TITLE: Liquid-liquid extraction of 3,3&#39;-dichlorobiphenyl (PCB11) and its hydroxylated metabolites from animal tissues
AUTHORS: Xueshu Li, Nicole M. Breese, Hansjoachim Lehmler

[DESCRIPTION]
This protocol describes a method to extract PCB11 (3,3'-dichlorobiphenyl) and its monohydoxylated metabolites from mouse tissues via a liquid-liquid extraction followed by solid-phase extraction clean-up. The level of PCB11 and OH-PCB11s in the tissue are subsequently measured by gas chromatography-tandem mass spectrometry. 

[BEFORE_START]
Always wear proper Personal Protective Equipment and work in a fume hood when working with hexanes, hydrochloric acid, MTBE, diazomethane, polychlorinated biphenyl (PCBs) and their derivatives, and strong acids and bases. 

Note that this protocol involves work with experimental animals and requires prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.

[STEPS]
SECTION: Tissue homogenization
7. 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.
SECTION: Addition of Analytical Standards
11. 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
SECTION: Extraction
17. Invert tube-A on the tube rotator and centrifuge.
SECTION: Derivatization of OH-PCBs - Day 1
27. Add 5 drops of methanol to tube-C all the samples except Reference standard and vortex for 5 s.
SECTION: Base clean-up step
36. Add 3 mL of hexane with a repeater pipettor and vortex.  
SECTION: Internal Standard (IS) Spiking IS and preparation for analysis
52. 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.
SECTION: ARCHIVING OF ALL SAMPLE EXTRACTS
61. Recap the samples after the GC-MS/MS or GC-ECD analysis
SECTION: Tissue homogenization
8. Add 3 mL of 2-propanol to all tube-A samples with repeater pipettor.
SECTION: Tissue homogenization
9. 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).
SECTION: Tissue homogenization
10. Add 1 mL of diethyl ether with a repeater pipettor in a fume hood to each sample, recap, and vortex gently. 
SECTION: Addition of Analytical Standards
12. 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
SECTION: Addition of Analytical Standards
13. 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)
SECTION: Addition of Analytical Standards
14. Cap tube-A and the Reference standard.     
SECTION: Extraction
18. Add 5 mL of phosphoric acid (H3PO4) in NaCl with repeater pipettor to tube-B.
SECTION: Extraction
20. 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.
SECTION: Extraction
21. Transfer the supernatant from tube-A to tube-B.
SECTION: Extraction
23. Transfer the organic layer (top layer) to a new tube-C with new long pipettes
SECTION: Extraction
24. Re-extract tube-B with 3 mL of hexane-diethyl ether (9:1), vortex, and centrifuge
SECTION: Extraction
26. Evaporate solvent in tube-C under a gentle stream of nitrogen to ~100 µL. 

Note: Do not evaporate to dryness.
SECTION: Derivatization of OH-PCBs - Day 1
28. 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.
SECTION: Derivatization of OH-PCBs - Day 1
29. 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).
SECTION: Derivatization of OH-PCBs - Day 2
35. Evaporate the Reference standard to near dryness (~ 50 μ L) and put it aside for the IS spiking step.

SECTION: Derivatization of OH-PCBs - Day 2
34. Evaporate the excess of ether and diazomethane in a fume hood under a gentle N2 flow (no yellow color, ~200 µL).
SECTION: Base clean-up step
37. Add 2 mL of KOH-95% EtOH solution with a repeater pipettor and vortex. 
SECTION: Base clean-up step
38. Heat for 1 hour in a water bath at 50 °C, vortexing the samples approximately every 15 mins.
SECTION: Base clean-up step
43. Evaporate the solvent under a gentle stream of nitrogen to ~0.5 mL
SECTION: Base clean-up step
41. Add 2 mL of hexane using a repeater pipettor to tube-C and vortex. 
SECTION: Base clean-up step
39. Add 7 mL of Mili-Q water with repeater pipettor, invert, and centrifuge. 
SECTION: Internal Standard (IS) Spiking IS and preparation for analysis
54. Transfer extracts from tube-E and the Reference standard with a long pipette to a combusted crimp-style GC vial. 
SECTION: Lipid removal (tube-E, volume ~10mL)
50. Concentrate the samples in tube-E under a gentle stream of nitrogen to ~200 µL.
SECTION: Lipid removal (tube-E, volume ~10mL)
46. 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.
SECTION: Lipid removal (tube-E, volume ~10mL)
45. 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.
SECTION: Quality control samples
4. 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. 
SECTION: Preparation
1. Combust silica gel, glass wool, all glassware (cartridges, short and long pipettes, tubes, beaker/volumetric flask, funnel), scalpel, forceps, and spatula.
SECTION: Preparation
2. Print the working sheet (see attachment for an example).
SECTION: DAY 1: TISSUE HOMOGENIZATION, EXTRACTION, AND DERIVATIZATION
5. Remove the analytical standards for the ORP and the SS from the freezer and allow to warm to room temperature (~ 30 min). 

SECTION: Tissue homogenization
6. Label tubes as A1, A2 etc, and Reference standard.
SECTION: Extraction
16. 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.  
SECTION: Extraction
19. Transfer the supernatant from tube-A to tube-B with a long pipet. 
SECTION: Base clean-up step
40. Transfer the top layer to tube-D with a short pipette. 
SECTION: Base clean-up step
42. Let samples sit on the lab bench for 15 mins, centrifuge, and transfer the top layer to combine with tube-D (volume ~5 mL). 
SECTION: Lipid removal (tube-E, volume ~10mL)
47. Rinse tube-D twice (2 x 0.5 mL) with hexane:DCM (1:1) and pass through the cartridge each time.
SECTION: Lipid removal (tube-E, volume ~10mL)
49. Turn off the manifold vacuum and remove tube-E from the SPE manifold.
SECTION: Lipid removal (tube-E, volume ~10mL)
51. 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.
SECTION: DISPOSAL
60. 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. 
SECTION: ARCHIVING OF ALL SAMPLE EXTRACTS
62. 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.
SECTION: Internal Standard (IS) Spiking IS and preparation for analysis
58. 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.
SECTION: Internal Standard (IS) Spiking IS and preparation for analysis
56. Prepare the solvent blank by filling a GC vial with hexane. 

Note: Use the same hexane used for the extractions.
SECTION: Internal Standard (IS) Spiking IS and preparation for analysis
57. Cap all vials immediately with a crimp cap and crimper to seal the cap. 
SECTION: Internal Standard (IS) Spiking IS and preparation for analysis
55. Rinse tube-E with hexane and combine with to the GC vial to give a final volume of ~0.6 mL. 
SECTION: Internal Standard (IS) Spiking IS and preparation for analysis
53. Vortex each tube-E.
SECTION: Preparation
3. Label all sample tubes (Tube-A, Tube-B, Tube-C, Tube-D, and Tube-E).
SECTION: Lipid removal (tube-E, volume ~10mL)
48. Repeatedly adding hexane:DCM (1:1) mixture (total volume ~ 9 mL) till the eluent in tube-E reaches 10 mL mark.
SECTION: DAY 2: BASE CLEANUP, LIPID REMOVAL, AND INTERNAL STANDARD SPIKING
31. Make fresh KOH-95% EtOH solution and set it aside for the base cleanup step.
SECTION: DAY 2: BASE CLEANUP, LIPID REMOVAL, AND INTERNAL STANDARD SPIKING
32. Remove the IS standards from the freezer and allow to warm to room temperature (~ 30 min).
SECTION: DAY 2: BASE CLEANUP, LIPID REMOVAL, AND INTERNAL STANDARD SPIKING
30. Label GC vials.
SECTION: Lipid removal (tube-E, volume ~10mL)
44. Prepare the SPE cartridges.

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. 
SECTION: Lipid removal (tube-E, volume ~10mL)
44.1. Add uncombusted tubes to the bottom of the SPE manifold to collect waste eluent from the SPE cartridge.
SECTION: Lipid removal (tube-E, volume ~10mL)
44.2. Replace the top and place the clean SPE cartridges into each port.
SECTION: Lipid removal (tube-E, volume ~10mL)
44.3. Add glass wool to the SPE cartridge and pack it down with the end of a glass pipet. 
SECTION: Lipid removal (tube-E, volume ~10mL)
44.4. Add 0.2 g of silica gel with a combusted small glass funnel to the SPE cartridges.
SECTION: Lipid removal (tube-E, volume ~10mL)
44.5. Add 2 mL of hexane:DCM (1:1) with a long pipette to rinse to the SPE cartridge.
SECTION: Lipid removal (tube-E, volume ~10mL)
44.6. 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.
SECTION: Lipid removal (tube-E, volume ~10mL)
44.7. Mix the suspension with a short glass pipette tip to eliminate air bubbles.
SECTION: Lipid removal (tube-E, volume ~10mL)
44.8. 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.
SECTION: Extraction
22. Invert tube-B for 5 min and centrifuge.  
SECTION: Extraction
25. Transfer the top layer to combine with the extract in tube-C (~ 7 mL).
SECTION: Addition of Analytical Standards
15. Put the Reference standard aside for the derivatization step.
SECTION: Internal Standard (IS) Spiking IS and preparation for analysis
59. Use the Excel worksheet included with this protocol to calculate the levels of PCB11 and its hydroxylated metabolites after gas chromatographic analysis.
SECTION: DAY 2: BASE CLEANUP, LIPID REMOVAL, AND INTERNAL STANDARD SPIKING
33. Turn on the water bath with setting of 50 °C.