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Extraction of Acrylamide from Coffee using ISOLUTESLE+ | Page 1

Application Note ANBiotage

Extraction of Acrylamide from Coffee UsingISOLUTESLE+ Prior to LC-MS/MS Analysis

This application note describes a Supported Liquid Extraction (SLE) protocol for the extraction of acrylamide

from coffee using ISOLUTESLE+ columns with LC-MS/MS detection.

Application Note AN

IntroductionThe method described in this application note achieves high

recoveries of acrylamide in coffee. The method is sensi-

tive enough to measure levels as low as ng/mL in coffee

(solution), ppm in ground coffee (solid) or ppm in

instant coffee (solid, traditional or decaffeinated) and gives

good selectivity from what is a challenging matrix.

ISOLUTE SLE+ products provide clean, rapid, robust and

efficient extraction solutions for a wide range of analytes.

AnalyteAcrylamide

Format: ISOLUTESLE+ mL Columns, part number --C

Sample Pre-treatment: Coffee was prepared in the same way that it would normally be consumed. In the case of ground

coffee, g of ground coffee was percolated with mL of boiling water. For instant coffee g of

instant coffee powder was dissolved in mL of boiling water. This resulted in solutions containingcoffee solid concentrations of mg/mL for ground coffee and mg/mL for instant coffee. Once

prepared the coffee was left to reach room temperature.

Calibration Line

Preparation:

A ng/mL acrylamide coffee over-spiked solution was prepared by diluting .L of a g/mL

aqueous acrylamide solution to mL with control coffee.

This was then serially diluted seven times by transferring .mL, diluting with .mL of control

coffee, mixing, and then transferring .mL of this mixture; repeating the procedure until a solution

with an over-spiked level of ng/mL had been reached.

.6mL aliquots were transferred to wells containing L of a g/mL Cacrylamide solution in

water and .L of a saturated solution of ammonium hydroxide in water.

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Sample Preparation Procedure


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Sample work-up: Samples (.mL) were transferred to tubes containing L of a g/mL C

acrylamide solution in water and .L of a saturated solution of ammonium hydroxide

in water. The tube was briefly shaken and then .mL of the mixture transferred to a mL

capacity ISOLUTESLE+ column.

Sample loading: Load pre-treated sample (.mL) onto each well. Apply a pulse of vacuum (VacMaster-

or Sample Processing Manifold, -or -) or positive pressure (Pressure+Positive Pressure Manifold, PPM-) to initiate flow. Allow the sample to absorb for

minutes.

Analyte Elution: Elute with ethyl acetate: tetrahydrofuran, (: , v/v, x .mL) and allow to flow under

gravity into a tube already containing L ethylene glycol. Apply vacuum or positive

pressure to elute any remaining extraction solvent.

Post Elution: Dry the volatile constituents of the eluate in a stream of air or nitrogen using an SPE Dry

(ambient, to L min-), (SD--DHS or SD--DHS) or TurboVap LV, (Cor

C) (bar at ambient for hr). Reconstitute in water (L).

Instrument: Waters Acquity

Column: Phenomenex Hydro, m x mm Ccolumn with a Cguard cartridge and

on-line filter

Mobile Phase: A: .% formic acid in water

B: .% formic acid in methanol

Flow rate: .mL min-

Injection: L

Gradient: Initial % A, hold till .min

linear ramp to % B over .min (.min), hold .min (.min)

linear ramp to % A in .min (.min), hold .min (min)

Column temperature: C

Sample temperature: C

Supported Liquid Extraction

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Recovery and RSD calculations based on extractions of blank

matrix spiked at ng/mL without using an internal standard.

The blank acrylamide response was subtracted from both

extracted and fortified quantities prior to calculating both

recovery and RSD.

Instrument: Waters Quattro Premier

Ionization mode: ES+

Desolvation temp.: C

Source temp: C

MS Conditions

Ions were selected in order to achieve maximum sensitivity using multiple reaction monitoring

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The addition of the ammonia solution results in thecoffee changing from a mid-brown to a darker brownappearance. Although the sample added to the ISOLUTESLE+ column is darker as a result of the basification,the final extract appears visibly cleaner than withuntreated coffee. The image below shows .mLuntreated ground coffee (lef t), coffee combined with %concentrated ammonia solution (middle) and the SLEextract diluted to an identical volume with water (right).

The majority of the coffee dyes are removed bybeing trapped on the SLE material. The image belowshows an unused mL SLE column (lef t) comparedto a column that has undergone a full extractionincluding the removal of acrylamide (right).

A calibration line extracted in water gave similar propertiesto that extracted in coffee but without the interceptdue to the lack of any process derived acrylamide.Preparing a calibration line in this solvent could beapplied for ultra low level acrylamide determinations.

Ethylene Glycol was added in a small quantity prior to theextraction step to avoid the evaporated sample dryingcompletely. Without this additive being present themajority of the acr ylamide would be lost at this stage.

A % aqueous mobile phase was required togive retention to the polar analyte. This requireda column that was designed to work under theseconditions and the method included a relativelylong equilibrium time between samples.

Additional Notes


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