A LC-MS/MS instrument composes of two components: a UPLC chromatography with a sample introduction system, and a triple quadruple mass spectrometer. A mass spectrometrist takes sensitivity, resolution, interference, and so on into consideration. They start with default instrumental parameters of each component, then fine-tune them to reach the best output they could get in a requested time frame. These parameters could be: flow (F), gradient (G), and injection volume (V_inj) of the UPLC chromatography; cone voltage (E_com), collision energy (E_col) of the mass spectrometer.

The method was optimized with respect to four responses: separation of peaks (Sep), peak area (A_peak), length of the analysis (T) and the signal to noise ratio (S/N). A quadratic model, namely central composite face (CCF) featuring 29 runs was used instead of a less powerful linear model