Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis

UAMC-3203 detection in plasma, whole blood, and tissue homogenates (lysed in PBS using a Precellys 24 Tissue Homogenizer (Bertin Instruments)) was performed on an Agilent 1200 series LC system connected to a 6410 triple quadrupole mass spectrometer from Agilent Technologies (Waldbronn, Germany) with electrospray ionization (ESI) interface operated in positive ionization mode. Chromatographic separation was carried out on a Kinetex Biphenyl column (100 × 2.1 mm, 2.6 μm; Phenomenex (Utrecht, the Netherlands)). The mobile phase consisted of (A) ultrapure water with 0.1% formic acid and (B) acetonitrile/ultrapure water (90/10) with 0.1% formic acid, in gradient at 0.3 mL/min. The ESI source parameters were gas temperature 350 °C, gas flow 10 L/min, nebulizer pressure 35 psi, and capillary voltage 4000 V. Data acquisition was done in multiple reaction monitoring modes (MRM). Confirmation of UAMC-3203 was done using three MRM transitions; the most abundant transition was used as a quantifier (Q) and the other two were used as a qualifier (q). Qualifier/quantifier ratios (q/Q) were calculated for each sample and had to be within ±20% of the q/Q ratio observed in the calibrators. In addition, the retention time of the compound in samples could not deviate >10% of the retention time observed in the calibrators.

UAMC-3203 and nordiazepam-D₅ (Cerilliant Corporation; Texas, US) as internal standard (IS) were diluted in LC-MS grade methanol (Fisher Scientific). A volume of 100 µL sample was spiked with 20 µL IS (200 ng/mL), followed by the addition of 150 µL acetonitrile for plasma and blood. For tissue, 500 µL acetonitrile with 0.1% formic acid was added. Afterward, the mixture was vortexed (2 min, 2000 rpm) and centrifuged (10 min, 9168 g resp. 17968 gfor plasma and blood resp. tissue). The supernatant of plasma and whole blood was then transferred to a 2 mL tube with a 0.20 μm centrifugal filter (VWR, Avantor, Randor, PA, USA). The supernatant of tissue was evaporated under a stream of nitrogen at 40 °C, reconstituted in 100 μL acetonitrile/ultrapure water (90/10) with 0.1% formic acid, and transferred to a 2 mL tube with a 0.20 μm centrifugal filter. All samples were then centrifuged (5 min, 9168 g resp. 10 min, 17968 g for plasma and blood resp. tissue), after which the final extract was transferred to an autosampler vial with a glass insert. Seven-level calibration curves were prepared in blank mouse plasma or whole blood, covering a linear range from 10 ng/mL to 700 ng/mL. Five-level calibration curves were prepared in blank homogenized mouse tissue matrix covering a linear range from 20 ng/mL to 4000 ng/mL. The measured concentrations in ng/mL were further normalized using the weight of the tissue used for homogenization to obtain final concentrations of UAMC-3203 expressed in µg/g.