Analysis of 34 mycotoxins using Agilent 6460C LC-MS/MS

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

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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.

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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

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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).

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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.

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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.

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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.

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