Whatman 903tm paper cards

Whole venous blood was collected using Li-heparin vacutainer tubes (Becton-Dickinson #367884, Franklin Lakes, NJ, USA) or ethylenediaminetetraacetic acid (EDTA) vacutainer tubes (Beckton-Dickinson #367861) mixed by inversion 10 times. Plasma was obtained by centrifugation of the whole blood at 900× g for 10 min at room temperature (RT). The DBS samples were prepared by pipetting 70 µL of Li-heparin whole blood (Li-hep DBS) or EDTA whole blood (EDTA DBS) on to Whatman 903^TM paper cards (GE Healthcare, #10534612, Chicago, IL, USA). Dried plasma spot (DPS) samples were prepared by spotting 40 µL of Li-hep plasma onto Whatman 903^TM paper cards. Additionally, blood via fingerstick was taken using a sterile lancet and directly spotted onto the circles of a paper card (Fingerstick DBS). One to two drops was sufficient to fill the 12-mm inner diameter of the dotted circle when applied in the center. The DBS cards were air-dried at room temperature (RT) for 24 h. The disks were punched out from the DBS using a ¼ inch diameter (6.3 mm) one-hole puncher (ACCO, Catalog: 74005). Punching from an empty area of the sampling card (Blank disks) was performed in between every sample.

Blood spotting volumes on Whatman 903^TM paper cards were optimized. Five blood spotting volumes (40, 50, 60, 70, and 80 µL) were tested. The volumes of extraction buffer (100, 200, 300, 400, 500 µL) and the extraction methods (20 min on ice, overnight at RT, and sonication for 5 min on ice) were also optimized. The potential cross-contamination was assessed by comparing the level of a metabolite in a DBS sample with the levels in the following 2 blank paper punches. Each condition was measured in triplicate.