Liquid-liquid extraction of 3,3'-dichlorobiphenyl (PCB11) and its hydroxylated metabolites from animal tissues

Combust silica gel, glass wool, all glassware (cartridges, short and long pipettes, tubes, beaker/volumetric flask, funnel), scalpel, forceps, and spatula.

Print the working sheet (see attachment for an example).

Label all sample tubes ( Tube-A , Tube-B , Tube-C , Tube-D , and Tube-E ).

Quality control samples include:

• Method blank (MB): x2 or x3 to make sample number even
• Tissue blank (TB): tissue from control animals
• Tissue samples: tissue from exposed animals
• Ongoing Precision and Recovery (OPR): tissue blank spiked with surrogate standard (SS), internal standard (IS), and analytes
• Reference standard: includes SS, IS, and analytes
• Instrument blank or solvent: hexane only

Note: For the spike test, use only blank tissue. For actual tests, use blank tissue plus exposed samples. Note: For the spike test, use only blank tissue. For actual tests, use blank tissue plus exposed samples.

Remove the analytical standards for the ORP and the SS from the freezer and allow to warm to room temperature (~ 30 min).

Label tubes as A1, A2 etc, and Reference standard .

Place tissues (liver 100-200 mg, brain 200 mg, adipose 50-80 mg) in tube-A . Record weight on the working sheet.

Note: Wash scalpel, forceps, and spatula between samples with water, acetone, and hexane subsequently. Note: Wash scalpel, forceps, and spatula between samples with water, acetone, and hexane subsequently.

Add 3 mL of 2-propanol to all tube-A samples with repeater pipettor.

Homogenize the sample (~30-60 s).

Note: Rinse the TissueRuptor blade between samples with water in a beaker, followed by 2-propanol (~ 5 mL in a test tube). Note: Rinse the TissueRuptor blade between samples with water in a beaker, followed by 2-propanol (~ 5 mL in a test tube).

Add 1 mL of diethyl ether with a repeater pipettor in a fume hood to each sample, recap, and vortex gently.

Spike all tube-A samples (MB, TB, tissue, OPR, and Reference standard ) with SS_PCB (10 ng d-PCB65 and 10 ng PCB14).

• d-PCB65, 100 µL x 100 ng/mL = 10 ng each
• PCB14, 100 µL x 100 ng/mL = 10 ng each

Spike all tube-A samples (MB, TB, tissue, OPR, and Reference standard ) with SS_OH-PCB (10 ng 4’-OH-PCB159).

• 4’-OH-PCB159, 100 µL x 100 ng/mL = 10 ng each

Spike the following analytes only to tube-A OPR samples and the tube for the Reference standard .

• 100 µL of PCB11 standard (100 µL x 100 ng/mL = 10 ng)
• 100 µL of OH-PCB11 standard mixture (100 µL x 100 ng/mL = 10 ng each)

Cap tube-A and the Reference standard .

Put the Reference standard aside for the derivatization step.

Note: Note: For the entire method, all centrifugation steps are for 5 mins at 3000 rpm. Tubes are inverted on the tube rotator for 5 mins at 40 RPM.

Note: For the evaporation or “blow down” step with nitrogen, a warm water bath (35 °C) can be used if needed.

Invert tube-A on the tube rotator and centrifuge.

Add 5 mL of phosphoric acid (H₃PO₄) in NaCl with repeater pipettor to tube-B.

Transfer the supernatant from tube-A to tube-B with a long pipet.

Adding 1 mL of 2-propanol and 3 mL of hexane-diethyl ether (9:1) to tube-A with a repeater pipettor, vortex for 10 secs, and centrifuge.

Transfer the supernatant from tube-A to tube-B.

Invert tube-B for 5 min and centrifuge.

Transfer the organic layer (top layer) to a new tube-C with new long pipettes

Re-extract tube-B with 3 mL of hexane-diethyl ether (9:1), vortex, and centrifuge

Transfer the top layer to combine with the extract in tube-C (~ 7 mL).

Evaporate solvent in tube-C under a gentle stream of nitrogen to ~100 µL.

Note: Do not evaporate to dryness.

Add 5 drops of methanol to tube-C all the samples except Reference standard and vortex for 5 s.

In the fume hood, using a 10 mL serological pipette, add ~0.5 mL of diazomethane to each sample and 1 mL to the Reference standard .

Cap the tubes and keep them in a solvent-proof refrigerator ( NOT freezer ) at 4-8 °C for at least 3 h or overnight (approximately 16 h).

Label GC vials.

Make fresh KOH-95% EtOH solution and set it aside for the base cleanup step.

Remove the IS standards from the freezer and allow to warm to room temperature (~ 30 min).

Turn on the water bath with setting of 50 °C.

Evaporate the excess of ether and diazomethane in a fume hood under a gentle N2 flow (no yellow color, ~200 µL).

Evaporate the Reference standard to near dryness (~ 50 μ$$L) and put it aside for the IS spiking step.

Add 3 mL of hexane with a repeater pipettor and vortex.

Add 2 mL of KOH-95% EtOH solution with a repeater pipettor and vortex.

Heat for 1 hour in a water bath at 50 °C, vortexing the samples approximately every 15 mins.

Add 7 mL of Mili-Q water with repeater pipettor, invert, and centrifuge.

Transfer the top layer to tube-D with a short pipette.

Add 2 mL of hexane using a repeater pipettor to tube-C and vortex.

Let samples sit on the lab bench for 15 mins, centrifuge, and transfer the top layer to combine with tube-D (volume ~5 mL).

Evaporate the solvent under a gentle stream of nitrogen to ~0.5 mL

Prepare the SPE cartridges.

Note: Note: The SPE cartridge can also be prepared using the dry reagent loading method: Glass wool is placed on the bottom of the SPE cartridges, and 0.2 g of silica gel and 2 g of acidified silica gel are added. Then, 3 mL of hexane:DCM (1:1) mixture is passed through the cartridge to rinse the filled cartridge.

Note: DCM is toxic, need to work in fume hood with good ventilation condition. Note: DCM is toxic, need to work in fume hood with good ventilation condition.

Add uncombusted tubes to the bottom of the SPE manifold to collect waste eluent from the SPE cartridge.

Replace the top and place the clean SPE cartridges into each port.

Add glass wool to the SPE cartridge and pack it down with the end of a glass pipet.

Add 0.2 g of silica gel with a combusted small glass funnel to the SPE cartridges.

Add 2 mL of hexane:DCM (1:1) with a long pipette to rinse to the SPE cartridge.

In a small beaker, mix hexane:DCM (1:1) with acidified silica gel (5:1), swirl, and add the entire suspension with a short pipette on top of the silica gel in each SPE cartridge.

Mix the suspension with a short glass pipette tip to eliminate air bubbles.

Let the silica gel precipitate to the bottom before passing ~ 3 mL of hexane:DCM (1:1) into the bottom waste tube.

Note: Note: Always keep the solvent above the acidified silica gel.

Replace the waste tubes at the bottom with freshly labeled, combusted tube-E . Align tube-D order in the tube rack with the order of tube-E.

With long glass pipettes, slowly load the extracts from tube-D into the SPE cartridge and pass it through the cartridge for collection.

Notes:

• Adjust the vacuum of the manifold accordingly, and let the eluent drip drop by drop.
• Close off the vacuum once the gel is dry to avoid evaporating the sample liquid.
• Always keep the solvent level above silica gel.

Rinse tube-D twice (2 x 0.5 mL) with hexane:DCM (1:1) and pass through the cartridge each time.

Repeatedly adding hexane:DCM (1:1) mixture (total volume ~ 9 mL) till the eluent in tube-E reaches 10 mL mark.

Turn off the manifold vacuum and remove tube-E from the SPE manifold.

Concentrate the samples in tube-E under a gentle stream of nitrogen to ~200 µL.

Add 3 mL of hexane to tube-E , then continue to concentrate the sample under a gentle stream of nitrogen to ~50 µL.

Note: Do not evaporate to dryness.

Spike 100 µL of IS (PCB30+PCB204, 100 µL x 100 ng/mL = 10 ng each) to each tube-E and the Reference standard using a single channel pipette.

Vortex each tube-E .

Transfer extracts from tube-E and the Reference standard with a long pipette to a combusted crimp-style GC vial.

Rinse tube-E with hexane and combine with to the GC vial to give a final volume of ~0.6 mL.

Prepare the solvent blank by filling a GC vial with hexane.

Note: Use the same hexane used for the extractions. Note: Use the same hexane used for the extractions.

Cap all vials immediately with a crimp cap and crimper to seal the cap.

Store samples in solvent-proof -20°C freezer until instrument analysis.

Note: Do not store samples in a regular -20°C freezer because solvent vapors will destroy the freezer over time. Note: Do not store samples in a regular -20°C freezer because solvent vapors will destroy the freezer over time.

Use the Excel worksheet included with this protocol to calculate the levels of PCB11 and its hydroxylated metabolites after gas chromatographic analysis.

Dispose of all items as follows:

• Scalpel – sharps container
• Homogenizer probe – If actual test, dispose in the PCB container. If spike test, dispose in the hazard waste container.
• SPE Cartridges—Remove the cartridges from the SPE manifold and dispose of all content in the PCB waste container. Place the cartridges in the sink to be washed. Add the original waste tubes to the manifold, open the vacuum tube on the manifold, and turn on the vacuum. Clean the cartridge with acetone and then hexane. Turn off the vacuum and remove it. Let the top dry on its end, and once fully dry, place the manifold back on the shelf.
• Evaporator – Place needles in the beaker to be combusted.
• Tube-A , Tube-B , Tube-C , Tube-D , Tube-E , and other aqueous solutions – Aqueous hazardous waste
• Organic solvents (waste tubes with hexane and 2-propanol) – Organic hazardous waste
• All PCB-exposed disposable glassware, foil, GC vials, and gloves – Blue PCB hazardous waste container.
• Serological pipette – glass hazard container.

Recap the samples after the GC-MS/MS or GC-ECD analysis

Store all samples, including the solvent blank, in a solvent-proof -20°C freezer until the data are published.

Note: Do not store samples in a regular -20°C freezer because solvent vapors will destroy the freezer over time. Note: Do not store samples in a regular -20°C freezer because solvent vapors will destroy the freezer over time.