The matrix effect caused by the PCIS and the number of data points below the LLOQ in the elution area of the analytes-ofinterest also need evaluation to optimize the concentration. This requires the injection of biological samples while infusing the different PCIS concentrations. For the example method, as Fig. shows, the matrix effect caused by the PCIS showed a slight decreasing trend with increasing concentration, with a mean of 82% at 25 ng/mL to 73% at 300 ng/mL. It should be noted, that because no correction could be applied, there was high variation between the triplicate measurements. Still, we considered the matrix effect caused by the PCIS to be acceptable across all concentration levels. However, the number of data points below the LLOQ in the endocannabinoid elution area was >5 up to 100 ng/mL (Fig. ). As each of these data points potentially disturbs the correction, a concentration of 150 ng/mL was selected as a compromise between costs, induced matrix effect and reliable signal measurement. When doing method development with more than one PCIS, an equal-intensity (as opposed to equal-concentration) mix can be used at various levels to evaluate the matrix effect caused by the PCIS mix and to evaluate the number of data points below the LLOQ of the most suppressed signal.