Calibration curves. The calibration curves were freshly prepared on three different days to assess the linearity of the method. For this purpose, an 8-point calibration line was created by serially diluting the standards. The concentrations of these calibration points are presented in Table S2. Three types of calibration lines were prepared: 1) Neat solvents; 2) Spiking standards in HepG2 cells before performing the extraction as described in the sample preparation section; 3) Spiking standards in HepG2 cells after extraction. To determine the linear range, an unweighted linear regression model was employed. The calculation of various validation parameters was performed using cal-3 (low), cal-5 (medium), and cal-7(high) concentration levels.
Calibration curves. A minimum of 5 points are used for every calibration curve. If the R2 of the line is less than 0.995, the equipment is checked/adjusted and a new calibration curve is made. The calibration curve should always capture the range of results expected from the samples. If sample results are more than 10% above or below the highest or lowest calibration point, a new curve should be made or samples should be diluted and reanalyzed. The individual procedure for producing the calibration curves varies by instrument and is detailed in the specific SOP.
