Waters chromatography system m600e

The intracellular concentration of L-arginine was determined by HPLC using pre-column derivatization with o-phthalaldehyde (OPA) according to a previously published method,16 (link) with modifications. L-arginine (100 µM) was added to cell lysate (0.1 mM) as an internal standard. The samples were extracted on solid-phase extraction cartridges (CBA Bond Elute, Varian, Palo Alto, CA, USA). The recovery rate was 87.5±3.9%. Eluates were dried over nitrogen and resuspended in double-distilled water for HPLC analysis. HPLC was performed on a computer-controlled Waters chromatography system (M600E) consisting of an automatic injector (M7725i; Waters Co., Milford, MA, USA) and a fluorescence detector (FP-1520; Jasco Co., Portland, OR, USA). Samples were incubated for exactly 1 min with OPA reagent (5.4 mg/mL OPA in borate buffer, pH 8.4, containing 0.4% 2-mercaptoethanol) before automatic injection into the HPLC. The OPA derivatives of L-arginine and polyamine were separated on a 150×4.6 mm-5 µm Zorbax Eclipse XDB-C18 column (Watertown, MA, USA), with the fluorescence detector set at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. Samples were eluted from the column with 0.96% citric acid/methanol (70:30; pH 6.8) at a flow rate of 1.5 mL/min.

The intracellular concentration of L-arginine was determined via high-performance liquid chromatography (HPLC) using pre-column derivatization with o-phthalaldehyde (OPA) according to a modification of a previously published method.11 (link) L-arginine (100 µmol/L) was added to the cell lysate (0.5 mL) as an internal standard. The samples were extracted on solid-phase extraction cartridges (CBA Bond elute, Varian). Recovery rates were 87.5±3.9%. Eluates were dried over nitrogen and resuspended in double-distilled water for HPLC analysis. HPLC was performed on a computer-controlled Waters chromatography system (M600E) consisting of an automatic injector (M7725i, Waters Co., Victoria, Australia) and a fluorescence detector (FP-1520, Jasco Co., Queensland, Australia) in the Central Laboratory of Kangwon National University. Samples were incubated for exactly 1 minute with OPA reagent (5.4 mg/mL OPA in borate buffer, pH 8.4, containing 0.4% 2-mercaptoethanol) before automatic injection into the HPLC. The OPA derivative of L-arginine was separated on a 150×4.6 mm (3.5 µm) Zorbax Eclipse XDB-C18 column with a fluorescence detector set at Ex 340 nm and Em 450 nm. Samples were eluted from the column with 0.96% citric acid/methanol (70:30, pH 6.8) at a flow rate of 1.5 mL/min.