Amino Acids

pp 1–19 | Cite as

Development and validation of GC–MS methods for the comprehensive analysis of amino acids in plasma and urine and applications to the HELLP syndrome and pediatric kidney transplantation: evidence of altered methylation, transamidination, and arginase activity

  • Erik Hanff
  • Stephan Ruben
  • Martin Kreuzer
  • Alexander Bollenbach
  • Arslan Arinc Kayacelebi
  • Anibh Martin Das
  • Frauke von Versen-Höynck
  • Constantin von Kaisenberg
  • Dieter Haffner
  • Stefan Ückert
  • Dimitrios TsikasEmail author
Original Article


We developed and validated gas chromatography–mass spectrometry (GC–MS) methods for the simultaneous measurement of amino acids and their metabolites in 10-µL aliquots of human plasma and urine. De novo synthesized trideutero-methyl esters were used as internal standards. Plasma proteins were precipitated by acidified methanol and removed by centrifugation. Supernatants and native urine were evaporated to dryness. Amino acids were first esterified using 2 M HCl in methanol and then amidated using pentafluoropropionic anhydride for electron-capture negative-ion chemical ionization. Time programmes were used for the gas chromatograph oven and the selected-ion monitoring of specific anions. The GC–MS methods were applied in clinical studies on the HELLP syndrome and pediatric kidney transplantation (KTx) focusing on l-arginine-related pathways. We found lower sarcosine (N-methylglycine) and higher asymmetric dimethylarginine (ADMA) plasma concentrations in HELLP syndrome women (n = 7) compared to healthy pregnant women (n = 5) indicating altered methylation. In plasma of pediatric KTx patients, lower guanidinoacetate and homoarginine concentrations were found in plasma but not in urine samples of patients treated with standard mycophenolate mofetil-based immunosuppression (MMF; n = 22) in comparison to matched patients treated with MMF-free immunosuppression (n = 22). On average, the global arginine bioavailability ratio was by about 40% lower in the MMF group compared to the EVR group (P = 0.004). Mycophenolate, the major pharmacologically active metabolite of MMF, is likely to inhibit the arginine:glycine amidinotransferase (AGAT), and to enhance arginase activity in leukocytes and other types of cell of MMF-treated children.


AGAT Amino acids Derivatization GAMT GC–MS Immunosuppression Quantification Stable isotopes 



Amino acid


Asymmetric dimethylarginine


Arginine:glycine amidinotransferase


Chronic kidney disease


Calcineurin inhibitor


Dimethylarginine dimethylaminohydrolase




Electron-capture negative-ion chemical ionization






Global arginine bioavailability ratio


Guanidinoacetate N-methyltransferase


Glycine:arginine transamidinase


Gas chromatography–mass spectrometry


Glycine N-methyltransferase




Hemolysis, elevated liver enzymes, low platelet


Interquartile range


Internal standard


Kidney transplantation


Limit of detection


Limit of quantification






Mycophenolate mofetil


Mycophenolic acid


Mass-to-charge ratio


N-Methyl-d-aspartate glutamate receptor


Nitric oxide


Nitric oxide synthase






Pentafluoropropionic anhydride


Protein-arginine methyltransferase


Quality control


Relative low limit of quantification




Symmetric dimethylarginine


Selected-ion monitoring

S/N or SN

Signal-to-noise ratio


Total ion current


Urinary nitrate-to-nitrite molar ratio


Week of gestation


Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interest.

Ethical approval

Both studies were conducted in accordance with the ethical principles of Good Clinical Practice (GCP) that have their origins in the Declaration of Helsinki.

Supplementary material

726_2018_2688_MOESM1_ESM.pdf (1.5 mb)
Supplementary material 1 (PDF 1504 kb)


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Erik Hanff
    • 1
  • Stephan Ruben
    • 2
  • Martin Kreuzer
    • 2
  • Alexander Bollenbach
    • 1
  • Arslan Arinc Kayacelebi
    • 1
  • Anibh Martin Das
    • 3
  • Frauke von Versen-Höynck
    • 4
  • Constantin von Kaisenberg
    • 4
  • Dieter Haffner
    • 2
  • Stefan Ückert
    • 5
  • Dimitrios Tsikas
    • 1
    Email author
  1. 1.Core Unit Proteomics, Hannover Medical SchoolInstitute of ToxicologyHannoverGermany
  2. 2.Department of Pediatric Kidney, Liver and Metabolic DiseasesHannover Medical SchoolHannoverGermany
  3. 3.Department of PediatricsHannover Medical SchoolHannoverGermany
  4. 4.Department of Obstetrics and GynecologyHannover Medical SchoolHannoverGermany
  5. 5.Department of Urology and Urological OncologyHannover Medical SchoolHannoverGermany

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