Capillary electrophoretic and micellar electrokinetic separations of asymmetric dimethyl‐L‐arginine and structurally related amino acids: Quantitation in human plasma

Abstract

We report the development of efficient electrophoretic methods for the separation and quantification of L-arginine and six naturally occurring derivatives that are structurally and functionally related. Capillary electrophoresis (CE) employing a concentrated borate buffer at pH 9.4 achieves the separation of mixtures containing dimethyl-L-arginine, NG-monomethyl-L-arginine, L-arginine, L-homoarginine, L-ornithine, and L-citrulline as 4-fluoro-7-nitrobenzofurazan derivatives. In addition, the separation of the isomeric dimethyl-L-arginine derivatives (symmetric and asymmetric) is attained with baseline resolution by micellar electrokinetic chromatography (MEKC) when a high concentration of deoxycholic acid is added as a surfactant to the same running buffer. The influence of buffer type, concentration, and pH on the separation was studied to optimize separation conditions. The limit of quantitation (LOQ) for asymmetric dimethyl-L-arginine in aqueous solution was determined to be 20 microM using UV absorption in a CE separation and 0.1 microM using laser induced fluorescence (LIF) detection in an MEKC separation. This newly developed method was successfully applied for the quantitation of asymmetric dimethyl-L-arginine and L-arginine in human plasma samples at levels that might be used as a clinical diagnostic for cardiovascular disease (0.125 microM LOQ).

Determination of amino acids by micellar EKC: Recent advances in method development and novel applications to different matrices

“…As previously indicated (see Section 2.2), the analysis in CSF of sulfur-containing excitatory amino acids in the presence of neurotransmitters Glu and Asp was performed by Becker et al [58] in 0.1 M borate, 50 mM SDS, and 5% v/v MeOH. Trapp et al [97] have suggested the use of 250 mM borate buffer containing 50 mM SDS to obtain the separation of mixtures containing dimethyl-L-Arg, monomethyl-L-Arg, L-Arg, L-homoArg, L-Orn, and L-Cit as 4-fluoro-7-nitrobenzofurazan derivatives. The addition of deoxycholic acid to the same running buffer allowed also the separation of the isomeric dimethyl-L-Arg (symmetric and asymmetric) derivatives.…”

Recent advances in amino acid analysis by capillary electrophoresis

“…L-ornithine, and L-citrulline as 4-NBD-F derivatives was achieved by Trapp et al [79] using a concentrated borate buffer at pH 9.4. In addition, the separation of the isomeric dimethyl-L-arginine derivatives (symmetric and asymmetric) was attained with baseline resolution by MEKC when a high concentration of deoxycholic acid is added like a surfactant to the same running buffer.…”

Bioanalytical profile of the l-arginine/nitric oxide pathway and its evaluation by capillary electrophoresis

“…The effect of ADMA infusion in humans and analytical approaches used in the quantification of ADMA have recently been reviewed [202,203]. Effective HPCE techniques for the quantification of L-arginine derivatives, including ADMA, in biomedical samples have been reported [204,205]. Example of CE-LIF analysis of ADMA in serum samples from healthy and memorialized human subjects are shown in Fig.…”

“…Micellar electrokinetic chromatography (MEKC) is almost exclusively used for analysis of small molecules such as drug and their metabolites. MEKC is a very potent technique to determine small organic members of the NO pathway including L-arginine, ADMA and SDMA [204,205,241]. Other HPCE modes, including capillary isoelectric focusing (CIEF), capillary electrochromatography (CEC) and gel-sieving capillary electrophoresis generally used for separation of medium and large size molecules, and specific analytical tasks were not included in this review.…”

“…Other biochemical parameters of the L-arginine/NO pathway became increasingly used in assessing disease risk factors and in laboratory diagnostics of samples from patients and laboratory mammalian models. This set includes ADMA [203][204][205], S-nitrosothiols [410][411][412][413][414][415][416], and 3-nitrotyrosine [179,292,417,418].…”