In vitro release, extraction, and analysis of PCBs and metabolites from polymeric implants

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

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

Place vials in a shaking water bath at 37 °C at a speed of 40 rpm

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

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

Record time of media transfers (1 hour, 4 hours, 12 hours, 24 hours, and every 24 hours)

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)

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

Acidify all 3 mL media samples by adding 1 mL of 2 M HCl and vortex the sample vials (Vial_A)

Add 4 mL of hexane:MTBE (9:1) to the vials (Vial_A)

Cap the vials (Vial_A) and shake vigorously

If an emulsion forms, add 0.5 g of NaCl to break the emulsion. Otherwise, skip this step

Invert vials (Vial_A) for 5 min and centrifuge vials at 1811 g (3,000 rpm) for 5 min

Transfer the organic phase (top layer) to new vials (Vial_B)

Cap the vials containing to organic extract (Vial_B) and store them at -20°C until analysis

Transfer 2 mL of hexane:MTBE (9:1), calibration standards, or organic sample extract (vial_B) to a clean quartz cuvette

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

Use 2 mL of hexane:MTBE (9:1, v/v) to zero the instrument as the first sample

Analyze 2 mL of the extract from all calibration standards to generate a calibration curve for quantification purposes

<img src="https://static.yanyin.tech/literature_test/protocol_io_true/protocols.io.kqdg3ppj1l25/Calibration_curve_figure.jpg" alt="Figure 1. Calibration curve of 4-OH-PCB 52. This matrix-matched calibration curve was prepared by adding known amounts of 4-OH-PCB 52 into the fresh medium. As described in the protocol, aliquots of these standard samples underwent the same extraction process as samples from the implant release study. The absorption was then measured spectrophotometrically using UV/Vis at 285 nm. The file "Calibration_curve_data.xlsx" contains the corresponding data." loading="lazy" title="Figure 1. Calibration curve of 4-OH-PCB 52. This matrix-matched calibration curve was prepared by adding known amounts of 4-OH-PCB 52 into the fresh medium. As described in the protocol, aliquots of these standard samples underwent the same extraction process as samples from the implant release study. The absorption was then measured spectrophotometrically using UV/Vis at 285 nm. The file "Calibration_curve_data.xlsx" contains the corresponding data."/>

Calibration_curve_data.xlsx

Analyze 2 mL of all sample extracts

Analyze 2 mL of the extract from all calibration standards for a second time after all samples have been analyzed

Rinse and use the same cuvette (minimize differences between cuvette walls = refraction differences) for each sample.

Repeat steps 15 to 17 two more times. Analyzing all extracts in triplicate will provide more robust and reproducible results

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.

Example_release_data.xlsx