Agilent 6460 triple quadrupole mass spectrometer

The analysis was performed on an Agilent 1290 series liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA) that included a binary pump, an on-line vacuum degasser, a surveyor autosampling system, a column temperature controller and an Agilent 6460 triple-quadrupole mass spectrometer (Agilent Technologies, CA, USA) with Turbo Ion spray, which is connected to the liquid chromatography system. The Agilent MassHunter B 4.0 (Agilent Technologies, CA, USA) software was used to control the equipment and for data acquisition. The chromatographic analysis of AC was performed on a Waters X-Bridge C18 column (Milford, MA, USA) (3.0 × 100 mm, i.d.; 3.5 μm) at room temperature. The mobile phase was water (containing 0.1% formic acid) and acetonitrile (30:70, v:v) at a flow rate of 0.4 mL/min.
The mass scan mode was the positive MRM mode. The precursor ion and product ion were m/z 559.2 → 440.2 for AC and m/z 419.9 → 199.4 for the simvastatin, respectively. The collision energy for AC and simvastatin were 30 and 25 eV, respectively. The MS/MS conditions were optimized as follows: fragmentor, 140 V; capillary voltage, 4 kV; nozzle voltage, 500 V; nebulizer gas pressure (N₂), 40 psig; drying gas flow (N₂), 10 L/min; gas temperature, 350 °C; sheath gas temperature, 400 °C; and sheath gas flow, 11 L/min.

Targeted UHPLC-MS/MS of choline and its downstream metabolites measurements was performed by Shanghai Biotree Biomedical Technology Co., Ltd. (Shanghai, China). The serum was mixed with a 4× volume of 0.1% formic acid aqueous solution and a 20× volume of acetonitrile (0.1% formic acid, containing isotopically labeled internal standard mixture), vortexed for 30 s, and then sonicated for 15 min in an ice-water bath, followed by incubation at –40°C for 60 min. After centrifugation at 12,000 × g and 4°C for 15 min, an 80-μL aliquot of the clear supernatant was transferred to an autosampler vial for UHPLC-MS/MS analysis. UHPLC separation was carried out using an Agilent 1290 Infinity II Series UHPLC System (Agilent Technologies) equipped with ACQUITY UPLC BEH Amide (100 × 2.1 mm, 1.7 μm; Waters). An Agilent 6460 triple-quadrupole mass spectrometer (Agilent Technologies) equipped with an AJS ESI interface was applied for assay development. The multiple reaction monitoring (MRM) parameters for each of the targeted analytes were optimized using flow injection analysis by injecting the standard solutions of the individual analytes into the API source of the mass spectrometer. Agilent MassHunter Work Station Software (B.08.00; Agilent Technologies) was used for MRM data acquisition and processing.

The glucosides produced by the recombinant proteins were dissolved in methanol and analyzed by the Agilent 1290 Infinity LC System (binary pump G4220A, diode array UV/VIS detector G4212A; Agilent Technologies) coupled with a SunFire C18 analytical column (5 μm, 4.6 × 250 mm; Waters, USA). The chromatograms were obtained and detected between 200 nm and 400 nm. HPLC were performed at a flow rate of 1 ml min⁻¹ with 100% water as solvent A and 100% acetonitrile as solvent B. The injection volume of samples was 20 ml. The column was first equilibrated with 10% solvent B and eluted with a linear gradient program from 30% to 70% solvent B for 15 min, then washed with 100% solvent A for 15 min. The MS analyses were performed with an Agilent6460 triple quadrupole mass spectrometer equipped with negative ion electrospray ionization (ESI) MS in full scan mode. The scan range was 100–1000 m/z, and the nebulizer pressure was set as 45 psi. Identification of glucoside was based on the HPLC retention time and MS spectral data. Linalyl-β-d-glucoside was quantified by absorption at 210 nm, and the m/z value was 315 [M-H]⁻ according to Bönisch et al. (2014) (link).

LC‐MS/MS analysis was carried out on an Agilent 1290 series chromatographic system and an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies) with electrospray ionization (ESI). Separation of negatively charged hormones was achieved in a Waters Xbridge BEH C18 column (2.1 mm × 150 mm, 3.5 μm), whereas separation of positively charged hormones was achieved in a Phenomenex Gemini C18 column (2.0 mm × 100 mm, 3.0 μm). The mobile‐phase gradient was composed of solvent A (0.1% ammonia) and solvent B (acetonitrile) with a flow rate of 300 μl/min and was performed as follows: 0–0.5 min (80% A, 20% B), 1–6 min (60% A, 40% B), 6–7 min (55% A, 45% B), and 7–9 min (10% A, 90% B). The injection volume was 15 μl, and the column was set at 40°C. For mass spectrometric analysis, the ESI settings were as follows: capillary voltage 3.0 kV; desolvation gas flow rate 9 L/min at 350°C; and nebulizer pressure 45 psi. MS‐MS acquisition was performed in multiple‐reaction‐monitoring (MRM) mode. The optimized settings for product ions of each hormone are shown in Table 1.