Determination of Asymmetric Dimethylarginine and Symmetric Dimethylarginine in Biological Samples of Mice Using LC/MS/MS

Abstract

Herein, we present a novel method of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) determination within biological samples using protein precipitation and LC/MS/MS. Chromatographic separation of ADMA and SDMA was successfully performed using a silica column with optimized elution, or mobile phase, of 10 mM ammonium acetate buffer H₂O/methanol/acetonitrile (20/30/45, v/v) at pH 4. The calibration ranges were 0.50 – 50.0 μg●mL^-1, and good linearities were obtained for all compounds ( γ > 0.99). The intra- and inter-assay accuracies with recoveries and precisions at three concentration levels (<i>i.e</i>. 1.00, 5.00 and 25.0 μg●mL^-1) were better than 86.9% and 7.36%, respectively. The analytical performance of the method was evaluated by determination of compounds in plasma, urine and tissues from male BALBc/J mice. For the first time, we were able to characterize the distribution of ADMA, SDMA and ADMA/SDMA in plasma, urine, brain, heart, kidneys, liver, lungs, pancreas and spleen. Additionally, we demonstrated that the ADMA/SDMA ratio in the brain was approximately 10-fold lower than all the other biological samples. Only 10 μL of plasma, 1 μL of urine and about 25 mg of tissues were required. These results suggest that the developed methodology was useful in ADMA and SDMA determination within biological samples.