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Inter-laboratory validation of liquid chromatography–tandem mass spectrometry multi-mycotoxin determination in animal feed – method transfer from the reference laboratory to regional laboratories

Piotr Jedziniak
Piotr Jedziniak
, 
Łukasz Panasiuk
Łukasz Panasiuk
, 
Katarzyna Pietruszka
Katarzyna Pietruszka
, 
Krzysztof Kolano
Krzysztof Kolano
 e 
Radosław Zbytniewski
Radosław Zbytniewski
   | 20 set 2023
Journal of Veterinary Research's Cover Image
INFORMAZIONI SU QUESTO ARTICOLO
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Pubblicato online: 20 set 2023
Pagine: 397 - 406
Ricevuto: 23 feb 2023
Accettato: 18 lug 2023
DOI: https://doi.org/10.2478/jvetres-2023-0045
Parole chiave
© 2023 Piotr Jedziniak et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1.

Individual results of analysis of quality control materials DON AV – average deoxynivalenol; ZEN AV – average zearalenone; ZEN – zearalenone; DON – deoxynivalenol; AF B1 AV – average aflatoxin B1; OTA AV – average ochratoxin A; AF B1 – aflatoxin B1; OTA – ochratoxin A
Individual results of analysis of quality control materials DON AV – average deoxynivalenol; ZEN AV – average zearalenone; ZEN – zearalenone; DON – deoxynivalenol; AF B1 AV – average aflatoxin B1; OTA AV – average ochratoxin A; AF B1 – aflatoxin B1; OTA – ochratoxin A

Fig. 2.

Retention times of mycotoxins in LC-MS/MS analysis performed by participants and the NVRI DON – deoxynivalenol; AF B1 – aflatoxin B1; HT-2 – toxin HT-2; T-2 – toxin T-2; FB1 – fumonisin B1; – FB2 – fumonisin B2; OTA – ochratoxin A; ZEN – zearalenone
Retention times of mycotoxins in LC-MS/MS analysis performed by participants and the NVRI DON – deoxynivalenol; AF B1 – aflatoxin B1; HT-2 – toxin HT-2; T-2 – toxin T-2; FB1 – fumonisin B1; – FB2 – fumonisin B2; OTA – ochratoxin A; ZEN – zearalenone

Mycotoxin standard mixture designated MIX6 1VL

Analyte Concentration (μg/mL) Volume of solution (mL) Concentration in solution (μg/mL) Solvent Final volume (for one lab)
AF B1 1 1 0.1    
DON 100 1.8 18    
OTA 10 1 1 acetonitrile 10 mL
ZEN 10 2 2    
T-2 10 1 1    
HT-2 10 1 1    

Mycotoxin contamination of blank samples

Feed sample code Type of feed DON (μg/kg) ZEN (μg/kg)
P1 Fish feed ~50 ~20
P2 Broiler feed ~25 -
P3 Fish feed - -
P4 Swine feed - ~20

Isotopically labelled mycotoxin internal standard mixture designated MIX7 IS

Internal standard Concentration Solvent Final concentration
AF B1 13C17 0.5 μg/mL   0.02 μg/mL
DON 13C15 25 μg/mL   1 μg/mL
FB1 13C34 25 μg/mL   1 μg/mL
OTA 13C20 10 μg/mL acetonitrile 0.4 μg/mL
ZEN 13C18 25 μg/mL   1 μg/mL
T-2 13C24 25 μg/mL   1 μg/mL
HT-2 13C22 25 μg/mL   1 μg/mL

Liquid chromatography-tandem mass spectrometry conditions used in inter-laboratory validation

  Liquid chromatograph Column Mobile phase Flow rate temperature Mass spectrometer
Laboratory 1 Eksigent ekspert ultra LC 100-XL Phenomenex Kinetex Biphenyl, 50 × 2.1 mm, 2.6 μm A – methanolB – 2 mM ammonium formate + 0.1% formic acid 0.35 mL/min 40°C Sciex 5500
Laboratory 2 Agilent 1260 Agilent Poroshell 120 EC-C18, 4.6 × 50 mm, 2.7μm A – 0.05 mM ammonium formate + 0.1% formic acidB – methanol + 0.05 mM ammonium formate + 0.1% formic acid 0.5 mL/min 40°C Sciex 5500
Laboratory 3 Agilent 1200 Agilent Poroshell 120 EC-C18, 3.0 × 50 mm, 2.7 μm A – methanol (5%) + 0.02 M ammonium acetate + 0.1% acetic acid, 0.25% ammonium fluoride (95%)B – methanol (95%) + 0.02 M ammonium acetate, 0.1% acetic acid, 0.25% ammonium fluoride (5%) 0.8 mL/min 30°C Agilent 6410
Laboratory 4 Shimadzu Prominence Phenomenex Kinetex Biphenyl 2.6 μm, 100 × 2.1 mm A – 0.01 M ammonium acetate + 0.1% of acetic acid/ methanol (95: 5, v/v)B – 0.01 M ammonium acetate + 0.1% of acetic acid/methanol (5:95, v/v) 0.3 mL/min 40°C Shimadzu LCMS-8040
Laboratory 5 Agilent 1260 Agilent Poroshell 120 EC-C18, 4.6 × 50 mm, 2.7μm A – 1 mM ammonium formate + 0.1% formic acidB – methanol + 0.1% formic acid + 1 mM ammonium formate 0.8 mL/min 40°C Sciex 4500
Laboratory 6 Agilent 1260 Agilent Poroshell 120 EC-C18, 3.0 × 50 mm, 2.7 μm A – methanol/0.02 M ammonium acetate + 0.1% acetic acid, (5:95, v/v)B – methanol/0.02M ammonium acetate + 0.1% acetic acid, (95:5, v/v) 0.8 mL/min 30°C Agilent LC/MS 6460
Laboratory 7 Shimadzu Nexera X2 Phenomenex Kinetex Biphenyl 2.6 μm, 100 × 2.1 mm A – 0.01 M ammonium acetate +0.1% acetic acid/methanol (95:5, v/v)B – 0.01 M ammonium acetate + 0.1% of acetic acid/methanol (5:95, v/v) 0.3 mL/min 40°C Shimadzu LCMS-8050

Validation results for toxin HT-2 (HT-2), toxin T-2 (T-2), fumonisin B1 (FB1) and fumonisin B2 (FB2)

  HT-2 T-2 FB1 FB2
VL (μg/kg) 25 50 75 25 50 75 125 250 375 125 250 375
X (μg/kg) 23.9 51.4 71.2 23.7 52.7 78.8 127 300 363 126 263 359
Rec (%) 95.6 103 94.9 94.6 105 105 101 120 96.8 101 105 95.8
RSDR (%) 14.0 9.54 9.92 11.6 6.82 14.4 5.45 8.22 4.44 8.32 20.5 8.30
PRSD 27.9 25.1 23.6 27.9 25.1 23.6 21.9 19.7 18.5 21.9 19.7 18.5
HORRAT 0.2 0.4 0.3 0.2 0.4 0.3 0.2 0.4 0.2 0.4 1.0 0.4
U (%, k = 2) 28 23 48 59

Scheme of the inter-laboratory validation

Samples Laboratory
1 2 3 4 5 6
Calibration curve (sample P1, n = 6) (blank, 0.25 × VL, 0.5 × VL, 1.0 × VL, 1.5 × VL and 2.0 × VL)
Repeatability (sample P1, n = 6) 0.5 × VL 1.0 × VL 1.5 × VL 0.5 × VL 1.0 × VL 1.5 × VL
Reproducibility + recovery (sample P2, n = 2; P3, n = 2; P4, n = 2) 1.5 × VL 1.0 × VL 0.5 × VL 1.5 × VL 1.0 × VL 0.5 × VL
Trueness (quality control material QCM, n = 3) all labs

Levels of analytes in calibration curve (μg/kg)

Analyte 0.25 × VL 0.5 × VL 1.0 × VL 1.5 × VL 2.0 × VL
AF B1 1.25 2.5 5 7.5 10
DON 225 450 900 1350 1800
FB1 62.5 125 250 375 500
FB2 62.5 125 250 375 500
HT-2 12.5 25 50 75 100
OTA 12.5 25 50 75 100
T-2 12.5 25 50 75 100
ZEN 25 50 100 150 200

Validation results for aflatoxin B1 (AFB1), deoxynivalenol (DON), zearalenone (ZEN) and ochratoxin A (OTA)

  AF B1 DON ZEN OTA
VL (μg/kg) 2.5 5 7.5 450 900 1350 50 100 150 25 50 75
X (μg/kg) 2.40 5.08 7.24 401 883 1217 52.2 107 148 23.2 48.8 71.4
Rec (%) 95.9 102 96.5 89.0 98.1 90.2 104 107 98.7 92.9 97.6 95.2
RSDR (%) 3.76 5.13 5.41 9.17 5.76 7.48 16.4 10.6 6.67 5.92 8.90 6.51
PRSD 39.4 35.5 33.4 18.0 16.3 15.3 25.1 22.6 21.3 27.9 25.1 23.6
HORRAT 0.1 0.1 0.2 0.5 0.4 0.5 0.7 0.5 0.3 0.2 0.4 0.3
U (%; k = 2) 15 17 35 25

The list of minor changes to the sample preparation

  Sample weight Volume of the extraction Extraction mixture Volume of the extract taken for evaporation Reconstitution of the dry residue Centrifugation
Laboratory 1 1g 4 mL acetonitrile: water: formic acid (79:20:1, v/v/v) 0.1 mL 0.05 mL of methanol and 0.05 mL of water +
Laboratory 2 1g 4 mL acetonitrile: water: formic acid (48:50:2, v/v/v) 0.1 mL 0.05 mL of methanol and 0.05 mL of 0.01 M ammonium acetate + 0.1% acetic acid +
Laboratory 3 1g 4 mL acetonitrile: water: formic acid (79:20:1, v/v/v) 0.1 mL 0.1 mL of mobile phase B –
Laboratory 4 1g 4 mL acetonitrile: water: formic acid (79:20:1, v/v/v) 0.1 mL 0.1 mL of mobile phase B +
Laboratory 5 1g 4 mL acetonitrile: water: formic acid (79:20:1, v/v/v) 0.1 mL 0.05 mL of methanol and 0.05 mL of 0.01 M ammonium acetate + 0.1% acetic acid +
Laboratory 6 1g 4 mL acetonitrile: water: formic acid (79:20:1, v/v/v) 0.2 mL 0.2 mL of mobile phase B +
Laboratory 7 1g 4 mL acetonitrile: water: formic acid (79:20:1, v/v/v) 0.1 mL 0.05 mL of methanol and 0.05 mL of 0.01 M ammonium acetate + 0.1% acetic acid +

The tandem mass spectrometry parameters of mycotoxin analysis applied by the Polish National Veterinary Research Institute (NVRI)

Analyte Parent ion Daughter ions Dwell time (msec) Collision energy
Positive ionization
Aflatoxin B1 (M+H)+ 313 285*, 241, 269 52, 52, 52 -23, -38, -32
Aflatoxin B1 (IS) (M+H)+ 330 301 79 -24
Fumonisin B1 (M+H)+ 722 352, 334 65, 65 -37, -41
Fumonisin B2 (M+H)+ 706 336, 318 65, 65 -37, -40
Fumonisin B1 (IS) (M+H)+ 756 357 42 -42
Ochratoxin A (M+H)+ 404 239, 358 65, 65 -24, -15
Ochratoxin A (IS) (M+H)+ 424 250 133 -24
T-2 toxin (M+Na)+ 489 327, 387 42, 42 -24, -22
T-2 toxin (M+NH4)+ 484 215, 305 65, 65 -20, -16
T-2 toxin (IS) (M+NH4)+ 508 198 65 -23
  (M+Na)+ 447 345, 285 65, 65 -20, -21
HT-2 toxin (M+NH4)+ 442 263, 215 65, 65 -14, -14
HT-2 toxin (IS) (M+NH4)+ 464 278 65 -14
Negative Ionisation
Zearalenone (M–H)- 317 175, 131 71, 71 25, 30
Zearalenone (IS) (M–H)- 335 140 71 31
Deoxynivalenol (M–CH3COO)– 355 295, 265, 59 247, 247, 247 11, 16, 20
Deoxynivalenol (IS) (M–CH3COO)– 370 310 247 11
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