determination of benzene in soft drinks and other beverages

7/31/2019 Determination of Benzene in Soft Drinks and Other Beverages

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3.4 Methanol, HPLC grade.

4. STANDARDS

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 50L of benzene through the septum of the vial containing the methanol and shakevigorously or vortex. Reweigh the sealed vial and record the weight (W2) to thenearest 0.1 mg. Subtract W1 from W2 to determine the weight of benzenetransferred (W3). The stock standard concentration equals W3 divided by thetotal volume (20.05 mL).

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

4.2 Preparation of Benzene Intermediate Standard (ca. 54 g/mL): By using asyringe, transfer 0.5 mL of the ca. 2.20 mg/ml benzene stock standard to asealed headspace vial containing 20 mL methanol and shake vigorously. Theintermediate standard concentration equals ca. 1100 g divided by 20.5 mL.Prepare daily.

4.3 Preparation of Benzene Working Standard (ca. 0.5 g/mL): By using asyringe, transfer 200 L of theca. 54 g/ml benzene intermediate standard to asealed headspace vial containing 20 mL water and shake vigorously. Theworking standard concentration equals ca. 10.8 g divided by 20.2 mL. Prepare

daily. This standard is used to prepare a 6-point calibration curve from 0.5 to 20ng/g (Section 5.2).

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

4.5 Preparation ofd6-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 sealthe vial. Weigh the sealed vial (W1) to the nearest 0.1 mg. By using a syringe,transfer 50 L ofd6-benzene through the septum of the vial containing themethanol and shake vigorously or vortex. Reweigh the sealed vial and record theweight (W2) to the nearest 0.1 mg. Subtract W1 from W2 to determine the weightofd6-benzene transferred (W3). The stock standard concentration equals W3divided by the total volume (20.05 mL).


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

4.6 Preparation ofd6-Benzene Working IS (ca. 4.2 g/mL): By using a syringe,

transfer 36 L of theca.

2.36 mg/mld6

-benzene stock standard to a sealedheadspace vial containing 20.0 mL water and shake vigorously or vortex. Theworking 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 calibrationstandard is analyzed with each sample batch as a quality control check sample.Before sample analysis, the HS GC/MS linear range should be established byanalyzing an external standard curve as described in Section 5.2 .

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

5.2 Preparation of the six-point external standard calibration curve: Add 10 g ofdeionized 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 bytransferring with a syringe 10, 20, 50, 100, 200, and 400 L of the 0.5 g/mlbenzene working standard followed by 25 L of the 4.2 g/mL d6-benzeneworking standard. To avoid the loss of benzene, immediately seal vials followingthe addition of the benzene and d6-benzene working standards.

5.3 Preparation of 10 ng/g benzene/d6-benzene quality control calibrationstandard: Add 10 g deionized water to two headspace vials. Add 25 L each ofthe 4 g/ml benzene and d6-benzene working standards. To avoid the loss ofbenzene, immediately seal vials following the addition of the benzene andd6-benzene working standards.

6. APPARATUS

7. 6.1 Refrigerator at 6C.

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

6.3 Analytical balance capable of weighing to nearest 0.1 mg.

6.4 Static headspace autosampler

6.4.1 Perkin Elmer HS-40 (Section 7)


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6.4.2 Perkin Elmer Turbo Matrix 40 (Section 8)

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

6.6 GC columns

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

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

6.7 20 mL headspace vials with aluminum crimp seals and Teflon-faced siliconsepta. Store vials in 90C forced-air oven until ready for use.

6.8 Syringes

6.8.1 Two, 50 L syringes

6.8.2 Two, 100 L syringes

6.8.3 One, 250 L syringe

6.8.4 Two, 1 mL syringes

6.9 Hand crimper for sealing vials

6.10 Hand de-crimper for removing vial seals

8. METHOD 1 -- HEADSPACE GC/MS ANALYSIS WITH CRYOGENICFOCUSSING

7.1 Headspace sampling with Perkin Elmer HS-40 Autosampler

7.1.1 TemperaturesNeedle: 125CTransfer Line: 125COven: 75C

7.1.2 TimingInjection: 0.2 minutesPressurization: 0.5 minutesWithdrawal: 0.2 minutesThermo Equilibration: 15 minutesCycle Time: 5 minutes


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7.1.3 OptionsVial Vent: OnOperation Mode: ConstantInjection Mode: TimeHi PSI Injection: On

7.1.4 Programmed Pneumatic ControlInject: 30.0 psiColumn/Vial Head Pressure: 20.0 psi

7.2 Cryogenic Focusing followed by Ballistic Heating of GC Capillary Column

emperatures: -50C for 1 min and then the ballistically heat to 210C the focusedzone of the capillary column

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

split ratio

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7.3 GC ConditionsGC Column: Phenomenex, ZB-624, 30 m, 0.25 mm I.D., 1.4 m FT, FSOTGC Oven: 60C, 7.5C/min to 230C and hold 7.33 min. Run-time, 30 min.GC Column flow: 1.0 mL/min helium (constant flow).GC inlet temperature: 175C.Split ratio: 20:1 at 0.5 minGas saver: on

Under these conditions, retention times of benzene andd6

-benzene areca.

4-5min

7.4 MS parametersMS source temperature: 230CMS quad temperature: 150CMS transfer line: 230CScan range: m/z 25 to 250Scan time: 2.5 to 30 min

Threshold: 100 countsSamples: n=2Scans/sec: 6.10

10. METHOD 2 -- HEADSPACE GC/MS ANALYSIS WITHOUT CRYOGENICFOCUSSING

8.1 Headspace sampling with Perkin Elmer Turbo Matrix-40 Autosampler


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8.1.1 TemperaturesNeedle: 100CTransfer Line: 130C

Oven: 60C

8.1.2 TimingInjection: 0.2 minutesPressurization: 0.5 minutesWithdrawal: 0.2 minutesThermo Equilibration: 15 minutesCycle Time: 36 minutes

8.1.3 OptionsVial Vent: OnWater Trap: OffShaker: OffOperation Mode: ConstantInjection Mode: TimeHi PSI Injection: On

8.1.4 Programmed Pneumatic Control

Inject: 20.0 psiColumn/Vial Head Pressure: 10.0 psi

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8.2 GC ConditionsGC Column: Agilent, HP-Plot Q, 15 m, 0.32 mm I.D., 20 m filmGC Oven: 100C, 10C/min to 225C and hold 12.5 min. Run-time, 25 min.GC Column flow: 1.7 mL/min helium (constant flow).GC inlet temperature: 200C.Split ratio: 2:1

Gas saver: off.Under these conditions, retention times of benzene and d6-benzene are ca. 7-8min

8.3 MS parametersMS source temperature: 230C


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MS quad temperature: 150CMS transfer line: 225CMS mode: SIMMonitoring m/z 51, 77, and 78 for benzene and m/z 52 and 84 ford6-benzeneScan time: 5 to 9 min

Dwell time: 75 msecCycles/sec: 2.20

12. CALCULATIONS

9.1 Quantitation of Benzene

Determine the integrated responses for m/z 78 for benzene and m/z 84 ford6-benzene and calculate the response ratio, m/z 78 divided by m/z 84. Multiply theresponse ratio by the amount ofd6-benzene added to the calibration standardsand 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.

13.9.2 MS Confirmation of Benzene

9.2.1 Determine the integrated response for m/z 51, 77 and 78 for the testportions and calibration standards. Calculate the response ratios of m/z 51divided by m/z 78 and m/z 77 divided by m/z 78. The response ratios for the testportions should agree with the average of the response ratios for the calibrationstandards by 10 percent, relative difference. For example, the matchingwindow would be 18 to 22 percent for an ion ratio with a 20 percent relativeabundance.

9.2.2 The retention time (RT) for the test portions should agree with the averageRTs for the calibration standards by 2 percent.

14. SUITABILITY OF ANALYTICAL METHODS

Benzene formation can occur in soft drinks containing benzoate salts, particularlythose with added ascorbic acid, when exposed to elevated temperatures and/orUV light. In order to evaluate whether or not benzene formation occurred during

analysis, HS oven temperature studies were conducted with three carbonatedand noncarbonated beverages containing benzoate salts, ascorbic acid, and > 5ppb benzene. Test portions of each product were heated for 15 minutes in a HSoven at 35, 60, 80, and 105C. Benzene formation was not observed at any ofthese 4 temperatures, demonstrating that the analytical method will not generatebenzene in beverages.

15. WITHIN-LABORATORY PRECISION


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Within-laboratory precision was determined by comparing the amount of benzenefound in 16 samples by two analysts (one using Method 1 and the other Method2) on different days with different instruments. For the majority of thesebeverages, the amount of benzene found was less than 5 ppb, and the differencebetween the amounts found by each analyst was less than 28 percent.

determination of benzene in soft drinks and other beverages

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