2.3. NMR spectroscopy

Dried organic phases (lipids) were reconstituted in 220 µL of 100% methanol-d4 containing 0.05% v/v of tetramethylsilane (TMS) (Cambridge Isotopes Lab, Andover, MA), vortexed, and centrifuged at room temperature. 200 µL of the supernatant was transferred into a 3-mm NMR tube. All NMR spectra were recorded at 288 K on a Bruker Avance III HD 600-MHz spectrometer (Bruker BioSpin) equipped with a 5-mm Broad Band Observed (BBO) Prodigy probe. For each sample, one-dimensional (1D) ¹H-NMR experiments were acquired using the noesygppr1d pulse sequence with presaturation of the residual water resonance using a 25-Hz bandwidth, 512 transients, a 15-ppm spectral width, a 4.0-s relaxation delay, and a 2.0-s acquisition time resulting in 44,640 data points. Prior to Fourier transformation, each ¹H spectrum was zero-filled to 128 K data points and apodized with a 1-Hz exponential line-broadening function. All spectra were recorded and transformed with the use of TopSpin 3.6.2 software (Bruker BioSpin, USA) and processed (phased and baseline corrected) using MestReNova software (MNova v12.0.3, Spain). Spectra were internally calibrated to the methyl group of the TMS at 0 ppm. Representative lipid families (glycerophospholipids, sterols, sphingolipids, glycerophospholipids, and fatty acids) were identified and assigned by using in-house databases, pure standards, and literature reports (16, 20, 21). Additionally, for selecting samples, 2D ¹H-¹H TOtal Correlation SpectroscopY (TOCSY) experiments were recorded to facilitate and confirm the identification of analytes. The area of each assigned lipid class was manually integrated using the global spectra deconvolution (GSD) algorithm available in MestReNova software (MNova v12.0.3, Spain), as previously described (18).