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MYCOTOXINS DETECTION Unleashing the potential of the LC-MS/MS BASED METHOD

Hunor Farkaš, Jog Raj, and Marko Vasiljević PATENT CO, DOO., Vlade Ćetkovića 1A, 24 211, Mišićevo, Serbia

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What are mycotoxins and why should we control them? Animal feed can be contaminated with microorganisms, mycotoxins, animal by-products, organic pollutants and toxic metals. This contamination has negative effects, both on animal and human health. Among these contaminants, mycotoxins are emerging as a major contaminant of feed and food. Mycotoxins are produced as secondary metabolites by various fungi. The major fungus producing mycotoxins are:

Aspergillus Fusarium Penicillium Mycotoxins cause mycotoxicosis and cause significant economic losses in the livestock industry due to reduced productivity, increased disease incidence and decreased reproductive performance. The mycotoxins that pose a higher threat due to their toxicity and occurrence are:

Aflatoxin B1 (AFB1) Deoxynivalenol (DON) Reduced productivity

Ochratoxin A (OTA) Zearalenone (ZEA) Fumonisins (FB1 and FB2)

CONSEQUENCES OF MYCOTOXICOSIS

T-2 toxins

â‚Ź

Increased disease incidence

Decreased reproductive performance

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How can we detect mycotoxins? Most of these methods employ solid phase column clean-up of extracts and immunoaffinity techniques to remove interferences and improve the measurement of mycotoxins.

There are many methods available for the detection of mycotoxins. Conventional methods for mycotoxins analysis include: ELISA Thin-layer chromatography (TLC) High-performance liquid chromatography (HPLC) Gas chromatography (GC)

A QUICK PEAK INTO THE LC-MS/MS BASED MULTI-MYCOTOXINS METHOD

ELISA is a method of choice when rapid analysis is required but requires confirmatory analysis by LC-MS/MS. LC-MS/MS is the most sensitive and preferred method of analysis for detecting and quantifying mycotoxins in food and feed samples.

Since it is necessary to test animal feeds for mycotoxin contamination, PATENT CO has developed and perfected a fast and simple LC-MS/MS based method for the determination and quantification of all mycotoxins (Aflatoxins B1, B2, G1, and G2; Deoxynivalenol, Zearalenone, Fumonisin B1 and B2, T-2, HT-2, and Ochratoxin A) regulated in feed (EU Directive 2002/32/ EC, 2006/576/EC and 2013/165/EU). The method is based on “dilute and shoot� principle, which involves a two-step extraction phase and the centrifugation of the extracts. To compensate the matrix effects* in electrospray ionization, the extracts are mixed with [13C] labelled internal standards for each group of mycotoxins (13C AB1, 13C DON, 13C ZON, 13C OTA, 13C FB1 and 13C T-2) before injection onto LC-MS/MS. *Effect on an analytical method caused by all other components of the sample except the specific compound to be quantified.

HOW DOES THE LC-MS/MS BASED METHOD WORK?

The LC-MS/MS method combines the separating power of liquid chromatography (LC) with the highly sensitive and selective analysis capability of mass spectrometry (MS). This method involves the following steps in order to quantify the mycotoxins present in the sample.

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2. INJECTION TO HPLC

The first step of the technique consists of the extraction of the mycotoxins that are present in the sample (Figure 1).

1. EXTRACTION

5.00 g homogenized sample. The extract is injected into the LC column that contains a stationary phase, passing through a mobile phase at high pressure (See parameters on Table 1).

Extraction with 20ml ACN/Water/ Formic acid=80:19.9:0.1; 60 min; room temperature.

The chemical interactions that take place between the components in the sample and the stationary and mobile phases determine their different migration rates, allowing for their separation.

Centrifugation at 4200 rpm for 5 min. Extraction with 20ml ACN/Water/ Formic acid=20:89.9:0.1; 60min; room temperature. Centrifugation at 4200 rpm for 5 min.

HPLC Column

Eclipse XDB-C18 4.6x50mm, 1,8µm (p/n:927975-902)

HPLC Guard Column

Eclipse Plus C18 Grd.2.1 x12.5mm.5m (p/n:821125-936)

Mobile phase

A: 0.1% formic acid, 5mM ammonium formate in water

B: 0.1% formic acid, 5mM ammonium formate in methanol

Gradient program

Combination of supernatants and centrifugation at 4200rpm for 5 min. Transfer of 500 µL filtered final extract to HPLC vial and addition of 20µL mix of 13C-labeled internal standards working solution. Inject 5µL Figure 1. HPLC extraction procedure.

Time [Min]

B [%]

0,00 45

4,00 45

4,10 75

13,00 75

15,00 100

17,00 100

Flow rate Injection volume

17,01 45 Post time

5min

0.22 mL/min 5 µL

Table 1. Parameters for the HPLC: Composition, Gradient Program, Flow rate and Injection volume.


3. STABLE ISOTOPE DILUTION ASSAY (SIDA)

Figure 2. Stable Isotope Dilution Assay (SIDA): Chromatogram

Mycotoxin [13C] Toxin

Calibration range [ng/ml]

Linear working range (µg/kg)

AB1 AB2 [13C] AB1

0,05 - 5

0,4 - 40

AG1 AG2 DON [13C] DON

8,0 - 800

Please [13C] Enable

0,2 - 20

64 - 6400 1.6 - 160

Sun [13C] Sun

2 - 200

16 - 1600

T-2 [13C] T-2

1,2 - 120

9,6 - 960

HT-2 FB1 [ C] FB1 13

5 - 500

40 - 4000

FB2

Table 2. Labelled internal standards for each group of mycotoxins.

4. MASS SPECTROMETRY

To compensate the matrix effects in electrospray ionization, the extracts are mixed with [13C] labelled internal standards for each group of mycotoxins (Table 2).

The sample is nebulized and ionized in order to obtain charged particles that migrate under high vacuum through a series of mass analysers by applying magnetic fields.

Results are expressed as μg/kg or ppb relative to a feed with a moisture content of 12 %

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WHICH INGREDIENTS CAN BE ANALYSED AND HOW RELIABLE ARE THE RESULTS? The LC-MS/MS based method has been successfully validated for:

Corn

Compound feed

Wheat Barley

Soya meal

Wheat bran

Sunflower meal

TMR (Total Mixed Ration)

In order to validate this method, performance parameters were obtained by in-house validation and the blank samples were spiked with a mixture of 11 mycotoxin standards on two levels (LOQ and 10xLOQ) in 12 replicates. The RSDr (Relative Standard Deviation of Repeatability) of the method were between 2,5% and 13,4% and the apparent recoveries were between 62% and 115% for all analytes.

RSDr:

2,5-13,4% Apparent recoveries:

62-115%

It is concluded that the “dilute and shoot� method with addition of [13C] labelled internal standards is capable for determining all EU regulated mycotoxins in animal feed and compound feed.

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Mycotoxins detection - Unleashing the potential of the LC-MS/MS based method  

Mycotoxins detection - Unleashing the potential of the LC-MS/MS based method