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

摘要

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 A mu L) 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.