Our method development follows a four-stage engineering process — each stage defined by a specific scientific question, not a checkbox.
Stage 1: UV Absorption Profiling and λmax Selection
Full 190–400 nm UV absorption scan on DAD to identify λmax and confirm no overlap with mobile phase solvent cut-offs. For non-chromophoric compounds, we candidly recommend LC-MS/MS.
Stage 2: Stationary Phase and Mobile Phase Optimization
We systematically evaluate retention and selectivity across C18, C8, HILIC, phenyl-hexyl, and PGC chemistries. Mobile phase pH (2.0–7.5), organic modifier (acetonitrile vs methanol), gradient or isocratic composition, flow rate (0.3–1.5 mL/min), and column temperature (25–45 °C) are optimized targeting resolution Rs > 2.0, tailing factor 0.8–1.5, and retention time reproducibility ±2% across six replicates.
Stage 3: Sample Preparation Strategy
PPT with acetonitrile/methanol for plasma (> 85% recovery), LLE with MTBE or ethyl acetate for lipophilic compounds (> 90%), SPE on Oasis HLB/C18/mixed-mode for tissue and bile, and 96-well SLE for high-throughput. ≤100 µL per replicate for rodent PK. Structural analog IS is sufficient — no costly SIL-IS required.
Stage 4: Fit-for-Purpose Method Validation
Validation follows ICH M10-aligned fit-for-purpose principles. The core package includes calibration linearity (≥6 standards, R2 > 0.99), intra-day and inter-day accuracy/precision at four QC levels (CV < 15%, < 20% at LLOQ), extraction recovery (> 85% with CV < 10%), selectivity screening across six blank matrix lots, and bench-top / freeze-thaw / autosampler stability. Each validation report includes raw chromatograms, calibration plots, and QC back-calculation tables.
