Vortex genie 2

Pinostrobin (10 μg/mL final concentration) was added to 120 μL of all serum samples with the exception of the 0 h sample. Samples were vortexed for 1 min using a Vortex Genie-2 (VWR Scientific, West Chester, PA). One (1.0) mL of cold HPLC grade acetonitrile (pre-stored at -20 C) was added to samples to precipitate plasma proteins, vortexed for 5 min (Vortex Genie-2, VWR Scientific), and centrifuged at 15,000 rpm for 15 min before the supernatant was transferred to new, labeled 2 mL centrifuge tubes. Samples were evaporated to dryness using a Savant SPD1010 SpeedVac Concentrator without heat (Thermo Fisher Scientific, Inc., Asheville, NC). The residue was reconstituted with 100 µL of mobile phase, vortexed for 1 min and centrifuged at 15,000 rpm for 5 min; the supernatant was transferred to HPLC vials and 120 µL was injected into the HPLC system.

All extraction work was conducted in a dedicated lab only used for processing sensitive samples with low DNA content and for pre-PCR work. The filters were extracted following the DNEasy PowerWater Kit protocol (Qiagen AG, Hombrechtikon, Switzerland) with the following adjustments: after adding 1 ml of solution PW 1 and shredding of the filter into small pieces using a pipette tip, the tubes were incubated at 65°C for 10 minutes in an oven (VWR Peqlab, Dietikon, Switzerland). After vortexing for 5 minutes at full speed on a Vortex-Genie 2 (VWR, Dietikon, Switzerland) with a 5 ml tube adapter (QIAGEN AG, Hombrechtikon, Switzerland) samples were subjected to an additional incubation period of 10 minutes at 65°C. Samples were then centrifuged at 8000 x g with an Eppendorf 5427 R centrifuge and rotor FA-45-12-17 for 5 ml tubes (VWR, Dietikon, Switzerland). The extraction protocol used is described in detail in the Supplement (Supplementary data 2). A no-template extraction control containing only a clean filter paper was included in all extraction runs. The processed samples were stored at -20°C until further analysis.
2.6. Real-time quantitative qPCR