Acquity uplc system

Untargeted metabolomic analysis to identify the differential metabolites between the different Jiupei samples was done using GC–MS and LC–MS. GC–MS analysis was performed using a 7890A gas chromatograph (Agilent) coupled to a PEGASUS HT mass selective detector (LECO). LC–MS analysis was performed on an Acquity UPLC system (Thermo Fisher Scientific) coupled with a Q Exactive HFX (Thermo Fisher Scientific). The detailed sample preparation and MS analysis methods are described in Text S1 (Sun et al., 2020 (link)).

Methods for metabolite level determination were previously described [38 (link)]. Metabolite extracts were prepared from 50 μL of fasting plasma stored at −80 °C via protein precipitation with 250 μL of acetonitrile, evaporation under nitrogen, and reconstitution in 50 μL of water. Three reference human plasma samples were prepared in each analytical batch as a control within and between analyses. UPLC-MS was performed using a Waters Acquity UPLC system coupled to a Thermo Scientific Q Exactive mass spectrometer. A proportion of values were below the lower limits of quantification (LLOQ) for 5HT (21.4%; LLOQ: 0.00113 μM) and 5HIAA (3.6%; 0.0131 μM) and were imputed as LLOQ/√2 (5HT:0.000799 μM; 5HIAA:0.00926 μM), as previously described [38 (link)].

Unless otherwise noted, all reagents from commercial sources were used without further purification. Reactions were monitored by thin-layer chromatography (TLC) using TLC plates precoated with TLC silica gel 60 F254 (Merck KGaA). Spots on TLC were visualized either directly with an ultraviolet light or after staining with p-anisaldehyde stain. Flash column chromatography was conducted on 40–63 µm silica gel. Unless otherwise stated, NMR spectra were obtained on a Varian Mercury 300 (300 MHz) in CDCl₃. The reported chemical shifts for the ¹H NMR spectra were recorded in parts per million (ppm) on the δ scale from an internal tetramethylsilane standard (0.0 ppm) and reported consecutively as position, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, m = multiplet, and br = broad), coupling constant (J/Hz), relative integral, and assignment. High-resolution mass spectra (HRMS) were obtained on a Waters Acquity UPLC system equipped with a ThermoFisher QExactive mass spectrometer with electrospray ionization (ESI) operated in positive ionization mode. The purity of the final products was determined by HPLC.

Each sample extract designated for LC–MS analysis was split into two aliquots, dried, and reconstituted in acidic or basic LC-compatible solvents, each of which contained eight or more injection standards at fixed concentrations to ensure injection and chromatographic consistency. The analysis of LC/MS samples performed on the platform using a Waters ACQUITY UPLC system and a Thermo Fisher Scientific Orbitrap Elite high resolution/accurate mass spectrometer, which was composed of a heated electrospray ionization (HESI) source and an orbitrap mass analyzer operating at 30,000 mass resolution. Samples were analyzed using acidic positive ion optimized conditions (one aliquot) and basic negative ion optimized conditions (second aliquot) in two independent injections on separate dedicated columns. Extracts reconstituted in acidic conditions were gradient eluted using water and methanol containing 0.1% formic acid, while the basic extracts used water/methanol containing 6.5 mM ammonium bicarbonate. The MS analysis alternated between MS and data-dependent MS² scans using dynamic exclusion.

The analyses were performed on an Acquity UPLC system interfaced to an Orbitrap Q-Exactive Focus mass spectrometer (Thermo Scientific) using a heated electrospray ionization source (HESI-II) and an Acquity UPLC PDA detector. Thermo Scientific Xcalibur 2.1 software was employed for instrument control. The detailed conditions are presented in Supplementary Section S9.

The whole brain cortex was also used to measure several different acylcarnitine metabolites using an UPLC-MS method as previously described^{43 (link)}. In short, 5 mg of pulverized tissue was homogenized in 50 µl of PBS before 25 µl of deuterated labeled internal standards was added. Proteins were removed by adding a solution of methanol/dichloromethane (v/v, 600 µl) to the sample mixture. The sample was centrifuged at 18,000×g for 15 min at 4 °C, and then the supernatant was transferred to a 1 dram vial, and dried under N2 stream. Samples were reconstituted and analyzed on a Waters Acquity UPLC system (Milford, MA) coupled with a Thermo Quantiva tandem mass spectrometer (West Palm Beach, FL) in positive (H)ESI mode. Concentrations of carnitine (162.1 > 85.0 m/z), acetylcarnitine (204.1 > 85.0 m/z), propionylcarnitine (218.1 > 85.0 m/z), butyrylcarnitine (232.1 > 85.0 m/z), isovalerylcarnitine (246.1 > 85.0 m/z), octanoylcarnitine (288.2 > 85.0 m/z), lauroylcarnitine (344.3 > 85.0 m/z), myristoylcarnitine 372.3 > 85.0 m/z), palmitoylcarnitine (400.4 > 85.0 m/z), oleoylcarnitine (426.4 > 85.0 m/z), and stearoylcarnitine (438.4 > 85.0 m/z) were measured against a 11-point calibration curve that underwent the same preparation.