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

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 ).

Note Note: If the whole blood is used for the study, the whole blood samples need to be collected with a medium tube with 80 µL of ethylenediaminetetraacetic acid solution (EDTA, 7.5% w/w), and stored at −20 °C until extraction.

Quality control samples should include:

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

Note: For the spike test, use only blank blood/serum. For actual tests, use blank blood/serum and exposed samples. Note: For the spike test, use only blank blood/serum. For actual tests, use blank blood/serum and 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 whole blood (0.3-0.5 g) or serum (~ 0.2 g) in tube-A . Record weight on the working sheet.

Add 3 mL of 1% KCl solution to tube A containing blank samples.

Add 1% KCl solution to the remaining tube A to a final volume of ~ 3 mL.

Note: Do not add 1% KCl solution to the tube for the Reference standard. Note: Do not add 1% KCl solution to the tube for the Reference standard .

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) 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 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: 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 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.

Add 1 mL of 6 M HCl to tube-A , vortex for 10 s.

Add 5 mL of 2-propanol to tube-A , vortex for 10 s.

Add 5 mL of hexane-MTBE mixture 1:1 (v/v) to tube-A .

Cap the tube-A with caps with a PTFE septum.

Invert tube-A on the tube rotator and centrifuge.

Transfer the organic phase (top layer) from tube-A to tube-B with a short glass pipette.

Re-extract tube-A with 3 mL of hexane-MTBE (1:1, v/v), vortex for 10 s, and centrifuge.

Transfer the organic phase (top layer) from tube-A to tube-B with a short pipette.

Add 3 mL of 1% KCl solution to tube-B .

Invert tube-B for 5 min and centrifuge.

Transfer the organic layer (top layer) form tube-B to tube-C .

Re-extract tube-B with 3 mL of hexane, vortex, and centrifuge.

Transfer the organic layer (top layer) form tube-B to tube-C .

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

Note: Do not evaporate to dryness.

Get samples, ice box, and serological pipettes ready in a fume hood.

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

Take the diazomethane vial out of freezer and place in an ice box in the fume hood.

Add ~0.5 mL of diazomethane to each sample and 1 mL of diazomethane to the Reference standard using a 10 mL serological pipette.

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 nitrogen 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 repeat pipettor and vortex.

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

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

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

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

Add 2 mL of hexane using a repeat 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 e: 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 the waste eluent from the SPE cartridges.

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 pipette.

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: Always keep the solvent above the acidified silica gel. Note e: 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 cartridges and pass them through the cartridges for collection.

Notes 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 add the hexane:DCM (1:1) mixture (total volume ~ 9 mL) until the eluent in tube-E reaches 10 mL.

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 concentrate the sample under a gentle stream of nitrogen to ~50 µL.

Note: Do not evaporate to dryness. 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 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 with insert.

Rinse tube-E with hexane (~150 µL) and combine with to the GC vial insert to give a final volume of ~300 µL.

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

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

Dispose of all items as follows:

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