![]() It provides the limit of quantitation (LOQ) of 30 pg itaconate on column with a 4.5-minute run time. The current protocol allows baseline separation of itaconate, citraconate, and cis-aconitate without derivatization and direct analysis of analytes in 80% methanol/water solution to avoid the dry-down step. An ion pairing LC-MS/MS method using tributylamine/formic acid as ion pairing agents and a Hypercarb TM guard column we proposed demonstrated better peak shape and better sensitivity for itaconate. Multiple HPLC and derivatization methods were tested. A rapid and sensitive method for measurement of itaconate will benefit the study of IRG1 biology. The biology associated with IRG1 and itaconate is not fully understood. Accumulation of Immune Responsive Gene 1(IRG1) in macrophage induced by lipopolysaccharide (LPS) and interferon gamma (IFN-γ) leads to production of itaconate by decarboxylation of cis-aconitate.
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