Milli q pf plus system

Sphingolipids were extracted as described (de Wit et al., 2016 (link), 2019 ). In short, frozen tissue samples were weighed and homogenized in cold Millipore water (MQ, 18.2 MΩ cm filter) from a Milli-Q^® PF Plus system (Merck Millipore B.V., Amsterdam, Netherlands). To 10 μL tissue homogenates and plasma samples, the internal standards Cer(d18:1/17:0), Cer(d17:0/24:1), and S1P(d18:1)-D7 were added (10 μL of 2, 2 and 0.2 μg/mL in methanol, respectively; IS: Avanti Polar Lipids, Alabaster, AL, United States; methanol: Merck Millipore B.V.). After addition of 10 μL of 10% TEA solution [triethylamine (10/90, v/v) in methanol/dichloromethane (DCM) (50/50, v/v); TEA: Merck Millipore B.V., DCM: Merck Millipore B.V.]. Lipids were extracted with 450 μL methanol/DCM (50/50, v/v). Samples were vortexed and incubated under constant agitation for 30 min at 4°C followed by centrifugation at 18,500 g for 20 min at 4°C (Hettich mikro 200R, Geldermalsen, Netherlands). Supernatants were transferred to glass vials, freeze dried and reconstituted in 100 μL methanol prior to liquid chromatography-tandem mass spectrometry (LC-MSMS).

LC-MS-grade
acetonitrile (ACN) and methanol (MeOH) were purchased from Actu-all
chemicals (Randmeer, The Netherlands). Methyl tert-butyl ether (MTBE, ≥99.8%) and sodium hydroxide were purchased
from Sigma-Aldrich (St. Louis, Missouri, United States). Formic acid
(FA) was purchased from Biosolve B.V. (Valkenswaard, Netherlands),
and hydrochloric acid (37% solution in water) was purchased from Acros
Organics (Geel, Belgium). Purified water was obtained from a Milli-Q
PF Plus system (Merck Millipore, Burlington, Massachusetts, United
States). Most chemical standards and stable isotopically labeled standards
(SILs) were purchased from CDN Isotopes (C/D/N Isotopes Inc., Quebec,
Canada), Cambridge Isotope Laboratories (Tewksbury, MA, USA), and
TRC (Toronto Research Chemicals, Toronto, Canada). Table S1 provides the supplier details of all standards. Pooled
EDTA plasma was obtained from Innovative Research (Peary Court Novi,
Michigan, United States), pooled male and female ETDA plasma was purchased
from Sanquin (Sanquin, Amsterdam, The Netherlands), and ETDA plasma
from individual donors was purchased from BioIVT (Westbury, New York,
United STates). Fecal samples were collected from four healthy adults,
including three female volunteers and one male volunteer (age range:
23–35 years old).

Bupivacaine free base and articaine hydrochloride were purchased from Wuhan Hezhong Biochemical Manufacturing Co. Ltd, (Wuhan, China) and SCI Pharmtech (Taoyuan, Taiwan), respectively. Lidocaine hydrochloride (Lopaine 2%) was purchased from Ethical agents Ltd., Auckland, New Zealand. Sucrose acetate isobutyrate (90%) in ethanol, N-Methylpyrrolidone (NMP) and acetonitrile were purchased from Merck, Auckland, New Zealand. Heparin sodium and normal saline were purchased from Pfizer New Zealand Limited (Auckland, New Zealand) and Baxter Healthcare Pty Ltd., (Old Toongabbie, NSW, Australia), respectively. Reagent grade potassium dihydrogen phosphate was obtained from Merck KGaA (Darmstadt, Germany). Phosphoric acid was obtained from Thermo Fisher Scientific Ltd (Scoresby, VIC, Australia). Milli-Q water was produced by the Milli-q PFplus system (Millipore Corporation, Bedford, MA, USA).