Unfractionated heparin (500 U) was added to the jars for anticoagulation. Fentanyl (1.5 ��g), morphine (7.5 ��g), midazolam below (7.5 ��g), meropenem (0.75 mg), vancomycin (3 mg), propofol (75 ��g), dexmedetomidine (0.375 ��g), thiopentone (1.5 mg), ceftriaxone (3.75 mg), linezolid (0.75 mg), ciprofloxacin (0.375 mg), fluconazole (0.75 mg), and caspofungin (0.375 mg) were added to the control jars after collection of baseline blood samples. These amounts were chosen in order to produce study drug concentrations that were similar to those achieved in the ECMO circuit. The jars were then placed in an incubator at 37��C and rocked continuously to ensure even distribution of the drugs.Blood sample collectionPost-oxygenator blood was collected into lithium heparin tubes (5 mL) at baseline and at 2, 5, 15, 30, 60, 120, and 360 minutes and at 12 and 24 hours after addition of the drugs to the circuit.

Blood samples (5 mL) were also obtained from the control jars at time intervals identical to that of the circuit. All blood samples were stored on ice and centrifuged (10 minutes at 3,000g), and the plasma was separated and stored in clean pre-labeled polypropylene cryogenic vials and stored at -80��C until analysis.Measurement of drugs in plasma samplesAn on-line solid-phase extraction (SPE) Symbiosis Pharma system (Spark Holland, Emmen, The Netherlands) was used to extract the analytes of interest (fentanyl, morphine, and midazolam) and two internal standards (morphine-d3 and 1-hydroxymidazolam-d5) from plasma samples simultaneously [17].

Mass spectrometry in ESI (electrospray ionization) mode (API 5500; AB Sciex, Framingham, MA, USA) triple quadropole system was used as the detector. Liquid chromatography and extraction method were created by Symbiosis Pro for Analyst (version 2.1.0.0) and submitted to the MS controlling software (Analyst 1.5.1). Meropenem and vancomycin concentrations in the collected plasma samples were determined by separate validated chromatographic assay methods. Meropenem and the internal standard (cefotaxime) were detected by ultraviolet absorbance at 304 nm. Vancomycin analysis was by liquid chromatography-tandem mass spectrometry on an Applied Biosystems API2000 (Applied Biosystems, Foster City, CA, USA) with Shimadzu autosampler (Shimadzu Corporation, Kyoto, Japan). Vancomycin and the internal standard (teicoplanin) were detected by positive-mode MRM (multiple reaction monitoring).

All samples were assayed alongside calibration standards and quality control samples and met the acceptance criteria.Statistical analysisLinear mixed effects modeling was used to examine the change in concentration over time. This model accounts for the repeated responses from the same circuit by using a random AV-951 intercept. The mixed effects model was fitted by using the R statistical software [18] version 2.13.2 and the ‘lme4′ library.