Salvatore Sotgia1, Angelo Zinellu2, Panagiotis Paliogiannis2, Gerard A Pinna3, Arduino A Mangoni4, Luciano Milanesi5, Ciriaco Carru6. 1. Department of Biomedical Sciences, School of Medicine, University of Sassari, Sassari, Italy. Electronic address: ssotgia@uniss.it. 2. Department of Biomedical Sciences, School of Medicine, University of Sassari, Sassari, Italy. 3. Department of Chemistry and Pharmacy, University of Sassari, Sassari, Italy. 4. Department of Clinical Pharmacology, College of Medicine and Public Health, Flinders University and Flinders Medical Centre, Adelaide, Australia. 5. National Research Council (CNR), Institute for Biomedical Technologies, Segrate, Italy. 6. Department of Biomedical Sciences, School of Medicine, University of Sassari, Sassari, Italy; Quality Control Unit, University Hospital of Sassari (AOU-SS), Sassari, Italy.
Abstract
BACKGROUND: Changes in NO metabolism correlate with cardiovascular risk factors and are associated with endothelial dysfunction. NO availability is regulated by nitric oxide synthase (NOS) and arginine and some chemically related metabolites and analogs have the capacity to alter NOS activity. Hence the need for analytical methods for the simultaneous assessment of these analytes. METHODS: Analytes (L-arginine (Arg), NG-monomethyl-L-arginine (MMA), L-homoarginine (hArg), asymmetric dimethyl-L-arginine (ADMA), symmetric dimethyl-L-arginine (SDMA), and L-citrulline (CIT)) were isolated from human plasma by thermal coagulation of plasma followed by a derivatization with diethylpyrocarbonate. Carbetoxy derivatives were separated on a C18 reversed-phase column in <10 min using an aqueous solution of 0.4% v/v formic acid and acetonitrile (95:5, v/v) mixture as a mobile phase. Positive electrospray ionization and tandem mass spectrometry in combination with specific multiple reaction monitoring transitions were used for detection of analytes and three deuterated forms of the analytes used as internal standards. RESULTS: Intra- and inter-day precision %RSD values ranged between 3 and 5.5% and percentage recoveries were close to 100% for all analytes. Plasma concentrations in 20 healthy male volunteers were 58.62 ± 8.81 μmol/L for Arg, 105.08 ± 21.66 nmol/L for MMA, 1.88 ± 0.57 μmol/L for hArg, 0.612 ± 0.140 μmol/L for ADMA, 0.581 ± 0.172 μmol/L for SDMA, and 28.62 ± 11.60 μmol/L for Cit, respectively. CONCLUSION: This LC-MS/MS method provides the capacity to quantify the plasma concentrations of arginine and some of its chemically related metabolites. Sample preparation was simple, inexpensive and effortless. Overall, given the short sample preparation and chromatographic run time, the method may be suitable for the fast and reproducible quantitative determination of the analytes in large clinical trials and routine analysis.
BACKGROUND: Changes in NO metabolism correlate with cardiovascular risk factors and are associated with endothelial dysfunction. NO availability is regulated by nitric oxide synthase (NOS) and arginine and some chemically related metabolites and analogs have the capacity to alter NOS activity. Hence the need for analytical methods for the simultaneous assessment of these analytes. METHODS: Analytes (L-arginine (Arg), NG-monomethyl-L-arginine (MMA), L-homoarginine (hArg), asymmetric dimethyl-L-arginine (ADMA), symmetric dimethyl-L-arginine (SDMA), and L-citrulline (CIT)) were isolated from human plasma by thermal coagulation of plasma followed by a derivatization with diethylpyrocarbonate. Carbetoxy derivatives were separated on a C18 reversed-phase column in <10 min using an aqueous solution of 0.4% v/v formic acid and acetonitrile (95:5, v/v) mixture as a mobile phase. Positive electrospray ionization and tandem mass spectrometry in combination with specific multiple reaction monitoring transitions were used for detection of analytes and three deuterated forms of the analytes used as internal standards. RESULTS: Intra- and inter-day precision %RSD values ranged between 3 and 5.5% and percentage recoveries were close to 100% for all analytes. Plasma concentrations in 20 healthy male volunteers were 58.62 ± 8.81 μmol/L for Arg, 105.08 ± 21.66 nmol/L for MMA, 1.88 ± 0.57 μmol/L for hArg, 0.612 ± 0.140 μmol/L for ADMA, 0.581 ± 0.172 μmol/L for SDMA, and 28.62 ± 11.60 μmol/L for Cit, respectively. CONCLUSION: This LC-MS/MS method provides the capacity to quantify the plasma concentrations of arginine and some of its chemically related metabolites. Sample preparation was simple, inexpensive and effortless. Overall, given the short sample preparation and chromatographic run time, the method may be suitable for the fast and reproducible quantitative determination of the analytes in large clinical trials and routine analysis.