Extraction of Aflatoxins and Ochratoxin from Dried Chili Using ISOLUTE? Myco Prior to LC–MS–MS Analysis

October 2, 2014
Adam Senior

The Application Notebook

Volume 0, Issue 0
Page Number: 10

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.

Sample Preparation Procedure

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.

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 (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.

HPLC Conditions

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

MS Conditions

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 ISOLUTE® 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.

Biotage AB

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