Quantitative and isotopic analysis of amino acids, allantoin, and allantoic acid in soybeans by LC-MS using the atmospheric pressure chemical ionization method

Abstract

In this report, we determined whether liquid chromatography mass spectrometry (LC-MS) using the atmospheric pressure chemical ionization (APCI) method could be applied for isotopic analysis of the high polar compounds, allantoin, and allantoic acid, which are the major transport forms of N from soybean nodules. The peak areas of the ion chromatogram of authentic allantoin and allantoic acid obtained by selected ion monitoring (SIM) were proportional to the contents of the compounds. The data obtained from the ion intensity using the scan mode and the area of the ion chromatogram using the SIM mode were entirely consistent with the isotope ratio of the compounds. The allantoin and allantoic acid concentrations in the xylem sap of soybeans at various growth stages were measured by the following two methods using LC-MS; an absolute calibration method and an isotopelabeled internal standard method. The values estimated by the isotope-labeled internal standard method were more consistent with those obtained by the colorimetric method. Furthermore, amino acids could be detected under the same analytical conditions. The 15N ratio of asparagine and aspartic acid in the non-nodulating soybean xylem sap which had been supplied with 15N-labeled nitrate could be measured. These results indicate that LCMS (APCI) is a suitable method for isotopic analysis of high polar compounds, including allantoin and allantoic acid in plant xylem sap and tissues.