title: GC-MS and GC-MS/MS measurement of the cardiovascular risk factor homoarginine in biological samples.
abstract: L-Homoarginine (hArg) has recently emerged as a novel cardiovascular risk factor and to herald a poor prognosis in heart failure patients. Here, we report on the development and thorough validation of gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) methods for the quantitative determination of hArg in biological samples, including human plasma, urine and sputum. For plasma and serum samples, ultrafiltrate (10 µL; cutoff, 10 kDa) was used. For urine samples, native urine (10 µL) was used. For sputum, protein precipitation by acetone was performed. hArg is derivatized to its methyl ester tri(N-pentafluoropropionyl) derivative; de novo synthesized trideutero-methyl ester hArg is used as the internal standard (IS). Alternatively, [guanidino-(15)N2]-arginine can be used as an IS. Quantitative analyses were performed after electron-capture negative-ion chemical ionization by selected-ion monitoring in GC-MS and selected-reaction monitoring in GC-MS/MS. We obtained very similar hArg concentrations by GC-MS and GC-MS/MS, suggesting that GC-MS suffices for accurate and precise quantification of hArg in biological samples. In plasma and serum samples of the same subjects very close hArg concentrations were measured. The plasma-to-serum hArg concentration ratio was determined to be 1.12 ± 0.21 (RSD, 19 %), suggesting that blood anticoagulation is not a major preanalytical concern in hArg analysis. In healthy subjects, the creatinine-corrected urinary excretion of hArg varies considerably (0.18 ± 0.22 µmol/mmol, mean ± SD, n = 19) unlike asymmetric dimethylarginine (ADMA, 2.89 ± 0.89 µmol/mmol). In urine, hArg correlated with ADMA (r = 0.475, P = 0.040); in average, subjects excreted in the urine about 17.5 times more ADMA than hArg. In plasma of healthy humans, the concentration of hArg is of the order of 2 µM. hArg may be a low-abundance constituent of human plasma proteins. The GC-MS and GC-MS/MS methods we report in this article are useful to study the physiology and pathology of hArg in experimental and clinical settings.
authors.affiliation: Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany.
authors.forename: Arslan Arinc
authors.initials: AA
authors.lastname: Kayacelebi
authors.forename: Bibiana
authors.initials: B
authors.lastname: Beckmann
authors.forename: Frank-Mathias
authors.initials: FM
authors.lastname: Gutzki
authors.forename: Jens
authors.initials: J
authors.lastname: Jordan
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Tsikas
mesh_terms: D001120:Arginine / Q000031:analogs & derivatives / Q000097:blood / Q000652:urine; D002318:Cardiovascular Diseases / Q000097:blood / Q000652:urine; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006709:Homoarginine / Q000097:blood / Q000652:urine; D006801:Humans; D008297:Male; D012307:Risk Factors; D013183:Sputum / Q000378:metabolism*
chemical_list: D006709:Homoarginine; C018524:N,N-dimethylarginine; D001120:Arginine