Sample preparation and analysis
DBS samples were stored at -20°C (after drying for a minimum of
30 minutes at room temperature) until analysis. DBS samples collected at
home were sent to the hospital by mail in a sealed plastic bag and
envelope. Venous blood samples were centrifuged and the plasma was
stored at -80°C until analysis. The quantification of concentrations of tezacaftor, tezacaftor-M1, ivacaftor, ivacaftor-M1 and ivacaftor-M6 in
plasma and DBS was performed using liquid chromatography-tandem mass
spectrometry (LC-MS/MS), utilizing the method detailed in our previous
publications. (7, 8) For all compounds the respective lower and
upper limit of quantification (LLOQ/ULOQ) was 0.01 mg/L and 10 mg/L in
plasma and DBS, except for tezacaftor-M1 (LLOQ 0.025 mg/L and ULOQ 12.5
mg/L). DBS concentrations were converted to their ‘estimated
plasma concentrations’ (EPC) by the Passing-Bablok regression equation
as described in our previous article. (8)