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Measurement of unlabeled and stable isotope-labeled homoarginine, arginine and their metabolites in biological samples by GC–MS and GC–MS/MS

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Abstract

Circulating and excretory l-homoarginine (hArg) and asymmetric dimethylarginine (ADMA) are cardiovascular risk factors. l-Arginine (Arg) is the common precursor of hArg and ADMA. This protocol describes gas chromatography–mass spectrometry (GC–MS) and gas chromatography–mass spectrometry–mass spectrometry (GC–MS/MS) methods for the quantitative determination of hArg, Arg and ADMA in biological samples, including human plasma, urine and sputum. Aliquots (10 µL) of native urine, plasma or serum ultrafiltrate (cutoff, 10 kDa), and acetone-deproteinized sputum samples are evaporated to dryness. Then, amino acids are derivatized to their methyl ester N-pentafluoropropionyl derivatives. In parallel, trideuteromethyl ester N-pentafluoropropionyl derivatives of hArg, Arg and ADMA are de novo synthesized from the unlabelled amino acids and used as internal standards. Alternatively, commercially available stable isotope-labeled analogs of hArg, Arg and ADMA are used as internal standards, and they are added to the native biological samples. Quantification is performed by selected ion monitoring in GC–MS and selected reaction monitoring in GC–MS/MS. By these protocols, unlabelled and stable isotope-labeled hArg, Arg and their metabolites including ADMA and ornithine can be measured equally accurately and precisely by GC–MS and GC–MS/MS in several different biological fluids in experimental and clinical settings.

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Abbreviations

ADMA:

Asymmetric dimethylarginine (N G,N G-dimethyl-l-arginine)

AGAT:

Arginine:glycine amidinotransferase

Arg:

Arginine

CID:

Collision-induced dissociation

EA:

Ethyl acetate

ECNICI:

Electron-capture negative-ion chemical ionization

GC–MS:

Gas chromatography–mass spectrometry

GC–MS/MS:

Gas chromatography–mass spectrometry–mass spectrometry

hArg:

Homoarginine

ID:

Internal diameter

IS:

Internal standard

Me:

Methyl

MeOH:

Methanol

NO:

Nitric oxide

NOS:

Nitric oxide synthase

Orn:

Ornithine

PFP:

Pentafluoropropionyl

PFPA:

Pentafluoropropionic anhydride

QC:

Quality control

SDMA:

Symmetric dimethylarginine (N G,N G′-dimethyl-l-arginine)

SIM:

Selected-ion monitoring

SRM:

Selected-reaction monitoring

TSQ:

Triple-stage quadrupole

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Acknowledgments

This work was in part funded by the DFG; SFB 738 (to GW).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standard

All animals received humane care in compliance with the German animal protection legislation, the Principles of Laboratory Animal Care, and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council. Principles of laboratory animal care, and the guide for the care and use of laboratory animals. Washington, DC: National Academies Press; 1996). The study was approved by the government board for animal welfare of Lower Saxony, Germany.

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Authors and Affiliations

  1. Bioanalytical Research Laboratory for NO, Oxidative Stress and Eicosanoids (BIOFORNOX20), Centre of Pharmacology and Toxicology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany

    Arslan Arinc Kayacelebi, Bibiana Beckmann, Erik Hanff & Dimitrios Tsikas

  2. Division of Cardiac, Thoracic, Transplantation, and Vascular Surgery, Hannover Medical School, Hannover, Germany

    Ann-Kathrin Knöfel & Gregor Warnecke

  3. German Center for Lung Research, BREATH Site, Hannover, Germany

    Gregor Warnecke

Authors
  1. Arslan Arinc Kayacelebi
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  2. Ann-Kathrin Knöfel
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  3. Bibiana Beckmann
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  4. Erik Hanff
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  5. Gregor Warnecke
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  6. Dimitrios Tsikas
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Correspondence to Dimitrios Tsikas.

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Kayacelebi, A.A., Knöfel, AK., Beckmann, B. et al. Measurement of unlabeled and stable isotope-labeled homoarginine, arginine and their metabolites in biological samples by GC–MS and GC–MS/MS. Amino Acids 47, 2023–2034 (2015). https://doi.org/10.1007/s00726-015-1984-3

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  • DOI: https://doi.org/10.1007/s00726-015-1984-3

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