Simultaneous Sensitive MEKC–LIF Determination of Homocysteine, Homoarginine, and Six Arginine Metabolic Derivatives in Fluids from Type 2 Diabetics with Peptic Ulcer Bleeding

Abstract

The suitability of micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection for simultaneous determination of two arginine analogues (homocysteine and homoarginine) and five closely related metabolites (asymmetric dimethyl-l-arginine, dimethyl-l-arginine, monomethyl-l-arginine, citrullin, and ornithine) in fluids from type 2 diabetics with peptic ulcer bleeding (PUB) has been investigated. 5-Carboxyfluorescein succinimidyl ester (CFSE) was chosen as the fluorescent labeling reagent and non-endogenous phenylpropanolamine (PPA) as the internal standard. Conditions affecting derivatization and separation were optimized. Under the optimum conditions, maximum derivatization could be achieved in 20 min at room temperature. Complete baseline separation was achieved in 10 min, and the relative standard deviations (RSD) of migration times and corrected peak areas were <3 % for intra-day assay and <5 % for inter-day assay. Limits of detection (LODs) were 0.12–1.70 nM for the eight analytes, which are well below the concentrations expected in real fluids. Compared with previously reported methods, 5 to 600-fold improvements in sensitivity were achieved by use of LIF detection. Sample preparation and analysis time were short and the derivatives of the analytes were highly stable. The method was fully validated with real plasma and urine and recoveries of spiked compounds were 95–102 % with the RSD <4 %.