A novel liquid–liquid extraction and stable isotope dilution NCI‐GC‐MS method for quantitation of agmatine in postmortem brain cortex

Abstract

The group of biologically important amines includes putrescine, spermidine and spermine, as well as agmatine, which is a guanidino-amine. There is considerable evidence supporting a role of these amines in the etiology and pathology of mental disorders. We have previously developed a quantitative GC-MS method for simultaneous measurement of three major polyamines to support our studies linking polyamines to mental disorders. However, a unique GC-MS method is required for agmatine. To efficiently extract agmatine from postmortem brain tissues, we developed an isopropanol based liquid-liquid extraction protocol using potassium carbonate as a salting-out agent which showed a much greater recovery than n-butanol used in earlier methods. The GC-MS analysis employed hexafluoroacetylacetone as derivatization reagent and was carried out using negative chemical ionization with total ion and selected ion monitoring. (15)N(4)-agmatine was synthesized from (15)N(4)-L-arginine and used as internal standard in a conventional stable isotope dilution assay. This method accurately measures the level of agmatine from very small quantities (10-20 mg) of postmortem brain tissue, with a quantitation limit down to 1 ng/g of wet tissue. The limit of detection is 0.01 ng/g of wet tissue.