Reference method for dibenzofuran and dibenzo-p-dioxin in defoamers: section 3

Section 3: Procedures

• 3.1 Extraction
• 3.2 Cleanup
• 3.3 Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
• 3.4 Confirmation of Identity

3.1 Extraction

The following are steps in the extraction procedure.

1. Shake sample well just before a subsample is taken for analysis.*
   
2. To 5.00 g defoamer sample in a 500 mL round-bottom flask, add 50 μL of a mixture of DBF-d₈ and DBF-d₈, internal standard surrogates of 5 ng/μL each in iso-octane, 200 mL of reagent water, and boiling chips.**
   
3. Place the flask into a heating mantle and attach a steam distillation condenser.
   
4. Add 3 mL of water and 2 mL of iso-octane inside the condenser.
   
5. Once a steady flow of cooling water is passing through the condenser, adjust the heater control of the mantle to bring the suspension to a vigorous boiling without bumping for 3 hours.
   
6. At the end of the extraction, let the condenser cool down to room temperature. Carefully drain off as much water as possible before draining the organic extract from the condenser into a 15 mL centrifuge tube.
   
7. Using a Pasteur pipet, transfer the organic extract into a second centrifuge tube containing a small amount of anhydrous sodium sulphate, while retaining the water in the first tube.
   
8. Rinse the condenser and the first tube twice with 2 mL aliquots of petroleum ether (P.E.) and transfer the rinsings to the second tube again.
   
9. Evaporate the combined extract down to 2 mL using a gentle stream of nitrogen and a water bath of 45°C.

* Since some defoamers are supplied in the form of a suspension, the sample should be homogenized by shaking to ensure a representative subsample is taken for analysis. A larger sample (i.e., 5 g instead of 1 g or less) also helps to minimize this potential inhomogeneity problem.

** In order to obtain quantitative recovery of DBF and DBF by steam distillation, about 100 boiling chips (0.6 to 0.7 g) were added to the water/defoamer sample to achieve vigorous boiling without bumping.

3.2 Cleanup

The following are steps in the cleanup procedure.

1. Plug a 400 × 10 mm (id) glass column with a piece of glass wool. Add 1 cm of granular anhydrous sodium sulphate to the bottom.
   
2. Fill the column with 5.00 g of activated basic alumina and then add 1 cm of anhydrous sodium sulphate to the top.
   
3. Elute the column with 20 mL of P.E. and discard this fraction.
   
4. Quantitatively transfer the sample extract obtained in step i. (subsection 3.1) to the column, elute the column with 50 L of P.E., and discard this fraction as well.
   
5. Continue the elution with 50 mL of 8% (v/v) dichloromethane in P.E. and collect this fraction in a 250 mL round-bottom flask as it contains all the native and deuterated DBF and DBF.
   
6. Evaporate the solvent down to about 5 mL with a three-stage Snyder column and a heating mantle.*
   
7. After cooling, transfer the extract to a 15 mL centrifuge tube and add 1 mL of iso-octane. Rinse the Snyder column and the flask with 2 × 2 mL of P.E. and combine the rinses in the above tube.
   
8. Using a gentle stream of nitrogen and a 45°C water bath, evaporate the solvent down to just below 0.5 mL.
   
9. After cooling, add 10 μL of a 25 ng/μL solution of hexamethylbenzene recovery standard in iso-octane, and adjust volume to 0.5 mL before GC-MS analysis.

* Other techniques can be used for evaporating solutions containing DBF and DBF The analyst must demonstrate, however, that losses of these compounds and their surrogates are negligible in the evaporative steps.

3.3 Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

1. The following is an example of the GC-MS operating conditions for analyzing DBD and DBF.

   Instrument:

   HP5880A GC, HP5970B MSD and data system

   Column:

   30 m × 0.25 mm, 0.25 μm film thickness

   Carrier gas:

   Helium with a head pressure of 69 kPa(10 psi), linear velocity 32 cm/s

   Injection:

   2 μL splitless (valve time 0.75 min)

   Injector temperature:

   250°C

   Oven program:

   70°C for 0.75 min then programmed to 140°C at 30°C/min, followed immediately by a 2°C/min temperature increase to 180°C. At the end of the run, bake the column at 280°C for 15 min. **

   Ionization:

   Electron impact (70 eV)

   Source temperature:

   200°C

   Dwell time:

   100 ms (milliseconds)

   EM voltage:

   200 V above autotune value

   Ions monitored:

   • m/z 147^a for hexamethylbenzene
   • m/z 168^a and 139^b for DBF
   • m/z 176^a for DBF-d₈
   • m/z 184^a and 155^b for DBF
   • m/z 192^a for DBF-d₈

   where:

   • ^a = quantitation ion
   • ^b = confirmation ion

   
   ** To avoid interference by the high boiling co extractives in the GC-MS analysis, the capillary column trust be baked at 280°C for 15 minutes before injecting the next defoamer extract.
   
2. Prepare a series of standards in iso-octane that cover the expected concentration range of DBF and DBF in the sample extracts. Each solution must also contain DBF-d₈, DBF-d₈ and hexamethylbenzene at a concentration of 500 pg/μL.
   
3. To maximize sensitivity, divide the ions into three groups or retention time windows. Monitor m/z 147(hexamethylbenzene) in group l; m/z 139, 168 (DBF) and 176 (DBF-d₈) in group 2; and m/z 155, 184 (DBF), and 192 (DBF-d₈) in group 3.
   
4. Inject 2 μL of the standard. Analyze the standard by GC-MS in the selected ion-monitoring (SIM) mode using the masses given in step c. A typical chromatogram is depicted in Figure 2 and the order of elution is: hexamethylbenzene, DBF-d₈, DBF, DBF-d₈, and DBF.
   
5. Analyze the samples in the same way as the standards.*

* If excessive interference is experienced in analyzing sample extracts, the use of a GC-MS system operating at a resolution of 5000 or higher is recommended. In this case, the ions monitored are: 147.1174 for HMB; 168.0575 and 139.0548 for DBF; 176.1077 for DBF-d₈; 184.0524 and 155.0497 for DBF; and 192.1026 for DBF-d₈

Figure 2: Typical Chromatogram

3.4 Confirmation of Identity

1. Integrate the reconstructed ion chromatograms for the quantitation ions (m/z 168 for DBF and m/z 184 for DBF) and confirmation ions (m/z 139 for DBF and m/z 155 for DBF) in the sample. If the ratio of peak areas for the quantitation and confirmation ions at the expected retention time in the sample is within ±20% of that of an authentic standard, then the presence of the parameter is confirmed.
   
2. To confirm DBF and DBF concentrations of ≤ 10 ng/g in samples, further evaporate the final extract to 100 μL or less.