Sampling Procedure and Analysis of Antibiotic Concentrations
Drain fluid and venous blood samples were collected simultaneously at random time points after surgery until the drains were removed as part of the standard treatment. The drain samples were collected at random time points that varied among patients to obtain high-resolution concentrations over time for the entire patient population. Before sampling, the volume of fluid in the drain container was registered and discarded. Then, 10 mL of the newly formed fluid was aspirated for analysis from the drain tube. As a standard, the drain sample was obtained from the inframammary drain. After anonymization, samples from the breasts that received placebo were discarded by an unblinded member of the coordinating trial unit of the BREAST-AB trial. In addition, a venous blood sample was obtained at the time of the drain sample. The blood and drain samples were collected in EDTA tubes, kept at 5 °C, and centrifuged within 30 minutes at a speed of 3000The concentrations of gentamicin, cefazolin, and vancomycin were analyzed using high-performance liquid chromatography–mass spectrometry with a limit of quantitation of 0.05 μg/mL. Calibration and quality control samples were prepared in blank human plasma and 0.1% saline, respectively. Benzylpenicillin was used as an internal standard, and plasma samples were precipitated by using 250 μg/mL of plasma on a 30-kDa molecular weight cutoff filter followed by dilution of the filtrate. For a more detailed description of the sample analysis, see the eMethods in 