A quantitative LC-MS/MS method for residues analysis of 62 plant growth regulators and the investigation of their effects on the quality of traditional Chinese medicinal: Codonopsis Radix as a case study

Plant growth regulators (PGRs) enhance the biosynthesis of plant secondary metabolites but can cause environmental pollution and health risks, especially if synthetic or overused. Here, we developed a simple, high-throughput method using salting-out extraction and LC-MS/MS to analyze 62 PGR residues. The extraction, chromatographic conditions, and spectrometric parameters were systematically optimized. The extraction process was performed with acetonitrile-water (1:1), EN15662 extraction salt and d-SPE sorbent. This method was applied to analyze commercial and field trial Codonopsis Radix (CR) samples. The limit of quantification (LOQ), for 62 PGRs ranged from 0.03 to 82.50 μg/kg, and the limit of detection (LOD) ranged from 0.01 to 18.58 μg/kg. Furthermore, we employed plant metabolomics to assess changes in secondary metabolites in CR following fertilizer application and conducted a correlation analysis to explore the relationship between PGRs and secondary metabolites. In commercial samples, residues of 10 PGRs were detected, while in field trial samples, residues of 7 PGRs were found. In plant metabolomics, the arrangement of CR samples, which have been exposed to different fertilization levels, along the axes of partial least squares-discriminant analysis (PLS-DA) indicates that the chemical composition of CR experiences substantial alterations once a particular fertilization threshold is surpassed. The correlation analysis showed that PGRs boost amino acid metabolite synthesis and inhibit alkaloid biosynthesis. This study focuses on quality and safety concerns from the unchecked use of PGRs in CR production. It offers a framework for standardized cultivation and quality control to ensure the sustainable development of Traditional Chinese Medicine.