Combined second harmonic and linear light scattering as a tool for solubility and aggregation screening.

Poor aqueous solubility and complex supersaturation behaviour of small-molecule drugs require rapid, material-sparing analytical tools that capture incipient aggregation and phase separation events under non-equilibrium conditions. This work evaluated a miniaturised optical platform that combined second harmonic scattering (SHS) with linear light scattering (LLS) driven by an ultrafast 1030 nm laser to monitor supersaturated solutions of 12 poorly water-soluble drugs prepared by DMSO solvent shift in 384-well plates. LLS and SHS signals were recorded after 1 h and 24 h and compared with apparent solubilities from a parallel solvent-shift HPLC assay. LLS, which reports on particle size/number, detected precipitation onsets typically close to or below HPLC values and could resolve particle formation down to ∼ 0.1 µM for pimozide, substantially exceeding the sensitivity of standard nephelometric methods. SHS, which is sensitive to interfacial asymmetry and nonlinear susceptibility, often showed onsets and complex peak-drop-rise patterns that deviated from LLS, particularly for lopinavir, loratadine, and ritonavir, indicating structurally distinct, largely centrosymmetric drug-rich assemblies above the solubility limit. Orthogonal characterization by scanning electron microscopy, Capflex (capillary flow with a hydrophobic fluorescent tracer), and confocal fluorescence microscopy provided independent evidence for droplet-like or amorphous drug-rich domains that preceded bulk precipitation. Together, the results showed that combined SHS-LLS enabled highly sensitive detection of precipitation, while revealing multistep, non-classical aggregation pathways in pharmaceutical supersaturated systems.