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; Łukasz Panasiuk; Katarzyna Pietruszka; Krzysztof Kolano; Radosław Zbytniewski

doi:10.2478/jvetres-2023-0045

Figures & Tables

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

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
RSD_R (%)	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
Samples	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
RSD_R (%)	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

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

Figures & Tables

Fig. 1.

Fig. 2.

Mycotoxin standard mixture designated MIX6 1VL

Mycotoxin contamination of blank samples

Isotopically labelled mycotoxin internal standard mixture designated MIX7 IS

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

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

Scheme of the inter-laboratory validation

Levels of analytes in calibration curve (μg/kg)

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

The list of minor changes to the sample preparation

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

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