A valve-assisted 4-in-1 polarity-partitioned LC-QTOF-MS workflow reveals amino acid dysregulation in human chlorfenapyr poisoning.

Untargeted metabolomics of clinical toxicology samples is often constrained by limited sample volume, incomplete metabolome coverage, and technical variability introduced by multiple LC-MS injections. Here, we applied and evaluated a valve-assisted 4-in-1 polarity-partitioned LC-QTOF-MS workflow for single-injection plasma metabolomic profiling. The term "4-in-1" refers to four complementary LC-ionization data channels acquired from a single injection: HILIC-ESI(+), HILIC-ESI(-), C8-ESI(+), and C8-ESI(-). The workflow combines valve-controlled collection and transfer of weakly retained HILIC effluent with sequential HILIC and C8 analyses and dual-polarity MS acquisition. Quality-control analyses demonstrated stable retention behavior and reproducible feature detection, and the single-injection design reduced the need for multiple separate LC-MS injections. As a clinical toxicology application, the workflow was applied to plasma samples from patients with chlorfenapyr poisoning and healthy controls, with poisoned patients further stratified according to plasma tralopyril concentration. PCA and OPLS-DA were used as exploratory tools to visualize global metabolic differences. Differential LC-MS features were screened using multivariate and univariate statistical criteria, followed by metabolite annotation and pathway enrichment analysis based on annotated differential metabolites. Prominent perturbations were observed in amino acid metabolism, the urea cycle, and energy-related pathways. Several annotated amino acids, including glutamine, asparagine, alanine, and threonine, differed among the exposure groups. These exploratory findings support the feasibility of the workflow for limited-volume clinical plasma metabolomics and identify candidate metabolic alterations consistent with mitochondrial metabolic stress in chlorfenapyr poisoning.