Two key parameters namely collision energy (CE) and declustering potential (DP, a voltage applied to the orifice to minimize solvent clusters and help on desolvation of ions) were optimized by either direct infusion (DP) or using a LC-MS method (CE). For DP, sub-mixes of all compounds at either 10 or 100 ng/mL (depending on ionization efficiency) of all standards were constantly injected in the source using a syringe pump set at 5µL/min. Automatic DP optimization was performed in SCIEX OS in the range from 10 to 300 V. For CE optimization, MS 2 spectra was first acquired using DDA with inclusion list for each of the sub-mixes at in the range of 100 to 1000 ng/mL. MS 2 spectra were acquired using the collision energy spread (CES) at 35±15 V. This allows for a combined (and representative) spectra of 3 collision energies: 20, 35 and 50 V. Next, the 5 most intense fragments (or fragments known to work best in our targeted approach using low-resolution instruments ) were selected for further optimization. Finally, an MRM-HR method was created with all selected transitions, and sub-mixes were injected multiple times using the above described LC-MS method with one specific CE for each injection, in the range from 15 to 60 V, with steps of 5 V (total of 9 injections per submix). Peak areas were used to select the best fragment and CE (Table )