Quantification of Arginine and its Methylated Derivatives in Healthy Children by Liquid Chromatography-Tandem Mass Spectrometry

Asymmetric dimethylarginine (ADMA) is a competitive inhibitor of nitric oxide synthase, which is responsible for most of the vascular nitric oxide (NO) produced. NO is an important physiological mediator of vascular tone and structure in normally functioning endothelial cells. We report the optimization of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of arginine (Arg) and its derivatives in biological samples. Chromatographic separation and mass detection were performed by reverse phase chromatography coupled with tandem mass spectrometry. For sample preparation, plasma proteins were removed by centrifugal filters. Positive electrospray ionization was performed and analytes were detected by multiple reaction monitoring. Inter- and intra-day repeatability, accuracy, recovery, and limits of detection and quantification were evaluated to validate the method. Plasma and urine levels were measured in healthy children to establish control values: 52.2-124.7 µM for Arg, 0.06-0.16 µM for MMA, 0.42-1.10 µM for ADMA and 0.41-0.96 µM for SDMA in plasma. Quantification of Arg and its methylated derivatives by LC-MS/MS can be carried out without the need of organic solvents for sample preparation, and be used as a valuable tool in research on endothelial dysfunction.