A high-throughput hydrophilic interaction liquid chromatography coupled with a charged aerosol detector method to assess trisulfides in IgG1 monoclonal antibodies using tris(2-carboxyethyl)phosphine reaction products: Tris(2-carboxyethyl)phosphine-oxide and tris(2-carboxyethyl)phosphine-sulfide

•A chromatographic HILICCAD method to measure levels of mAb trisulfide modifications.•Comparable results to the alternative method—peptide map with LC/MS detection.•Method applicable to cysteine-linked ADC production and cell culture process development. A robust, high-throughput method using hydrophilic interaction liquid chromatography (HILIC) coupled with a charged aerosol detector (CAD) is reported as a novel approach for trisulfide quantitation in monoclonal antibodies (mAbs). The products of mAb reduction using tris(2-carboxyethyl)phosphine (TCEP) include a species (TCEP(S)) that is stoichiometrically produced from trisulfides. The TCEP reaction products are chromatographically separated, detected, and quantified by the HILICCAD method. The method was qualified to quantify trisulfides across a range of 1–40% (mol trisulfide/mol mAb). In all tested matrix components, assay linearity and intermediate precision were established with correlation coefficients (R2)>0.99, and relative standard deviations (RSD)