Amino Acids

, Volume 51, Issue 4, pp 627–640 | Cite as

GC–MS measurement of biological NG-hydroxy-l-arginine, a stepmotherly investigated endogenous nitric oxide synthase substrate and arginase inhibitor

  • Alexander Bollenbach
  • Stephan J. L. Bakker
  • Dimitrios TsikasEmail author
Original Article


l-Arginine is converted by nitric oxide synthase (NOS) to l-citrulline and nitric oxide (NO). NG-Hydroxy-l-arginine (NOHA) is the isolable intermediate of this reaction. NOHA has been identified in biological samples by gas chromatography–mass spectrometry (GC–MS) and quantified by high-performance liquid chromatography (HPLC). Reportedly, NOHA concentrations in human plasma and serum range over four orders of magnitude (e.g., 2 nM–34 µM). The natural occurrence of NOHA in urine has not been reported thus far. Here, we report a validated stable-isotope dilution GC–MS method for the quantitative determination of NOHA in 10-µL aliquots of human serum and urine samples. The method is based on a two-step derivatization of NOHA to the methyl ester pentafluoropropionyl (PFP) derivatives using newly synthesized trideuteromethyl ester NOHA (d3Me-NOHA) as the internal standard and GC–MS quantification. NOHA was found to form a methyl ester-NG,Nδ,Nα-pentafluoropropionyl derivative, i.e., Me-(PFP)3 (M, 642) with the NG-hydroxy group remaining non-derivatized. Selected-ion monitoring of mass-to-charge (m/z) ratio of 458 for endogenous NOHA and m/z 461 for d3Me-NOHA in the negative-ion chemical ionization mode revealed NOHA concentrations of the order of 0.2 µM in human serum and 3 µM in urine samples. Accuracy (recovery, %) was 91.6 ± 1.6% in serum and 39.9 ± 4.5% in urine. Inorganic nitrate was found to decrease NOHA recovery from urine presumably through the reaction of the OH group of NOHA with nitric acid. Imprecision (RSD,  %) ranged between 1.4 and 14.8% in serum, and between 5.3 and 18.4% in urine in the investigated concentration range (0–15 µM NOHA). Ten healthy kidney donors excreted in the urine (mean ± SEM) 13.9 ± 1.81 µmol NOHA per day before and 10.9 ± 1.4 µmol NOHA per day after kidney donation (P = 0.24). Similar results were observed for dimethylamine (DMA), the major urinary metabolite of asymmetric dimethylarginine (ADMA). Changes in NOHA and DMA correlated positively (r = 0.718, P = 0.019). This is the first report on the occurrence and measurement of NOHA in human urine and on the effect of human unilateral nephrectomy on urinary NOHA and DMA. Healthy kidney donation may be useful as a model for kidney disease.


Arginase l-Arginine Asymmetric dimethylarginine Dimethylamine NG-Hydroxy-l-arginine Mass spectrometry Nitrate Nitric oxide Nitric oxide synthase 



Asymmetric dimethylarginine


Dimethylarginine dimethylaminohydrolase




Electron ionization


Gas chromatography–mass spectrometry


Internal standard


Liquid chromatography–mass spectrometry


Liquid chromatography–tandem mass spectrometry


Lower limit of quantitation








Guanidine nitrogen


Nitric oxide




Nitric oxide synthase


Inducible nitric oxide synthase




Peak area ratio




Pentafluoropropionic anhydride


Protein arginine methyltransferase


Relative standard deviation


Symmetric dimethylarginine


Tumor necrosis factor alpha


Compliance with ethical standards

Conflicts of interest

All authors report no conflicts of interest.

Ethical statement

The Institutional Review Board approved the study protocol (METc 2008/186) which was in adherence to the Declaration of Helsinki.

Supplementary material

726_2018_2695_MOESM1_ESM.docx (273 kb)
Supplementary material 1 (DOCX 273 kb)


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Copyright information

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

Authors and Affiliations

  • Alexander Bollenbach
    • 1
  • Stephan J. L. Bakker
    • 2
    • 3
  • Dimitrios Tsikas
    • 1
    Email author
  1. 1.Institute of ToxicologyCore Unit Proteomics, Hannover Medical SchoolHannoverGermany
  2. 2.Division of Nephrology, Department of Internal MedicineUniversity Medical CenterGroningenThe Netherlands
  3. 3.Groningen Kidney CenterGroningenThe Netherlands

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