Analytical methods - Acrylamide

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prEN 16618 Food analysis – Determination of acrylamide in food by liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS) (under approval – publication date March 2015)


  • Scope

This document specifies a method for the determination of acrylamide in bakery ware such as bread, toasted bread, crisp bread, butter cookies and biscuits, as well as potato products such as potato chips, potato crisps and potate pan cake and roasted coffee, by liquid chromatography in combination electrospray ionisation and tandem mass spectrometry (LC-ESI-MS/MS). This method has been validated in an interlaboratory study via the analysis of both naturally contaminated and spiked samples, ranging from 14,3 µg/kg to 9 083 µg/kg.

The limit of quantification (LOQ) depends on the type of instrument used and on the actual performance of the instrument. The majority of the laboratories participating in the validation study was able to determine acrylamide in a butter cookie sample at a level of 14,3 µg/kg. The validation by interlaboratory study showed that it can be expected to be in the range below 15 µg/kg and 30 µg/kg.


  • Principle

- Extraction of acrylamide with water

- Addition of isotopic labelled acrylamide

- Centrifugation of the extract

- Cleanup of the supernatans on two SPE columns:

  • SPE with silica-based C18 groups as well as anion and cation exchangers: acrylamide is not retained
  • SPE with a polymer-based phase: high capacity to bind acrylamide
    • Elution with 60 % methanol in water

- Evaporation of methanol

- LC-MS/MS analysis

  • HPLC column with graphitised carbon as stationary phase


  • Key steps
    • Never exceed 40 °C during extraction or the work-up process. Acrylamide has been found to be formed as an artefact in some analytical procedures for acrylamide.
    • Extraction
      • Make sure that particles are small enough before extraction (< 1 mm)
      • Extraction for bakery ware and potato product samples
        • Test portion: 2.0 g ± 0.01 g
        • Addition of 40 mL of water and 400 µL of internal standard solution
        • Shaking of the mixture by hand, by vortex mixer and finally on a mechanical shaker
        • Centrifugation in a cooled centrifuge at 10 °C
        • Collection of 10 mL of aqueous phase
      • Extraction for coffee samples
        • Test portion: 2.0 g ± 0.01 g
        • Addition of 5 mL of n-hexane, 40 mL of water and 400 µL of internal standard solution
        • Shaking of the mixture by hand, by vortex mixer and finally on a mechanical shaker
        • Centrifugation in a cooled centrifuge at 10 °C to induce a proper phase separation of n-hexane, aqueous and solid phase
        • Removal of the organic solvent phase and collection of 10 mL of aqueous phase
        • Cleanup
          • Cleanup for bakery and potato product samples
            • SPE on ISOLUTE Multimode SPE column
              • Pass 10 mL of aqueous extract through column and collection of eluate
        • SPE on ISOLUTE ENV+ SPE column
          • 10 mL of aqueous extract (from previous column)
          • Elution of acrylamide with 60 % methanol in water
        • Evaporation of methanol from the extract (never exceeding 40 °C!) to not less than approximately 500 µL
        • Transfer of sample into autosampler vial
      • Cleanup for coffee samples
        • SPE on ISOLUTE Multimode SPE column
          • Pass 2 mL of aqueous extract through column, followed by 3 mL of water
          • Collection of the combined eluate (5 mL)
        • SPE on ISOLUTE ENV+ SPE column
          • 5 mL of aqueous extract (from previous column)
          • Elution of acrylamide with 60 % methanol in water
          • Evaporation of methanol from the extract (never exceeding 40 °C) to not less than approximately 500 µL
          • Transfer of sample into autosampler vial
          • LC-MS/MS
            • HPLC system comprising a thermostated column compartment, an injection system (injection of 10 µL), a mobile phase pump and a data acquisition and analysis system
            • Column: Hypercarbä column (5 µm particle size; 50*2.1 mm). Stationary phase is graphitized carbon
            • Mass spectrometer: triple quadrupole MS, operating in positive electrospray ionisation and selected reaction monitoring (SRM)
            • Mobile phase: 0.1 % glacial acetic acid in water
            • SRM transitions: m/z 72>55, 72>54, 72>44 and 75>58
            • Identification
              • Confirmation of peak identity by comparison of peak area ratios for transitions 72>54/72>55, and 72>44/72>55 from sample extracts and standard solutions (difference not more than ± 20 %)
            • Determination of concentrations
              • Internal standardization
              • Calculation of the response factor Rf based on the calibration solutions
              • Calculation of the average amount of acrylamide, extracted from the sample, for the N replicate injections of the respective sample
              • Calculation of mass fraction of acrylamide, in µg/kg, in the sample

       

      CEN/TC 275 N1061 Foodstuffs – Determination of acrylamide in potato-based products, cereal-based products and coffee with GC-MS (under drafting)