Determination of Benzene in Soft Drinks and Other Beverages by Isotope Dilution Headspace Gas Chromatography/Mass Spectrometry

ISBN: 978-0-662-47337-4 (PDF version)
Catalogue number: H164-61/2007E-PDF (PDF version)

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• 1.0 Scope and Applicability

  This method is for determination of benzene in carbonated and non-carbonated soft drinks and other beverages.

• 2.0 Definitions
• 3.0 Equipment and Supplies
  • 3.1 Equipment
    • 3.1.1 Agilent 6890 gas chromatograph equipped with cryogenic oven cooling.
    • 3.1.2 Agilent 5973N mass selective detector, coupled to Agilent 6890 gas chromatograph.
    • 3.1.3 Gerstel MPS 2 MultiPurpose autosampler, or equivalent, connected to Agilent 6890 gas chromatograph.
    • 3.1.4 EASYpure RODI water purification system (Barnstead, Iowa), or equivalent.
    • 3.1.5 Oven from VWR, or equivalent.
    • 3.1.6 Balances.
  • 3.2 Chemicals
    • 3.2.1 Methanol (HPLC grade) from EM Science (Gibbstown, NJ).
    • 3.2.2 Benzene (99.8 %) Sigma-Aldrich (St. Louis, MO).
    • 3.2.3 Benzene-d₆ with isotopic purity of 99.95 atom % D from Sigma-Aldrich (St. Louis, MO).
    • 3.2.4 Sodium sulfate (ACS reagent, >99.0%, granular) from Sigma-Aldrich (St. Louis, MO).
  • 3.3 Materials
    • 3.3.1 100 µL syringe for autosampler from Agilent Technologies.
    • 3.3.2 5, 25, 50, 100, 200 µL syringes from Supelco
    • 3.3.3 2 mL autosampler amber vials from Canadian Life Science.
    • 3.3.4 40 mL amber vials from VWR.
    • 3.3.5 DB-624 capillary column (30 m x 0.25 mm x 1.4 µm) from Agilent Technologies.
    • 3.3.6 1 mL Eppendorf pipettor.
    • 3.3.7 Pipettor tips for 1 mL Eppendorf pipettor.
    • 3.3.8 UHP Helium from BOC.
    • 3.3.9 Liquid CO₂ from BOC.
• 4.0 Health and Safety
  • 4.1 Benzene is a known human carcinogen and, to prevent exposure, its solutions should be handled in a fume hood using appropriate precautions.
• 5.0 Responsibilities
• 6.0 Procedures
  • 6.1 Preparation of Standard Solutions
    • 6.1.1 Benzene stock #1
      • 6.1.1.1 Add 20 mL methanol using a 20 mL volumetric pipette to a 20 mL amber vial, cap and weigh the vial.
      • 6.1.1.2 Add 100 µL benzene through the septum to methanol, and weigh the vial to determine the exact amount of benzene added.
      • 6.1.1.3 Cool the vial in the refrigerator, and then replace the septum.
      • 6.1.1.4 Benzene concentration is 4437.8 µg/mL. Calculation must include the volume of benzene added.
    • 6.1.2 Benzene-d₆ stock #1
      • 6.1.2.1 Add 20 mL methanol using a 20 mL volumetric pipette to a 20 mL amber vial, cap and weigh the vial.
      • 6.1.2.2 Add 100 µL benzene-d₆ through the septum to methanol, and weigh the vial to determine the exact amount of benzene-d₆ added.
      • 6.1.2.3 Cool the vial in the refrigerator, and then replace the septum.
      • 6.1.2.4 Benzene-d₆ concentration is 4925.4 µg/mL. Calculation must include the volume of benzene-d₆ added.
    • 6.1.3 Benzene stock #2
      • 6.1.3.1 Add 20 mL methanol using a 20 mL volumetric pipette to a 20 mL amber vial, cap the vial.
      • 6.1.3.2 Add 200 µL benzene stock #1 (6.1.1) through the septum to methanol.
      • 6.1.3.3 Cool the vial in the refrigerator, and then replace the septum.
      • 6.1.3.4 Benzene concentration is 43.9 µg/mL. Calculation must include the volume of benzene stock #1 added.
    • 6.1.4 Benzene-d₆ stock #2
      • 6.1.4.1 Add 20 mL methanol using a 20 mL volumetric pipette to a 20 mL amber vial, and cap the vial.
      • 6.1.4.2 Add 200 µL benzene-d₆ stock #1 (6.1.2) through the septum to methanol.
      • 6.1.4.3 Cool the vial in the refrigerator, and then replace the septum.
      • 6.1.4.4 Benzene-d₆ concentration is 48.8 µg/mL. Calculation must include the volume of benzene-d₆ stock #1 added.
    • 6.1.5 Benzene stock #3
      • 6.1.5.1 Add 20 mL methanol using a 20 mL volumetric pipette to a 20 mL amber vial, cap the vial.
      • 6.1.5.2 Add 100 µL benzene stock #2 (6.1.3) through the septum to methanol.
      • 6.1.5.3 Cool the vial in the refrigerator, and then replace the septum.
      • 6.1.5.4 Benzene concentration is 0.219 µg/mL. Calculation must include the volume of benzene stock #2 added.
    • 6.1.6 Benzene-d₆ stock #3
      • 6.1.6.1 Add 20 mL methanol using a 20 mL volumetric pipette to a 20 mL amber vial, and cap the vial.
      • 6.1.6.2 Add 410 µL benzene-d₆ stock #2 (6.1.4) through the septum to methanol.
      • 6.1.6.3 Cool the vial in the refrigerator, and then replace the septum.
      • 6.1.6.4 Benzene-d₆ concentration is 0.98 µg/mL. Calculation must include the volume of benzene-d₆ stock #2 added.
    • 6.1.7 Benzene standard working solutions in methanol
      • 6.1.7.1 Add 20 mL methanol using a 20 mL volumetric pipette to a 20 mL amber vial, and cap the vial.
      • 6.1.7.2 Add 20 µL benzene-d₆ stock #1 (6.1.2) through the septum to methanol.
      • 6.1.7.3 Add 50 and 200 µL of benzene stock #2 for STD-1 and STD-2, or add 10, 25, 50, 100, and 200 µL of benzene stock #1 for STD-3, STD-4, STD-5, STD-6, STD-7, through the septum to methanol.
      • 6.1.7.4 Cool the vial in the refrigerator, and then replace the septum.
      • 6.1.7.5 Benzene-d₆ concentration is 4.9 µg/mL, and benzene concentrations are 0.11, 0.43, 2.22, 5.53, 11.06, 22.06, and 43.9 µg/mL for STD-1 to STD-7, respectively. Calculation must include the volumes of benzene stock #1 or #2 and benzene-d₆ stock #2 added.
    • 6.1.8 Benzene standard working solutions in water
      • 6.1.8.1 Weigh a 40 mL amber vial with cap.
      • 6.1.8.2 Add deionized water to the 40 mL vial within 2 mm from the brim, and cap the vial.
      • 6.1.8.3 Weigh the vial to determine the exact amount of water added using water density.
      • 6.1.8.4 Add 40 µL of STD-1 to STD-7 benzene standard working solutions in methanol through the septum to water to generate STD-1 to STD-7 benzene standard working solutions in water with benzene-d₆ at 4.8 µg/L and benzene at 0.107, 0.429, 2.197, 5.454, 11.007, 21.716, and 43.481 µg/L for STD-1 to STD-7, respectively.
      • 6.1.8.5 Cool the vials in the refrigerator, and replace the septa.
  • 6.2 Sample Preparation
    • 6.2.1 Heat sodium sulfate in a beaker in the oven at 200°C for at least 24 hours.
    • 6.2.2 Cool the samples to be analysed overnight in the refrigerator (0 to 4°C).
    • 6.2.3 Weigh 0.4 g sodium sulfate (pre-heated at 200°C for at least 24 hrs) into a 2 mL autosampler vial.
    • 6.2.4 Heat the vials containing 0.4 g Na₂SO₄ at 200°C for 2 hrs to remove any adsorption of benzene from lab air during the weighing process
    • 6.2.5 Remove the vials from the oven, and cap them as soon as they can be handled comfortably.
    • 6.2.6 Label the vials and then cool them in the refrigerator (0 to 4°C).
    • 6.2.7 Place the vials on metal tray and the samples in crushed ice.
    • 6.2.8 Add 1 mL portion of the sample via the pipettor into the 2 mL pre-cooled (0 to 4°C) autosampler vial containing 0.4 g Na₂SO₄.
    • 6.2.9 Spike with 5 µL of methanolic solution of benzene-d₆ directly into the sample and cap the vial with Teflon-lined silicone septum.
    • 6.2.10 This sampling and spiking steps in 6.2.8 and 6.2.9 should be completed within 10-15 seconds.
    • 6.2.11 Vortex the vial containing the sample for 1-2 mins to speed up dissolving Na₂SO₄ in the sample until saturation.
    • 6.2.12 Weigh the vial to determine the exact amount of sample in the vial. Record the amount.
    • 6.2.13 Samples should be equilibrated at room temperature for 3 hours before analysis.
  • 6.3 Instrument Conditions
    • 6.3.1 Flow rate of helium carrier gas: 1.5 mL/min.
    • 6.3.2 Injector temperature: 150°C
    • 6.3.3 Sample injection volume: 100 µL, through a 1.5 mm i.d. liner in splitless mode.
    • 6.3.4 GC column: DB-624 capillary column (30 m x 0.25 mm x 1.4 µm).
    • 6.3.5 GC oven temperature program: -5°C for 1 min., raised to 110°C at 10°C/min., then raised to 250°C at 40°C/min., and hold for 5 min.
    • 6.3.6 Headspace sampling was conducted at room temperature (22-24°C).
    • 6.3.7 The syringe was flushed repeatedly with ambient air before and after an injection.
    • 6.3.8 The mass selective detector was operated in selected ion monitoring (SIM) mode.
    • 6.3.9 Three ions selected for benzene: 78 (quantification ion), 77 and 51.
    • 6.3.10 Three ions for benzene-d₆: 84 (quantification ion), 82 and 52.
    • 6.3.11 Dwell time was 50 ms for each ion.
    • 6.3.12 The gas chromatograph/mass selective detector interface temperature: 280°C.
    • 6.3.13 Mass selective detector source temperature: 230°C
  • 6.4 Calculations
    • 6.4.1 Benzene concentration in samples was calculated by using an isotope dilution methodology with benzene-d₆ being the internal standard.
• 7.0 Record Keeping
• 8.0 Quality Control and Quality Assurance
  • 8.1 For each sequence of analysis, 7 standard solutions in water should be analysed at the beginning. An air sample is analysed after the last standard to check any carry-over. A blank sample (de-ionized water with 0.4 g sodium sulfate) and a standard solution in water (one of the 7 standard solutions in water) are analysed after every 15 samples.
  • 8.2 Linearity: The R² values for the calibration plots with benzene peak areas normalized to benzene-d₆ versus concentrations (0.1 to 40 μg/L) should be better than 0.999.
  • 8.3 Repeatability: All samples should be analysed in triplicates, and should be re-analysed if the relative standard deviations of the results are greater than 10%.
  • 8.4 Method Detection Limit: The method detection limit (MDL) was estimated using the U.S. EPA (Environmental Protection Agency) approach:

    MDL = t(n-1, 1-α=0.99) x SD

    Where t(n-1, 1-α=0.99) is the Student's t-value at 99% confidence level, and is 2.998 when eight replicates are used. SD is the standard deviation of n replicates.

    Eight samples of deionized water blanks with 0.4 g sodium sulfate were analysed for benzene, and the average blank benzene level is 0.1 µg/L with standard deviation of 0.0054. Thus the MDL is estimated as 0.016 µg/L.

  • 8.5 Recovery: The possible losses of benzene during sample preparation were evaluated by analysing deionized water samples spiked with benzene at three levels, 1, 5, and 10 µg/L, and the recoveries were 97.7±2.8%, 103.1±2.7%, and 100.4±2.9%, respectively.
• 9.0 Associated Documents
  • 9.1 References

    X.-L. Cao; V. Casey; S. Seaman; B. Tague; A. Becalski (2007) Determination of benzene in soft drinks and other beverages by isotope dilution headspace gas chromatography/mass spectrometry. Journal of AOAC International, Vol. 90, No. 2, 479-484.

    X.-L. Cao; V. Casey (2007) An improved method for determination of benzene in soft drinks at sub-ppb levels. Food Additives and Contaminants, in press.