Food

Determination of Furan in Foods

May 7, 2004; Updated June 2, 2005, and October 27, 2006

UPDATE INFORMATION

This section will identify differences between the present method and the last method posted on the web site. This is the second revision of the method.

Changes made to the first version (May 7, 2004) were as follows:

The test portion amount for liquids was changed from 5 to 10 grams, since liquids do not need to be diluted. Section 5.2, Sample Preparation and Precautions, was added to provide clarification on sample preparation for solid foods in general, and peanut butter in particular. Peanut butter ferments when diluted with water, and the resulting ethanol co-elutes with furan/d4-furan. Fermentation was prevented by diluting peanut butter with 5 mL of saturated NaCl.

Changes made to the second version (June 2, 2005) are as follows:

The headspace oven temperature was reduced from 80°C to 60°C to prevent low, ng/g levels of furan formation that can occur during the analysis of a few relatively high fat foods (Section 7.1.1). (See Method Ruggedness Testing for more information on the temperature change.) The headspace cycle time was reduced from 48 to 36 min to decrease the gas chromatography post-run time (Section 7.1.2). The scan range was changed from m/z 25 - 150 to m/z 35 - 150 to reduce low molecular weight noise associated with the mass spectrometry analysis (Section 7.3). Section 5.2.4, Low Moisture Solid Foods, was added to address method modifications required for the analysis of low moisture solid foods such as crackers, cookies, cereals and chips. Section 5.3, Preparation of Test Portions for Standard Additions Based on Internal Standard, was revised to include the test portion amounts and dilution volume for low moisture solid foods.

  1. SCOPE OF APPLICATION

    This method is for the determination of furan in foods.

  2. PRINCIPLE

    Semi-solid and solid foods are weighed into headspace vials, diluted with either water or saturated NaCl solution, fortified with internal standard (d4-furan), and the vials are sealed. Liquid foods are weighed into headspace vials, fortified with internal standard (d4-furan), and the vials are sealed. Automated headspace sampling followed by gas chromatography/mass spectrometry (GC/MS) analysis is used to detect furan and d4-furan in the scan mode. Furan is quantified by using a standard additions curve, where the concentration of furan in the fortified test portions is plotted versus the furan/d4-furan response factors.

  3. REAGENTS

    High purity standards and analytical grade reagents should be used.

    1. 3.1    Furan minimum purity 99% (Aldrich, St. Louis, MO, CAS# 110-00-9, density = 0.936 g/mL). Store in -20°C freezer.
    2. 3.2   d4-Furan minimum purity 99% (Aldrich, St. Louis, MO, CAS# 6142-90-1, density = 0.991 g/mL). Store in -20°C freezer.
    3. 3.3    Water, HPLC grade or purified by water purification system such as Milli-Q
    4. 3.4    Methanol, HPLC grade
    5. 3.5    5 M NaCl – Dissolve 145 g NaCl (99% minimum purity, VWR, Pittsburgh, PA) into 500 mL water, and mix with a magnetic stirrer until NaCl has dissolved.
  4. STANDARDS
    1. 4.1    Preparation of Furan Stock Standard (ca. 2.50 mg/mL): By using a volumetric pipet, place 20.0 mL methanol in a headspace vial and seal the vial. Weigh the sealed vial (W1) to the nearest 0.1 mg. By using a chilled 50 µL syringe, transfer 50 µL of furan through the septum of the vial containing the methanol and shake vigorously or vortex. Reweigh the sealed vial and record the weight (W2) to the nearest 0.1 mg. Subtract W1 from W2 to determine the weight of furan transferred (W3). The stock standard concentration equals W3 divided by the total volume (20.05 mL).

      The stock standard should be stored in a 4°C refrigerator to minimize loss of furan by evaporation. Once the septum on the stock standard has been pierced, it should be replaced daily. The furan stock standard is stable for two weeks.

    2. 4.2    Preparation of Furan Working Standard (ca. 30.9 µg/mL): By using a syringe, transfer 250 µL of the ca. 2.50 mg/ml furan stock standard to a sealed headspace vial containing 20 mL water and shake vigorously. The working standard concentration equals ca. 625 ng divided by 20.25 mL. Prepare daily.

      Note: A ca. 30.9 µg/mL furan working standard may not always be appropriate; the concentration may need to be adjusted depending on the target quantitation level.

    3. 4.3    Preparation of d4-Furan Stock Internal Standard (IS, ca. 2.50 mg/mL): By using a volumetric pipet, place 20.0 mL methanol in a headspace vial and seal the vial. Weigh the sealed vial (W1) to the nearest 0.1 mg. By using a chilled 50 µL syringe, transfer 50 µL of d4-furan through the septum of the vial containing the methanol and shake vigorously or vortex. Reweigh the sealed vial and record the weight (W2) to the nearest 0.1 mg. Subtract W1 from W2 to determine the weight of d4-furan transferred (W3). The stock standard concentration equals W3 divided by the total volume (20.05 mL).

      The IS should be stored in a 4°C refrigerator to minimize loss of d4-furan by evaporation. Once the septum on the IS has been pierced, it should be replaced daily. The d4-furan IS is stable for two weeks.

    4. 4.4    Preparation of d4-Furan Working IS (ca. 30.9 µg/mL): By using a syringe, transfer 250 µL of the ca. 2.50 mg/ml d4-furan stock standard to a sealed headspace vial containing 20.0 mL water and shake vigorously or vortex. The working IS concentration equals ca. 625 ng divided by 20.25 mL. Prepare daily.

      Note: A ca. 30.9 µg/mL d4-furan working IS may not always be appropriate and may need to be adjusted depending on the target quantitation level.

  5. PREPARATION OF TEST PORTIONS

    Quantification is based on a standard additions curve. The amounts of furan and/or d4-furan added to the test portions are based on an estimate (x0) of the furan concentration in the food.

    1. 5.1    Estimate of x0: A one-point estimate of the concentration of furan in the food can be made by fortifying a test portion with working IS, and analyzing by headspace GC/MS as described in Section 6. By using the integrated response ratio for furan/d4-furan (m/z 68/72) and the ng amount of IS, the ng amount of furan in the food can be estimated.
    2. 5.2    Sample Preparation and Precautions
      1. 5.2.1    Homogenization of Food: For foods that are not homogenous, chill the entire container of food in a refrigerator for ca. 4 hr. Transfer contents to a beaker immersed in an ice bath. Homogenize using a Telemar Tissumizer or similar benchtop homogenizer. Quickly transfer test portions to tared headspace vials. To avoid loss of furan, it is essential that the homogenization be done as quickly as possible, and that the sample be kept chilled until test portions are transferred to headspace vials.
      2. 5.2.2    Solid Foods: For solid foods, chill entire container of food in a 4°C refrigerator for ca. 4 h. Transfer contents to a chilled bowl of a food processor and pulverize sample. Quickly transfer test portions to tared headspace vials. To avoid loss of furan, it is essential that the sample is pulverized as quickly as possible, and that the sample is kept chilled until test portions are transferred to headspace vials. (See Section 5.2.4, Low Moisture Solid Foods.)
      3. 5.2.3    Peanut Butter and Nuts: Peanut butter will ferment when diluted with water. Test portions of peanut butter and other nuts should be diluted with saturated NaCl solution to prevent ethanol formation that will co-elute with furan/d4-furan.
      4. 5.2.4   Low Moisture Solid Foods:  Pulverize sample in a food processor. Test portions of foods similar to potato chips (36% total fat) can separate into solid, aqueous, and lipid phases and adversely affect the vapor phase equilibrium. To avoid this, vortex the sealed test portions, let them stand at room temperature for 10 min, and vortex again for 30 sec.
    3. 5.3   Preparation of Test Portions for Standard Additions Based on Internal Standard: Fortify the first three vials with d4-furan working IS at ca. 2x0. Seal the vials after each fortification. Fortify the remaining four vials with d4-furan working IS at ca. 2x0 and furan working standard as follows: two vials at ca. 0.5x0, one vial at ca. x0, and one vial at ca. 2x0. Seal the vials after each fortification.
      1. 5.3.1    For liquid foods: Transfer 10 g portions of liquid food into tared headspace vials.
      2. 5.3.2    For low moisture solid foods such as crackers, cookies, cereals and chips: Transfer 1.5 g portions into tared headspace vials and dilute with 11 mL saturated NaCl solution.
      3. 5.3.3   For low moisture solid foods with high fat content such as potato chips (36% total fat): Transfer 1 g portions into tared headspace vials and dilute with 11 mL saturated NaCl solution (see Section 5.2.4).
      4. 5.3.4    For other solid and semi-solid foods: Transfer 5 g portions into tared headspace vials and dilute with 5 mL water. For peanut butter and nuts, dilute with 5 mL saturated NaCl (see Section 5.2.3).
    4. 5.4    Example of Standard Addition Fortification in Semi-Solid Food: If the estimated concentration of furan in the food, x0, is ca. 20 ng/g:
      1. 5.4.1    Prepare furan and d4-furan working standards at ca. 5 µg/mL by transferring, with a 50 µL syringe, 40 µL of ca. 2.50 mg/mL stock standards into individual sealed headspace vials containing 20.0 mL of water. The resulting furan and d4-furan working standard concentrations equal ca. 100 ng divided by 20.04 mL.
      2. 5.4.2    Accurately weigh 5.00 g portions of the food into seven headspace vials and add 5 mL water. Using two 50 µL syringes, fortify each vial with the furan and d4-furan working standards as indicated in the following table:
        5 g test portion µL 5 µg/mL
        furan
        µL 5 µg/mL
        d4-furan
        ppb furan added
        0x0 -- 40 0
        0x0 -- 40 0
        0x0 -- 40 0
        0.5x0 10 40 10
        0.5x0 10 40 10
        1x0 20 40 20
        2x0 40 40 40
  6. APPARATUS
    1. 6.1    Refrigerator at 4°C.
    2. 6.2    Freezer at -20°C.
    3. 6.3    Top pan balance capable of weighing to nearest 0.01 g.
    4. 6.4    Analytical balance capable of weighing to nearest 0.1 mg.
    5. 6.5    Dynamic headspace autosampler (Perkin Elmer Turbo Matrix 40, or equivalent).
    6. 6.6    GC/MS (Agilent 6890N GC with Agilent 5973N MSD, or equivalent).
    7. 6.7    GC column: HP-Plot Q, 15 m, 0.32 mm I.D., 20 µm film.
    8. 6.8    20 mL headspace vials with aluminum crimp seals and Teflon-faced silicon septa. Store vials in 90°C forced-air oven until ready for use.
    9. 6.9    Syringes (gas-tight syringes are recommended)
      1. 6.9.1    Two, 50 µL syringes
      2. 6.9.2    Two, 100 µL syringes
      3. 6.9.3    One, 200 µL syringe
      4. 6.9.4    Two, 1 mL syringes
    10. 6.10    Hand crimper for sealing vials
    11. 6.11    Hand de-crimper for removing vial seals
    12. 6.12    Homogenizer equipped with 30 × 150 mm open slotted generator (Telemar Tissumizer or equivalent)
    13. 6.13    Benchtop food processor (Robot Coupe or equivalent)
  7. HEADSPACE GC/MS ANALYSIS
    1. 7.1    Headspace sampling
      1. 7.1.1    Temperatures:
        1.   Needle - 100°C
            Transfer Line - 130°C
            Oven - 60°C
      2. 7.1.2    Timing:
        1.   Injection - 0.2 minutes
            Pressurization - 0.5 minutes
            Withdrawal - 0.2 minutes
            Thermo Equilibration - 30 minutes
            Cycle Time - 36 minutes
      3. 7.1.3    Options:
        1.   Vial Vent - On
            Water Trap - Off
            Shaker - On
            Operation Mode - Constant
            Injection Mode - Time
            Hi PSI Injection - On
      4. 7.1.4    Programmed Pneumatic Control
        1.   Inject - 20.0 psi
            Column/Vial Head Pressure - 10.0 psi
    2. 7.2    GC Conditions
      1. GC Oven - 50°C, 10°C/min to 225°C and hold 12.5 min. Run-time, 30 min.
        GC Column flow, 1.7 mL/min helium (constant flow).
        GC inlet temperature, 200°C.
        Split ratio - 2:1
        Gas saver - off.
        Under these conditions, retention times of furan and d4-furan are ca. 6-7 min
    3. 7.3    MS parameters
      1. MS source temperature - 230°C
        MS quad temperature - 150°C
        MS transfer line - 225°C
        Scan range - m/z 35 to 150
        Scan time - 2.5 to 25 min
        Threshold - 100 counts
        Samples - n=3
        Scans/sec - 5.56
      2.   7.3.1    MS Confirmation of Furan
        Determine the integrated response for m/z 39 and 68 for the test portions and calibration standards and calculate the response ratio of m/z 39 divided by m/z 68. The response ratio for the test portions should agree with the average of the response ratios for the calibration standards by ± 10 percent, and the retention time (RT) for the test portions should agree with the average RTs for the calibration standards by ± 2 percent.
  8. CALCULATIONS
    1. 8.1    Determine the integrated responses for m/z 68 for furan and m/z 72 for d4-furan and calculate the response ratio, m/z 68 divided by m/z 72.
    2. 8.2    Standard Additions Calibration Curve with Internal Standard: For each sample, subject all test portions (0x0) and calibration standards (0.5x0, x0, and 2x0) to a linear regression analysis where the x equals the ng amount of furan added to the test portion and the y equals the response ratios. Determine the slope and intercept of the calibration curve and solve for 0x0 at y equal to zero. Divide 0x0 by the test portion amount in grams to determine the ppb amount of furan in the sample.

    Chemical Name: furan
    IUPAC International Chemical Identifier: InChi=1/C4H4O/c1-2-4-5-3-1/h1-4H

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