Summary
This application note describes a Solid Phase Extraction (SPE) protocol for the extraction of a range of mycotoxins from wheat flour, wheat, maize and barley using ISOLUTE® Myco with LC-MS/MS.
Introduction
Mycotoxins are toxic metabolites produced by fungal molds on food crops. Regulation and legislation for testing of mycotoxin contamination has established which mycotoxins are preva- lent 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 grain food crops.
Figure 1. Structures of Aflatoxin B1 and ZearalenoneThe method described in this application note achieves high recoveries of all relevant mycotoxins from a range of different grain matrices with %RSDs and LOQs that all meet the require- ments set in European regulations for measurement of these analytes in grains. ISOLUTE Myco solid phase extraction columns provide robust, reliable sample preparation for multiple mycotoxin classes from a wide range of foodstuffs. Using a single, easy to use sample preparation product, along with optimized matrix specific application notes, scientists can prepare diverse food/crop samples for analysis by LCMSMS.
Analytes
Aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ergocryptine, ergocornine, ochratoxin A, fumonisin B1, zearalenone, T-2 mycotoxin, HT-2 mycotoxin
Sample Preparation Procedure
Column configuration Sample pre-treatment:
ISOLUTE Myco 60 mg/3 mL column (Tabless) Part Number 150-0006-BG Sample pre-treatment:
- Sample processing: Grind the sample (wheat, maize, barley, 50 g). Store ground sample in a sealed container at room temperature until required.
- Extraction: Mix the ground whole grain (or flour) sample (5 g) with 50% acetonitrile (aq) (20 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.
- Dilution: Take the supernatant (8 mL), transfer to a new 50 mL centrifuge tube and dilute with water (32 mL). Centrifuge diluted extract at 3000 g for a further 10 minutes.
Solid Phase Extraction
Use flow rates of 1 mL min-1 throughout Condition:
Condition the column with acetonitrile (2 mL) Equilibration:
Equilibrate column with water (2 mL) Sample loading:
Load pre-treated sample (3 mL) onto the column at a maximum flow rate of 1 mL min-1 (gravity load is recommended) Interference wash 1:
Wash the column with water (3 mL) Interference wash 2:
Wash the column with 10% acetonitrile (3 mL) Drying:
Dry the column for 30 seconds at maximum vacuum Elution 1:
Elute with 0.1% formic acid in acetonitrile (2 mL) Elution 2:
Elute with methanol (2 mL) Post elution:
The combined eluate is dried in a stream of air or nitrogen using a SPE Dry (35 °C, 20 to 40 L min-1) or TurboVap LV (15 bar at 35 °C for 40 min). Reconstitute in 0.1 % acetic acid in 20% acetonitrile : methanol (1 mL, 1:1, v/v). Syringe-filter using a 0.2 μm PTFE membrane prior to analysis.
HPLC Conditions
Instrument:
Shimadzu Nexera UHPLC (Shimadzu Europe Gmbh) Column:
Kinetex XB-C18 50 x 2.1 mm 2.6 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:
0.45 mL min-1 Injection:
20 μL Gradient:
Initial 20 % B, hold 1.0 min
linear ramp to 73 % B in 6 min
linear ramp to 100 % B in 0.2 min, hold 2.3 min
linear ramp to initial conditions in 0.2 min
hold 2.3 min, total run time 10.0 min Column temperature:
40 °C Sample temperature:
15 °C
MS Conditions
Ions were selected in order to achieve maximum sensitivity, and 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 tempurature: 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
Figure 2: Extracted ion chromatograms in negative ion mode using ISOLUTE Myco protocol at 50 μg kg-1 from wheat
Figure 3. Extracted ion chromatograms in positive ion mode using ISOLUTE Myco protocol at 5 μg kg-1 (aflatoxins and ochratoxin A) and 50 μg μg kg-1 (others) from wheat grainValidation Criteria
Method linearity was determined using matrix-matched calibration standards in six replicates over a minimum of five levels (the majority were determined with seven levels); the ranges are shown below.LOQ was determined from the lowest matrix-matched standard meeting EU repeatability and recovery criteria. Where no criteria were specified the LOQ criteria were estimated by correlation to similar analytes. Repeatability (%RSDr) was determined from single acquisitions of 5 SPE replicates of a single sample extraction. The RSDs generated gave close agreement when a single sample was extracted and processed using ISOLUTE Myco from three separate sorbent batches. Recovery was determined as a % of ISOLUTE Myco extract spike before sample prep to spike after at the EU MRL.
Results
The extracted ion chromatograms in figures 2 and 3 demonstrate chromatography at 5 μg kg-1 (aflatoxins and ochratoxin A) and 50 μg kg-1 for all other analytes from a spiked extraction of 10 g ground wheat. Good linearity was achieved for all analytes in all the different matrices as demonstrated in the example charts shown in figures 4 and 5.
Figure 4: Calibration curve for aflatoxin B1 from ground wheat using the ISOLUTE Myco protocol from 0.1 – 10 ngmL-1
Figure 5. Calibration curve for T2 from ground wheat using the ISOLUTE® Myco protocol from 5 – 200 ngmL-1All analytes extracted using the ISOLUTE Myco protocol achieved the limits of quantities and recovery required by the current European standards for mycotoxin analysis as shown in tables 4, 5 and 6.
Ordering Information
Download the PDF of this App Note here.
For the latest application notes and more information about ISOLUTE® Myco, please visit www.biotage.com/isolutemyco
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