LC-SPE-NMR/MS
The analyses were performed by in-line instruments of interfacing liquid chromatography with parallel NMR and mass spectrometry. The 60 dried microsome pellets described above were each re-constituted in 300 µL of methanol and sonicated for 5 min. Supernatants were pooled into one 50 mL falcon tube. A second extraction of the microsome residue was performed by adding an additional 300 µL of acetonitrile followed by sonication for 5 min. The supernatant from the second extraction was then transferred to the same 50 mL falcon tube described above. The pooled supernatant was evaporated under nitrogen gas to a final volume of 200 µL and transferred to an HPLC vial. The extract (33 µL) was injected into the HPLC (Agilent 1260) at a flow rate of 0.5 mL/min at 25°C on a 250×4.6mm, 5µm Kinetex EVO C18 column (Phenomenex). The mobile phase A was water with 0.1% formic acid-d2 (CDOOD) and mobile phase B was acetonitrile with 0.1% CDOOD. The elution gradient was started at 50% mobile phase B for 2 min (0.0-2.0 min), 50-100 %B for 33 min (2.0-35.0 min), 100%B for 5 min (35.0-40.0 min). UV detection was done at 240 nm. An MS Bridge interface (Bruker Biospin) was used to split a small portion of the effluent from the HPLC column and direct it to the ion source of a MicrOTOF-QII mass spectrometer (Bruker Daltonik, Bremen, Germany) using an acetonitrile make-up flow of 70 µL/min. The mass spectrometer was operated in positive ionization mode with a scan range at m/z 50 to 1,000. Mass calibration was done with sodium acetate infused at the beginning of the chromatography. The isolated metabolites were trapped post-column on 2×10 mm solid phase extraction cartridges filled with HySphere GP resin using the Prospekt 2 SPE interface from Spark Holland (Emmen, the Netherlands). The peaks of 3 injections (each 33 µL) were combined on individual cartridges (multi trapping). In total 2x3 trappings were performed. The make-up flow rate for the trapping was 1.5 mL/min. After chromatography the cartridges including the metabolites were dried with nitrogen gas and the two cartridges containing the metabolites were eluted with each 300uL of acetonitrile-d3 (CD3CN) into 5 mm NMR tubes. In total, the six trappings were finally transferred to the NMR tube for each metabolite resulting in a total volume of 600 µL. The API reference sample was measured with a 500 MHz AVANCE III NMR spectrometer equipped with a nitrogen cooled 5 mm Prodigy TCI (triple resonance inverse configuration of the coils with a cooled carbon channel) cryo probe. Post-column SPE fractions of the metabolites were measured first with the 500 MHz spectrometer and later with an 800 MHz Neo NMR spectrometer equipped with a helium cooled 5 mm TCI cryo probe (Bruker Biospin, Rheinstetten, Germany). SPE fractions were analyzed with the CMC-se software using optimized parameter sets including the Proton-1D, edited hetero nuclear single coherence spectroscopy (HSQC) and hetero nuclear multiple bond coherence spectroscopy (HMBC). In addition, selective HMBC experiments were performed for areas of closely resonating carbon resonances. Ivermectin (5.2 mg) was dissolved in deuterated acetonitrile (1 mL) and transferred to a 5 mm NMR tube and run as a reference compound.