Extraction of Acrylamide from Coffee Using ISOLUTE® SLE+ Prior to LC-MS/MS Analysis

Summary

This application note describes a Supported Liquid Extraction (SLE) protocol for the extraction of acrylamide from coffee using ISOLUTE® SLE+ columns with LC-MS/MS detection.

Application Note AN796

Introduction

The method described in this application note achieves high recoveries of acrylamide in coffee. The method is sensitive enough to measure levels as low as 1 ng/mL in coffee (solution), 25 ppm in ground coffee (solid) or 125 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.

Figure 1. Structure of Acrylamide

Analyte

Acrylamide

Sample Preparation Procedure

Format: ISOLUTE® SLE+ 1 mL Columns, part number 820-0140-C Sample Pre-treatment: Coffee was prepared in the same way that it would normally be consumed. In the case of ground coffee, 60 g of ground coffee was percolated with 1500 mL of boiling water. For instant coffee 2 g of instant coffee powder was dissolved in 250 mL of boiling water. This resulted in solutions containing coffee ‘solid’ concentrations of 40 mg/mL for ground coffee and 8 mg/mL for instant coffee. Once prepared the coffee was left to reach room temperature. Calibration Line Preparation: A 128 ng/mL acrylamide coffee over-spiked solution was  prepared by diluting 25.6 μL of a 10 μg/mL aqueous acrylamide solution to 2 mL with control coffee. This was then serially diluted seven times by transferring 0.8 mL, diluting with 0.8 mL of control coffee, mixing, and then transferring 0.8 mL of this mixture; repeating the procedure until a solution with an over-spiked level of 1 ng/mL had been reached. 0.625 mL aliquots were transferred to wells containing 10 μL of a 4 μg/mL ¹³C₃ acrylamide solution in water and 12.75 μL of a saturated solution of ammonium hydroxide in water.

Supported Liquid Extraction

Sample work-up: Samples (0.625 mL) were transferred to tubes containing 10 μL of a 4 μg/mL ¹³C₃ acrylamide solution in water and 12.75 μL of a saturated solution of ammonium hydroxide in water. The tube was briefly shaken and then 0.5 mL of the mixture transferred to a 1 mL capacity ISOLUTE® SLE+ column. Sample loading: Load pre-treated sample (0.5 mL) onto each well. Apply a pulse of vacuum (VacMaster-10 or 20 Sample Processing Manifold, 121-1016 or 121-2016) or positive pressure (Pressure+Positive Pressure Manifold, PPM-48) to initiate flow. Allow the sample to absorb for 5 minutes. Analyte Elution: Elute with ethyl acetate: tetrahydrofuran, (1 : 1, v/v, 2x 2.5 mL) and allow to flow under gravity into a tube already containing 2 μL 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, 20 to 40 L min ^-1), (SD-9600-DHS or SD2-9600-DHS) or TurboVap LV, (C103198 or C103199) (15 bar at ambient for 1 hr). Reconstitute in water (200 μL).

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