An automated analyzer for methylated arginines in rat plasma by high-performance liquid chromatography with post-column fluorescence reaction

Abstract

A fully automated analyzer for methylated L-arginine metabolites [N,N-dimethyl-L-arginine (ADMA), N-methylarginine (NMMA) and N,N′-dimethyl-L-arginine (SDMA)] by high-performance liquid chromatography with post-column fluorescence derivatization was developed. This system consists of an on-line extraction, a separation on a reversed phase ion-pair chromatograph, a post-column derivatization by o-phthaladehyde (OPA) and thiol reaction, and fluorescence detection. NMMA, ADMA and SDMA were separated in 40 min with isocratic elution by a combination of octanoate and cyclohexane carboxylate as ion-pair reagents. The eluate was monitored at 450 nm with excitation at 337 nm. The calibration curves for NMMA, ADMA and SDMA showed linearity over the range from 0.05 μmol l⁻¹ (0.5 pmol on column) to 5.0 μmol l⁻¹ (50 pmol on column). This method does not require any time-consuming pre-treatment and requires only 10 μl of plasma sample for assay.