Amino Acid Analysis in Physiological Samples by GC–MS with Propyl Chloroformate Derivatization and iTRAQ–LC–MS/MS

Abstract

Two mass spectrometry-based methods for the quantitative analysis of free amino acids are described. The first method uses propyl chloroformate/propanol derivatization and gas chromatography-quadrupole mass spectrometry (GC-qMS) analysis in single-ion monitoring mode. Derivatization is carried out directly in aqueous samples, thereby allowing automation of the entire procedure, including addition of reagents, extraction, and injection into the GC-MS. The method delivers the quantification of 26 amino acids. The isobaric tagging for relative and absolute quantification (iTRAQ) method employs the labeling of amino acids with isobaric iTRAQ tags. The tags contain two different cleavable reporter ions, one for the sample and one for the standard, which are detected by fragmentation in a tandem mass spectrometer. Reversed-phase liquid chromatography of the labeled amino acids is performed prior to mass spectrometric analysis to separate isobaric amino acids. The commercial iTRAQ kit allows for the analysis of 42 physiological amino acids with a respective isotope-labeled standard for each of these 42 amino acids.

Evaluation of Physiological Amino Acids Profiling by Tandem Mass Spectrometry

“…Nowadays, identification and quantification of amino acids by mass spectrometry are rather uncommon in clinical laboratories. Only a few methods have already been developed and validated and are based either on native (Piraud et al 2003(Piraud et al , 2005Waterval et al 2009) or on derivatized (Dettmer et al 2012;Dietzen et al 2008;Harder et al 2011;Held et al 2011) metabolites identification.…”

An UPLC-ESI-MS/MS Assay Using 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Derivatization for Targeted Amino Acid Analysis: Application to Screening of Arabidopsis thaliana Mutants

“…This new generation of reagents has the additional advantage of rendering amino acid adducts with desirable features for LC-MS/MS analysis. These reagents include N -hydroxysuccinimide-activated N -alkylnicotinic acid esters (Cn-NA-NHS) [35], p-N,N,N -trimethyl- -ammonioanilyl N’ -hydroxysuccinimidyl carbamate iodide (TAHS) [36], 3-aminopyridyl- N -hydroxysuccinimidyl carbamate (APDS) [37,38], (5- N -succinimidoxy-5-oxopentyl)- triphenylphosphonium bromide (SPTPP) [25], and iTRAQ (isobaric tag for relative and absolute quantitation) [39,40]. Although highly sensitive and selective detection of amino acids is attained by LC-MS/MS when employing these new generation of reagents, unfortunately the reagents are not commercially available (iTRAQ being the exception but it is prohibitively expensive) and some derivatization procedures are still complex and time-consuming.…”

“…Although highly sensitive and selective detection of amino acids is attained by LC-MS/MS when employing these new generation of reagents, unfortunately the reagents are not commercially available (iTRAQ being the exception but it is prohibitively expensive) and some derivatization procedures are still complex and time-consuming. Advantages and shortcomings of these pre-column derivatization methods can be found in the literature [25,35,36,37,38,39,40]. …”

Stepwise extraction, chemical modification, GC–MS separation, and determination of amino acids in human plasma#

“…GC–MS is a reliable and effective method for such measurements. This technology has proved to be an effective and dependable instrumental tool for the comprehensive qualitative and quantitative analysis of complex mixtures of the metabolome based on its robustness, speed, high sensitivity, specificity and reproducibility . The non‐volatile nature and the zwitterionic structure of AA due to their polar (such as amino and carboxyl) groups preclude direct GC–MS experiments, and their conversion to volatile derivatives before GC–MS experiments is required .…”