17:15 〜 18:30
[MIS14-P18] GC-MSによる水溶液試料中の13C安定同位体ラベル化有機酸の分析法
キーワード：GC-MS、安定同位体トレーサー、13Cラベル、酢酸、ギ酸
Organic acids, especially formic acid and acetic acid, have been predicted to form abiotically from inorganic precursors under certain aqueous conditions (e.g., McCollom, 2013, Reviews in Mineralogy & Geochemistry 75: pp. 467-494; Preiner et al., 2020, Nature ecology & evolution 4.4: pp. 534-542). Stable isotope tracers are powerful tool for visualizing a specific reaction pathway and distinguishing between experimental products and contamination. Laboratory experiments with stable-isotopically labeled starting materials require the determination of isotope enrichment of product materials. Gas chromatography-Mass spectrometry (GC-MS) technique with direct injection of aqueous sample has been a simple and rapid method for determining the isotope enrichment of underivatized acetic acid (Mulat and Feilberg, 2015, Talanta 143: pp. 56-63). However, the applicability of 13C-tracer analysis by GC-MS to formic acid has still not been investigated adequately. Furthermore, the previous calculation method which estimates the 13C-labeled mass isotopomer ratio of acetic acid by using ion intensities of m/z 60, 61 and 62 (Mulat and Feilberg, 2015) cannot distinguish between two kinds of singly labeled isotopomers, i.e., CH313COOH and 13CH3COOH. In this study, I investigate a determination method for 13C-labeled isotopomer ratio of formic acid and acetic acid in aqueous solution by using GC-MS data.
Standard solutions were prepared from sodium formate and sodium acetate reagents. The unlabeled sodium salts with natural isotope abundance were purchased from Sigma-Aldrich. The 13C-labeled sodium salts, which were artificially enriched in 13C atom in some specific position with isotopic purity of 99%, were purchased from Cambridge Isotope Laboratories, Inc. Pure standard solutions (50 to 1000 μmol/L) were individually prepared; unlabeled formic acid (HCOOH), labeled formic acid (H13COOH), unlabeled acetic acid (CH3COOH), singly labeled acetic acids (CH313COOH and 13CH3COOH) and doubly labeled acetic acid (13CH313COOH). Then, mixture solutions in which two or more isotopomers presented were prepared by mixing of these pure standards. The aqueous samples were analyzed by GC-MS (7890B gas chromatograph and 5977B mass spectrometer, Agilent Technologies) which was placed at the dedicated analysis laboratory for samples labeled with heavy stable isotopes such as 2H, 13C and 15N…etc. in the Geomicrobiology Research Group (Geological Survey of Japan, AIST). Prior to direct injection of aqueous sample into the GC-MS system, sample was acidified to about pH 2 with phosphoric acid to improve peak sharpness and reduce ghost peaks (Pind et al., 2003, Biotechnology and Bioengineering 82: pp. 791-801; Mulat and Feilberg, 2015). Dissolved formic acid and acetic acid were detected with good peak sharpness at minimum concentration of 50 μmol/L and 4 μmol/L, respectively.
In this presentation, I will report the following results and discussions.
- Comparison of results from different data acquisition modes to assess the optimal GC-MS configuration; Profile mode vs. Selected Ion Monitoring (SIM) mode.
- Effectiveness assessment of new calculation approach by least-square method with GC-MS data for determining each mixing ratio of 13C isotopomeric tracers of formic acid and acetic acid.
Standard solutions were prepared from sodium formate and sodium acetate reagents. The unlabeled sodium salts with natural isotope abundance were purchased from Sigma-Aldrich. The 13C-labeled sodium salts, which were artificially enriched in 13C atom in some specific position with isotopic purity of 99%, were purchased from Cambridge Isotope Laboratories, Inc. Pure standard solutions (50 to 1000 μmol/L) were individually prepared; unlabeled formic acid (HCOOH), labeled formic acid (H13COOH), unlabeled acetic acid (CH3COOH), singly labeled acetic acids (CH313COOH and 13CH3COOH) and doubly labeled acetic acid (13CH313COOH). Then, mixture solutions in which two or more isotopomers presented were prepared by mixing of these pure standards. The aqueous samples were analyzed by GC-MS (7890B gas chromatograph and 5977B mass spectrometer, Agilent Technologies) which was placed at the dedicated analysis laboratory for samples labeled with heavy stable isotopes such as 2H, 13C and 15N…etc. in the Geomicrobiology Research Group (Geological Survey of Japan, AIST). Prior to direct injection of aqueous sample into the GC-MS system, sample was acidified to about pH 2 with phosphoric acid to improve peak sharpness and reduce ghost peaks (Pind et al., 2003, Biotechnology and Bioengineering 82: pp. 791-801; Mulat and Feilberg, 2015). Dissolved formic acid and acetic acid were detected with good peak sharpness at minimum concentration of 50 μmol/L and 4 μmol/L, respectively.
In this presentation, I will report the following results and discussions.
- Comparison of results from different data acquisition modes to assess the optimal GC-MS configuration; Profile mode vs. Selected Ion Monitoring (SIM) mode.
- Effectiveness assessment of new calculation approach by least-square method with GC-MS data for determining each mixing ratio of 13C isotopomeric tracers of formic acid and acetic acid.