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Automated Parallel Derivatization of Metabolites with SWATH-MS Data Acquisition

For metabolite profiling chemical derivatization has been used to improve MS sensitivity and LC retention. However, for multi-analytes quantification, the number of available isotopically labelled internal standards is limited. Besides, there is no single workflow which can provide large-scale metabolomics coverage in particular for polar metabolites. Differential Isotope Labeling (DIL) was adopted by derivatizing carbonyl, amino and phenol metabolites with two isotopic forms. Urine samples were derivatized with 12C-dansyl chloride (DnsCl) and 12C-dansylhydrazine (DnsHz). Suitable quantification standards were generated by derivatized with labelled 13C2-dansyl chloride and 13C2dansylhydrazine. The derivatization of the standards and the urine sample was performed using a PAL RTC autosampler in-line to column-switching LC-HRMS analysis with data independent acquisition (SWATH-MS). The combination of dual DIL with SWATH-MS acquisition enables post-identification of unknown metabolites and quantitation at precursor (MS1) and specific tag fragment (MS2) levels .