Amino Acids

, Volume 46, Issue 9, pp 2205–2217 | Cite as

GC–MS and GC–MS/MS measurement of the cardiovascular risk factor homoarginine in biological samples

  • Arslan Arinc Kayacelebi
  • Bibiana Beckmann
  • Frank-Mathias Gutzki
  • Jens Jordan
  • Dimitrios TsikasEmail author
Original Article


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-15N2]-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.


GC–MS Homoarginine Plasma Stable isotopes Urine Validation 



N G ,N G -Dimethyl-l-arginine


Arginine:glycine amidinotransferase


Collision-induced dissociation


Dimethyl amine


Double-stage quadrupole


Electron-capture negative-ion chemical ionization


Gas chromatography–mass spectrometry


Gas chromatography–tandem mass spectrometry




Internal standard


Liquid chromatography–tandem mass spectrometry


Nitric oxide


Nitric oxide synthase




Pentafluoropropionic anhydride


Symmetric dimethylarginine


Selected-ion monitoring


Selected-reaction monitoring


Triple-stage quadrupole



We thank the staff of the Institute of Clinical Pharmacology, Hannover Medical School, for providing us with spot urine samples for the analysis of hArg and its relatives.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Albsmeier J, Schwedhelm E, Schulze F, Kastner M, Böger RH (2004) Determination of NG, NG-dimethyl-l-arginine, an endogenous NO synthase inhibitor, by gas chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 809(1):59–65Google Scholar
  2. Atzler D, Mieth M, Maas R, Böger RH, Schwedhelm E (2011) Stable isotope dilution assay for liquid chromatography-tandem mass spectrometric determination of l-homoarginine in human plasma. J Chromatogr B 879:2294–2298CrossRefGoogle Scholar
  3. Bretscher LE, Li H, Poulos TL, Griffith OW (2003) Structural characterization and kinetics of nitric-oxide synthase inhibition by novel N5-(iminoalkyl)- and N5- (iminoalkenyl)-ornithines. J Biol Chem 278:46789–46797PubMedCrossRefGoogle Scholar
  4. Choe CU, Atzler D, Wild PS, Carter AM, Böger RH, Ojeda F, Simova O, Stockebrand M, Lackner K, Nabuurs C, Marescau B, Streichert T, Muller C, Luneburg N, De Deyn PP, Benndorf RA, Baldus S, Gerloff C, Blankenberg S, Heerschap A, Grant PJ, Magnus T, Zeller T, Isbrandt D, Schwedhelm E (2013) Homoarginine levels are regulated by l-arginine:glycine amidinotransferase and affect stroke outcome: results from human and murine studies. Circulation 128:1451–1461PubMedCrossRefGoogle Scholar
  5. Dai Z, Wu Z, Jia S, Wu G (2014) Analysis of amino acid composition in proteins of animal tissues and foods as pre-column o-phthaldialdehyde derivatives by HPLC with fluorescence detection. J Chromatogr B. doi: 10.1016/j.jchromb.2014.03.025 Google Scholar
  6. Davids M, Ndika JD, Salomons GS, Blom HJ, Teerlink T (2012a) Promiscuous activity of arginine:glycine amidinotransferase is responsible for the synthesis of the novel cardiovascular risk factor homoarginine. FEBS Lett 586:3653–3657PubMedCrossRefGoogle Scholar
  7. Davids M, Swieringa E, Palm F, Smith DE, Smulders YM, Scheffer PG, Blom HJ, Teerlink T (2012b) Simultaneous determination of asymmetric and symmetric dimethylarginine, L-monomethylarginine, l-arginine, and L-homoarginine in biological samples using stable isotope dilution liquid chromatography tandem mass spectrometry. J Chromatogr B 900:38–47CrossRefGoogle Scholar
  8. Drechsler C, Meinitzer A, Pilz S, Krane V, Tomaschitz A, Ritz E, März W, Wanner C (2011) Homoarginine, heart failure, and sudden cardiac death in haemodialysis patients. Eur J Heart Fail 13:852–859PubMedCentralPubMedCrossRefGoogle Scholar
  9. Engeli S, Tsikas D, Lehmann AC, Böhnke J, Haas V, Strauß A, Janke J, Gorzelniak K, Luft FC, Jordan J (2012) Influence of dietary fat ingestion on asymmetrical dimethylarginine in lean and obese human subjects. Nutr Metab Cardiovasc Dis 22:720–726PubMedCrossRefGoogle Scholar
  10. Horowitz JD, Heresztyn T (2007) An overview of plasma concentrations of asymmetric dimethylarginine (ADMA) in health and disease and in clinical studies: methodological considerations. J Chromatogr B 851:42–50CrossRefGoogle Scholar
  11. Kayacelebi AA, Nguyen TH, Neil C, Horowitz JD, Jordan J, Tsikas D (2014) Homoarginine and 3-nitrotyrosine in patients with takotsubo cardiomyopathy. Int J Cardiol. doi: 10.1016/j.ijcard.2014.03.080 PubMedGoogle Scholar
  12. Khalil AA, Tsikas D, Akolekar R, Jordan J, Nicolaides KH (2013) Asymmetric dimethylarginine, arginine and homoarginine at 11–13 weeks gestation and preeclampsia: a case-control study. J Hum Hypertens 27:38–43PubMedCrossRefGoogle Scholar
  13. Leiper J, Vallance P (1999) Biological significance of endogenous methylarginines that inhibit nitric oxide synthases. Cardiovasc Res 43:542–548PubMedCrossRefGoogle Scholar
  14. Martens-Lobenhoffer J, Bode-Böger SM (2014) Mass spectrometric quantification of l-arginine and its pathway related substances in biofluids: the road to maturity. J Chromatogr B. doi: 10.1016/j.jchromb.2013.10.030 Google Scholar
  15. Martens-Lobenhoffer J, Schwedhelm E, Tsikas D (2009) Quantification of arginine and its mono- and dimethylated analogs NMMA, ADMA and SDMA in biological fluids by LC-MS/MS: is LC superfluous? J Chromatogr B 877:3261–3266CrossRefGoogle Scholar
  16. März W, Meinitzer A, Drechsler C, Pilz S, Krane V, Kleber ME, Fischer J, Winkelmann BR, Böhm BO, Ritz E, Wanner C (2010) Homoarginine, cardiovascular risk, and mortality. Circulation 122:967–975PubMedCrossRefGoogle Scholar
  17. Moali C, Boucher JL, Sari MA, Stuehr DJ, Mansuy D (1998) Substrate specificity of NO synthases: detailed comparison of l-arginine, homo-l-arginine, their N omega-hydroxy derivatives, and N omega-hydroxynor-l-arginine. Biochemistry 37:10453–10460PubMedCrossRefGoogle Scholar
  18. Moali C, Brollo M, Custot J, Sari MA, Boucher JL, Stuehr DJ, Mansuy D (2000) Recognition of alpha-amino acids bearing various C = NOH functions by nitric oxide synthase and arginase involves very different structural determinants. Biochemistry 39:8208–8218PubMedCrossRefGoogle Scholar
  19. Moncada S, Higgs A (1993) The l-arginine-nitric oxide pathway. New Engl J Med 329:2002–2012PubMedCrossRefGoogle Scholar
  20. Pilz S, Tomaschitz A, Meinitzer A, Drechsler C, Ritz E, Krane V, Wanner C, Bohm BO, März W (2011) Low serum homoarginine is a novel risk factor for fatal strokes in patients undergoing coronary angiography. Stroke 42:1132–1134PubMedCrossRefGoogle Scholar
  21. Ryan WL, Wells IC (1964) Homocitrulline and homoarginine synthesis from lysine. Science 144:1122–1127PubMedCrossRefGoogle Scholar
  22. Ryan WL, Johnson RJ, Dimari S (1969) Homoarginine synthesis by rat kidney. Arch Biochem Biophys 131:521–526PubMedCrossRefGoogle Scholar
  23. Schwedhelm E (2005) Quantification of ADMA: analytical approaches. Vascular Med (London, England) 10(Suppl 1):S89–S95CrossRefGoogle Scholar
  24. Schwedhelm E, Maas R, Tan-Andresen J, Schulze F, Riederer U, Böger RH (2007) High-throughput liquid chromatographic-tandem mass spectrometric determination of arginine and dimethylated arginine derivatives in human and mouse plasma. J Chromatogr B 851:211–219CrossRefGoogle Scholar
  25. Sobczak A, Prokopowicz A, Radek M, Szula M, Zaciera M, Kurek J, Goniewicz ML (2014) Tobacco smoking decreases plasma concentration of the emerging cardiovascular risk marker, l-homoarginine. Circ J 78:1254–1258PubMedCrossRefGoogle Scholar
  26. Teerlink T (2007) HPLC analysis of ADMA and other methylated l-arginine analogs in biological fluids. J Chromatogr B 851:21–29CrossRefGoogle Scholar
  27. Tsikas D (2008) A critical review and discussion of analytical methods in the l-arginine/nitric oxide area of basic and clinical research. Anal Biochem 379:139–163PubMedCrossRefGoogle Scholar
  28. Tsikas D, Beckmann B (2009) Albumin from human serum does not contain asymmetric dimethylarginine (ADMA). Clin Biochem 42:1739–1740PubMedCrossRefGoogle Scholar
  29. Tsikas D, Kayacelebi AA (2014) Do homoarginine and asymmetric dimethylarginine act antagonistically in the cardiovascular system? Circ J 20Google Scholar
  30. Tsikas D, Schubert B, Gutzki FM, Sandmann J, Frölich JC (2003) Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative. J Chromatogr B 798:87–99CrossRefGoogle Scholar
  31. Tsikas D, Engeli S, Beckmann B, Jordan J (2010) Asymmetric dimethylarginine (ADMA) is present in plasma proteins of healthy subjects at the low nmol-per-g-level. Nitric Oxide 22:316–317PubMedCrossRefGoogle Scholar
  32. Tsikas D, Beckmann B, Gutzki FM, Jordan J (2011) Simultaneous gas chromatography-tandem mass spectrometry quantification of symmetric and asymmetric dimethylarginine in human urine. Anal Biochem 413:60–62PubMedCrossRefGoogle Scholar
  33. Valtonen P, Laitinen T, Lyyra-Laitinen T, Raitakari OT, Juonala M, Viikari JS, Heiskanen N, Vanninen E, Punnonen K, Heinonen S (2008) Serum l-homoarginine concentration is elevated during normal pregnancy and is related to flow-mediated vasodilatation. Circ J 72:1879–1884PubMedCrossRefGoogle Scholar
  34. Wu G, Bazer FW, Davis TA, Kim SW, Li P, Marc Rhoads J, Carey Satterfield M, Smith SB, Spencer TE, Yin Y (2009) Arginine metabolism and nutrition in growth, health and disease. Amino Acids 37:153–168PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Arslan Arinc Kayacelebi
    • 1
  • Bibiana Beckmann
    • 1
  • Frank-Mathias Gutzki
    • 1
  • Jens Jordan
    • 1
  • Dimitrios Tsikas
    • 1
    Email author
  1. 1.Institute of Clinical Pharmacology, Hannover Medical SchoolHannoverGermany

Personalised recommendations