Media were extracted 1:1 with dichloromethane overnight, after which the solvent was separated and dried. Extracts (equivalent to 1 mL media) were resuspended in dichloromethane, spotted on Si-gel 60 F254 plates (EMD Millipore, Burlington, MA, USA, cat #1.05735.0001), and run in a mobile phase of 5:11:4 dichloromethane–hexane–acetone plus 0.5% v/v acetic acid. Plates were stained with p-anisaldehyde reagent (75 mL ethanol, 2.5 mL sulfuric acid, 1 mL acetic acid, and 2 mL p-anisaldehyde (Sigma Aldrich, St. Louis, MO, USA, A88107-100G)) and heated for 10 min at 105 °C. This reagent stains all ART drugs dark blue–purple, except for ART (dark pink) and dART (brown). ART, dART, AS, and DHA were quantified in fibroblast media after two-phase extraction with dichloromethane using gas chromatography–mass spectrometry as previously detailed in Kane et al. [44 (link)] on an Agilent GC-MS system (GC, Agilent, Santa Clara, CA, USA, 7890A; MS, Agilent 5975 C).
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