C . C . Honeycutt et al .: J Extra Corpor Technol 2023 , 55 , 159 – 166 161
Figure 1 . ECMO and CRRT circuit configurations . ( A ) ECMO complete circuit . ( B ) ECMO Oxygenator circuit . ( C ) ECMO Pump circuit . ( D ) CRRT circuit . ECMO – extracorporeal membrane oxygenation ; CRRT – continuous renal replacement therapy .
net 0 mL / h . The 0 mL / h patient fluid removal setting induced the system to remove the extra fluid added by the pre-blood pump ( 700 mL / h ) and replacement fluids ( 300 mL / h ) via the effluent pump ( Q EFF ).
Control setup
For ECMO and CRRT circuits , six controls were analyzed to determine drug degradation during the experiments . For these controls , 45 mL of blood prime solution was drawn from the primed circuit before drug administration but after at least 5 min of circulation to ensure adequate mixing and transferred to polypropylene centrifuge tubes ( 229,426 , CELLTREAT , Pepperell , MA ). The control samples were capped and maintained at 37 ° Cinawaterbath .
Drug administration and sample collection
Meropenem was dosed into the ECMO and CRRT circuits via arterial ports to achieve a concentration of 20 lg / mL and 50 lg / mL , respectively , both within the therapeutic range and above the minimum inhibitory concentration of meropenem [ 33 – 35 ]. The drug was administered at time = 0 . The controls for ECMO and CRRT were also dosed to achieve concentrations of 20 lg / mL and 50 lg / mL , respectively . In controls , the drug was administered at time = �5 min . The test tubes were then capped and placed into a gentle rotator at room temperature . The control tubes were placed into a water bath at 37 ° C at time = 0 .
For ECMO circuits and controls , samples were collected at 1 , 5 , 15 , and 30 min and 1 , 2 , 3 , 4 , 8 , 12 , and 24 h . For CRRT circuits and controls , samples were collected at 1 , 5 , 15 , and 30 min and 1 , 2 , 3 , and 4 h . In both ECMO and CRRT circuits , ~ 3 mL of circuit blood was drawn as “ waste ” with a syringe prior to sample collection and returned afterward . Samples were collected with syringes and then transferred to untreated microcentrifuge tubes . In CRRT circuits , hemofiltrate samples were collected at each time point just before the effluent bag . After sample collection in ECMO and CRRT circuits , the blood was centrifuged at 3,000 g for 10 min at 4 ° C . Plasma was then pipetted into a cryovial ( Fisher Scientific , Pittsburgh , PA ) and stored at �80 ° C . Hemofiltrate samples were transferred to a cryovial and stored at �80 ° C after collection .
Analysis
Meropenem concentrations in plasma and hemofiltrate were measured at OpAns Laboratory ( Durham , NC ) using highperformance liquid chromatography tandem mass spectrometry ( HPLC-MS / MS ) with [ 2H6 ] meropenem as an internal standard . Briefly , meropenem was extracted from 10 lL of a sample using methanol protein precipitation before analysis on an Agilent 6400 Series Triple Quadrupole Mass Spectrometer . Reverse phase chromatography using 0.1 % ( v / v ) formic acid in water or methanol with a Poroshell 120 EC-C18 ( Agilent ) column preceded electrospray ionization in positive ion mode with multiple reaction monitoring ( precursor and product ion m / z of 384 / 390 and 141 / 147 for meropenem and [ 2H6 ] -Meropenem , respectively ). The assay was validated using standard curves achieving coefficients of determination ( R 2 ) > 0.9956 with coefficients of variation < 6.38 % for concentrations across the range of the standard curves