Simultaneous bioanalysis of L-arginine, L-citrulline, and dimethylarginines by LC-MS/MS

Abstract

Purpose: L-Arginine (ARG) is converted to nitric oxide (NO) and L-citrulline (CIT) by endothelial nitric oxide synthase which is competitively inhibited by asymmetric dimethylarginine (ADMA). We have developed a liquid chromatography-mass spectrometric method for the simultaneous determination of endogenous ARG, labeled ARG (¹⁵N₄-ARG), CIT, ADMA, and its inactive isomer, symmetric dimethylarginine (SDMA) in biological samples.

Methods: Concentrations of unlabeled ARG, ¹⁵N₄-ARG, CIT, ADMA, and SDMA in EA.hy926 human endothelial cell lysate, cell incubation media, rat plasma or rat urine were measured by hydrophilic-interaction liquid chromatography electrospray tandem mass spectrometry. ¹³C₆-ARG, D₄-CIT and D₇-ADMA were used as internal standards for ARG and ¹⁵N₄-ARG, CIT, and dimethylarginines, respectively.

Results: The calibration curves of ARG, ¹⁵N₄-ARG, CIT, ADMA, and SDMA were linear and independent of several sample matrices. Intra- and inter-day variabilities for the quantification of all the compounds were below 15% in quality control samples. Application of this method to determine the uptake as well as efflux of these compounds was illustrated through in vitro cell study by exposing human endothelial cells to ¹⁵N₄-ARG, which allowed the observation of generation of ¹⁵N₃-CIT and ¹⁵N₃-ARG in the cell lyate. Use of these isotopes adds insights into the cellular handling of endogenous vs. exogenous ARG. Application of this method for rat plasma and rat urine assays was demonstrated after ARG oral supplementation in rats.

Conclusion: An LC-MS/MS method was developed to quantify 6 ARG-related compounds simultaneously, utilizing 3 separate internal standards. This assay allows concurrent monitoring of uptake, efflux and metabolic processes when isotope-labeled ARG and CIT are measured, and can be applied for determination of these compounds in rat plasma and rat urine.

Figure 1

The ion chromatograms of (A) ¹⁵N₃-CIT and (B) ¹⁵N₃-ARG in cell lysate samples after cells were incubated with 100 μM of ¹⁵N₄-ARG. A peak with m/z of 179.2→71.1 and a retention time of 2.83 min (panel A) was assigned as that for ¹⁵N₃-CIT. A peak with m/z of 178.2→71.1 and a retention time of 3.33 minutes (panel B) was assigned as ¹⁵N₃-ARG based on their theoretical m/z and retention times.

Figure 2

Cellular concentrations of (A) ¹⁵N₄-ARG, (B) ARG, (C) ADMA, and (D) CIT after incubation with different concentrations of ¹⁵N₄-ARG for 2 hours. Each point represents mean ± SD (n=3). *p<0.05 vs Control (i.e. ¹⁵N₄-ARG = 0 μM).

Figure 3

Extracellular concentrations of (A) ¹⁵N₄-ARG, (B) ARG, (C) ADMA, (D) CIT, and (E) SDMA after incubation with different concentrations of ¹⁵N₄-ARG for 2 hours. Each point represents mean ± SD (n=3). *p<0.05 vs Control (i.e. ¹⁵N_4-ARG = 0 μM).