Acquity uplc ms system

The samples were analyzed in a Waters 2695 HPLC system (Waters Co., Milford, MA, USA) fitted with a Diomansil C18 column (4.6 mm × 250 mm). The chromatographic conditions were as follows: mobile phase A (NaAc-HAc buffer (50 mM, pH 4.2):acetonitrile = 50:50); mobile phase B (acetonitrile); gradient elution program; flow rate, 1 mL min⁻¹; column temperature, 25 °C; injection volume, 10 µL. Post-column derivatization was conducted with Fmoc-Cl [44 (link)]. The Fmoc-Cl derivatives of the amino acids were detected at 263 nm.
LC-MS analysis was conducted in a Waters ACQUITY UPLC-MS system fitted with a Waters ACQUITY UPLC HSS C18 reverse-phase column (inner diameter, 1.8 µm) (Waters Co., Milford, MA, USA). The inlet, MS transfer line, and ion source temperatures were set to 280, 280, and 230 °C, respectively.

The reaction products were detected with the ACQUITY UPLC/MS system (Waters, Milford, MA, USA) in electrospray ionization negative ion mode with selected ion monitoring, as described in Method S5. The amount of reaction products was determined as the peak area using MassLynx software (Waters). The kinetic parameters were calculated by fitting a Michaelis–Menten curve to the raw kinetic data using GraphPad Prism 6.0h software (GraphPad Software, San Diego, CA, USA). The raw data were calibrated beforehand with standard curves of authentic glycyrrhizin and glucoglycyrrhizin.

Extracted metabolites were detected using the ACQUITY UPLC/MS system (Waters, Milford, MA, USA) with an ACQUITY UPLC HSS C18 column (2.1 × 150-mm column and 2.1 × 5-mm VanGuard pre-column; particle size, 1.8 μm, Waters, Milford, MA, USA) and an ACQUITY TQ Detector (Waters) in electrospray ionisation negative-ion mode with selected-ion monitoring (SIM). The mobile phase was composed of 0.025% (v/v) acetic acid in water (solvent 1) and 0.025% (v/v) acetic acid in acetonitrile (solvent 2). Samples were separated via gradient elution with 30% solvent 2 for 6 min to 100% over 22 min (40% at 6 min, 50% at 18 min and 100% at 28 min) at a flow rate of 0.20 mL/min. The final condition was maintained for 3.5 min and returned to the initial condition, resulting in a total chromatography run time of 38.5 min. The sample manager and the column were kept at 15 °C and 30 °C, respectively. For MS detection, the capillary voltage was set to 2.5 kV, cone voltage to 80 V, extractor voltage to 3 V, source temperature to 150 °C, desolvation temperature to 350 °C, cone gas flow to 50 L/h and desolvation gas flow to 600 L/h. The quantities of extracted compounds of interest were determined from the peak areas using MassLynx software (Waters). The values used for SIM for each assay are listed in Supplementary Table 4.

The aqueous kinetic
solubility was determined from a 10 mM DMSO stock solution of test
compound (39 or 50) in PBS buffer at pH
7.4. The study was performed by incubating an aliquot of 10 mM DMSO
stock solution in PBS (pH 7.4) to a target concentration of 250 μM
(2.5% DMSO). The incubation was carried out under shaking at 25 °C
for 24 h, followed by centrifugation at 21,100g for
30 min. The supernatant was further diluted (4:1) with CH₃CN, and the dissolved test compound was quantified by UV at 215 nm
on a Waters ACQUITY UPLC-MS system consisting of a single quadrupole
detector (SQD) mass spectrometer equipped with an electrospray ionization
interface and a photodiode array detector (PDA) from Waters Inc. (Milford,
MA, USA). Electrospray ionization in positive mode was used in the
mass scan range 100–500 Da. The PDA range was 210–400
nm. The analyses were run on an ACQUITY UPLC BEH C18 column (50 ×
2.1 mm ID, particle size 1.7 μm) with a VanGuard BEH C18 precolumn
(5 × 2.1 mm ID, particle size 1.7 μm), using 10 mM NH₄OAc in H₂O at pH 5 adjusted with AcOH (A) and 10
mM NH₄OAc in CN–H₂O (95:5) at pH 5 (B)
as the mobile phase. The aqueous kinetic solubility (in μM)
was calculated by dividing the peak areas of dissolved test compound
and test compound in the reference (250 μM of test compound
in CH₃CN) and multiplying by the target concentration and
dilution factor.

All the
tested compounds
were >95% pure. Compound 5 displayed ≥99% purity as determined
by UPLC/MS analysis (Supporting Information Purity of compound 5). 10 mM stock solution of Compound 5 was prepared
in DMSO-d₆ and further diluted 20-fold
with CH₃CN–H₂O (1:1) for analysis. The
QC analyses were performed on a Waters ACQUITY UPLC/MS system consisting
of a SQD (Single Quadrupole Detector) Mass Spectrometer equipped with
an electrospray ionization interface and a photodiode array detector.
Electrospray ionization in positive and negative modes was applied
in the mass scan range 100–500 Da. The PDA range was 210–400
nm. The analyses were run on an ACQUITY UPLC BEH C18 column (100 ×
2.1 mm ID, particle size 1.7 μm) with a VanGuard BEH C18 pre-column
(5 × 2.1 mm ID, particle size 1.7 μm). The mobile phase
was 10 mM NH₄OAc in H₂O at pH 5 adjusted with
AcOH (A) and 10 mM NH₄OAc in CH₃CN–H₂O (95:5) at pH 5 (B) with 0.5 mL/min as flow rate. A linear
gradient was applied: 0–0.2 min: 10% B, 0.2–6.2 min:
10–90% B, 6.2–6.3 min: 90–100%, 6.3–7.0
min: 100% B.