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.