Analysis of LPA species

The LPA species of saturated fatty acids palmitic acid (16:0-LPA) and stearic acid (18:0-LPA), the LPA of the monounsaturated fatty acid oleic acid (18:1-LPA), and the LPA species of the polyunsaturated fatty acids linoleic acid (18:2-LPA) and arachidonic acid (20:4-LPA) were determined using an extraction protocol followed by LC–MS–MS separation and quantification. Briefly, 0.2 mL of plasma were spiked with 100 ng of a mathanolic solution of 17:0-LPA (IS). A liquid–liquid extraction was performed after the addition of 200 μL of butanol. The organic phase was evaporated and reconstituted in 100 μL of mobile phase (80A:20B, see below) prior to analysis.

Stock solutions (100 μg/mL) for each analyte were independently prepared by diluting adequate amounts of standards in methanol. The working solutions were prepared by mixture of the stock solutions and dilution in methanol. The linearity of calibration curves containing the following concentrations for all the target analytes: 0.2, 0.5, 1, 1.5, 2, 4, 6, 8, 10 μg/mL was verified being the coefficient of determination r² > 0.99 in all cases.

Before the quantification of real samples and in order to verify matrix effect and recovery of the analytical method for each analyte, calibration curves were prepared in both plasma and water samples. In all cases, matrix effects lower than 6% and recoveries higher than 66% were achieved. At this point, calibration curves to perform quantification of real samples were prepared in water, and were added in duplicate in each analytical batch.

The procedure of lipid analysis in plasma was performed by a validated method previously described in clinical samples⁴¹. Quantification of LPA species in human plasma was performed using an ACQUITY UPLC system (Waters Associates, Milford, MA, USA) for the chromatographic separation coupled to a triple quadrupole (Xevo TQ-S micro) mass spectrometer provided with an orthogonal Z-spray-electrospray interface (ESI) (Waters Associates, Milford, MA, USA). The drying and nebulizing gas was nitrogen. The desolvation gas flow was set to 1200 L/h and the cone gas flow to 50 L/h. A capillary voltage of 3 kV was used in negative ionization mode. The nitrogen desolvation temperature was set to 600 °C and the source temperature to 150 °C. Collision gas was argon and the injection volume was 5 μL.

The chromatographic separation was achieved at 30 °C using an ACQUITY UPLC BEH C18 column (2.1 × 100 mm × 1.7 µm) (Waters Associates, Milford, MA, USA), at a flow rate of 300 µL/min. Mobile phase A was ammonium formate 1 mM with formic acid (0.01% v/v) dissolved in methanol. Mobile phase B was ammonium formate 1 mM with formic acid (0.01% v/v) in water. A gradient program was employed for the separation of the analytes; the percentage of mobile phase B linearly changed as follows: 0 min, 20%; 0.2 min, 20%; 6 min, 10%; 6.5 min, 10%; 7 min, 20%; 8 min, 20%. Total run time was 8 min. Analytes were determined by a Selected Reaction Monitoring (SRM) method by acquiring two transitions for each compound as specified (Supplementary Table ^S1). The most specific transition was selected for quantitative purposes. MassLynx software V4.1 and TargetLynx XS were used for data management. Finally, the LPA species plasma concentrations were recalculated to molar concentration (nmol/L).