A novel mass spectrometry-based method for simultaneous determination of asymmetric and symmetric dimethylarginine, l-arginine and l-citrulline optimized for LC-MS-TOF and LC-MS/MS

Biomed Chromatogr. 2017 Nov;31(11). doi: 10.1002/bmc.3994. Epub 2017 May 24.

Abstract

Nitric oxide (NO) is a regulatory molecule involved in many biological processes. NO is produced by nitric oxide synthase by conversion of l-arginine to l-citrulline. l-Arginine methylated derivatives, asymmetric and symmetric dimethylarginines (asymmetric dimethylarginine, ADMA, and symmetric dimethylarginine, SDMA), regulate l-arginine availability and the activity of nitric oxide synthase. As such, they have been frequently investigated as potential biomarkers in pathologies associated with dysfunctions in NO synthesis. Here, we present a new multistep analytical methodology based on liquid chromatography combined with mass spectrometry for the accurate identification of l-arginine, l-citrulline, ADMA and SDMA. Compounds are measured as stable 2,3,4,5,6-pentafluorobenzoyl chloride derivatives, which allows for simultaneous analysis of all compounds through chromatographic separation of ADMA and SDMA using a reverse-phase column. Serum aliquots (100 μL) were spiked with isotope-labeled internal standards and sodium carbonate buffer. The derivatization process was carried out at 25°C for 10 minu using pentafluorobenzoyl chloride as derivatization reagent. Calibration demonstrated good linearity (R2 = 0.9966-0.9986) for all derivatized compounds. Good accuracy (94.67-99.91%) and precision (1.92-11.8%) were observed for the quality control samples. The applicability of the method was evaluated in a cohort of angiological patients and healthy volunteers. The method discerned significantly lower l-arginine and l-citrulline in angiologic patients. This robust and fast LC-ESI-MS method may be a useful tool in quantitative analysis of l-arginine, ADMA, SDMA and l-citrulline.

Keywords: ADMA; amino acids derivatization; electrospray ionization; liquid chromatography-mass spectrometry; nitric oxide; quantitative analysis.

MeSH terms

  • Arginine / analogs & derivatives*
  • Arginine / blood*
  • Arginine / chemistry
  • Arginine / metabolism
  • Chromatography, Liquid / methods
  • Citrulline / blood*
  • Citrulline / chemistry
  • Citrulline / metabolism
  • Humans
  • Limit of Detection
  • Linear Models
  • Nitric Oxide / metabolism
  • Reproducibility of Results
  • Tandem Mass Spectrometry / methods

Substances

  • Citrulline
  • Nitric Oxide
  • Arginine