Triple tof 5600 mass spectrometer system

For LC-MS/MS analysis, 0.3 g Quanduzhong capsule powder was added to 25 mL deionized water and ultrasonically dissolved for 30 min. Then, 1 ml of the solution was taken, diluted 100 times, separated by high-speed centrifugation, and filtered through a 0.22 μm filter membrane.
The identification experiment was performed using ultra-performance liquid chromatography-quadrupole-time of flight (UPLC-Q-TOF) mass spectrometry (MS) [AB SCIEX Triple TOF 5600 + mass spectrometer system (AB SCIEX, Foster City, CA, United States)]. A C₁₈ column (100 × 2.1 mm, 1.8 μm) was used with a flow rate of 0.25 ml/min. The mobile phases used were A) acetonitrile and B) 0.1% aqueous formic acid under the following gradient elution: 10% A from 0.1 to 2 min; 10%–25% A from 2 to 5 min; 25%–40% A from 5 to 15 min; 40%–90% A from 15 to 23 min; 90% A from 23 to 27 min. The electrospray ionization (ESI) source was used in negative ion mode. For the compounds of interest, a scan range of m/z 120–1500 was chosen. Other conditions were as follows: atomization temperature, 600°C; spray voltage, −4500 V; declustering potential, −80 V. MS data were collected in TOF-MS-IDA-MS/MS mode.

Sample analysis was performed by UPLC-Q-TOF-MS using a triple TOF™ 5600⁺ mass spectrometer system (AB SCIEX, Foster City, CA) coupled with a Shimadzu UPLC system (Nexera, UHPLC LC-30A, Japan). This UPLC system contained a binary pump, an auto sampler, and a column oven. The samples were loaded onto a C18 column (1.9 μm, 2.1 × 100 mm; Shimadzu), with a column temperature maintained at 30°C, a flow rate of 0.2 mL/min, and an injection volume of 4 μL. Mobile phases A and B were methanol and 0.1% formic acid (v/v) in water, respectively. The UPLC elution conditions were optimized as follows: linear gradient from 95% to 86% B (0–6 min), 86% to 51.8% (6–12 min), 51.8% to 50% (12–20 min), 50% to 15% (20–23 min), and isocratic 15% B (23–26.7 min).
The optimal parameters of the MS/MS detector were as follows: ion spray voltage, 5500 V; ion source temperature, 550°C; declustering potential (DP), 110 V; and collision energy (CE), ±45 V. An electrospray ionization (ESI) source was operated in both positive and negative ion modes. The nebulizer gas (gas 1), heater gas (gas 2), and curtain gas (gas 3) were set to 55, 55, and 35 psi, respectively. MS data were collected in the full-time scan mode within a mass range of 50–1200 Da. Data acquisition was conducted using the PeakView Software TM V1.1 (AB SCIEX, Foster City, CA).

The LC fractions were analyzed using a Triple TOF 5600 mass spectrometer system equipped with nanoflow reversed-phase liquid chromatography (RPLC) system (AB SCIEX). The peptide fractions were loaded onto a nanobored C18 column at a flow rate of 0.20 μL/min. An elution gradient of 5%~ 40% ACN (0.1% FA) within a 120 min gradient was used. The mass spectroscopy data were acquired using an ion spray voltage of 2.5 kV, curtain gas of 30 PSI, nebulizer gas of 5 PSI, and an interface heater temperature of 150 °C. We used an information-dependent acquisition (IDA) mode to acquire MS/MS data. Survey scans were acquired in 250 ms intervals, and as many as 35 product ion scans were collected with a 20 s exclusion window and a total cycle time of 2.5 s. A rolling collision energy setting was applied to all precursor ions for CID. The data acquisition rate was 4 s per spectrum.

A clear aqueous solution of the urine extract (5 μl) was injected onto an Eksigent reverse-phase C₁₈ pre-column cartridge (0.5 cm x 200 μm ID) in 0.1% formic acid. Metabolites bound to the cartridge were eluted with a 20-min linear gradient of 0–80% acetonitrile in 0.1% formic acid onto an Eksigent ChipLC C₁₈ column (15 cm x 200 μm ID) at a flow rate of 1 μl/min. The ChipLC column was placed in an Eksigent Nanoflex (SCIEX, Concord, ON, Canada) operating at 45°C. At the end of each analytical run, the column was washed with acetonitrile:0.1% formic acid for 1 min followed by re-equilibration with 0.1% formic acid for 4 min. Eluate from the ChipLC column was passed into the nanoelectrospray ionization interface of a SCIEX Triple TOF™ 5600 Mass Spectrometer System operating in the negative and positive ion modes. The collision energy was set to 35 eV with a 15 eV collision spread, curtain gas to 20, GS1 to 15, spray voltage to 2300 V (positive ion mode)/2200 V (negative ion mode), and temperature to 120°C. In each duty cycle lasting 1.25 s, high mass resolution MS spectra were collected for 250 ms followed by 50 ms MS/MS spectra of the top 20 most intense molecular ions. Ions producing successful MS/MS spectra were put onto an exclusion list for the next 60 s.

The LC-MS system comprised a Vanquish UHPLC system (Thermo Fisher Scientific, Sunnyvale, CA, USA) with an Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.7 μm; Waters) and a Triple TOF 5600⁺ mass spectrometer system (Sciex, Foster City, CA, USA). The ultrahigh performance liquid chromatography (UHPLC) system used 0.05% formic acid and 2.5 mM ammonium formate in water as eluent A and acetonitrile as eluent B. The optimized elution program was as follows: 0–2.5 min (1% B), 2.5–3.0 min (1–10% B), 3.0–6.0 min (10–19% B), 6.0–9.0 min (19–22% B), 9.0–14.0 min (22–25% B), 14.0–17.0 min (25–70% B), 17.0–19.0 min (70–100% B), 19.0–22.0 min (100% B), and equilibration with 1% B for 3 min at a flow rate of 0.4 mL/min. The column was maintained at 40 °C, and the auto-sampler was held at 4 °C. The injection volume of each sample solution was 1 μL. The MS/MS data were acquired by an information-dependent acquisition scan at positive-ion mode, and the parameters were as follows: mass range 50–1500 m/z, ion spray voltage, 4.5 kV; source temperature, 450 °C; declustering potential, 50 V; nitrogen as nebulizer gas, 50 L/min; heater gas, 50 L/min; curtain gas, 25 L/min; and collision energy, 10 eV.