Multifunctional and Highly Sensitive Precolumn Reagents for Amino Acids in Liquid Chromatography/Tandem Mass Spectrometry

Abstract

We have developed novel precolumn derivatization reagent, p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS), for sensitive analyses of amino acids using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). TAHS, an activated carbamate, was reacted briefly with the amino group to form a ureide bond under mild condition. The derivatives provided selective cleavage at the binding site between the reagent and the amino acid in the collision cell of the mass spectrometer and produced a characteristic fragment derived from the reagent moiety. Using the precursor ion scan mode of the tandem mass spectrometry, amino acids derivatized with the reagents were simultaneously measured on the chromatogram. Selective cleavage also enabled the straightforward isotope ratio analysis of amino acids by the selected reaction monitoring mode, which was applicable in (13)C metabolic flux analysis. TAHS, which contains a cationic quaternary amine, achieved subfemtomole to attomole levels of amino acids detection by measurement in the selected reaction monitoring mode. We also synthesized trideuteriummethyl-substituted TAHS, TAHS-d(3), and demonstrated that the combination of TAHS and TAHS-d(3) is useful in comparing amino acid concentrations between two different samples using a single LC/MS/MS measurement.

Role of dietary amino acid balance in diet restriction‐mediated lifespan extension, renoprotection, and muscle weakness in aged mice

“…The water‐soluble fractions were concentrated 10‐fold using a centrifugal evaporator and applied to the amino acid analysis. Amino acid concentrations in kidney homogenates were measured using high performance liquid chromatography‐electron spray ionization‐mass spectrometry (MS)‐MS (HPLC‐ESI‐MS/MS), followed by precolumn derivatization (Kume et al., 2014; Miyagi et al., 2011; Shimbo, Yahashi, Hirayama, Nakazawa, & Miyano, 2009). …”

Plasma amino acid profiles in healthy East Asian subpopulations living in Japan

“…Subsequently, plasma samples were deproteinized with acetonitrile at a final concentration of 80%. The samples were then subjected to precolumn derivatization followed by high‐performance liquid chromatography (HPLC)‐electrospray ionization (ESI)‐mass spectrometry (MS) for AA quantification, which was performed as previously described (Shimbo et al, 2009a; Shimbo et al, 2009b; Yoshida et al, 2015). The following 21 AAs were quantified: alanine (Ala), alpha‐aminobutyric acid (α‐ABA), arginine (Arg), asparagine (Asn), citrulline (Cit), glutamate (Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).…”

Amino acid analysis of sub-picomolar amounts of proteins by precolumn fluorescence derivatization with 6-aminoquinolyl-<i>N</i>-hydroxysuccinimidyl carbamate

“…Furthermore, precolumn derivatization has enabled the sub-picomolar quantitation of amino acids with the development of fluorescent reagents such as OPA, 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F), 9-fluorenylmethyl-chloroformate (FMOCCl), and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) (See details in Section 2.2.). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection of pre-labeled amino acids has been also reported including pre-labeling by aminereactive isobaric tagging reagents that include different mass group of stable isotope (7,8), by p-N,N,Ntrimethylammonioanilyl-N'-hydroxysuccinimidyl carbamate iodide (9), and by AQC (10).…”