Overview of Method Development Exercises Analyte Information • Structure • Properties Method Optimization QC Preparation Appropriate to Range Evaluation of Extraction Strategies (Protein Precipitation, LLE/SLE, SPE) • Phospholipid impact evaluation • Recovery • Matrix Stability • Extract Stability • Initial Carryover Evaluation Post-column infusion experiment Mock Validation Run • Accuracy and Precision • Carryover • Freeze / Thaw Stability • Bench Top (pre-processed) Stability • Long Term Stability • Recovery • Matrix Effect • Selectivity Length of run should mimic longest expected sample set, padded with blanks

Table 1: Overview of Method Development Exercises.