OR WAIT null SECS
Biotage GB Ltd
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.
Analytes: Aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A.
Format: ISOLUTE® Myco 60 mg/3 mL columns (Tabless), part number 150-0006-BG.
Sample 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 min. Transfer the extract to a 50 mL centrifuge tube and centrifuge at 4000 g for 10 min.
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 min.
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 (2 × 2.5 mL).
Interference wash 2: Wash the column with 10% acetonitrile (aq) (2 × 2.5 mL).
Drying: Dry the column for 30 s 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, 1 mL). Syringe-filter using a 0.2 μm PTFE membrane prior to analysis.
Instrument: Shimadzu Nexera UHPLC (Shimadzu Europe Gmbh)
Column: 50 × 2.1 mm, 2.6-μm dp Kinetex XB-C18 (Phenomenex)
Mobile phase A: 1 mM ammonium acetate, 0.5% acetic acid
Mobile phase 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 ESIDesolvation temperature: 500 °C
Curtain gas: 30 psi Spray voltage: +5.0 kV
Gas 1: 60 psi Gas 2: 60 psi Collision gas: 7 psi
Table 1: Analyte recovery (n = 5) and limit of quantitation data for a range of mycotoxins from chili using the ISOLUTEÂ® Myco protocol.
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 1.
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