Development and validation of a fast quantitative method for plasma dimethylarginines analysis using liquid chromatography-tandem mass spectrometry.

OBJECTIVES: The aim of this work was to implement a fast, accurate and simple method to quantify plasma ADMA and SDMA, in a run time suitable for routine analysis. DESIGN AND METHODS: We developed and validated a hydrophilic interaction chromatographic method coupled to tandem mass spectrometry (HILIC-MS/MS) for separation and simultaneous quantification of Arginine (Arg) and its dimethylarginines, ADMA and SDMA, with a short run time (less than 5 min) using a small volume of human plasma (0.02 mL). RESULTS: Correlation coefficients (r) of the calibration curves ranged from 0.9926 to 0.9984. Within-day and between-day imprecision (CV%) and inaccuracy (%), carry-over and recovery were also evaluated for validation. Preliminary data of Arg, ADMA and SDMA from 30 apparently healthy subjects and type 2 diabetic patients (n=33) with and without kidney dysfunction were calculated and some statistical differences occurred among them (p<0.05). CONCLUSIONS: Data from calibration curves and quality controls reveal that the method is accurate and precise. Healthy subjects and diabetic patients' values are in agreement with those reported in other studies.

Determination of dimethylarginine levels in rats using HILIC-MS/MS: an in vivo microdialysis study.

Nitric oxide (NO) is one of the most important mediators and neurotransmitters and its levels change under pathological conditions. NO production may be regulated by endogenous nitric oxide synthase (NOS) inhibitors, in particular asymmetric dimethylarginine (ADMA). Most of the interest is focused on ADMA, since this compound is present in plasma and urine and accumulation of ADMA has been described in many disease states but little is known about cerebrospinal fluid (CSF) concentrations of this compound and of its structural isomer symmetric dimethylarginine (SDMA). To determine the levels of methylarginines, we here present a new hydrophilic interaction chromatography (HILIC)-MS/MS method for the precise determination of these substances in CSF from microdialysis samples of rat prefrontal cortex (PFC). The method requires only minimal sample preparation and features isotope-labelled internal standards.

Evaluation of mobile phase, ion pairing, and temperature influence on an HILIC-MS/MS method for L-arginine and its dimethylated derivatives detection.

Asymmetric N(G),-N(G)-dimethylarginine (ADMA) increases in diseases such as renal failure, diabetes mellitus, and hypercholesterolemia. The feasibility and utility of a hydrophilic interaction chromatography (HILIC) method for the separation of free L-arginine (Arg), ADMA, and symmetric N(G),-N(G')-dimethylarginine (SDMA) on a typical silica column were explored and the impact of some experimental parameters on the chromatographic behavior of these analytes was investigated. The effect of water and TFA content in mobile phase and of column temperature was investigated during the development of a fast and simple HILIC-MS/MS method that might be suitable for the quantification of free Arg, ADMA, and SDMA in plasma for routine analysis. Our results show that a good compromise between efficiency and peak shape with acceptable retention and total chromatographic run time is achieved using an ACN/water (90:10) mobile phase with TFA% as additive ranging from 0.015 to 0.025% and column temperature ranging from 25 to 30 degrees C.