Microtof q mass spectrometer

HPLC/Q-TOF-MS analytical procedures were performed on an Agilent 1200 system (Billerica, MA) coupled with a Bruker Daltonics microTOF-Q mass spectrometer. The HPLC separation was achieved on a Venusil ASB C₈ column (150 mm × 4.6 mm, 5 μm, Bonna-Agela, Tianjin, China) and preceded by a C₁₈ guard column (4.0 mm × 3.0 mm, 5 μm, Phenomenex, Torrance, CA). The mobile phase consisted of acetonitrile (solution A) and 0.1% formic acid in water (solution B). The gradient elution condition was optimized as follows: linear gradient from 5% to 15% A (0–5 min), 15–30% A (5–18 min), 30–65% A (18–24 min), 65–95% A (24–34 min), 95% A (34–41 min) and then back to 5% A in 2 min. The solvent flow rate was 0.8 mL/min. The column temperature was maintained at 35 °C.
The Q-TOF MS was operated in both positive and negative ion modes with an ESI source. The optimized ionization conditions were as follows: capillary voltage was 4.5 kV (ESI⁺) and 3.8 kV (ESI⁻). The nebulizer pressure was maintained at 1.2 bar. Nitrogen was used as the desolation and nebulizing gas at 180 °C by gas flow of 8.0 L/min. The full scan range was set at m/z 100–1000. Formic sodium was used for mass correction.

The identification of the secondary metabolites of rue essential oil was carried out following the methodology established by Díaz-Montes et al. [40 (link)]. An ultra-high-performance liquid chromatography (UHPLC) system (Thermo Scientific, Waltham, MA, USA) equipped with a photo diode array detector (PDA) was used for the analysis. The chromatographic separation was performed on a Hypersil C18 column (50 mm × 2.1 mm and 1.8 μm particle size) (Thermo Scientific, Waltham, MA, USA). The samples (0.3 μL) were injected into the system. The mobile phases were A: acetonitrile (20%) and B: water–trifluoracetic acid pH 2 (80%) in isocratic conditions, with a flow rate of 104 μL/min for a running time of 9.87 min. The UHPLC system was coupled with a micro TOF-Q mass spectrometer (Bruker Daltonics, Karlsruhe, Germany). The conditions of the micro TOF-Q were as follows: 2.7 kV capillary voltage; ionization source in negative electrospray mode (ESI⁻ and ESI⁺); scan range, m/z 50–3000; set collision cell RF, 200.0 Vpp; set nebulizer, 0.4 bar; desolvation gas flow set at 4.0 L/min; and source temperature, 180 °C. The mass data were processed using Bruker Compass Data Analysis version 4.1 software (Bruker Datonics, Karlsruhe, Germany).

All the ESI-MS experiments were performed on a micrOTOF-Q mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a standard ESI source. The instruments were used in the positive-ion mode and calibrated with the Tunemix™ mixture (Agilent Technologies, Palo Alto, CA, USA). The mass accuracy was higher than 5 ppm. The analyte solutions (70 μl) were introduced at a flow rate of 3 μL/min. The instrument parameters were as follows: the scan range of the micrOTOF-Q MS was 50–1600 m/z; nitrogen was used as the drying gas; the flow rate was 4.0 L/min, the temperature was 200°C; the potential between the spray needle and the orifice was 4.2 kV.

NMR data were collected in Bruker ARX-400 and ARX-600 spectrometers, and TMS was used as the internal standard. HR-ESI-MS spectrum was recorded in m/z (rel. %) mode by Bruker micro TOF-Q mass spectrometer. Silica gel used in the experiment was purchased from Qingdao Ocean Chemical Group Co. of China. HPLC separations were carried out on Shimadzu HPLC apparatus (Shimadzu RID-20A UV detector and Shimadzu LC-6AD series pumping system) with a YMC-pack ODS column (250 × 20 mm). Primary antibodies against Bcl-2, Bax, caspase 3 and cleaved caspase-3; Dimethyl sulfoxide (DMSO), thiazolyl blue (MTT), CDCl3, DMSO-d₆ and Pyridine-d₅ were afforded by Sigma-Aldrich Company (St. Louis, MO, USA). Cyclophosphamide (CTX) was ordered from HengRui Medicinal Limited Compony (Jiangsu, China). Sodium chloride injection (Batch No. 100232) was bought from Collen Cornell pharmaceutical compony (Jilin, China). DDP was purchase from Sigma-Aldrich Company (St. Louis, MO, USA). RPMI-1640 medium was purchased from GIBCO (NY, U.S.A.)

The LC-MS analyses were performed in the Laboratory of Mass Spectrometry at the Faculty of Chemistry, University of Wroclaw using an Agilent 1200 HPLC system coupled to a micrOTOF-Q mass spectrometer (Bruker Daltonics, Germany). The micrOTOF-Q instrument equipped with an ESI source with an ion funnel was operated in the positive ion mode and calibrated before each analysis with the Tunemix™ mixture (Bruker Daltonics, Germany) in a quadratic method. Argon was used as a collision gas. For separation, an Aeris PEPTIDE, Phenomenex (50 × 2.1 mm, 3.6 μm) column was used with elution gradient of 0–100 % B in A (A = 0.1 % HCOOH in water; B = 0.1 % HCOOH in acetonitrile) over 62 min (flow rate 0.05 ml/min, room temperature). In LC-MS/MS experiments, the collision energy eV was selected after CID studies on the synthetic model peptides.

The formation of the drug-polymer conjugates in ring opening polymerization was confirmed with electrospray mass spectrometry in acetonitrile on a micrOTOF-Q mass spectrometer (Bruker Daltonics, Bremen, Germany). Isotopic distribution of the peaks found by the experiment was compared to corresponding distributions of drug-tagged polymeric chains simulated by Mmass software. Proton nuclear magnetic resonance analysis (¹H NMR) in CDCl₃ was performed on Bruker 300 ultrashield NMR system (Bruker, Billerica, MA, USA).