Table 2.

Goals to independent and dependent variables for analytical method optimization

Variables	Goal	Lower limit	Upper limit
Independent variables
Organic phase ratio (%)	In range	60	80
Buffer concentration (mM)	In range	10	20
Flow rate (mL/min)	In range	0.8	1.2
Dependent variables
Theoretical plate number	In range	8000	-
Retention time (min)	In range	-	10
Peak area (LU·s) *	Maximize	2.5
Resolution 1	In range	2	-
Resolution 2	In range	2	-

^* The optimization objective was to maximize the peak area, while the design space was constrained to ensure a minimum response of 2.5 LU·s, which is sufficient to meet the sensitivity required for achieving a LLOQ of 5 ng/mL