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U.S. Department of Health and Human Services

Food

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Determination of Benzene in Soft Drinks and Other Beverages

May 19, 2006

  1. SCOPE OF APPLICATION

    This method is for the determination of benzene in soft drinks and other beverages.

  2. PRINCIPLE

    Ten gram test portions are weighed into headspace vials, fortified with internal standard (d6-benzene), and the vials sealed. Automated static headspace (HS) sampling followed by gas chromatography/mass spectrometry (GC/MS) analysis is used to detect benzene and d6-benzene in the scan mode (Section 7) or by single ion monitoring (SIM, Section 8). Benzene is quantified by isotope dilution using the ng amount and integrated area for d6-benzene. 10 ng/g quality control calibration standards are analyzed at the beginning and end of each sample batch. As a quality control check, the 10 ng/g calibration standards are compared with a six-point calibration curve previously determined to establish the linear range of the HS GC/MS system.

    Method 1 reported in Section 7 required that the sampled headspace be cryogenically focused at -50°C prior to GC/MS analysis. In order to eliminate cryogenic focusing and speed up the analysis, the method was modified to incorporate GC separations on a PLOT-Q capillary column as described in Section 8 (Method 2). For Method 1, the estimated limit of detection (LOD) is 0.2 ppb when benzene is determined with cryogenic focusing using the m/z 78 ion in the scan mode. For Method 2, the estimated LOD is 0.02 ppb when benzene is determined without cryogenic focusing using the m/z 78 ion in the SIM mode. The reportable limit of quantitation (LOQ) with confirmation is estimated to be approximately 1 ppb for benzene in beverages.

  3. REAGENTS

    High purity standards and analytical grade reagents should be used.

    1. 3.1 Benzene minimum purity 99.0% (Fisher Scientific, Pittsburgh, PA, CAS# 71-43-2, density = 0.876 g/mL). Store in 6°C refrigerator.

    2. 3.2 d6-Benzene minimum purity 99.96% (Aldrich, St. Louis, MO, CAS# 1076-43-3, density = 0.950 g/mL). Store in 6°C refrigerator.

    3. 3.3 Water, HPLC grade or purified by water purification system such as Milli-Q.

    4. 3.4 Methanol, HPLC grade.

  4. STANDARDS 

    1. 4.1 Preparation of Benzene Stock Standard (ca. 2.20 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 syringe, transfer 50 µL of benzene 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 benzene transferred (W3). The stock standard concentration equals W3 divided by the total volume (20.05 mL).

      Store the stock standard at room temperature. Once the septum on the stock standard has been pierced, it should be replaced daily. The benzene stock standard is stable for at least two weeks.

    2. 4.2 Preparation of Benzene Intermediate Standard (ca. 54 µg/mL): By using a syringe, transfer 0.5 mL of the ca. 2.20 mg/ml benzene stock standard to a sealed headspace vial containing 20 mL methanol and shake vigorously. The intermediate standard concentration equals ca. 1100 µg divided by 20.5 mL. Prepare daily.

    3. 4.3 Preparation of Benzene Working Standard (ca. 0.5 µg/mL): By using a syringe, transfer 200 µL of the ca. 54 µg/ml benzene intermediate standard to a sealed headspace vial containing 20 mL water and shake vigorously. The working standard concentration equals ca. 10.8 µg divided by 20.2 mL. Prepare daily. This standard is used to prepare a 6-point calibration curve from 0.5 to 20 ng/g (Section 5.2).

    4. 4.4 Preparation of Benzene Working Standard (ca. 4 µg/mL): By using a syringe, transfer 36 µL of the ca. 2.2 mg/ml benzene stock standard to a sealed headspace vial containing 20 mL water and shake vigorously. The working standard concentration equals ca. 79 µg divided by 20.036 mL. Prepare daily. This standard is used to prepare the 10 ng/g quality control calibration standard (Section 5.3).

    5. 4.5 Preparation of d6-Benzene Stock Internal Standard (IS, ca. 2.36 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 syringe, transfer 50 µL of d6-benzene 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 d6-benzene transferred (W3). The stock standard concentration equals W3 divided by the total volume (20.05 mL).

      Store the stock IS at room temperature. Once the septum on the IS has been pierced, it should be replaced daily. The d6-benzene IS is stable for at least two weeks.

    6. 4.6 Preparation of d6-Benzene Working IS (ca. 4.2 µg/mL): By using a syringe, transfer 36 µL of the ca. 2.36 mg/ml d6-benzene stock standard to a sealed headspace vial containing 20.0 mL water and shake vigorously or vortex. The working IS concentration equals ca. 85 ng divided by 20.036 mL. Prepare daily.

  5. PREPARATION OF TEST PORTIONS

    Quantification is based on isotope dilution with d6-benzene. A 10 ng/g calibration standard is analyzed with each sample batch as a quality control check sample. Before sample analysis, the HS GC/MS linear range should be established by analyzing an external standard curve as described in Section 5.2 .

    1. 5.1 Sample Preparation: To avoid loss of benzene, quickly transfer 10 g portions of sample into tared headspace vials, fortify with 25 µL of the 4 µg/mL d6-benzene working standard, seal the vial, and mix by shaking or vortexing.

    2. 5.2 Preparation of the six-point external standard calibration curve: Add 10 g of deionized water to 6 headspace vials. Calibration standards equivalent to ca. 0.5, 1, 2.5, 5, 10, and 20 ng/g benzene and 10 ng/g d6-benzene are prepared by transferring with a syringe 10, 20, 50, 100, 200, and 400 µL of the 0.5 µg/ml benzene working standard followed by 25 µL of the 4.2 µg/mL d6-benzene working standard. To avoid the loss of benzene, immediately seal vials following the addition of the benzene and d6-benzene working standards.

    3. 5.3 Preparation of 10 ng/g benzene/d6-benzene quality control calibration standard: Add 10 g deionized water to two headspace vials. Add 25 µL each of the 4 µg/ml benzene and d6-benzene working standards. To avoid the loss of benzene, immediately seal vials following the addition of the benzene and d6-benzene working standards.

  6. APPARATUS
    1. 6.1 Refrigerator at 6°C.

    2. 6.2 Top pan balance capable of weighing to nearest 0.01 g.

    3. 6.3 Analytical balance capable of weighing to nearest 0.1 mg.

    4. 6.4 Static headspace autosampler

      1. 6.4.1 Perkin Elmer HS-40 (Section 7)

      2. 6.4.2 Perkin Elmer Turbo Matrix 40 (Section 8)

    5. 6.5 GC/MS (Agilent 6890N GC with Agilent 5973N MSD, or equivalent).

    6. 6.6 GC columns

      1. 6.6.1 Phenomenex, ZB-624, 30 m, 0.25 mm I.D., 1.4 µm FT, FSOT (Section 7)

      2. 6.6.2 Agilent, HP-Plot Q, 15 m, 0.32 mm I.D., 20 µm film (Section 8)

    7. 6.7 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.

    8. 6.8 Syringes

      1. 6.8.1 Two, 50 µL syringes

      2. 6.8.2 Two, 100 µL syringes

      3. 6.8.3 One, 250 µL syringe

      4. 6.8.4 Two, 1 mL syringes

    9. 6.9 Hand crimper for sealing vials

    10. 6.10 Hand de-crimper for removing vial seals
       

  7. METHOD 1 — HEADSPACE GC/MS ANALYSIS WITH CRYOGENIC FOCUSSING

    1. 7.1 Headspace sampling with Perkin Elmer HS-40 Autosampler

      1. 7.1.1 Temperatures
        Needle: 125°C
        Transfer Line: 125°C
        Oven: 75°C

      2. 7.1.2 Timing
        Injection: 0.2 minutes
        Pressurization: 0.5 minutes
        Withdrawal: 0.2 minutes
        Thermo Equilibration: 15 minutes
        Cycle Time: 5 minutes

      3. 7.1.3 Options
        Vial Vent: On
        Operation Mode: Constant
        Injection Mode: Time
        Hi PSI Injection: On

      4. 7.1.4 Programmed Pneumatic Control
        Inject: 30.0 psi
        Column/Vial Head Pressure: 20.0 psi

    2. 7.2 Cryogenic Focusing followed by Ballistic Heating of GC Capillary Column temperatures: -50°C for 1 min and then the ballistically heat to 210°C the focused zone of the capillary column

      GC Inlet Split vent: Closed 45 sec prior to injection and then opened with a 20:1 split ratio

    3. 7.3 GC Conditions
      GC Column: Phenomenex, ZB-624, 30 m, 0.25 mm I.D., 1.4 µm FT, FSOT
      GC Oven: 60°C, 7.5°C/min to 230°C and hold 7.33 min. Run-time, 30 min.
      GC Column flow: 1.0 mL/min helium (constant flow).
      GC inlet temperature: 175°C.
      Split ratio: 20:1 at 0.5 min
      Gas saver: on
      Under these conditions, retention times of benzene and d6-benzene are ca. 4-5 min

    4. 7.4 MS parameters
      MS source temperature: 230°C
      MS quad temperature: 150°C
      MS transfer line: 230°C
      Scan range: m/z 25 to 250
      Scan time: 2.5 to 30 min
      Threshold: 100 counts
      Samples: n=2
      Scans/sec: 6.10
       
  8. METHOD 2 — HEADSPACE GC/MS ANALYSIS WITHOUT CRYOGENIC FOCUSSING

    1. 8.1 Headspace sampling with Perkin Elmer Turbo Matrix-40 Autosampler

      1. 8.1.1 Temperatures
        Needle: 100°C
        Transfer Line: 130°C
        Oven: 60°C

      2. 8.1.2 Timing
        Injection: 0.2 minutes
        Pressurization: 0.5 minutes
        Withdrawal: 0.2 minutes
        Thermo Equilibration: 15 minutes
        Cycle Time: 36 minutes

      3. 8.1.3 Options

        Vial Vent: On
        Water Trap: Off
        Shaker: Off
        Operation Mode: Constant
        Injection Mode: Time
        Hi PSI Injection: On

      4. 8.1.4 Programmed Pneumatic Control
        Inject: 20.0 psi
        Column/Vial Head Pressure: 10.0 psi

    2. 8.2 GC Conditions
      GC Column: Agilent, HP-Plot Q, 15 m, 0.32 mm I.D., 20 µm film
      GC Oven: 100°C, 10°C/min to 225°C and hold 12.5 min. Run-time, 25 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 benzene and d6-benzene are ca. 7-8 min
       

    3. 8.3 MS parameters
      MS source temperature: 230°C
      MS quad temperature: 150°C
      MS transfer line: 225°C
      MS mode: SIM
      Monitoring m/z 51, 77, and 78 for benzene and m/z 52 and 84 for d6-benzene
      Scan time: 5 to 9 min
      Dwell time: 75 msec
      Cycles/sec: 2.20

  9. CALCULATIONS

    1. 9.1 Quantitation of Benzene

      Determine the integrated responses for m/z 78 for benzene and m/z 84 for d6-benzene and calculate the response ratio, m/z 78 divided by m/z 84. Multiply the response ratio by the amount of d6-benzene added to the calibration standards and test portions. Divide ng amount of benzene found by the test portion amount in grams to determine ng/g amount of benzene in the sample.

    2. 9.2 MS Confirmation of Benzene        

      1. 9.2.1 Determine the integrated response for m/z 51, 77 and 78 for the test portions and calibration standards. Calculate the response ratios of m/z 51 divided by m/z 78 and m/z 77 divided by m/z 78. The response ratios for the test portions should agree with the average of the response ratios for the calibration standards by ± 10 percent, relative difference. For example, the matching window would be 18 to 22 percent for an ion ratio with a 20 percent relative abundance.

      2. 9.2.2 The retention time (RT) for the test portions should agree with the average RTs for the calibration standards by ± 2 percent.
  10. SUITABILITY OF ANALYTICAL METHODS

    Benzene formation can occur in soft drinks containing benzoate salts, particularly those with added ascorbic acid, when exposed to elevated temperatures and/or UV light. In order to evaluate whether or not benzene formation occurred during analysis, HS oven temperature studies were conducted with three carbonated and noncarbonated beverages containing benzoate salts, ascorbic acid, and > 5 ppb benzene. Test portions of each product were heated for 15 minutes in a HS oven at 35, 60, 80, and 105°C. Benzene formation was not observed at any of these 4 temperatures, demonstrating that the analytical method will not generate benzene in beverages.

  11. WITHIN-LABORATORY PRECISION

    Within-laboratory precision was determined by comparing the amount of benzene found in 16 samples by two analysts (one using Method 1 and the other Method 2) on different days with different instruments. For the majority of these beverages, the amount of benzene found was less than 5 ppb, and the difference between the amounts found by each analyst was less than 28 percent.

  12.  

    Questions and Answers on the Occurrence of Benzene in Soft Drinks and Other Beverages May 19, 2006

    Data on Benzene in Soft Drinks and Other Beverages May 19, 2006

    Chemical Name: benzene
    IUPAC International Chemical Identifier: InChI=1/C6H6/c1-2-4-6-5-3-1/h1-6H