Isolation of underivatized amino acids by ion-pair high performance liquid chromatography for precise measurement of nitrogen isotopic composition of amino acids: Development of comprehensive LC × GC/C/IRMS method

Abstract

a b s t r a c tNitrogen isotopic composition of amino acids has been widely applied to biochemical, ecological, archeological, and biogeochemical studies in an attempt to trace nitrogen source and transformation processes. For accurate isotope analysis of individual amino acids, we validated a preparative method involving the isolation of underivatized amino acids by ion-pair chromatographic separation and confirmed the consistency of nitrogen isotope composition. Ion-pair reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI-MS) and gas chromatography/combustion coupled with isotope ratio mass spectrometry (GC/C/IRMS) were conducted for the purpose of separation of underivatized amino acids and nitrogen isotopic analysis, respectively. Firstly, we confirmed the resolution of proteinogenic and non-proteinogenic amino acids by the preparative ion-pair LC separation. Diagnostic product ions determined by mass spectrometry can support the rapid identification of individual amino acids in screening analyses. Secondly, we observed no dependency on nitrogen isotopic composition for the injection amount of underivatized amino acids and even for different chemical formula including neutral, acidic, sulfur-containing, heterocyclic, and aromatic species. The recovery during the LC was 91.7 ± 4.3% (n = 3). The present method and strategy of LC coupled with GC/C/IRMS (i.e., comprehensive LC × GC/C/IRMS) are useful for the high precision determination of the nitrogen isotopic composition of amino acids, in conjunction with an appropriate pre-treatment of cation-exchange chromatographic procedures.

Further characterization of carbonaceous materials in Hayabusa‐returned samples to understand their origin

“…; Takano et al. ; Sugahara et al. ), as well as in situ molecular analysis by desorption electrospray ionization coupled with high‐resolution mass spectrometry (Naraoka and Hashiguchi ).…”

“…Once carbonaceous materials are contaminated by terrestrial materials, they are difficult to distinguish from each other. In future work, we will establish a state-of-the-art analytical scheme including combustion and/or dissolution methods for the analysis of higher molecular structures, including high-precision, compound-specific measurement of amino acids with the corresponding stereoisomers (Naraoka et al 2012;Hamase et al 2014;Takano et al 2015;Sugahara et al 2018), as well as in situ molecular analysis by desorption electrospray ionization coupled with high-resolution mass spectrometry (Naraoka and Hashiguchi 2018). witness plate (silicon and fluoro rubber), by FE-SEM-EDS, STXM-XANES, and TEM.…”

A new analytical method for determination of the nitrogen isotopic composition of methionine: Its application to aquatic ecosystems with mixed resources

“…To ensure the precision and accuracy of the δ 15 N Met measurements, we used two independent methods. In the first method, half the underivatized AA fractions from the gastropod samples (i.e., HCl‐hydrolyzed soft tissue of Haliotis discus ) were injected into an HPLC apparatus (Agilent 1260 series, Agilent Technologies, Palo Alto, California, U.S.A.) and separated with the modified method of Takano et al (). We used a Hypercarb column (4.6 × 150 mm, particle size 5 μ m; Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.) and a guard column (4.6 × 10 mm, 5 μ m; Thermo Fisher Scientific) with a Cool Pocket column cooler (Thermo Fisher Scientific) stabilized at 10.0°C.…”

“…Increasing the amount of AAs injected during GC‐C‐IRMS does not improve detection, as nearby peaks (Met and Glu) coelute and are indistinguishable on the chromatograms. The Glu peak and the level of background impurities must be reduced by with open‐column chromatography before GC injection (Takano et al ) or by isolating Met with HPLC followed by EA‐IRMS (Broek and McCarthy ; Takano et al ) to improve the analytical stability and sensitivity of the δ 15 N Met analysis. Alternatively, the β Glu/Phe value must be corrected with an independent approach (e.g., using fatty acid profiles; Hebert et al ).…”

A Method for Detection of Trace Concentrations of Underivatized Amino Acid in Hydrothermal Fluids by Ion-Pairing Reversed-Phase UPLC-ESI-QTOF-MS

“…These pioneers in ion-pairing HPLC-ESI-MS/MS achieved separation in about 13 min and obtained LOD from 0.03 to 3 µM depending on the amino acid. The technique was used later on but LOD were similar or not reported and retention times were higher [37] [38]. Adaptation of the original method to the current most efficient chromatographic instrument (UPLC) enhanced the analysis time down to 6.5 min [39].…”