Molecular Average and Intramolecular δ¹³C Measurements of Pyruvic Acid and Acetic Acid Using Gas Chromatography Orbitrap Mass Spectrometry (GC Orbitrap-MS)

We developed a novel method using gas chromatography – Orbitrap mass spectrometry (GC Orbitrap MS) to measure the intramolecular carbon isotope value of pyruvic acid and acetic acid with high mass accuracy and mass resolution with nanomole (10–100) injections of analyte. Previous efforts to analyze intramolecular isotope patterns of small organic acids have been limited by labor-intensive chemical degradation steps, a narrow potential analyte pool, or large sample mass requirements. We present a new way to trap an analyte peak, which is then tested by measuring the molecular average and intramolecular carbon isotope values of pyruvic acid and acetic acid standards. Molecular average (MA) δ¹³C measurements with our method agreed with complementary methods within 2.2–2.6‰ relative mean standard deviation (RMSD) for 0.1 micromoles of compound, whereas intramolecular δ¹³C values varied from the complementary method from 1.9 to 4.9‰ RMSD. Intramolecular δ¹³C measurements of pyruvic acid and acetic acid yielded acquisition errors (σ_AE) of better than 0.2‰ and an experimental reproducibility (σ_ER) of <2.8‰ and <3.4‰, respectively, for 0.1 micromoles of analyte. This method enables measurements of the intramolecular δ¹³C values of small organic acids sufficiently precise to differentiate synthesis pathways for these compounds, including for pathways within the central metabolism, and in extracts of carbonaceous meteorites, which are theorized to have formed in interstellar ices.