Xevo tqd

The analysis of C26:0-LPC is performed using the Neobase 2 newborn screening kit (PerkinElmer) following the manufacturer’s instructions. Single 3.2 mm discs are punched from DBS and transferred into 96-well plates. 125 μL of the PerkinElmer Neobase 2 extraction working solution (EWS) is added to each well. The microplate is covered with an adhesive microplate cover and shaken for 30 min with 650–750 rpm at 45°C. The microplate cover is removed and 100 μL is transferred to a new microplate and covered with an adhesive microplate cover before being analyzed with a Waters Xevo TQD.

DMS273 cells were seeded at 4 × 10⁵ cells per tube in 100 μM MG and the inhibitor (0 or 10 μM) in DPBS (pH 7.4) (n = 4). After 2 h incubation at 37 °C, sample was centrifuged, and supernatant was collected. Extracellular d-lactate was measured by analysis of the supernatant with Q-dsAMC. After collection of supernatants, the cells were washed with DPBS (pH 7.4) and lysed by vortexing with MeOH/H₂O (80/20, v/v). The lysate was centrifuged, and the supernatant was collected. Intracellular S-D-lactoylglutathione (SLG) was measured by analysis of the cell extract with LC-MS/MS. For detection of intracellular GSH, the cell extract (15 μL) was mixed with 15 μL 5 mM Dansyl-Cl in MeCN and 15 μL 100 mM borate buffer (pH 9.1) at r.t. for 30 min, then quenched by adding 15 μL MeCN containing 10% formic acid. The mixture was subjected to LC-MS/MS. LC-MS/MS analysis was performed on an Acquity UPLC H-Class system (Waters) equipped with an Acquity UPLC BEH C18 1.7 μm (2.1 × 50 mm) column (Waters) and an MS detector (Xevo TQD, Waters). Detection was performed in the positive mode. For SLG, the fragment of m/z = 366.2 > 170.1 was used (cone voltage: 25 V, collision voltage: 25 V). For GSH, the fragment m/z = 367.2 > 170.1 was used (cone voltage: 25 V, collision voltage: 25 V).

The DIMS analysis for the evaluation of metabolite profile in tear strip samples was performed using an Ultra Performance Liquid Chromatography Tandem Quadrupole Mass Spectrometry (UPLC/MS/MS) system (Acquity UPLC I-Class coupled to a Xevo TQD, Waters Corp., Manchester, UK). Mass spectra were acquired in positive electrospray ionization, using multiple reaction monitoring (MRM) as acquisition mode. Data are processed by using MassLynx V4.1 and NeoLynx Software (Waters Corp.). 10 μL were injected into the ion source and the run time was 1.1 minutes, injection-to-injection. The specific mass spectrometer setting conditions are reported in our previous works [37 (link),38 (link)]. A detailed list of analyzed metabolites, their ionization source settings, and their abbreviations as used in the text are available in Supplementary Table S5. The mass spectrometer ionization source settings were optimized for maximum ion yields for each analyte.

NMR spectra were recorded using a JEOL JNM-ECZ400 spectrometer at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR. Mass spectra (MS) were obtained using JEOL JMS-T100LP AccuTOF LC-plus 4 G (ESI).
Preparative HPLC was performed on an Inertsil ODS-3 (10.0 × 250 mm) column (GL Sciences Inc.) using an HPLC system composed of a pump (PU-2080, JASCO) and a detector (MD-2015). Preparative MPLC was performed on an Isolera One purification system (Biotage) equipped with a Biotage SNAP Ultra C18 column (for reverse phase separation) or on an MPLC system comprising a pump and detector (EPCLC AI-580S, Yamazen) and equipped with a silica gel column (silica gel 40 μm or Amino 40 μm, Yamazen) (for normal phase separation). LC-MS analysis was performed on an Acquity UPLC H-Class system (Waters) equipped with an Acquity UPLC BEH C18 1.7 μm (2.1 × 50 mm) column (Waters) and an MS detector (QDa or Xevo TQD, Waters).

The human, rat, and mice microsomes employed were purchased from Tebu-Xenotech. The compound was incubated at 37 °C with the microsomes in a 50 mM phosphate buffer (pH = 7.4) containing 30 mM MgCl₂, 10 mM NADP, 100 mM glucose-6-phosphate and 20 U/mL glucose-6-phosphate-dehydrogenase. Samples (75 µL) were taken from each well at 0, 10, 20, 40, and 60 min and transferred to a plate containing 4 °C 75 µL acetonitrile. Then, 30 µL of 0.5% formic acid in water was added to improve the chromatographic conditions. The plate was centrifuged (46,000× g, 30 min) and supernatants were taken and analyzed in a UPLC-MS/MS (Xevo-TQD, Waters) by employing a BEH C18 column and an isocratic gradient of 0.1% formic acid in water: 0.1% formic acid acetonitrile (60:40) for 5 minutes and a flow of 0.25 mL/min. The metabolic stability of the compounds was calculated from the logarithm of remaining compounds at each time point studied.