Quantitative UHPLC-MS/MS analysis

The samples derived from YES plates was followed by dilution of 1 + 9 in acetonitrile/water/acetic acid 49.5/49.5/1 (v/v/v). Further dilutions of 1 + 49 and 1 + 999 were performed and re-analyzed in cases of distorted peak shapes due to column overloading caused by large analyte concentrations. The method used in this study is an extension of the version described in detail elsewhere [30 (link),31 (link),32 ]. Briefly, a QTrap 5500 MS/MS system (Sciex, Foster City, CA, USA) equipped with a TurboV electrospray ionization (ESI) source was coupled to a 1290 series UHPLC system (Agilent Technologies, Waldbronn, Germany). Chromatographic separation was performed at 25 °C on a Gemini C18-column, 150 × 4.6 mm i.d., 5 μm particle size, equipped with a C18 security guard cartridge, 4 × 3 mm i.d. (both Phenomenex, Torrance, CA, USA). Two MS/MS transitions were acquired per analyte with the exception of moniliformin and 3-nitropropionic acid that yield only one product ion. For confirmation of a positive identification, the ion ratio had to agree with the related values of the related authentic standard within 30% as stated in official guidelines [32 ], whereas for the retention time, a more strict in-house criterion of ± 0.03 min was applied.