Agilent 6545 qtof

Secondary amines were synthesized according to the described procedure^{24 (link)}. The appropriate aldehyde and primary amine derivatives were commercially available from Sigma Aldrich. Formaldehyde was available as a 37% solution in water from Fisher Bioreagents. Sodium triacetoxyborohydride was purchased from Apollo Scientific. Reactions were accomplished in an Anton Paar BM500 vibrational ball mill by using stainless-steel milling beakers (5 mL) and stainless-steel milling balls. ¹H NMR spectra were recorded on a Varian Unity Inova 500 (500 MHz) spectrometer. Chemical shifts δ are reported in parts per million relative to the residual solvent peak (DMSO-d₆ = 2.49 ppm for 1 H). Coupling constants are given in Hertz. High resolution mass spectra were recorded on Agilent 6545 Q-TOF. Thin-layer chromatography (TLC) was carried out with Polygram SIL G/UV254, silica gel (Macherey–Nagel GmbH & Co. KG, Duren, Germany). Compounds were visualized by means of irradiation with UV light and/or by treatment with a solution of ninhydrin. Flash chromatography was carried out using Büchi FlashPure Cartridges (silica gel 40 μm irregular) on a Büchi Pure Chromatography System with an integrated UV and ELSD detector. Melting points (uncorrected) were determined using a Stuart Scientific SMP30 apparatus. Infrared (IR) spectra were recorded using a Nicolet 8700 spectrometer.

Fifty microliters of each sample, QC, and process blank solution were added to individual PTFE auto sampler vials and randomized prior to analysis. An Agilent 1290 UPLC coupled to an Agilent 6545 Q-ToF (Agilent Technologies, Inc) was used for chromatography and mass spectrometry. Chromatography was performed with a Waters BEH amide 2.1 × 100 mm column with BEH amide precolumn (Waters Corporation). An initial concentration of 99% buffer A (ACN with 5% 10 mM NH4OAc ddH2O) and 1% buffer B (10 mM NH4OAc in ddH2O) was held for 1.66 min at a flow rate of 0.4 ml/min. B was decreased to 70% over 4.75 min then to 40% over 1.5 min. Finally, B was decreased to 30% over 0.68 min and held for 3.75 min. The system was allowed to re-equilibrate for 5.16 min between runs. Samples were analyzed by an Agilent 6545 with a Dual Jetstream ESI source in positive and negative modes with the following source parameters: Gas Temp = 275 °C, Drying Gas = 12 l/min, Nebulizer = 35 psig, Sheath Gas Temp 325 °C, Sheath Gas Flow 12 l/min, VCap = 3500 V, Nozzle Voltage = 250 V, Fragmentor = 100 V, Skimmer = 65 V. Data analysis was performed using Agilent Profinder and Quantitative Analysis software (Agilent Technologies, Inc).

Metabolite analysis was performed by LC–MS, using hydrophilic interaction (HILIC) LC and high resolution QTOF mass spectrometry. Sample extracts (10 μL) were injected onto an Agilent 1290 LC fitted with a ZIC-pHILIC column (5 μm, 2.1 × 150 mm; Merck), and 20 mM ammonium carbonate (A) and acetonitrile (B) as the mobile phases. A 14 min gradient starting from 90% B to 40% B over 12 min, held for 2 min followed by washing at 5% B for 3 min and re-equilibration at 90% B, was used. Mass spectrometry utilized an Agilent 6545 QTOF with heated electrospray source operating in negative ionization mode and scan range m/z 50–1700. Conditioning was performed before each batch using 2–3 blanks and 5 mixtures of authentic standards (234 metabolites), which were analyzed in data-dependent MS/MS mode to facilitate downstream metabolite identification where necessary. PBQC samples were analyzed periodically throughout the analysis.

The dried lipids were dissolved in 150 µL of methanol/chloroform (1:1, v/v). We employed an ultra-performance liquid chromatograph quadrupole time-of-flight mass spectrometer (UPLC/Q-TOF-MS, Agilent Technologies, Santa Clara, CA, USA; Metabolomics Research Center for Functional Materials, Kyungsung University, Busan, Republic of Korea) equipped with an electrospray ion source (ESI) for the analysis. Chromatographic separation was achieved using a ZORBAX Eclipse Plus C18 Column (95 Å, 1.8 µm, 2.1 mm × 100 mm, Agilent Technologies), with the column temperature maintained at 50 °C. A binary mobile phase system was utilized: mobile phase A consisting of water/methanol (90:10) with 10 mM ammonium acetate, and mobile phase B consisting of acetonitrile/methanol/isopropanol (20:20:60) with 10 mM ammonium acetate. Gradient elution with a flow rate of 0.3 mL/min was conducted as follows: 0 min, 55% B; 5 min, 57% B; 25 min, 100% B; 27 min, 100% B; 27.1 min, 55% B; 30 min, 55% B. For mass spectrometry, an Agilent 6545 Q-TOF (Agilent Technologies) equipped with positive and negative electrospray ionization (ESI) sources was set as follows: capillary voltage 4 kV, fragmentor voltage 160 V, gas temperature 250 °C, drying gas 10 L/min, maximum pressure of nebulizer with 35 psi, sheath gas temperature 300 °C, sheath gas flow 12 L/min, and RF voltage 750 V.

This study conducted a quantitative analysis of the lipids of Crassostrea hongkongensis fresh and dried products through HPLC/Q-TOF-MS technology. The HPLC separation was carried out using an Agilent 1,290 series HPLC System (NYSE: A, USA). The experiment utilized the 100^*2.1 mm of the Acquity UPLC Beh C18 (Waters, USA), and the filler particles are 1.7 μm BEH particles. The mobile phase A consisted of a water solution with 0.1% formic acid in it. The mobile phase C consisted of 10% acetonitrile and 90% isopropanol formate. The column temperature was 45°C. The two groups of experiments were maintained at the column temperature of 40°C, the flow rate was 0.4 ml/min, and the injection volume was 4 μl.
Gradient program of the mobile phase: 0–2 min, 30–60% B; 2–8 min, 60–85% B; 8–10 min, 85–98% B; 10–15 min, 98% B; 15–16 min, 98–30% B; 16–20 min 30% B.
Q-TOF-MS is used to experiment with Agilent 6,545 Q-TOF (NYSE: A, USA), set the capillary voltage to 4,500 V, control the drying air temperature at 325°C and turn on the dry gas at a flow rate of 10 L/min. Keep the spray pressure at 20 PSIG, set the craft of the breaker to 120 V, and finally, collect the mass spectrometry data in the 200–2,000 m/z range for subsequent research and analysis.