Development of quantitative method for agmatine and putrescine in frontal cortex of VPA model mouse using liquid chromatography tandem mass spectrometry

Abstract

Agmatine, known as a metabolite of arginine, has been reported to block excessive excitatory synaptic transmission at NMDA receptors in the brain. The objective of this study was to develop and validate analytical methods for L-Arginine metabolites [Agmatine, Putrescine] in animal brain. After dissolution with 0.1% formic acid in water and then precipitated in acetonitrile, chromatographic separation of analytes in mouse brain was performed using a HILIC column with the gradient elution of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Samples were fortified with hexamethylenediamine as internal standard. Quantification was performed using multiple-reaction monitoring of the transitions at m/z 131.1→ 114.1 for agmatine, m/z 89.1 → 72.1 for putrescine and 117.1 → 100.1 for internal standard, respectively. The flow rate of the mobile phase was 300 μL/min and the retention time of agmatine, putrescine and internal standard was 9.21, 9.26 and 9.22 min, respectively. The calibration curves were linear over the range of 9.8-625 ng/g and 24-2500 ng/g for agmatine and putrescine. The lower limits of quantification for agmatine and putrescine were 9.8 and 24.5 ng/g respectively. The mean accuracy and precision for agmatine and putrescine were acceptable range of 4.4-9.3% and 1.3-6.5% RSD. Proven methods have been successfully applied for quantitative analysis in mouse brain.