Extraction of multiple mycotoxins from infant cereal using ISOLUTE® Myco prior to LC-MS/MS analysis

Introduction

This application note describes a solid-phase extraction (SPE) protocol for the extraction of a range of mycotoxins from cereal based infant food using ISOLUTE® Myco with LC-MS/MS.

Mycotoxins are toxic metabolites produced by fungal molds on food crops. Regulation and legislation for testing of mycotoxin contamination has established which mycotoxins are prevalent on a wide variety of food crops. This application note describes an SPE protocol appropriate for LC-MS/MS analysis of a range of mycotoxins found on cereal based infant food.

The method described in this application note achieves high recoveries of relevant mycotoxins from a cereal based infant food with low %RSDs and LOQs.

ISOLUTE® Myco solid-phase extraction cartridges provide robust, reliable sample preparation for multiple mycotoxin classes from a wide range of foodstuffs. Using a single, easy to use sample preparation product, scientists can prepare diverse food/crop samples for analysis by LC-MS/MS.

Figure 1. Structures of aflatoxin B1 and zearalenone

Analytes

Aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, fumonisin B1, zearalenone, HT-2 toxin, T-2 toxin

Sample preparation procedure

Cartridge configuration

ISOLUTE® Myco 60 mg/3 mL column (Tabless)

Sample pre-treatment

1. Sample processing: Grind the sample (50 g, Cow&Gate® Sunny Start Baby Wheat Flakes were used in this application note). Store the ground sample in a dry sealed container at temperature < 25°C until required.
2. Extraction: Mix the ground sample (5 g) with NaCl (1g) and 60% methanol (aq) (25 mL) and place on a shaking table for 30 minutes. Transfer the extract to a 50 mL centrifuge tube and centrifuge at 3000 g for 10 minutes.
3. Dilution: Take the supernatant (7 mL), transfer to a new 50 mL centrifuge tube and dilute with water (35 mL). Centrifuge diluted extract at 3000 g for a further 10 minutes.

Solid-phase extraction

Use flow rates of 1 mL/min throughout

Condition

Condition the cartridge with acetonitrile (2 mL)

Equilibration

Equilibrate cartridge with reagent water (2 mL)

Sample loading

Load pre-treated sample (2 x 2.5 mL) onto the cartridge at a maximum flow rate of 1 mL/min (gravity load is recommended)

Interference wash 1

Wash the cartridge with reagent water (2 x 2.5 mL)

Interference wash 2

Wash the cartridge with 10% acetonitrile (2 x 2.5 mL)

Drying

Dry the cartridge for 30 seconds at maximum vacuum or positive pressure

Elution 1

Elute with 0.1% formic acid in 60% aqueous acetonitrile (2 mL)

Elution 2

Elute with 0.1% formic acid in methanol (2 mL)

Post-elution

The combined eluates are dried in a stream of nitrogen using a TurboVap® LV. Reconstitute with 0.1 % acetic acid in acetonitrile:methanol:reagent water (1 mL, 1:1:8, v/v/v). Filter using a 0.2 μm PTFE membrane filter prior to analysis.

HPLC conditions

Conditions used to generate the data in this application note. Other HPLC conditions may be appropriate.

Instrument

Shimadzu Nexera UHPLC (Shimadzu Europe Gmbh) 

Column

Kinetex XB-C18 50 x 2.1 mm 1.7 µm dp (Phenomenex, Macclesfield, UK)

Mobile phase

A: 1 mM ammonium acetate, 0.5% acetic acid 
B: 1 mM ammonium acetate, 0.5% acetic acid in 95% methanol (aq)

Flow Rate

0.6 mL/min

Injection

20 µL

Gradient

Initial 20 % B, hold 0.8 min

Iinear ramp to 73 % B in 5 min

linear ramp to 100 % B in 1.2 min, hold 1.0 min

linear ramp to initial conditions in 0.2 min

hold 1.0 min, total run time 8.2 min

Column temperature

40°C

Sample temperature

15°C

Table 1. Typical retention time for a range of mycotoxins using LC-MS/MS method described

MS conditions

Ions were selected in order to achieve maximum sensitivity. The MS was operated in dual polarity (+ve/-ve switching) mode, using multiple reaction monitoring.

Instrument

AB Sciex Triple Quad 5500 (Warrington, UK)

Source

Turbo-V ESI

Desolvation temperature

500°C

Curtain gas

30 psi

Spray voltage

+5.0 kV / -4.5 kV

Gas 1

60 psi

Gas 2

60 psi

Collision gas

7 psi

Table 2. Negative ion mode - MRM parameters. Note: MRM detection window 60 s / target scan time 0.25 s / settling time 50 ms / scan pause 5 ms

Figure 2. Extracted ion chromatograms in negative ion mode using ISOLUTE® Myco protocol at 100 μg/kg from infant cereal

Table 3. Positive ion mode - MRM parameters. Note: MRM detection window 60 s / target scan time 0.2 s / settling time 50 ms / scan pause 5 ms

Figure 3. Extracted ion chromatograms in positive ion mode using ISOLUTE® Myco
protocol at 10 μg/kg (aflatoxins and ochratoxin A) and 100 μg/kg (T-2) from infant cereal

Validation criteria

Method linearity was determined using matrix-matched calibration standards in four replicates over five levels with a 1/x weighting; the ranges are shown in table 4 below. Note: linearity data was not generated for HT-2 

Table 4. Working range for determination of various mycotoxins in infant cereal (µg/kg) along with corresponding LC-MS/MS on column matrix-matched calibration range (pg/µL)

Limit of quantitation (LOQ) was determined from the lowest pre-SPE spike meeting EU repeatability and recovery criteria. Where no criteria were specified, the LOQ were estimated by correlation to similar analytes.

Repeatability (%RSDr) was determined from single acquisitions of 5 SPE replicates of a single sample extraction.

Recovery was determined as a % of ISOLUTE® Myco extract spike before sample prep to spike after at the EU minimum reporting limit (MRL), close to the analytical LOQ.

Results

The extracted ion chromatograms in figures 2 and 3 demonstrate chromatography at 2 μg/kg (aflatoxins and ochratoxin A) and 20 μg/kg for all other analytes from a spiked extraction of 5 g ground infant cereal. Linearity is demonstrated in the example charts shown in figures 4 and 5.

Figure 4. Calibration curve for AB1 from infant cereal using the ISOLUTE® Myco protocol from 0.033 to 5.0 ng/mL

Figure 5. Calibration curve for T-2 toxin from infant cereal using the ISOLUTE® Myco protocol from 0.333 to 50 ng/mL

This work was performed following the relevant analytical performance criteria referenced in commission implementing regulation (EC) 401/2006. The analytes extracted using the ISOLUTE® Myco protocol achieved the limits of quantitation and recovery required by the European standards for mycotoxin analysis. Table 5 displays results for LOQs, %RSD_r, and recovery % against corresponding target performance criteria referenced in section 4.3.1.(subparts a - g) of commission implementing regulation (EC) 401/2006.

Table 5. Analyte recovery and limit of quantitation data for a range of mycotoxins from infant cereal using the ISOLUTE® Myco protocol. 

Reference literature:

European Commission. (2006). Commission Regulation (EC) No 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the European Union, L70, 12-34. 

European Commission. (2023). Commission Regulation (EU) 2023/915 of 25 April 2023 on maximum levels for certain contaminants in food and repealing Regulation (EC) No 1881/2006. Official Journal of the European Union, L 119, 103-157. 

Literature number: AN823

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