Uplc ms ms

Quantification of TMAO in plasma samples was performed as described previously^{61 (link)}. Briefly, for the sample extraction, 25 µL of plasma was mixed with 80 µL of methanol with labelled IS working solution (TMAO-d9; Cambridge Isotope Laboratories, Massachusetts, USA) and mixed 30 seconds to precipitate proteins. The samples were subjected to centrifugation at 9000 rpm for 5 min at room temperature, and the supernatants were diluted with 150 uL of MilliQ water. Diluted samples were filtered with PVDF filters 0.22 µm and transferred into HPLC vials for analysis. The analysis was performed by liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) (Waters, Milford, MA, USA) using a column Acquity UPLC BEH HILIC (1,7 µm 2,1 × 100 mm).

The microalgal cells were centrifuged at 5000× g for 5 min at 4 °C. Then, pre-chilled methanol solution (80%, v/v, methanol in water) was added into the pellet for 3 min to quench microalgal metabolism. Afterward, cells were collected after centrifugation at 5000× g for 5 min at 4 °C, and the pellets were resuspended in pre-chilled methanol solution (80%) and frozen with liquid nitrogen. After 20 min incubation at −20 °C, the supernatant was collected after centrifugation for 5 min at 4 °C. The resuspension was then repeated twice. The merged supernatants were vacuum dried, and the residuals were dissolved in 80% methanol and analyzed using UPLC-MS/MS (Waters, Milford, MA, USA). The tandem system was equipped with an HSS T3 column (C18, 2.1 mm × 100 mm, 1.8 μm particle size) with a flow rate set as 0.4 mL/min, and the linear gradient procedure was set as follows: (a) 100% solvent B (H₂O/HCOOH/0.2 M NH₄Ac in water, 94.9:0.1:5, v/v/v) to 60% solvent A (C₂H₃N/HCOOH/0.2 M NH₄Ac in water, 94.9:0.1:5, v/v/v); (b) 100% solvent A, held for 1 min; and (c) 100% solvent B and held for 4 min. The system was operated in the positive and negative ESI modes, and quantification was performed using the multiple reaction monitoring (MRM) mode. The optimal operating conditions were set according to our previous study [4 (link)].

The metabolomic analysis was performed using untargeted ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS, Waters ACQUITY, Milford, MA, United States), as described previously (Vizioli et al., 2021 (link)). Briefly, the fecal samples were prepared using the automated MicroLab STAR system (Hamilton Company, Franklin, MA, United States) and extracted at a constant per-mass basis. Proteins were removed using methanol precipitation (Glen Mills GenoGrinder 2000), followed by centrifugation. The samples were processed using four methods: reverse phase (RP)-UPLC/MS/MS with electrospray ionization (ESI), in both positive (optimized for hydrophilic and hydrophobic compounds, respectively) and negative modes, and hydrophilic interaction chromatography (HILIC)-UPLC/MS/MS-ESI in negative ion mode. The raw UPLC/MS/MS data were integrated into ion peaks organized by mass, retention time/index, and peak area. Metabolites were annotated by comparison of individual spectra to a standard reference library, and area-under-the-curve analysis was performed for peak quantification.

The blood samples for measuring PCZ C_min were collected for routine care. Only children having achieved steady-state were included in the analysis and PCZ was considered to have achieved a steady-state plasma concentration after at least 7 days of dosing (Lai et al., 2020 (link)). Dose adjustments were taken according to the current guidelines and the manufacturer’s recommendations (Vicenzi et al., 2018 (link)). PCZ trough plasma concentrations were determined using a previously described ultra-high-performance liquid chromatograph-tandem mass spectrometry method (UPLC-MS/MS, Waters, United States) (Jia et al., 2020 (link)). The analytical range was 0.025–5.00 μg mL⁻¹. For the purpose of this study, plasma concentrations ≥0.7 μg mL⁻¹ were considered therapeutic for prophylaxis and ≥1.0 μg mL⁻¹ for treatment (Bernardo et al., 2020 (link)).