Development of the sensitive determination method for the endogenous NO synthetase inhibitor and analysis of homeostasis of blood pressure.

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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 in-line extraction, a separation on a reversed phase ion-pair chromatography, 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 cyclohexanecarboxylate as ion-pair reagents. The eluate was monitored at 450 nm with the excitation at 337 nm. The calibration curves for NMMA, ADMA and SDMA showed linearity over the range from 0.05 μmol 1^<-1> (0.5 pmol on column) to 5.0 μmol 1^<-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.