 4 d 5 (Nitrophenyl derivative of AOZ-d 4 ) + 240>134 NP-SEM [ N 2 ; 13 C] (Nitrophenyl derivative of SEM-[ > N 2 ; 13 C]) Samples to be tested: The standards used are in the matrix. For all standards pig muscles are used. The following table provides the standards concentration used: Table B1-b: Matrix calibration standards and their concentrations Standard Concentration Analytes Concentration Internal standard B1-Std µg/kg 2.5 µg/kg B1-Std µg/kg 2.5 µg/kg B1-Std µg/kg 2.5 µg/kg B1-Std µg/kg 2.5 µg/kg B1-Std µg/kg 2.5 µg/kg B1-Std µg/kg 2.5 µg/kg Matrix blank and 5 matrix calibration standards (B1-Std 0 to B1-Std 5) are provided for the test. The matrix blank should be included in the calibration curve. Results According to A.3. Bilag 3, Appendix B, Function tests Page 3 of 11

4 B2 Function test for Antibiotics in milk. The test solutions consist of a matrix blank and 5 matrix calibration standards (milk). The milk is cleaned up by SPE and dissolved in 15% Methanol. A standard without matrix is also provided (B2- LC-standard). UPLC/UHPLC-MS/MS method An important parameter for the choice of instrument is the runtime of analysis. Basically it is required that analysis of the compounds listed in Table B2-a (Antibiotics) can be done in one run of less than 10 minutes (positive and negative mode in the same run). Table B2-a: Compounds and proposals for transitions and polarity Compound Polarity MRM 3-O-acetylosin > > Amoxicillin > > Ampicillin > > Cefalexin > > Cefapirin > > Cefquinom > > Cefoperazone > > Ceftiofur > > Chloramphenicol > > Chlortetracycline > > Ciprofloxacin > > Clindamycin > > Cloxacillin > > Colistin A > and > or >384.9 and >379.2 Colistin B > > Danofloxacin > > Bilag 3, Appendix B, Function tests Page 4 of 11

5 Compound Polarity MRM Dapsone > > Demeclocyklin (IS) > > Dicloxacillin > > Difloxacin > > Dihydrostromycin > > Doxycycline > > Enrofloxacin > > Erythromycin > > Florfenicol > > Florfenicol amine > > Flumequin > > Gamithromycin > > Lincomycin > > Marbofloxacin > > Nafcillin > > Nalidixinic acid > > Norfloxacin > > Ormethoprim > > Oxacillin > > Oxolinic acid > > Oxytetracycline > > Penicillin G > > Bilag 3, Appendix B, Function tests Page 5 of 11

6 Compound Polarity MRM Penicillin V > > Pirlimycin > > Sarafloxacin > > Spectinomycin > > Spiramycin > > Streptomycin > > Sulfachlorpyridazin > > Sulfaclozin > > Sulfadiazine > > Sulfadimethoxin > > Sulfadoxin > > Sulfamerazin > > Sulfamethazin > > Sulfamethizol > > Sulfamethoxazol > > Sulfamethoxypyridazin > > Sulfamonomethoxin > > Sulfaquinoxalin > > 108 Sulfathiazol > > Tetracycline > > Thiamphenicol > > 78.9 Tiamulin > > Bilag 3, Appendix B, Function tests Page 6 of 11

7 Compound Polarity MRM Tildipirosine > > Tilmicosin > > Trimethoprim > > Tulathromycin > > Tylosin > > Tylvalosin > > Valnemulin > > Virginiamycin M > > Virginiamycin S > > Samples to be tested Matrix blank and 5 matrix calibration standards (B2-S0 to B2-S5) are provided for the test. The matrix blank should be included in the calibration curve. Each standard includes the compounds listed in Table B2-a. The compounds are divided into 13 groups of concentration levels (A-K) according to Table B2-b and B2-c. Bilag 3, Appendix B, Function tests Page 7 of 11

8 Table B2-b: Overview of compounds Group Compounds A B C D E F G H I J K L M Chloramphenicol Amoxicillin, Ampicillin, Penicillin G Dapson 3-OAT, Clindamycin, Difloxacin, Doxycycline, Florfenicol, Florfenicol amine, Gamithromycin, Nalidixic acid, Norfloxacin, Ormethoprim, Oxolinic acid, Penicillin V (Phenoxymethylpenicillin), Sarafloxacin, Tiamulin, Tildipirosine, Tulathromycin, Tylvalosin, Valnemulin, Virginiamycin S1 and Virginiamycin M1 Cefquinome Cloxacillin, Danofloxacin, Dicloxacillin, Nafcillin, Oxacillin Erythomycin Cefoperazone, Colistine, Flumequine, Thiamphenicol, Tilmicosin, Trimethoprim, Tylosin. Cefapirin Marbofloxacin Cefalexin, Ceftiofur, Chlortetracycline, Ciprofloxacin, Enrofloxacin, Oxytetracycline, Pirlimycin, Sulfachlorpyridazin, Sulfaclozin, Sulfadiazin, Sulfadimethoxin, Sulfadoxin, Sulfamerazin, Sulfamethazin, Sulfamethizol, Sulfamethoxazol, Sulfamethoxypyridazin, Sulfamonomethoxin, Sulfaquinoxaline, Sulfathiazol, Tetracycline Lincomycin Dihydrostreptomycin, Spectinomycin, Spiramycin, Streptomycin Table B2-c: Concentration levels of each compound group Group B2-S0 (blank) B2-S1 B2-S2 B2-S3 B2-S4 B2-S5 A B C D E F G H I J K L M Results According to A.3. Bilag 3, Appendix B, Function tests Page 8 of 11

9 B3 Function test for Mycotoxins in flour. The test must be performed for the mycotoxins listed in Table B3-a below. The test includes the analysis of diluted standards and blank / spiked flour samples. Samples and standards are desolved in methanol/water 40/60 (v/v). UPLC/UHPLC-MS/MS method An important parameter for the choice of instrument is the runtime of analysis. Basically it is required that analysis of the compounds listed in Table B3-a (mycotoxins) can be done in less than 10 minutes (positive and negative mode in the same run). Table B3-a: Compound and proposals for transitions and polarity for mycotoxins. Note that Esi + transitions are based on Na + adducts Compound (abbreviation) Polaritet (ESI) M/Z MRM Quantifier (m/z) MRM Qualifier (m/z) Nivalenol (NIV) Deoxynivalenol (DON) Fusarenon-X (FUS-X) Acetyl-DON (15-ac-DON) Acetyl-DON (3-ac-DON) T HT C-DON C-T C-HT C-Zearalenon Zearalenon (ZON) Enniatin B (ENN-B) Enniatin B1 (ENN-B1) Enniatin A1 (ENN-A1) Enniatin A (ENN-A) Beauvericin (BEAU) Bilag 3, Appendix B, Function tests Page 9 of 11

10 Mycotoxins to be tested Flour blank, flour blank spiked on 1 level and 9 calibration standards (Table B3-b, B3-c and B3-d) are provided for the test. Table B3-b: Calibration standards and their concentrations Standard DON, NIV, FUS-X, 3-ac- DON, 15-ac-DON T-2, HT-2, ZON, ENN- A/A1/B/B1, BEAU B3-S0 / Blank 0 0 B3-S B3-S B3-S B3-S B3-S B3-S B3-S B3-S Table B3-c: Internal standards and their concentrations Standard In all calibration std. and Samples 13C-DON 13C-T-2, 13-C HT-2, 13C- ZON Table B3-d: Spike concentrations Spike DON, NIV, FUS-X, 3-ac- DON, 15-ac-DON T-2, HT-2, ZON, ENN- A/A1/B/B1, BEAU Blank spiked Bilag 3, Appendix B, Function tests Page 10 of 11

11 Results According to A.3 B4 Function test for Ergot alkaloids in flour The test must be performed for the ergot alkaloids listed below. The flour is cleaned up by SPE and dissolved in acetonitrile/water/0.2m ammonium hydrogen carbonate (50/25/25). UPLC/UHPLC-MS/MS method An important parameter for the choice of instrument is the runtime of analysis. Basically it is required that analysis of the compounds listed in Table B4-a (ergot alkaloids) can be performed in one run of less than 10 minutes. Table B4-a: Compound and proposals for transitions and polarity for ergot alkaloids Compound Polaritet (ESI) M/Z MRM Quantifier (m/z) MRM Qualifier (m/z) Ergometrine Ergotamine Ergocristine Ergocristinine Ergosine Ergosinine Ergocryptine Ergocryptinine Ergocornine Ergocorninine Flour samples to be tested: 6 matrix calibration standards (Table B4-b) are provided for the test. Table B4-b: Calibration standards and their concentrations Standard Concentration () B4-S0/blank 0 B4-S1 5 B4-S2 10 B4-S3 50 B4-S4 100 B4-S5 200 Results According to A.3. Bilag 3, Appendix B, Function tests Page 11 of 11

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