Sample preparation and UHPLC-MS analysis

The solvents and eluent modifiers used in this work were purchased from Sigma-Aldrich (Steinheim, Germany): HPLC-grade chloroform, LC-MS grade acetonitrile (ACN), isopropanol (IPA), water (H₂O), methanol (MeOH), ammonium acetate (NH₄Ac), analytical grade formic acid (HCOOH) and sodium chloride (NaCl).

The following internal standards were purchased for quality control (QC) and calibration purposes: 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (PE(17:0/17:0)), N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine (SM(d18:1/17:0)), N-heptadecanoyl-D-erythro-sphingosine (Cer(d18:1/17:0)), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC(17:0/17:0)), 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(17:0)) and 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (PC(16:0/d31/18:1)) from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA), 1,2-Dimyristoyl-sn-glycero-3-phospho(choline-d₁₃) (PC(14:0/d13)) from Sigma-Aldrich and Tripalmitin-1,1,1-13C3 (TG(16:0/16:0/16:0)-13C3) and Trioctanoin-1,1,1-13C3 (TG(8:0/8:0/8:0)-13C3) from Larodan (Solna, Sweden). Stock solutions (1.0 mg mL⁻¹) of the internal standards were prepared by dissolving the analytes in CHCl₃:MeOH (2:1, v/v). The working standard solutions were prepared by further diluting the stock solutions in CHCl₃:MeOH (2:1, v/v) to achieve concentrations of 250 ng mL⁻¹ and 3.5 μg mL⁻¹.

Calibration curves using 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(16:0e/18:1(9Z))), 1-(1Z-octadecenyl)-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(18:0p/18:1(9Z))), 1-octadecanoyl-sn-glycero-3-phosphocholine (LPC(18:0)), 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PC(18:0p/22:6)) and 1-stearoyl-2-linoleoyl-sn-glycerol (DG(18:0/20:4)) from Avanti Polar Lipids Inc., 1-Palmitoyl-2-Hydroxy-sn-Glycero-3-Phosphatidylcholine (LPC(16:0)) from Larodan, and 1,2,3-Triheptadecanoylglycerol (TG(17:0/17:0/17:0)) and 3β-Hydroxy-5-cholestene 3-linoleate (ChoE(18:2)) from Sigma-Aldrich were prepared to the following concentration levels: 100, 500, 1000, 1500, 2000 and 2500 ng mL⁻¹ (in CHCl₃:MeOH, 2:1, v/v) including 250 ng mL⁻¹ of each QC standard.

A total of 428 plasma samples were extracted in randomised order using a modified version of the Folch procedure: 10 μL of 0.9% NaCl, 40 μL of CHCl₃:MeOH (2:1, v/v) and 80 μL of the 3.5 μg mL⁻¹ working standards solution were added to 10 μL of each plasma sample. The samples were vortex mixed and incubated on ice for 30 min after which they were centrifuged (9400 × g, 3 min, 4 °C). From the lower layer of each sample, 60 μL was transferred to a glass vial with an insert and 60 μL of CHCl₃:MeOH (2:1, v/v) and added to each sample. The samples were re-randomised and stored at −80 °C until analysis on the UHPLC-QTOF-MS system.

The UHPLC system used was a 1290 Infinity system from Agilent Technologies (Santa Clara, California, USA). The system was equipped with a multisampler (maintained at 10 °C) using 10% DCM in MeOH and ACN:MeOH:IPA:H₂O (1:1:1:1, v/v/v/v) + 0.1% HCOOH as needle wash solutions after each injection for 7.5 s each, a quaternary solvent manager and a column thermostat (maintained at 50 °C). Separations were performed on an ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, particle size 1.7 μm) by Waters (Milford, USA). The flow rate was 0.4 mL min⁻¹ and the injection volume was 1 μL. H₂O + 1% NH₄Ac (1 M) + 0.1% HCOOH (A) and ACN:IPA (1:1, v/v) + 1% NH₄Ac + 0.1% HCOOH (B) were used as the mobile phases for the gradient elution. The gradient was as follows: from 0 to 2 min. 35-80% B, from 2 to 7 min. 80–100% B and from 7 to 14 min 100% B. Each run was followed by a 7 min re-equilibration period under the initial conditions (35% B).

The mass spectrometer coupled to the UHPLC was a 6550 iFunnel QTOF-MS from Agilent Technologies interfaced with a dual jet stream electrospray (dual ESI) ion source. Nitrogen generated by a nitrogen generator (PEAK Scientific, Scotland, UK) was used as the nebulising gas at a pressure of 21 psi, as the drying gas at a flow rate of 14 L min⁻¹ (at 193 °C) and as the sheath gas at a flow rate of 11 L min⁻¹ (at 379 °C). Pure nitrogen (6.0) from Praxair (Fredericia, Denmark) was used as the collision gas. The capillary voltage and the nozzle voltage were kept at 3643 V and 1500 V, respectively. The reference mass solution including ions at m/z 121.0509 and 922.0098 was prepared according to instructions by Agilent and was introduced to the mass spectrometer through the other nebuliser in the dual ESI ion source using a separate Agilent series 1290 isocratic pump at a constant flow rate of 4 mL min⁻¹ (split to 1:100 before the nebuliser). The acquisition mass range was m/z 100–1700 and the instrument was run in extended dynamic range mode with an approximate resolution of 30,000 FWHM measured at m/z 1521.9715 (which is included in the tune mixture) during calibration of the instrument. MassHunter B.06.01 software (Agilent) was used for data acquisition.