TITLE: Liquid-liquid extraction of 3,3&#39;-dichlorobiphenyl (PCB11) and its hydroxylated metabolites from animal blood/serum
AUTHORS: Xueshu Li, Maxwell W Shumaker, Hansjoachim Lehmler

[DESCRIPTION]
This protocol describes the extraction of 3,3'-dichlorobiphenyl (PCB11) and its monohydoxylated metabolites from mouse blood/serum using liquid-liquid extraction followed by solid-phase extraction clean-up. The levels of PCB11 and OH-PCB11s in the blood/serum samples 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: Sample preparaiton
8. Place whole blood (0.3-0.5 g) or serum (~ 0.2 g) in tube-A. Record weight on the working sheet.
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. Add 1 mL of 6 M HCl to tube-A, vortex for 10 s.
SECTION: Derivatization of OH-PCBs -  Day 1
31. Get samples, ice box, and serological pipettes ready in a fume hood.
SECTION: Base clean-up step
42. Add 3 mL of hexane with a repeat pipettor and vortex. 
SECTION: Lipid removal (tube-E, volume ~10 mL)
50. 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: Internal Standard (IS) Spiking IS and preparation for analysis
58. 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.
SECTION: Addition of Analytical Standards
12. 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
SECTION: Addition of Analytical Standards
13. 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)
SECTION: Addition of Analytical Standards
14. Cap tube-A and the Reference standard.   
SECTION: Extraction
22. Transfer the organic phase (top layer) from tube-A to tube-B with a short glass pipette.
SECTION: Extraction
29. Transfer the organic layer (top layer) form tube-B to tube-C.
SECTION: Derivatization of OH-PCBs -  Day 1
34. Add ~0.5 mL of diazomethane to each sample and 1 mL of diazomethane to the Reference standard using a 10 mL serological pipette.
SECTION: Derivatization of OH-PCBs -  Day 1
35. 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
41. 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
40. Evaporate the excess of ether and diazomethane in a fume hood under a gentle nitrogen flow (no yellow color, ~200 µL).
SECTION: Base clean-up step
43. Add 2 mL of KOH-95% EtOH solution with a repeat pipettor and vortex. 
SECTION: Base clean-up step
44. Heat for 1 hour in 50 °C water bath, vortexing the samples approximately every 15 min.
SECTION: Base clean-up step
45. Add 7 mL of Mili-Q water with a repeat pipettor, invert, and centrifuge. 
SECTION: Internal Standard (IS) Spiking IS and preparation for analysis
61. Rinse tube-E with hexane (~150 µL) and combine with to the GC vial insert to give a final volume of ~300 µL. 
SECTION: Lipid removal (tube-E, volume ~10 mL)
52. With long glass pipettes, slowly load the extracts from tube-D into the SPE cartridges and pass them through the cartridges 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 ~10 mL)
51. 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
5. 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. 
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: Sample preparaiton
7. 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
24. Transfer the organic phase (top layer) from tube-A to tube-B with a short pipette.
SECTION: Base clean-up step
49. Evaporate the solvent under a gentle stream of nitrogen to ~0.5 mL.
SECTION: Lipid removal (tube-E, volume ~10 mL)
53.  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 ~10 mL)
54. Repeatedly add the hexane:DCM (1:1) mixture (total volume ~ 9 mL) until the eluent in tube-E reaches 10 mL.
SECTION: Sample preparaiton
9. Add 3 mL of 1% KCl solution to tube A containing blank samples. 
SECTION: Extraction
18. Add 5 mL of 2-propanol to tube-A, vortex for 10 s.
SECTION: Extraction
19. Add 5 mL of hexane-MTBE mixture 1:1 (v/v) to tube-A.
SECTION: Extraction
20. Cap the tube-A with caps with a PTFE septum.

SECTION: Extraction
25. Add 3 mL of 1% KCl solution to tube-B.
SECTION: Extraction
26. Invert tube-B for 5 min and centrifuge.
SECTION: Extraction
27. Transfer the organic layer (top layer) form tube-B to tube-C.
SECTION: Extraction
28. Re-extract tube-B with 3 mL of hexane, vortex, and centrifuge.
SECTION: Extraction
30. Evaporate solvent in tube-C under a gentle stream of nitrogen to ~100 µL. 

Note: Do not evaporate to dryness.
SECTION: Internal Standard (IS) Spiking IS and preparation for analysis
62. Prepare the solvent blank by filling a GC vial with hexane. 

Note: Use the same hexane used for the extractions.
SECTION: ARCHIVING OF ALL SAMPLE EXTRACTS
66. Recap the samples after the GC-MS/MS or GC-ECD analysis.
SECTION: Preparation
3. Label all sample tubes (Tube-A, Tube-B, Tube-C, Tube-D, and Tube-E).
SECTION: Addition of Analytical Standards
15. Put the Reference standard aside for the derivatization step.
SECTION: Extraction
23. Re-extract tube-A with 3 mL of hexane-MTBE (1:1, v/v), vortex for 10 s, and centrifuge.
SECTION: Extraction
21. Invert tube-A on the tube rotator and centrifuge.
SECTION: DAY 1: SAMPLE PREPARATION, EXTRACTION, AND DERIVATIZATION
6. Remove the analytical standards for the ORP and the SS from the freezer and allow to warm to room temperature (~ 30 min).
SECTION: Preparation
4. 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. 
SECTION: DAY 2: BASE CLEANUP, LIPID REMOVAL, AND INTERNAL STANDARD SPIKING
36. Label GC vials.
SECTION: DAY 2: BASE CLEANUP, LIPID REMOVAL, AND INTERNAL STANDARD SPIKING
39. Turn on the water bath with setting of 50 °C.
SECTION: DAY 2: BASE CLEANUP, LIPID REMOVAL, AND INTERNAL STANDARD SPIKING
38. 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
37. Make fresh KOH-95% EtOH solution and set it aside for the base cleanup step.
SECTION: Base clean-up step
48. 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: Base clean-up step
47. Add 2 mL of hexane using a repeat pipettor to tube-C and vortex. 
SECTION: Base clean-up step
46. Transfer the top layer to tube-D with a short pipette. 
SECTION: Lipid removal (tube-E, volume ~10 mL)
50.6. 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 ~10 mL)
50.1. Add uncombusted tubes to the bottom of the SPE manifold to collect the waste eluent from the SPE cartridges.
SECTION: Lipid removal (tube-E, volume ~10 mL)
50.5. Add 2 mL of hexane:DCM (1:1) with a long pipette to rinse to the SPE cartridge.
SECTION: Lipid removal (tube-E, volume ~10 mL)
50.4. Add 0.2 g of silica gel with a combusted small glass funnel to the SPE cartridges.
SECTION: Lipid removal (tube-E, volume ~10 mL)
50.3. Add glass wool to the SPE cartridge and pack it down with the end of a glass pipette. 
SECTION: Lipid removal (tube-E, volume ~10 mL)
50.2. Replace the top and place the clean SPE cartridges into each port.
SECTION: Lipid removal (tube-E, volume ~10 mL)
50.7. Mix the suspension with a short glass pipette tip to eliminate air bubbles.
SECTION: Lipid removal (tube-E, volume ~10 mL)
50.8. 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: Lipid removal (tube-E, volume ~10 mL)
57. 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.
SECTION: Lipid removal (tube-E, volume ~10 mL)
56. Concentrate the samples in tube-E under a gentle stream of nitrogen to ~200 µL.
SECTION: Lipid removal (tube-E, volume ~10 mL)
55. Turn off the manifold vacuum and remove tube-E from the SPE manifold.
SECTION: Internal Standard (IS) Spiking IS and preparation for analysis
60. Transfer extracts from tube-E and the Reference standard with a long pipette to a combusted crimp-style GC vial with insert. 
SECTION: Internal Standard (IS) Spiking IS and preparation for analysis
59. Vortex each tube-E.
SECTION: Internal Standard (IS) Spiking IS and preparation for analysis
65. 
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.
SECTION: Internal Standard (IS) Spiking IS and preparation for analysis
64. 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
63. Cap all vials immediately with a crimp cap and crimper to seal the cap. 
SECTION: ARCHIVING OF ALL SAMPLE EXTRACTS
67. 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: Derivatization of OH-PCBs -  Day 1
33. Take the diazomethane vial out of freezer and place in an ice box in the fume hood.
SECTION: Sample preparaiton
10. 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.
SECTION: Derivatization of OH-PCBs -  Day 1
32. Add 5 drops of methanol to tube-C, except for the Reference standard, and vortex for 5 s.