Metabolite analysis was carried out using a 1290 Series HPLC System (Agilent, Waldbronn, Germany) coupled to a QTrap 5500 LC–MS/MS System (Applied Biosystems SCIEX, Foster City, CA, USA) equipped with Turbo Ion Spray electrospray ionization source as described earlier (Sulyok et al., 2020 (link)). 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 4 × 3 mm i.d. security guard cartridge (Phenomenex, Torrance, CA, USA). 5 μl of sterile‐filtered sample was directly injected without any further manipulation.
Confirmation of positive metabolite identification was carried out by the acquisition of two time scheduled multiple reaction monitoring which yielded 4.0 identification points according to the European Commission decision 2002/657. In addition, retention time and ion ratio had to agree to the related values of authentic standards within 0.03 min and 30% rel., respectively. Quantitation was based on external calibration using serial dilutions of a multi‐analyte stock solution. The accuracy of the method is verified on a continuous basis by participation in a proficiency testing scheme organized by BIPEA (Gennevilliers, France) with a current rate of z‐scores between −2 and 2 of >94% (>1300 results submitted).
Confirmation of positive metabolite identification was carried out by the acquisition of two time scheduled multiple reaction monitoring which yielded 4.0 identification points according to the European Commission decision 2002/657. In addition, retention time and ion ratio had to agree to the related values of authentic standards within 0.03 min and 30% rel., respectively. Quantitation was based on external calibration using serial dilutions of a multi‐analyte stock solution. The accuracy of the method is verified on a continuous basis by participation in a proficiency testing scheme organized by BIPEA (Gennevilliers, France) with a current rate of z‐scores between −2 and 2 of >94% (>1300 results submitted).
Free full text: Click here
