TITLE: In vitro release, extraction, and analysis of PCBs and metabolites from polymeric implants  
AUTHORS: Amanda J. Bullert, Hui Wang, Hansjoachim Lehmler

[DESCRIPTION]
This protocol describes a method to determine the release of 2,2’,5,5’-tetrachlorobiphenyl-4-ol (4-OH-PCB 52), a human-relevant metabolite of PCB 52 (2,2’,5,5’-tetrachlorobiphenyl), from polymeric implants in vitro. Implants containing 0%, 1%, 5%, or 10% of 4-OH-PCB 52 by weight were incubated in 10% bovine calf serum in PBS (pH = 7.4). The bovine calf serum/PBS mixture was replaced regularly, typically every 24 h, for up to 28 days. The 4-OH-PCB 52 released from the implants was extracted from an aliquot of the bovine calf serum/PBS mixture with hexanes, and levels of 4-OH-PCB 52 were quantified by UV/Vis spectroscopy against a matrix-matched standard calibration curve. This approach demonstrated the continuous release of 4-OH-PCB 52 from the implants over 28 days under physiological conditions. 

[BEFORE_START]
Always wear proper Personal Protective Equipment and work in a fume hood when working with hexanes, hydrochloric acid, MTBE, penicillin-streptomycin, and polychlorinated biphenyl (PCBs) derivatives. 

[STEPS]
SECTION: Extraction
7. Transfer 3 mL of each media sample from the in vitro release experiment described above into clean, well-labeled glass vials for extraction (Vial_A)
SECTION: Extraction
7.1. TIP: A seven-point calibration curve of the study compound is prepared and extracted concurrently. The calibration curve can then be used to check the instrument for drift. If you are running many samples, consider running the calibration curve before, during, and after all of the samples
SECTION: Extraction
8. Acidify all 3 mL media samples by adding 1 mL of 2 M HCl and vortex the sample vials (Vial_A)
SECTION: Extraction
9. Add 4 mL of hexane:MTBE (9:1) to the vials (Vial_A)
SECTION: Extraction
10. Cap the vials (Vial_A) and shake vigorously
SECTION: Extraction
11. If an emulsion forms, add 0.5 g of NaCl to break the emulsion. Otherwise, skip this step
SECTION: Extraction
12. Invert vials (Vial_A) for 5 min and centrifuge vials at 1811 g (3,000 rpm) for 5 min
SECTION: Extraction
13. Transfer the organic phase (top layer) to new vials (Vial_B)
SECTION: Extraction
14. Cap the vials containing to organic extract (Vial_B) and store them at -20°C until analysis
SECTION: UV/Vis samples
15. Transfer 2 mL of hexane:MTBE (9:1), calibration standards, or organic sample extract (vial_B) to a clean quartz cuvette
SECTION: UV/Vis samples
16. Analyze samples on the UV/Vis spectrometer at 285 nm. Keep the instrument/sample temperature at 25°C if a circulating water bath is available. Use the sample order shown in steps 16.1 to 16.4

Note: Dilute samples if the reading is over 1 in absorbance
SECTION: UV/Vis samples
17. Rinse and use the same cuvette (minimize differences between cuvette walls = refraction differences) for each sample.
SECTION: UV/Vis samples
18. Repeat steps 15 to 17 two more times. Analyzing all extracts in triplicate will provide more robust and reproducible results
SECTION: In vitro release of 2,2’,5,5’-tetrachlorobiphenyl-4-ol (4-OH-PCB 52) from implants
1. Add 10 ml of media (10% bovine serum in PBS containing 1% penicillin-streptomycin, v/v) to 20 mL amber vials with screw caps

Note: It is helpful to add penicillin-streptomycin to the media because the samples may be stored for several weeks before extraction and analysis
SECTION: In vitro release of 2,2’,5,5’-tetrachlorobiphenyl-4-ol (4-OH-PCB 52) from implants
2. Add a 2 cm piece of a polymeric implant containing either 0%, 1%, 5%, or 10% of 2,2’,5,5’-tetrachlorobiphenyl-4-ol (4-OH-PCB 52) per vial. Each concentration is analyzed in triplicate. Document the weight of each implant and the time point of media addition and collection
SECTION: In vitro release of 2,2’,5,5’-tetrachlorobiphenyl-4-ol (4-OH-PCB 52) from implants
3. Place vials in a shaking water bath at 37 °C at a speed of 40 rpm
SECTION: In vitro release of 2,2’,5,5’-tetrachlorobiphenyl-4-ol (4-OH-PCB 52) from implants
4. Replace the media at designated time points by transferring old media to 10 mL glass tubes and adding 10 mL of fresh media to the vials, leaving the implant in the vials
SECTION: In vitro release of 2,2’,5,5’-tetrachlorobiphenyl-4-ol (4-OH-PCB 52) from implants
6. Record time of media transfers (1 hour, 4 hours, 12 hours, 24 hours, and every 24 hours)
SECTION: In vitro release of 2,2’,5,5’-tetrachlorobiphenyl-4-ol (4-OH-PCB 52) from implants
5. Store old media capped at 4°C until extraction

Note: The media solutions must be stored in glass vials and PTFE-coated caps because PCB compounds can partition into plastic ware over time
SECTION: UV/Vis samples
16.2. Analyze 2 mL of the extract from all calibration standards to generate a calibration curve for quantification purposes

    
SECTION: UV/Vis samples
16.3. Analyze 2 mL of all sample extracts
SECTION: UV/Vis samples
16.4. Analyze 2 mL of the extract from all calibration standards for a second time after all samples have been analyzed
SECTION: UV/Vis samples
16.1. Use 2 mL of hexane:MTBE (9:1, v/v) to zero the instrument as the first sample
SECTION: UV/Vis samples
19. Calculate the concentrations of 4-OH-PCB 52 in the hexane:MTBE (9:1, v/v) extracts from the calibration curve. 

EXAMPLE: The file "Example_release_data.xlsx" contains release data from four implants containing 5% (sample 1 and 2) and 10% (sample 3 and 4) of 4-OH-PCB 52 from an in vitro release study. As outlined in this protocol, media samples were collected at various time points and extracted using hexanes:MTBE (9:1, v/v). The absorption was then measured spectrophotometrically using UV/Vis at 285 nm. The concentration of the test compound in the extract was calculated based on the equation from the calibration curve: concentration = (Absorption-0.0023)/0.0085; see Figure 1.