Supplementary data

Matrix-effect free multi-residue analysis of veterinary drugs in food samples of animal origin by nanoflow liquid chromatography high resolution mass spectrometry

Jaime Alcántara-Durán, David Moreno-González*, Bienvenida Gilbert-López, Antonio

Molina-Díaz and Juan F. García-Reyes

Analytical Chemistry Research Group, Department of Physical and Analytical Chemistry,

University of Jaén, 23071 Jaén Spain

Prepared for: Food Chemistry

Corresponding author: Dr. David Moreno-González, Analytical Chemistry Research Group,

University of Jaén, 23071 Jaén, Spain. Tel.: (+34) 953218758; Fax: (+34)953 212940. E-mail:

dmgonzal@ujaen.es

Contents

Sample treatment procedures

Table S1. Relative abundance (%) of fragments for each compound using high-energy collision dissociation (0, 20, 30 and 40V).

Table S2. Intraday and inter-day peak area precision (n=10) expressed as RSD (%) for milk samples.

Table S3. Comparison of the proposed method with other reported methods for the determination of veterinary drugs in food samples.

Figure S1. Optimization of gradient program.

Figure S2. Comparison of percentages of compounds with negligible, soft and medium ME using different dilution factors.

2

Sample treatment procedures

1. Salting-out supported liquid extraction (SOSLE) sample treatment for milk, beef muscle and egg samples.

Step 1 Salting-out acetonitrile/water extraction:

Fresh veal muscle samples were crushed and homogenized in a blender. In the case of egg

samples, they were homogenized by vortex agitation. After pretreatment (not required in

milk samples), 5 g of sample was weighed into 50 mL centrifuge tube. Then, 5 mL of

extraction buffer (detailed in section 2.1. of the manuscript) and 10 mL of MeCN were

added to the sample. This mixture was shaken for 30 s by hand and then centrifuged at

5000 rpm for 5 min. The supernatant was transferred into another 50 mL centrifuge tube, to

which 1 g of ammonium sulfate was added. The mixture was mixed for 2 min by hand, and

then was left to stand for 2 min. A phase separation was observed from the mixture. The

analytes with moderate polarity were in the organic upper layer, whereas the most polar

compounds were in the aqueous phase. Both phases were collected and transferred to

separate falcon tubes for subsequent cleaning-up by SPE.

Step 2 solid-phase extraction (SPE) Clean-up.

The aqueous phase collected from step 1 was loaded onto a HLB cartridge (previously

conditioned with 10 mL of MeOH, 10 mL of water, and 2 mL of extraction buffer) at a flow

rate of approximately 1 mL min-1. After aqueous phase loading, the cartridge was washed

with 2 mL of extraction buffer at a flow rate of 1 mL min -1. Subsequently, vacuum was

applied for 2 min. Subsequently, the polar analytes were eluted with the organic phase from

step 1 at flow rate of 1 mL min-1. Thus, this organic phase, which contains veterinary drugs

with moderate polarity, acts also as eluting solvent. This extract was diluted 1:20 by

dissolving 50 µL of each extract in 1950 µL of water and transferred to a 2-mL glass vial. In

3

the case of dilution 1:50, 100 µL of extract was dissolved with 150 µL of MeCN and 4750 µL

of water and transferred to a 2-mL glass vial.

2. Liquid-liquid partitioning with acetonitrile following by a dispersive solid-phase

extraction clean-up (QuEChERS for honey samples).

5 g of sample was weighed in a 50-mL plastic centrifuge tube and mixed with 10 mL of

water, being the tubes vigorously shaken for 1 minute. Then, 0.2g of EDTA chelating agent

solution was added. The mixture was homogenized by vortex for 10 s. Subsequently, 10 mL

of ethyl acetate were added and stirred vigorously for 10 min. Then, 5 g of sodium sulfate,

0.2 g of C18 and 0.2 g of PSA were added and shaken vigorously for 30 min. Subsequently,

the samples were centrifuged for 6 min at 3000rpm. This extract was diluted 1:20 by

dissolving 50 µL of each extract in 1950 µL of water and transferred to a 2-mL glass vial. In

the case of dilution 1:50, 100 µL of extract was dissolved with 150 µL of MeCN and 4750 µL

of water and transferred to a 2-mL glass vial.

4

Table S1. Relative abundance (%) of fragments for each compound using high-energy collision dissociation (0, 20, 30 and 40V).

CompoundElemental Composition(M)

AIFRelative Abundance0V (%)

Relative Abundance20V (%)

Relative Abundance30V (%)

Relative Abundance40V (%)

Acetamiprid C10H11ClN4 223.0745 100 0 0 0Acetamiprid F1 C6H5ClN 126.0105 3 100 100 100Acetamiprid F2 C5H4Cl 99.0001 0 12 13 54Acriflavine C14H14ClN3 260.0949 100 0 0 0Acriflavine F2 C14H13N3 224.11822 0 100 100 100Albendazole C12H15N3O2S 266.0958 100 16 0 11Albendazole F1 C11H12N3OS 234.0696 0 100 100 14Albendazole F2 C8H6N3OS 192.0226 0 0 42 39Albendazole F3 C10H7O2S 191.0161 0 0 53 100Albendazole F4 C10H7O2 159.0441 0 0 13 53Albendazole Sulfone C12H15N3O4S 298.0856 100 0 0 0Albendazole Sulfone F1 C11H12N3O3S 266.0592 0 100 91 0Albendazole Sulfone F2 C8H6N3O3S 224.0124 0 0 100 28Albendazole Sulfone F3 C10H7O2 159.0427 0 0 55 100Albendazole Sulfoxide C12H15N3O3S 282.0907 100 0 0 0Albendazole S. F1 C8H6N3O2S 208.0174 0 100 100 89Albendazole S. F2 C10H7O2 159.0420 0 0 22 100Alternogest C21H26O2 311.2006 100 100 16 0Alternogest F1 C16H19O 227.143 0 44 100 100Alternogest F2 C14H14O 198.1039 0 0 15 89Alternogest F3 C11H11O 159.0805 0 11 28 63Alternogest F4 C9H9O 133.0649 0 0 11 52Amoxicillin C16H19N3O5S 366.1118 0 0 0 0Amoxicillin F1 C13H15O2N2S 277.0869 100 0 0 0Amoxicillin F2 C14H17N2S 245.1105 0 23 47 24Amoxicillin F3 C9H5ON3S 203.0141 95 12 13 10Amoxicillin F4 C16H14OS 254.0762 0 100 57 20Amoxicillin F5 C5H10O4S 168.0462 0 25 42 36Amoxicillin F6 C8H16O2N 158.1188 0 45 100 100Amoxicillin F7 C8H8N2 132.0694 0 36 61 52Betamethasone C22H29FO5 393.2072 100 35 10 4Betamethasone F1 C22H29O5 373.2008 75 31 25 8Betamethasone F2 C19H28O5F 355.1903 9 100 7 7Betamethasone F3 C17H24O2F 279.1733 0 90 58 20Betamethasone F4 C9H12O2F 171.0803 0 31 56 61Betamethasone F5 C10H11O 147.084 0 58 100 100Carbosulfan C20H32N2O3S 381.2206 83 0 0 0Carbosulfan F1 C8H18NS 160.1155 0 25 27 9Carbosulfan F2 C8H17N2 141.1386 100 55 16 0Carbosulfan F3 C5H12NS 118.0688 0 44 100 97

5

Cefalexin C16H17N3O4S 348.1013 100 0 0 0Cefalexin F1 C16H17N2O3S 318.1623 0 100 44 0Cefalexin F2 C14H16N2OS 261.1037 0 0 100 100Cefalexin F3 CH9N3O4S 159.0305 75 0 0 0Cefazolin C14H14N8O4S3 455.0373 50 0 59 0Cefazolin F1 C10H15O2N8 279.1590 100 0 100 0Cefazolin F2 C10H14O2N6 250.1185 67 0 29 0Cefazolin F3 C7H15N5OS 217.1070 65 0 59 0Cefazolin F4 C7H11N7 183.1492 51 0 28 0Ceftiofur C19H17N5O7S3 524.0363 100 34 0 0Ceftiofur F1 H12N3O3S2 166.0312 0 0 18 54Ceftiofur F2 C5H6N3OS 156.023 0 0 22 50Ceftiofur F3 C8H15N3 149.02339 0 100 100 96Ceftiofur F4 C4H4N3S 126.0119 0 0 42 100Ceftiofur F5 C4H3N3S 125.0042 0 14 0 58Chloramphenicol C11H12O5N2Cl2 321.0051 100 100 32 0Chloramphenicol F1 C10H9Cl2N2O3 274.9985 0 50 100 100Chloramphenicol F2 C11H9Cl2NO 241.0056 0 21 64 78Chlorpyrifos ethyl C9H11Cl3NO3PS 349.9336 100 0 0 0Chlorpyrifos e. F1 C8Cl2O2 197.9275 4 65 54 43Chlorpyrifos e. F2 H4O3PS 114.9616 0 100 100 100Chlortetracycline C22H23ClN2O8 479.1216 28 0 0 0Chlortetracycline F1 C22H22O8N 428.135 0 70 26 0Chlortetracycline F2 C16H24O7N2 356.1576 0 100 23 0Chlortetracycline F3 C18H20O2N2Cl 331.1221 100 0 0 0Chlortetracycline F4 C20H19O3N 321.1356 40 35 23 0Chlortetracycline F5 C7H14O8N2 254.0751 0 74 57 21Ciprofloxacin C17H18FN3O3 332.1405 65 47 38 0Ciprofloxacin F1 C13H12O3N3F 277.0868 100 0 0 0Ciprofloxacin F2 C6H11O2F 254.0764 0 100 89 21Ciprofloxacin F3 C14H14ON2F 245.1105 0 0 96 24Clenbuterol C12H18Cl2N2O 277.0869 100 0 0 0Clenbuterol F1 C11H6NOCl 203.0136 0 100 0 13Clenbuterol F2 C8H16NO2 158.1188 0 48 100 100Clenbuterol F3 C8H8N2 132.0677 0 38 61 57Cloxacillin C19H18ClN3O5S 468.0991 100 0 0 0Cloxacillin F1 C12H2O2 178.0054 0 8 58 100Cloxacillin F2 C6H10O2NS 160.0427 0 100 100 74Cloxacillin F3 C5H8NS 114.0376 0 6 24 58Colchicine C22H25NO6 400.1755 100 100 71 0Colchicine F1 C20H24NO5 358.1647 0 12 86 0Colchicine F2 C19H20O4 326.1429 0 3 42 0Colchicine F3 C19H18O4 310.1191 0 4 96 22Cortisone C21H28O5 361.201 100 42 0 0Cortisone F1 C8H5O3 149.0233 0 100 100 100Cortisone F2 C7H5O2 121.0287 0 41 23 37

6

Cortisone F3 C8H9 105.0702 0 9 16 42Danofloxacin C19H20FN3O3 358.1561 100 69 100 100Danofloxacin F1 C16H11O2F 254.0747 0 100 0 44Danofloxacin F2 C14H14N2OF 245.1099 0 35 0 60Danofloxacin F3 C11H6O 168.0449 0 25 0 71Dapsone (H2NC6H4)2SO2 249.0692 100 0 0 0Dapsone F1 C11H2N 149.0234 0 100 59 99Dapsone F2 C6H5NO 108.0448 0 0 100 100Dicloxacillin C19H17N3Cl2O5S 470.0339 0 0 0 0Dicloxacillin F1 C16H16O4NCl 310.9982 9 50 22 5Dicloxacillin F2 C6H10O2NS 160.0426 29 100 100 100Dicloxacillin F6 C5H8NS 114.0375 0 19 32 100Difloxacin C21H19F2N3O3 400.1467 81 100 25 15Difloxacin F1 C18H17O2N2F2 331.1221 100 48 11 9Difloxacin F2 C8H13O2N 158.1188 0 48 100 100Dimetridazole C5H7N3O2 142.0611 100 100 0 0Dimetridazole F1 C5H8N2O 112.0631 0 30 100 0Dimetridazole F2 C4H5N2 81.0447 20 48 60 100Dimetridazole F3 C3H6N 56.0495 0 6 54 89Enoxacin C15H17FN4O3 321.1357 100 100 23 12Enoxacin F1 C8H7O2NF 168.0462 0 48 42 36Enoxacin F2 C8H10O2N 158.1188 0 100 100 100Enoxacin F3 C8H8N2 132.0684 0 80 61 57Enrofloxacin C19H22FN3O3 360.1718 100 100 34 20Enrofloxacin F1 C13H12FO3N3 277.0868 74 18 1 1Enrofloxacin F2 C14H14ON2F 245.1105 0 34 47 24Enrofloxacin F3 C8H16O2N 158.1179 0 70 100 100Eprinomectin C50H75NO14 914.526 100 100 20 10Eprinomectin F1 C9H16NO3 186.1125 0 60 100 100Eprinomectin F2 C8H12NO2 154.0863 0 0 60 75Febantel C20H23N4O6S 447.1333 100 100 100 100Febantel F1 C19H19N4O5S 415.1071 0 31 0 0Febantel F1 C20H15N4O4S 383.0809 0 32 0 0Febendazole C15H13N3O2S 300.0801 100 22 0 0Febendazole F1 C14H10N3OS 268.0541 0 100 100 30Febendazole F2 C8H5N3O 159.0419 0 0 32 100Fenthion C10H15O3PS2 279.0273 100 6 0 0Fenthion F1 C9H9S2 169.014 0 100 57 31Fleroxacin C17H18F3N3O3 370.1373 8 3 0 0Fleroxacin F1 C4H7F3N3O3 202.0432 100 100 100 11Fleroxacin F2 C3H6O3N2F3 175.0327 0 22 100 90Fleroxacin F3 C8H13O2N 158.1178 0 20 69 100Flumequine C14H12FNO3 262.0874 100 100 100 21Flumequine F1 C14H11FNO2 244.0766 6 59 69 13Flumequine F2 C11H7O3NF 220.0404 0 3 52 100Flumequine F3 C11H5O2NF 202.0298 0 3 30 53Imidacloprid C9H10ClN5O2 256.0596 100 0 0 0

7

Imidacloprid F1 C9H10ClN4 209.0589 15 85 89 100Imidacloprid F2 C9H11N4 175.0978 19 100 100 82Ivermectin C48H72O14 892.5417 100 0 0 0Ivermectin F1 C34H47O6 551.3367 0 9 20 12Ivermectin F2 C19H31O3 307.2268 0 64 75 100Ivermectin F3 C7H13O3 145.0859 0 6 28 53Josamycin C42H69NO15 828.474 16 23 13 0Josamycin F1 C38H67NO13 734.46802 68 20 0 0Josamycin F5 C19H24O11 428.13504 0 70 26 0Josamycin F6 C14H26NO12 400.14651 81 73 25 16Josamycin F7 C13H26O10N 356.1576 0 100 23 0Josamycin F8 C11H23O11 331.1221 100 0 11 0Josamycin F10 C13H17NO7 299.10086 0 11 50 46Josamycin F11 C12H14O6 254.0764 0 77 57 19Josamycin F12 C8H16NO2 158.11865 0 28 100 100Leucomalachite G. C23H26N2 331.2169 100 0 0 0Leucomalachite G. F1 C16H19N2 239.1542 0 100 100 100Levamisole C11H12N2S 205.0794 100 100 6 0Levamisole F1 C9H7N2S 175.0324 0 85 100 66Levamisole F2 C8H7N2 131.0604 0 8 33 100Levamisole F3 C10H12NS 178.0683 0 54 19 0Lincomycin C18H35N2O6S 407.2210 100 64 0 0Lincomycin F1 C8H16N 126.1278 0 100 100 100Malachite Green C23H24N2 329.2012 100 100 100 22Malachite G. F1 C22H21N2 313.1703 0 0 10 45Malachite G. F2 C15H14N 208.1124 0 0 7 14Malachite G. F3 C8H7N2 131.0607 0 0 64 100Marbofloxacin C17H20FN4O4 363.1463 100 100 100 100Marbofloxacin F1 C17H17FN4O3 345.1355 0 31 0 0Marbofloxacin F2 C15H15FN3O4 320.1041 0 32 0 0Mebendazole C16H13N3O3 296.103 100 21 3 0Mebendazole F1 C15H10O2N3 264.0767 0 95 100 74Mebendazole F2 C7H5O2 105.034 0 6 18 100Metronidazole C6H10N3O3 172.0717 100 0 0 0Metronidazole F1 C4H6O2N3 128.0455 30 100 0 0Minocycline C23H28N3O7 458.1922 0 0 0 0Minocycline F1 C16H18N3O 352.1469 100 91 100 0Minocycline F2 C8H16N 126.1284 0 100 0 100Nandrolone C18H26O2 275.2006 100 100 0 0Nandrolone F1 C18H23 239.1793 0 43 0 0Nandrolone F2 C7H10O 109.0651 0 47 100 100Norfloxacin C16H18FN3O3 320.1405 100 16 28 47Norfloxacin F1 C15H19FN3O 276.1511 0 100 9 0Norfloxacin F2 C13H14FN2O 233.1087 0 0 100 100Ofloxacin C18H20FN3O4 362.1511 100 39 0 0Ofloxacin F1 C17H20FN2O2 318.1611 0 100 44 0

8

Ofloxacin F2 C14FN2O2 261.0104 0 0 100 100Orbifloxacin C19H21F3N3O3 396.1530 22 9 0 0Orbifloxacin F1 C20H19F2N3O3 386.1311 31 20 27 16Orbifloxacin F2 C17H20ONF2 291.1451 100 100 42 36Orbifloxacin F3 C11H17O2N2F3 265.1158 0 19 56 16Orbifloxacin F4 C8H17N 126.1281 0 23 100 100Oxacillin C19H19N3O5S 402.1118 100 0 0 0Oxacillin F1 C14H11N2O3S 287.0485 0 9 20 12Oxacillin F2 C9H6ON 144.0447 0 64 75 100Oxacillin F3 C5H8NS 114.0375 0 6 28 53Oxfendazole C15H14N3O3S 316.0750 100 93 17 10Oxfendazole F1 C14H10N3O2S 284.0488 0 100 18 3Oxfendazole F2 C9H10N3O2 191.0685 0 91 55 11Oxfendazole F3 C8H6N3O 159.0419 0 25 100 100Oxolinic Acid C13H11NO5 262.071 100 100 100 50Oxolinic Acid F1 C13H10O4N 244.0603 0 53 57 0Oxolinic Acid F2 C11H8NOS 234.0396 0 0 21 67Oxolinic Acid F3 C10H6O3N 188.0349 0 0 0 42Oxolinic Acid F4 C9H6O2N 160.0395 0 0 0 100Oxybendazole C12H15N3O3 250.1186 100 12 0 0Oxybendazole F1 C11H12N3O2 218.0925 0 100 40 0Oxybendazole F2 C8H6N3O3 176.0454 0 18 100 100Oxytetracycline C22H24N2O9 461.1555 12 0 0 0Oxytetracycline F1 C3H10O8N 202.0433 100 100 69 4Oxytetracycline F2 C8H16O3N 174.1119 0 17 62 77Oxytetracycline F3 C8H16O2N 158.1171 0 28 100 67Oxytetracycline F4 C8H7N2 131.0599 0 0 56 63Paclobutrazol C15H20ClN3O 294.1368 100 100 4 0Paclobutrazol F1 C10H10Cl 165.0465 0 40 32 10Paclobutrazol F2 C7H6Cl 125.0155 0 42 100 100Penicillin G C16H18N2O4S 335.1060 100 100 100 100Penicillin G F1 C10H10NO2 176.0706 0 74 81 87Penicillin G F2 C6H10NO2 160.0427 0 80 76 54Penicillin G F3 C7H7 91.0542 0 2 12 41Penicillin V C16H18N2O5S 351.1009 100 100 0 0Penicillin V F1 C9H13O2NS 213.0692 0 26 100 100Phenylbutazone C19H20N2O2 309.1598 100 44 17 12Phenylbutazone F1 C13H11ON2 211.0869 0 49 36 24Phenylbutazone F2 C11H14N 160.1121 0 59 100 49Phenylbutazone F3 C7H6NO 120.0447 0 100 99 100Phenylbutazone F4 C7H8N 106.0656 0 8 43 78Phenylbutazone F5 C8H6NO 132.0449 0 15 36 65Ractopamine C18H23O3 302.1756 100 11 0 0Ractopamine F1 C8H16O2N 158.1178 0 100 100 100Ractopamine F2 C8H9 121.0653 0 96 38 36Rifaximin C43H51N3O11 786.3596 100 100 0 0

9

Rifaximin F2 C42H47N3O10 754.3331 0 59 100 79Rifaximin F4 C9H10O2 151.0764 0 0 0 100Ronidazole C6H8N4O4 201.0618 51 8 35 6Ronidazole F1 C5H6N3O2 140.0455 100 100 0 0Ronidazole F2 C5H6N2O 110.0476 0 0 100 100Roxithromycin C41H76N2O15 837.5318 100 6 0 0Roxithromycin F1 C41H77N2O15 679.4365 0 100 0 0Roxithromycin F2 C8H17NO2 158.1157 0 66 100 100Sarafloxacin C20H17F2N3O3 386.1311 31 20 63 44Sarafloxacin F1 C20H17F2N2O 299.0991 0 0 66 83Sarafloxacin F2 C20H12FN2 291.1153 100 100 100 100Spynosin A C41H65NO10 732.4681 100 59 32 1Spynosin A F1 C18H16ON 142.1227 0 35 100 100Sulfabenzamide C13H12N2O3S 156.0114 100 0 0 0Sulfabenzamide F1 C7H7O 107.0497 0 100 100 100Sulfacetamide C8H10N2O3S 215.0485 100 100 0 0Sulfacetamide F1 C6H6NO2S 156.0114 0 43 0 0Sulfacetamide F2 C6H6NO 108.0444 0 47 100 100Sulfachloropyridazine C10H11ClN4O2S 285.0208 100 7 9 11Sulfachloropyridazine F1 C6H6NO2S 156.0118 0 100 30 0Sulfachloropyridazine F2 C6H6NO 108.0457 0 43 100 100Sulfadiazine C10H12N4O2S 251.0597 100 6 0 0Sulfadiazine F1 C4H4N3O2S 158.0019 0 100 0 0Sulfadiazine F2 C6H6NO 108.0441 0 66 100 100Sulfadimethoxyn C12H14N4O4S 311.0809 100 100 60 25Sulfadimethoxyn F1 C12H13N4O2 245.1033 0 2 100 100Sulfadimethoxyn F2 C6H10N3O2 156.0114 0 0 33 64Sulfadoxine C12H14N4O4S 311.0809 100 100 2 0Sulfadoxine F1 C2H7N2O4 156.0114 0 0 100 100Sulfadoxine F2 C6H6NO 108.0444 0 0 10 25Sulfaguanidine C7H10N4O2S 215.0597 100 0 0 0Sulfaguanidine F1 C7H7O 107.0497 0 100 100 100Sulfamerazine C11H13N4O2S 265.0754 100 100 100 100Sulfamerazine F1 C11H2N4 190.0274 0 15 30 37Sulfamerazine F2 C6H6NO2S 156.0114 0 20 46 52Sulfameter C11H12N4O3S 265.0754 100 100 4 0Sulfameter F1 C11H2N4 190.0274 0 40 32 10Sulfameter F2 C6H6NO2S 156.0114 0 42 100 100Sulfamethazine C12H14N4O2S 279.091 100 50 0 0Sulfamethazine F1 C6H10N3 124.087 0 100 100 100Sulfamethazine F2 C6H10N3 121.087 0 90 60 54Sulfamethazine F3 C6H6ON 108.0447 0 56 88 97Sulfamethoxazole C10H13N3O3S 254.0594 100 0 0 0Sulfamethoxazole F1 C6H6NO2S 156.0113 0 73 18 0Sulfamethoxazole F2 C6H3O2N3 149.0234 0 100 100 100Sulfamethoxazole F3 C6H6NO 108.0441 0 47 90 45

10

Sulfamethoxypyridazine C11H12N4O3S 281.0703 100 50 0 0Sulfamethoxypyridazine F1 C6H6NO2 156.0114 0 100 100 100

Sulfamethoxypyridazine F2 C5H8N3O 126.0662 0 90 60 54

Sulfamonomethoxine C11H12N4O3S 281.0703 100 100 60 24Sulfamonomethoxine F1 C7H7O 107.0497 0 80 100 100Sulfamoxole C11H13N3O3S 268.0750 100 100 0 0Sulfamoxole F1 C6H6NO2S 156.0114 0 70 60 20Sulfamoxole F2 C5H9N2O 113.0709 0 47 100 100Sulfapyridine C11H11N3O2S 250.0645 100 34 5 2Sulfapyridine F1 C11H10NO3 184.0869 0 100 42 23Sulfapyridine F2 C6H6ON 108.0447 0 97 100 100Sulfaquinoxaline C14H12N4O2S 301.0754 100 0 0 0Sulfaquinoxaline F1 C6H6ON 108.0447 0 32 100 100Sulfaquinoxaline F2 C6H6O2SN 156.0114 0 100 41 6Sulfathiazole C9H11N3O2S2 256.0209 100 0 0 0Sulfathiazole F1 C6H6NO2S 156.0114 0 100 0 12Sulfathiazole F2 C8H15N 126.1279 0 25 100 37Sulfathiazole F3 C6H6NO 108.0448 0 61 52 100Sulfisoxazol C11H13N3O3S 268.0750 100 100 100 100Terbutaline C12H19NO3 226.1437 100 9 0 0Terbutaline F1 C8H16NO3 174.1128 0 61 62 100Terbutaline F2 C8H16NO2 158.1178 0 100 100 87Tetracycline C22H24N2O8 445.1605 34 0 0 0Tetracycline F1 C18H24O3N 302.1748 100 17 0 0Tetracycline F3 C2H9O8N 175.0327 0 18 100 63Tetracycline F4 C8H7N2 131.0607 0 6 38 100Thiabendazole C10H7N3S 202.0433 100 100 47 8Thiabendazole F1 C9H7N2S 175.0327 0 22 100 63Thiabendazole F2 C8H7N2 131.0607 0 2 38 100Thiamphenicol C12H16Cl2NO5S 356.0121 100 100 0 0Thiamphenicol F1 C12H14Cl2NO4S 338.0015 0 25 46 22Thiamphenicol F2 C11H12Cl2NO3S 307.9909 0 12 100 100Tilmicosin C46H80N2O13 869.5733 27 42 100 100Tilmicosin F1 C45H59O5 679.4371 100 100 5 0Tolfenamic Acid C14H12ClNO2 262.0629 55 0 0 0Tolfenamic Acid F1 C14H11NClO 244.0523 100 100 72 14Tolfenamic Acid F2 C14H11N 209.0834 0 9 100 100Trimethoprim C14H20N4O3 291.1452 100 100 42 36Trimethoprim F1 C8H16N 126.1284 0 23 100 100Trimethoprim F2 C7H9ON 123.067 0 14 37 41

11

Table S2. Intraday and inter-day peak area precision (n=10) expressed as RSD (%) for milk samples.

Compound IntradayRSD (%)

InterdayRSD (%)

Acetamiprid 5.6 5.1Acriflavine 4.3 5.3

Albendazole 5.7 6.0Albendazole

Sulfone 1.7 2.3

Albendazole Sulfoxide 7.0 8.1

Alternogest 4.0 5.7Amoxicillin 5.3 6.3

Betamethasone 7.0 7.3Carbosulfan 6.3 5.8

Cefalexin 4.6 5.2Cefazolin 5.3 7.1Ceftiofur 3.7 4.9

Chloramphenicol 5.4 5.0Chlorpyrifos ethyl 0.9 1.5Chlortetracycline 6.9 6.7

Ciprofloxacin 8.3 8.9Clenbuterol 5.3 7.5Cloxacillin 7.4 8.1Colchicine 4.7 5.1Cortisone 4.3 5.5

Danofloxacin 2.9 4.5Dapsone 2.4 3.7

Dicloxacillin 3.0 4.0Difloxacin 3.3 4.4

Dimetridazole 4.2 5.6Enoxacin 3.2 4.0

Enrofloxacin 3.2 4.7Eprinomectin 3.8 4.5

Febantel 2.6 4.2Febendazole 4.3 5.5

Fenthion 3.6 4.2Fleroxacin 5.6 5.9

Flumequine 4.9 5.7Imidacloprid 6.7 6.9Ivermectin 4.8 5.8Josamycin 4.0 5.2

Leucomalachite Green 5.9 5.8

Levamisole 5.7 6.5Lincomycin 5.2 5.9

12

Malachite Green 3.9 4.4Marbofloxacin 4.3 5.9Mebendazole 3.6 6.6Metronidazole 2.5 3.7

Minocycline 5.8 6.3Nandrolon 5.8 5.3Norfloxacin 3.1 4.2Ofloxacin 3.0 4.7

Orbifloxacin 2.1 5.6Oxacillin 6.4 7.4

Oxfendazole 3.8 4.8Oxolinic Acid 5.9 7.6

Oxybendazole 4.1 4.7Oxytetracycline 4.6 5.3Paclobutrazol 3.1 4.2

Penicillin G 4.0 5.1Penicillin V 5.2 6.8

Phenylbutazone 5.0 5.8Ractopamine 3.9 5.1

Rifaximin 4.8 5.3Ronidazole 5.7 5.9

Roxithromycin 3.9 4.9Sarafloxacin 4.7 4.4Spinosad A 4.5 5.4

Sulfabenzamide 3.2 4.4Sulfacetamide 4.2 4.5

Sulfachloropyri-dazine 5.0 5.6

Sulfadiazine 6.1 7.0Sulfadimethoxyn 5.5 6.1

Sulfadoxine 5.4 6.5Sulfaguanidine 5.3 6.7Sulfamerazine 5.0 6.5

Sulfameter 3.7 5.4Sulfamethazine 7.4 8.4

Sulfamethoxazole 3.9 5.6Sulfamethoxypyri-

dazine 5.7 5.4

Sulfamonome-thoxine 3.6 4.8

Sulfamoxole 6.1 5.8Sulfanitran 5.8 5.9

Sulfapyridine 5.6 7.0Sulfaquinoxaline 7.0 8.5

Sulfathiazole 5.5 4.6Sulfisoxazol 4.8 6.4Terbutaline 5.8 6.6

13

hemisulfateTetracycline 2.0 5.1

Thiabendazole 4.1 3.7Tolfenamic Acid 4.6 5.3Trimethoprim 4.3 5.0

14

Table S3. Comparison of the proposed method with other reported methods for the determination of veterinary drugs in food samples.

References Detection Sample Treatment MatrixLOQ

(µg kg–1)Interday precision

(RSD (%))Recovery

(%)Matrix Effect

(%)# Compounds

(Family)

Present Work

Q OrbitrapSOSLE for Milk, Veal

and EggQuEChERs for honey

Milk, Veal, honey and

egg0.1-1.0* 0.5 - 8 73- 120 Negligible

87(multiclass)

Aguilera-Luiz, M.M.

et al. (2008)

Tandem Quadrupole QuEChERs Milk 3 - 10 5 – 20.4 70.6-106.5 NP 18

(multiclass)

S-lker, A.A.M. et al. (2008).

TOF SPE (Strata-X) milk< 7 (90% of compound)

< 10(65% of compounds)

105 -110 NP> 100

(multiclass)

Aguilera-Luiz,

M.M., et al. (2013).

Exactive Orbitrap

SOSLE Honey ≤ 50 < 25 68-120 Strong40

(multiclass)

Kaufmann, A., et al. (2008).

TOF LLSE (acetonitrile) &

SPE (clean-up)Meat NP 0 – 10.0

8 – 11380% of compounds between 70 to 120

Negligible for 14% of compouds, Soft for 10%, Medium for 45% and strong for 25% of analytes

>100(multiclass)

Ortelli, D., et al.

(2009). A.TOF Ultrafiltration Milk NP 4 – 13.2 0.3 -61

Strong for 79% of analytes

150(multiclass)

Jedziniak, P., et al. (2016).

Q-Trap SPEbovine, swine

and horse muscle

1.25 – 125 7 - 250 – 100, Depending of

the cartridge used Soft

31(Anti-Inflammatory andGlucocortico-steroids)

Kaufmann, A., et al. (2011).

Q OrbitrapLiquid/Solid-Liquid

extractionBeef and milk

0.5 2.1 - 25.4

70 - 100 Negligible (strong

for avermectin)13 (anthelmintic)

Ortelli, D., et al.

(2009). B.

TOF Protein precipitation associated

with ultrafiltration

Milk NP 0.5 - 25 –< 70 (20% analytes)70 – 120 (53% analytes)

NP 150 (multiclass)

> 120 (27% analytes)

Kaufmann, A., et al. (2014).

Q Orbitrap QuEChERS, SPE and

SOSLEMilk 0.2- 6.0 1.2 - 10.9

– Analytes recovered in the range 70-120: 73% for QuEChERS, 83% for SPE and 91% for SOSLE

Negligible for 80% of compounds.

7% of compounds showed ME > 100

114(multiclass)

Petersa, R.J.B., et

al. (2009).TOF SPE

Egg, fish and meat

NP 15 - 20

–95% of compounds between 70 to 1202% of compounds

above 120

Strong (70 – 100)100

(multiclass)

Wang, J., et al.

(2015).Q Orbitrap SOSLE Milk 1.0 2.3 - 53.8 15 - 72.4

Medium for 75% of compounds and

Negligible for 23%

125(multiclass)

NP: Not Provided

* Corresponds to the lowest concentration level studied yielding S/N ratio equal or higher than 10.

1

References

1 Aguilera-Luiz, M.M., Martínez-Vidal, J.L., Romero-González, R. & Garrido-Frenich, A. (2008). Multi-residue determination of veterinary drugs in milk by ultra-high-pressure liquid chromatography-tandem mass spectrometry. Journal of Chromatography A, 1205, 10–16

2 S-lker, A.A.M., Rutgers, P., Oosterink, E., Lasaroms, J.J.P., Peters, R.J.B. & van Rhijn, J.A. (2008). Comprehensive screening and quantification of veterinary drugs in milk using UPLC-TOF-MS. Analytical and Bioanalytical Chemistry, 391, 2309–2322.

3 Aguilera-Luiz, M.M., Romero-González, R., Plaza-Bolaños, P., Martínez Vidal, J.L. & Garrido Frenich, A. (2013). Rapid and semiau-mated method for the analysis of veterinary drug residues in honey based on turbulentTOFlow liquid chroma-graphy coupled - ultrahigh-performance liquid chromatography-Orbitrap mass spectrometry (TFC-UHPLC-Orbitrap-MS). Journal of Agricultural and Food Chemistry, 61, 829–839.

4 Kaufmann, A., Butcher, P., Maden, K. & Widmer, M. (2008). Quantitative multiresidue method for about 100 veterinary drugs in different meat matrices by sub 2-µm particulate high-performance liquid chromatography coupled - time of flight mass spectrometry. Journal of Chromatography A, 1194, 66–79.

5 Ortelli, D., Cognard, E., Jan, P. & Edder, P. (2009). A. Comprehensive fast multiresidue screening of 150 veterinary drugs in milk by ultra-performance liquid chromatography coupled - time of flight mass spectrometry. Journal of Chromatography B, 877, 2363–2374

6 Jedziniak, P., Olejnik, M., Pietruk, K., Protasiuk, E., Szprengier-Juszkiewicz, T. & Zmudzki, J. (2016). Simultaneous Determination of Residues of Non-Steroidal Anti-Inflammary Drugs and Glucocorticosteroids in Animal Muscle by Liquid Chromatography-Tandem Mass Spectrometry. Food Analytical Methods, 9, 1837-1848.

7 Kaufmann, A., Butcher, P., Maden, K., Walker, S. & Widmer, M. (2011). Quantification of anthelmintic drug residues in milk and muscle tissues by liquid chromagratophy coupled - Orbitrap and liquid chroma-graphy coupled - tandem mass spectrometry. Talanta, 85, 991- 1000.

8 Ortelli, D., Cognard, E., Jan, P. & Edder, P. (2009). B. Comprehensive fast multiresidue screening of 150 veterinary drugs in milk by ultra-performance liquid chromatography coupled - time of flight mass Spectrometry. Journal of Chromatography B, 877, 2363–2374.

9 Kaufmann, A., Butcher, P., Maden, K., Walker, S. & Widmer, M. (2014). Multi-residue quantification of veterinary drugs in milk with a novel extraction and clean up technique: Salting out supported liquid extraction (SOSLE), Analytica Chimica Acta , 820, 56-68.

10 Petersa, R.J.B., Bolcka, Y.J.C., Rutgersa, P., S-lkera, A.A.M. & Nielen, M.W.F. (2009). Multi-residue screening of veterinary drugs in egg, fish and meat using high-resolution liquid chromatography accurate mass time-of Flight mass spectrometry. Journal of Chromatography A, 1216, 8206-16.

11 Wang, J., Leung, D., Chow, W., Chang, J. & Wong, J.W. (2015). Development and Validation of a Multiclass Method for Analysis of Veterinary Drug Residues in Milk Using Ultrahigh Performance Liquid Chromatography Electrospray Ionization Quadrupole Orbitrap Mass Spectrometry, J. Journal of Agricultural and Food Chemistry, 63, 9175-87.

Figure S1. Optimization of gradient program.

-10

10

30

50

70

90

110

0 10 20 30 40 50

% B

Time (min)

Gradientes

Gradiente 1 Gradiente 2 var 1 Gradiente 2 var 2Gradiente 3 Gradiente 4Gradient 1Gradient 4

Gradient 2Gradient 5

Gradient 3

1

Figure S2. Comparison of percentages of compounds with negligible, soft and medium ME using different dilution factors.

2