Analytical method of agmatine and putrescine in human embryonic kidney cell by LC-ESI-MS/MS

Abstract

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) for the analysis of L-Arginine metabolites [Agmatine, Putrescine] in human embryonic kidney cell has been successfully developed and validated. Analysis of cell pellet and cell supernatant samples from Hek293t cell was performed. Sample preparation was added 200 ？L of internal standard (1000 ng/mL of Hexamethylenediamine) to a 45 ？L of cell sample. After vortexing and centrifugation, the supernatant was dried with speed vaccum. The sample was then reconstituted in 200 ？L of acetonitrile:water (70:30, v/v). Agmatine and putrescine were separated with a HILIC column (150 × 2.1 mm, 2.5μm) using a mobile phase of 0.05% formic acid containing 5 mM ammonium formate buffer(water/ACN, 30/70, v/v) at a flow rate of 0.3 mL/min. 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 standard curve had good linearity (R2 > 0.99) in both the cell pellet and cell supernatant. The lower limits of quantification (LLOQ) were 15.625 ng/mL and 31.25 ng/mL in putrescine and agmatine respectively. The accuracy of the method in cell pellets was 88.3-108% and 88.5-104.7% for putrescine and agmatine, and those in cell supernatant was 94.6-105.3% and 92.1-106%, respectively. The developed methods have been successfully applied for quantitative analysis in cell.