Tsq altis plus

For the evaluation
of phase I metabolic stability, the compound (1 μM) was incubated
with 0.5 mg/mL pooled C57BL/6 mouse, Wistar rat liver microsomes (Xenotech,
Kansas City, USA), or human liver microsomes (Corning, New York, USA),
2 mM NADPH, and 10 mM MgCl₂ at 37 °C for 120 min on
a microplate shaker (Eppendorf, Hamburg, Germany). The metabolic stability
of testosterone, verapamil, and ketoconazole was determined in parallel
to confirm the enzymatic activity of mouse/rat liver microsomes; for
human liver microsomes, testosterone, diclofenac, and propranolol
were used. Incubation was stopped after defined time points by precipitation
of aliquots of enzymes with 2 volumes of cold acetonitrile containing
an internal standard (15 nM diphenhydramine). Samples were stored
on ice until the end of the incubation, and precipitated protein was
removed by centrifugation (15 min, 4 °C, and 4000g). The concentration of the remaining test compound at the different
time points was analyzed by HPLC-MS/MS (TSQ Altis Plus, Thermo Fisher,
Dreieich, Germany) and used to determine the half-life (t_1/2).

Plasma protein binding was determined
using a Rapid Equilibrium Dialysis (RED) system (Thermo Fisher Scientific,
Waltham MA, USA). Compounds were diluted in murine (CD-1), rat (Wistar),
or human plasma (Neo Biotech, Nanterre, France) to 10 μM and
added to the respective chamber according to the manufacturer’s
protocol followed by addition of PBS pH 7.4 to the opposite chamber.
Samples were taken immediately after addition to the plate as well
as after 2, 4, and 5 h by mixing 10 μL with 80 μL of ice-cold
acetonitrile containing 12.5 nM diphenhydramine as an internal standard
followed by addition of 10 μL of plasma to samples taken from
PBS and vice versa. Samples were stored on ice until the end of the
incubation, and precipitated protein was removed by centrifugation
(15 min, 4 °C, 4000g, and 2 centrifugation steps).
The concentration of the remaining test compound at the different
time points was analyzed by HPLC-MS/MS (TSQ Altis Plus, Thermo Fisher,
Dreieich, Germany). The amount of the compound bound to protein was
calculated using the equation PPB [%] = 100 – 100 × (amount
in the buffer chamber/amount in the plasma chamber).

The digested peptides were injected into a nanoflow DIONEX UltiMate 3000 RSLCnano System coupled with a TSQ Altis Plus (Thermo Fisher Scientific™). For each sample, 500 ng of peptide was separated across a 30 min LC gradient (10–30% buffer B: 98% ACN, 0.1% FA; buffer A: 2% ACN, 0.1% FA) at a flowrate of 300 nL/min (trapcolumn, 3 μm, 100 Å, 20 mm × 75 μm i.d.; analytical column, 1.9 μm, 120 Å, 150 mm × 75 μm i.d.). The cycle time was 2 s, and the resolutions of Q1 and Q3 were 0.7 and 1.2, respectively. The retention time was predicted via iRT, and the isolation time window was 5 min. The data acquired via the MRM experiment were analyzed using Skyline [20147306]. The peak area of each peptide was calculated to their Log values, and the relative expressions of the RF groups were normalized to their control groups.