Journal
BIOMEDICAL CHROMATOGRAPHY
Volume 31, Issue 11, Pages -Publisher
WILEY
DOI: 10.1002/bmc.3994
Keywords
ADMA; amino acids derivatization; electrospray ionization; liquid chromatography-mass spectrometry; nitric oxide; quantitative analysis
Categories
Funding
- Wroclaw Research Center EIT+ [POIG 01.01.02-02-003/08-00]
- WroVasc - Integrated Cardiovascular Center
- Wroclaw Medical University [18/Pbmn]
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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 (100L) were spiked with isotope-labeled internal standards and sodium carbonate buffer. The derivatization process was carried out at 25 degrees C for 10 minu using pentafluorobenzoyl chloride as derivatization reagent. Calibration demonstrated good linearity (R-2=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.
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