This application note describes a solid phase extraction (SPE) protocol for the extraction of a range of mycotoxins from dried chili (pimiento) using ISOLUTE® Myco with LC-MS/MS analysis. The method described achieves high recoveries of relevant mycotoxins from dried chili (pimiento) with %RSDs and LOQs that meet the requirements set in European Union regulations for measurement of these analytes.
By Adam Senior, Biotage GB Ltd, Hengoed, UK
Analytes: Aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A
Sample Preparation Procedure
Format: ISOLUTE® Myco 60 mg/3 mL Columns (Tabless), part number 150-0006-BGSample processing: Grind the sample (50 g) with a burr-grinder or equivalent device. Store ground sample in a sealed container at room temperature until required.
Extraction: Mix the ground sample (5 g) with 80% acetonitrile (aq) (20 mL). Place the sample pre-treatment tube on a shaking table for 30 mins. Transfer the extract to a 50 mL centrifuge tube and centrifuge at 4000 g for 10 minutes.
Dilution: Take the supernatant (2 mL), transfer to a new 50 mL centrifuge tube and dilute with water (32 mL). Centrifuge diluted extract at 4000 g for a further 10 minutes.
Solid Phase Extraction
Use flow rates of 1 mL min-1 throughoutCondition: 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 (2 x 2.5 mL)
Interference Wash 2: Wash the column with 10% acetonitrile (aq) (2 x 2.5 mL)
Drying: Dry the column for 30 seconds at maximum vacuum, -0.5 bar/7 psi
Elution 1: Elute with 0.1% formic acid in 40% acetonitrile (aq) (2 mL)
Elution 2: Elute with 1.0% ammonia (conc.) in methanol (2 mL)
Post Elution: Dry the eluate 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 acetonitrile : methanol : water (1 : 1 : 8, v/v/v, 1mL). Syringe-filter using a 0.2 μm PTFE membrane prior to analysis
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: 1mM 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
Ions were selected in order to achieve maximum sensitivity, and the MS was operated in positive ion polarity mode, using multiple reaction monitoring.
Instrument: AB Sciex Triple Quad 5500 (Warrington, UK)
Source: Turbo-V ESI
Desolvation temp.: 500 °C
Curtain gas: 30 psi
Spray voltage: +5.0 kV Gas 1: 60 psi Gas 2: 60 psi Collision gas: 7 psi
Results
All analytes extracted using the ISOLUTE Myco protocol achieved the limits of quantitation and recovery required by the current European standards for mycotoxin analysis. See table below
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