Analytical methods - PAHs

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prEN 16619 Food analysis - Determination of benzo(a)pyrene, benz(a)anthracene, chrysene and benzo(b)fluoranthene in foodstuffs by gas chromatography mass spectrometry (GC-MS) (under approval - publication date March 2015)

 

  • Scope

This European Standard specifies a method for the determination of 4 of the 15+1 EU priority polycyclic aromatic hydrocarbons (PAHs), identified as target PAHs. They are benz[a]anthracene (BaA), benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF) and chrysene (CHR). The method allows their quantification in the presence of the other 12 EU priority PAHs in extruded wheat flour, smoked fish, dry infant formula, sausage meat, freeze dried mussels, edible oil and wheat flour by gas chromatography-mass spectrometry (GC-MS). The extraction of PAHs from solid samples is performed by pressurised liquid extraction (PLE). Soxhlet extraction may be applied as alternative to PLE. The sample cleanup is performed by applying the following techniques in the reported sequence: size exclusion chromatography (SEC), and solid phase extraction (SPE).

 

The method has been validated in an interlaboratory study via the analysis of both naturally contaminated and spiked samples, ranging from 0.5 µg/kg to 11.9 µg/kg. However, linearity of the instrument response was proven for the concentration range 0,5 µg/kg to 20 µg/kg.

 

  • Principle

-          Homogenization of the sample

-          Mixing of a test portion with desiccant, sand and stable isotope labelled internal standard solution

-          Pressurized liquid extraction or alternatively Soxhlet extraction with n-hexane or cyclohexane

-          Evaporation of organic extract to small volume and filtration

-          Purification by SEC, using a mixture of ethyl acetate and cyclohexane as eluent

-          Addition of toluene as keeper to collected SEC fraction

-          Evaporation of SEC fraction

-          Cleanup by SPE on silica, using cyclohexane as eluent

-          Evaporation of sample extract

-          Addition of an injection standard solution prior to measurement by GC-MS

 

  • Key steps
    • Sample preparation
      • Test portion of the sample: 5.0 g ± 0.1 g
      • Homogenization and grinding of the sample with polyacrylic acid and sand
      • Addition of stable isotope labelled PAH internal standard solution
      • Transfer of the test portion into the PLE extraction cell
  • Extraction
    • Pressurized liquid extraction:
      • Solvent: n-hexane or cyclohexane
      • Pressure: 1500 psi
      • Temperature: 100 °C
      • 2 static cycles
    • Evaporation of the extract to small volume (0.5 mL)
    • Transfer of concentrated extract to test tube, washing of evaporation vessel with SEC eluent (cyclohexane:ethyl acetate 1:1) (combined with extract) and adjusting final volume to 5 mL with SEC eluent.
    • Alternative: Soxhlet extraction with n-hexane
  • Cleanup
    • SEC cleanup
    • Evaporation of the collected fraction to about 5 mL
    • Addition of 200 µL of toluene as keeper
    • Evaporation of extract to approximately 200 µL under gentle stream of nitrogen and addition of 800 µL of cyclohexane
    • SPE cleanup
      • Silica SPE column
      • 1000 µL of extract onto the column
      • Elution with 10 mL of cyclohexane
    • Evaporation of SPE extract to about 200 µL
    • Transfer into autosampler vial
    • Addition of 100 µL of injection standard solution (9-fluorobenzo[k]fluoranthene as injection standard)
  • GC-MS
    • GC-MS system consisting of programmed temperature vaporising (PTV) injector, GC oven, interface to the mass spectrometer, computer based instrument control system and data processing system
    • GC capillary column: mid-polar stationary phase with high selectivity for PAHS. For example: Select PAH or any column with comparable separation characteristics
    • Mass spectrometer with following characteristics:
      • Electron ionisation source
      • Ionisation energy of 70 eV
      • Mass resolution: at least 1 u
      • Temperature control devices for the ion source (300 °C), the quadrupole (150 °C), the GC-MS interface (325 °C)
      • Tuning stability of at least 48 h
      • Response linearity range of at least two orders of magnitude
    • Detection of target compounds in Single Ion Monitoring (SIM) mode
  • Identification and quantification
    • Identification based on the presence of quantifier and qualifier ion and the ratio between the peak areas of quantifier and qualifier within the acceptable range
    • Quantification: by comparison with stable isotope labelled analogues (internal standard method)

 

FprEN 16621 Food analysis - Determination of benzo(a)pyrene, benz(a)anthracene, chrysene and benz(b)fluoranthene in foodstuffs by high performance liquid chromatography with fluorescence detection (HPLC-FLD) (under approval - publication date March 2015)

 

  • Scope

This Technical Specification describes a method for the determination of benzo(a)pyrene, benzo(a)anthracene, benzo(b)fluoranthene and chrysene in several food matrices. The method is based on gel permeation chromatography (GPC) cleanup, followed by quantification with high performance liquid chromatography (HPLC) with programmable fluorescence detection.

The method has been in-house validated via the analysis of spiked samples of edible olive oil, fresh mussels, smoked fish, smoked meat products, processed cereal-based foods for young children, infant formulae, chocolate and food supplements. Furthermore, the method has been shown applicable to a variety of additional matrices as meat products, fresh fish, paprika, roasted coffee, bread, herbs, breakfast cereals, beer, sunflower oil, olives and fried tomato, with a limit of quantification of 0.5 µg/kg.

 

In addition, the method was tested in house and shown to be applicable also for the quantification of the other 12 PAHs of the 15+1 EU priority PAHs in all matrices listed above.

 

  • Principle

-          The PAHs are extracted from solid matrices with dichloromethane

-          In case of edible oils: dispersion in dichloromethane

-          Purification of aliquots of crude extracts by gel permeation chromatography (GPC)

-          Analysis of final extracts with HPLC with programmable fluorescence detection

 

  • Key steps
    • Extraction
      • Edible portions of the foodstuffs are grinded and homogenized
      • Addition of dichloromethane
      • An aliquot of the lower organic layer is filtered
      • Samples of edible oils: dispersion in dichloromethane
  • Cleanup
    • Equilibration of the GPC system by passing dichloromethane during ca. 30 minutes
    • Injection of the filtered extract into the GPC system
    • Collection of the fraction or fractions corresponding to the elution time of the PAHs
  • HPLC
    • HPLC system consisting of an injection system, mobile phase pump (gradient), programmable fluorescence detector and computer based data processing system
    • Column: reverse-phase HPLC specific for PAH separation column: C18, base deactivated octadecyl silane (ODS) (recommended 4.6 mm x 250 mm column with 5 µm particle size)
    • Mobile phase: water (solvent A), acetonitrile (solvent B)
    • For PAH-4: excitation wavelength 230 nm and emission wavelength 357 nm.
  • Calculation
    • Determination of the mass (ng) of each PAH in the aliquot of test solution injected onto the HPLC column, using the calibration curve
    • Calculation of the mass fraction of each PAH in µg/kg

 

Other methods available

-          ISO/DTR 24054 - Animal and vegetable fats and oils - Determination of polycyclic aromatic hydrocarbons (PAH) - Method using gas chromatography/mass spectrometry (GC/MS)

-          EN ISO 15753:2006 - Animal and vegetable fats and oils - Determination of polycyclic aromatic hydrocarbons

-          EN ISO 22959:2009 - Animal and vegetable fats and oils - Determination of polycyclic aromatic hydrocarbons by on-line donor-acceptor complex chromatography and HPLC with fluorescence detection

-          EN ISO 15302:2007 - Animal and vegetable fats and oils - Determination of benzo[a]pyrene - reverse-phase high performance liquid chromatography method

-          AOAC 973.30 - Polycyclic aromatic hydrocarbons and benzo[a]pyrene in food (1974)



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