ANALYSIS OF 34 MYCOTOXINS USING AGILENT 6460C LC-MS/MS
Hunor Farkaš, Jog Raj*, Svetlana Ćujić, Zdenka Jakovčević and Marko Vasiljević PATENT CO, DOO., Vlade Ćetkovića 1A, 24 211, Mišićevo, Serbia *jog.raj@patent-co.com
1
Mycotoxins are secondary metabolites produced by fungal species in food and feed materials. Most of them are produced by
Aspergillus, Fusarium, and Penicillium sp. Commonly, most people think of the six main mycotoxins that contaminate feed: Aflatoxins (AF) Deoxynivalenol (DON) T-2 toxin Fumonisins (FB) Ochratoxin Zearalenone (ZEN) The primary effects of these toxins on the performance and health of production animals are known, and regulatory guidance on threshold levels for these toxins in food and feed materials exists.
However, although testing and reporting on the prevalence of mycotoxins in feed has increased in recent years, many mycotoxins
One category of
continue to go undetected.
growing concern are emerging mycotoxins.
Just as DON, T-2 toxin, and zearalenone, emerging mycotoxins are also commonly produced by various Fusarium molds. Furthermore, because molds produce multiple mycotoxins under the same environmental stressors, emerging mycotoxins are likely to be frequent co-contaminants in feed together with the main mycotoxins.
This article describes a multi mycotoxin sample preparation
Therefore, a method for analysis of total mycotoxins,
and analysis method on
including emerging mycotoxins is highly desirable.
Agilent 6460c LC-MS/MS.
2
PROCEDURES 1. Sample preparation The laboratory sample should be finely ground and thoroughly mixed using a mill and mixer before a test portion is removed for analysis. Transfer 5 g of homogenized and finely ground sample with an accuracy of 0.1 g into a 50 ml conical tube and proceed to the extraction step.
5 g sample
2. Spiking procedure If recovery needs to be determined, execute the following in duplicates: Transfer 5 g of homogenized
5 g sample
and finely ground sample into a 50ml conical tube. Transfer 5 g of homogenized and finely ground sample into a 50ml conical tube and add 200 µl of the multi-mycotoxin spiking solution. Proceed to extraction step.
5 g sample + 200 µl multimycotoxin solution
3
3. Extraction Add 20 ml of extraction mixture and close the tube. Shake the mixture in an orbital shaker at 250 rpm for 90 minutes at room temperature. After extraction, centrifuge at 4200 x g for 5min. Transfer 450 μl of supernatant to a glass
20 ml extraction solution
vial and add 750 μl of ultrapure water to
250 rpm 90 min
the supernatant.
450 µl supernatant
750 µl ultrapure water
Vortex and analyse the sample using LC-MS/MS. Vortex well. Filter aliquot of 1200 µL of the prepared sample extract across a membrane syringe filter into a glass vial.
120 µl sample extract
Matrixes: corn, compound feed, wheat, barley, soya meal, wheat bran, sunflower meal, total mixed ration (TMR).
4
RESULTS Table 1 shows the mycotoxins analysed along with their corresponding Limits of Quantification (LOQ).
Mycotoxin family
Aflatoxins
Zearalenone + Metabolite
A-Trichothecenes
B-Trichothecenes
Fumonisins + Metabolite
Ochratoxin
Emerging mycotoxins
Penicillium toxins Ergot alkaloids
Alternaria toxin
Mycotoxin
Abbreviation
LOQ (μg/kg)
Aflatoxin B1
AFB1
< 0,4
Aflatoxin B2
AFB2
< 0,4
Aflatoxin G1
AFG1
< 0,4
Aflatoxin G2
AFG2
< 0,4
α – Zearalenol
α -ZEL
< 10
β – Zearalenol
β -ZEL
< 10
Zearalanone
ZAN
< 10
Zearalenone
ZEN
<5
Diacetoxyscirpenol
DAS
<3
HT-2 toxin
HT-2
< 9,6 < 9,6
T-2 toxin
T-2
Neosolaniol
NEO
< 10
3-Acetyl Deoxynivalenol
3-ADON
< 20
15-Acetyl Deoxynivalenol
15-ADON
< 20
Deoxynivalenol
DON
< 20
Nivalenol
NIV
< 20
Deoxynivalenol-3-Glucoside
D3G
< 30
Fumonisin B1
FB1
< 25
Fumonisin B2
FB2
< 25
Fumonisin B3
FB3
< 25
Fusaric Acid
FA
<3
Moniliformin
MON
<3
Fusarenon X
FX
< 40
Ochratoxin A
OTA
< 1,6
Beauvericin
BEA
<1
Enniatin A
ENN A
<1
Enniatin A1
ENN A1
<1
Enniatin B
ENNI B
<1
Enniatin B1
ENN B1
<1
Citrinin
CIT
< 50
Patulin
PAT
< 100
Ergosine
ES
<1
Ergocryptinine
ECR
<1
Alternariol
AOH
< 20
Table 1. Mycotoxins analysed and their LOQs.
5
Table 2 shows the recovery (%) of mycotoxins screened in corn, wheat, and barley.
RECOVERY (%) Analyte CORN
WHEAT
BARLEY 90
1.
Moniliformin
82
91
2.
Nivalenol
85
89
88
3.
Deoxynivalenol
103
108
105
4.
Fusaric Acid
102
96
109
5.
3-Acetil Deoxynivalenol
80
88
90
6.
15-Acetil Deoxynivalenol
83
92
91 99
7.
Aflatoxin G2
99
99
8.
Aflatoxin G1
98
98
92
9.
Aflatoxin B2
93
96
96
10.
Aflatoxin B1
96
93
104
11.
Diacetoxyscirpenol
96
101
103
12.
HT-2 toxin
92
93
99
13.
Fumonisin B1
103
96
94
14.
β – Zearalenol
101
96
89
15.
α – Zearalenol
87
104
99
16.
T-2 toxin
98
103
90
17.
Zearalanone
103
103
95
18.
Ochratoxin A
94
97
98
19.
Zearalenone
102
101
91
20.
Fumonisin B2
82
91
101
21.
Fumonisin B3
86
99
93
22.
Enniatin B
86
98
96
23.
Beauvericin
85
89
88
24.
Enniatin B1
81
89
82
25.
Enniatin A1
84
95
96 94
26.
Enniatin A
81
98
27.
Citrinin
84
88
81
28.
Patulin
72
75
71
29.
Fusarenon X
89
85
82
30.
Neosolaniol
94
90
85
31.
Ergosine
103
99
98
32.
Ergocryptinine
96
92
94
33.
Alternariol
79
84
81
34.
Deoxynivalenol-3-Glucoside
83
82
80
Table 2. Percentage of mycotoxins recovered in corn, wheat, and barley samples.
6
CONCLUSIONS Analysis of 34 mycotoxins using CORTECS C18 column in Agilent 6460c LC-MS/MS showed a recovery of 71-109% for various mycotoxins. This proves that it is a robust method for analysis of 34 mycotoxins.
The detection of mycotoxins in food and feed samples is a matter of great importance due to their inherent toxicity. These unavoidable contaminants cannot be eliminated and, therefore, the implementation of effective monitoring programs and stringent legal regulations can significantly contribute to the reduction and carry over of these mycotoxins in food chain.
7