2.2. Calibration of ion-selective electrodes (ISE)

Ion-selective electrodes (ISE) (Octens, Belgium) were employed to monitor and quantify the real-time changes in CVDL concentration in test media. ISE have previously been successfully utilized as in situ probes to accurately determine real-time drug concentrations in numerous varied media¹⁷, including complex media such as milk¹⁸ and simulated gastric fluids¹⁸. Prior to experiments, the electrodes were conditioned in base buffer (10 mM phosphate buffer, to pH 6.50) containing 50 µg/mL drug at 37 °C for 7 days, conditions previously determined to sufficiently condition the ISE to CVDL (data not shown). Calibration curves were generated at the beginning of each experiment, and were constructed by stepwise addition (5 × 20 µL additions) of a 10 mg/mL CVDL in methanol standard solution into a glass vial containing 20 mL test medium, achieving an end concentration of 50 µg/mL.