Elute uhplc

For negative ion mode, LC separation was achieved with Elute UHPLC (Bruker Daltonics, Germany) using mobile phase A: methanol/water (1:1; v/v) and mobile phase B: methanol/isopropanol (2:8; v/v), both containing 0.1% formic acid, 7.5 mM ammonium formate, and 0.1% triethylamine (TEA), respectively. For positive ion mode, the same solvent system was used without TEA. The LC method consisted of a 20 min run time with the following gradient: T0 to T1 – mobile phase A at 60% and mobile phase B at 40% which was then elevated to 90% mobile phase B and 10% mobile phase by T16 and up to 99% mobile phase B by T16.5 and then escalated back to 40% mobile phase B by T20. A constant flow rate of 0.2 mL/min was maintained throughout the runtime. Throughout the analysis, the autosampler was maintained at 4 °C. The samples were injected onto the column with a partial loop mode with an injection volume of 20 µL and 10 µL corresponding on the column to 1 µL plasma in negative ion mode and to 0.5 µl plasma in positive ion mode, respectively. For the separation, a C18 Luna Omega column (100 × 2.1 mm × 1.6 μm) (Phenomenex, Germany) was used and thermostated at 45 °C. Total run time including column wash was 20 min. No carry-over was observed in negative and positive ion mode when using wash-run protocol and analysis batch structure inferred from previous work^{22 (link),59} .

LC-MS was performed as previously described [21 (link)]. Chromatographic separation was performed using an Elute UHPLC (Bruker Daltonics GmbH, Bremen, Germany) equipped with a Millipore Chromolith Performance/PR-18e, C18 analytical column (100 mm × 2 mm, Merck, Darmstadt, Germany) with Chromolith^® RP-18 endcapped 5-3 guard cartridges (Merck, Darmstadt, Germany). Mass spectra were obtained on a Maxis Impact Q-TOF mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) equipped with an electrospray ionization source (Bruker Daltonics GmbH, Bremen, Germany) and operated in positive ionization mode. Mass spectra were analyzed using DataAnalysis^® and TASQ^® software (Bruker Daltonics GmbH, Bremen, Germany).

HPLC-ESI-MS analysis was performed on Impact II QqTOF high-resolution mass spectrometer (Bruker Daltonik, Germany) with Elute UHPLC (Bruker Daltonik, Germany) on Intensity Solo 1.8 C18-2 2.1 × 100 mm 1.8 μm 90 Å reverse-phase column (Bruker Daltonik, Germany) with the following conditions: column flow 0.25 ml/min, gradient elution from 5 to 70% B in 25 min (A: 0.1% formic acid in water, B: 0.1% formic acid in AcN), column temperature 40°C, injection volume 5 μl, ESI source in positive mode, HV capillary at 4.5 kV, spray gas–nitrogen at 2.1 bar, dry gas–nitrogen at 8 l/min 220°C, scan range m/z 50–2200, 2-Hz scan rate for full scan, automatic MS/MS mode (CID) with dynamic scan rate 2–8 Hz, nitrogen as collision gas, collision energy from 23 eV at m/z 300 to 65 eV at m/z >1300, and automatic internal calibration with ESI-L low concentration tuning mix (Agilent Technologies, USA). Spectra were processed with BioPharma Compass 3.1.1 (Bruker Daltonik, Germany).

HPLC-MS/MS analysis was performed as described earlier [3 (link)]. Impact II QqTOF high-resolution mass-spectrometer (Bruker Daltonik, Germany) equipped with Elute UHPLC (Bruker Daltonik, Germany) and Intensity Solo 1.8 C18-2 2.1*100 mm 1.8 μm 90 Å reverse-phase column (Bruker Daltonik, Germany) was used. Chromatography was performed in an acetonitrile gradient with 0.1% (v/v) formic acid as additive.

The UHPLC–MS/MS
analysis
was performed in a Maxis II ETD Q-TOF mass spectrometer (Bruker Daltonics,
Germany) using an electrospray ionization (ESI) source with either
an Elute UHPLC (Bruker Daltonics, Germany) or a 1260 Infinity II Binary
Pump (Agilent Technologies, USA) system. The separation was performed
on an Acquity UPLC HSS T3 column (1.8 μm, 100 × 2.1 mm)
from Waters Corporation. Milli-Q water with 0.1% formic acid was used
as mobile phase A, and LC–MS grade methanol with 0.1% formic
acid was used as mobile phase B. The column temperature was kept at
40 °C, and the autosampler temperature was kept at 4 °C.
The flow rate was set to 0.22 mL/min with an injection volume of 5
μL. The gradient used was as follows: 0–2 min, 0% B;
2–15 min, 0–100% B; 15–16 min, 100% B; 16–17
min, 100–0% B; 17–23 min, 0% B. The system was controlled
using the Compass HyStar software package from Bruker (Bruker Daltonics,
Germany). High-resolution mass spectra were acquired in negative mode
at a mass range of m/z 50–1200.
Data acquisition was performed in AutoMSMS mode (data-dependent acquisition,
DDA) with a cycle time of 0.5 s and a ramped collision energy from
20 to 50 eV. A solution of sodium formate [10 mM in a mixture of 2-propanol/water
(1/1, v/v)] was used for internal calibration at the beginning of
each run, in a segment between 0.10 and 0.31 min.

HPLC-MS/MS analysis was performed on Impact II QqTOF high-resolution mass-spectrometer (Bruker Daltonik, Germany) equipped with Elute UHPLC (Bruker Daltonik, Germany) on Intensity Solo 1.8 C18-2 2.1*100 mm 1.8 µm 90 Å reverse-phase column (Bruker Daltonik, Germany) with following conditions: column flow 0.25 mL/min, gradient elution from 30% to 100% B in 60 min (A: 0.1% (v/v) formic acid in water, B: 0.1% (v/v) formic acid in ACN), column temperature 40°C, injection volume 15 μL (1 µg/µL of dry sample in 60% (v/v) ACN), ESI source in positive mode, HV capillary at 4.5 kV, spray gas – nitrogen at 2.1 bar, dry gas – nitrogen at 8 L/min 220°C, scan range m/z 50-2200, 2 Hz scan rate for full scan, automatic MS/MS mode (CID) with dynamic scan rate 2-8 Hz, nitrogen as collision gas, collision energy from 23 eV at m/z 300 to 65 eV at m/z >1300, automatic internal calibration with ESI-L low concentration tuning mix (Agilent Technologies, USA). Spectra were processed with BioPharma Compass 3.1.1 (Bruker Daltonik, Germany).