Lipid profile variability in children at different ages measured in dried blood spots

Lipids were separated by a C18 reverse phase column, using an Ascentis® Express 90 Å C18 HPLC column (15 cm x 2.1 mm; 2.7 μm, Supelco®) inserted into an HPLC system (Ultimate 3000 Dionex, Thermo Fisher Scientific, Bremen, Germany) with an autosampler coupled online to a Q-Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany). A volume of 5 μL of each sample mixture was injected into the HPLC column, at a flow rate of 260 μL/min. The temperature of the column oven was maintained at 50 °C. Elution started with 32% of mobile phase B, and the gradient used was: 45% B (1.5 min), 52% B (4 min), 58% B (5 min), 68% B (8 min), 70% B (11 min), 85% B (14 min), 97% B (18 min, maintained for 7 min), and 32% B (25.01 min, followed by a re-equilibration period of 8 min prior next injection).The Q-Exactive™ orbitrap mass spectrometer with a heated electrospray ionization source was operated in the positive mode (electrospray voltage of 3.0 kV) and negative modes (electrospray voltage of −2.7 kV). The sheath gas flow was 35 U, the auxiliary gas was 3 U, the capillary temperature was 320 °C, the S-lenses RF was 50 U and the probe`s temperature was 300 °C. Full scans MS spectra were acquired both in positive and negative ionisation modes in an m/z range of 300-1600, with a resolution of 70.000, automatic gain control (AGC) target of 1x106 and maximum injection time of 100 ms. For tandem MS (MS/MS) experiments, a top-10 data-dependent method was used. The top 10 most abundant precursor ions in full MS were selected to be fragmented in the collision cell HCD. A stepped normalized collision energy™ scheme was used and ranged between 25 and 30 eV for positive ion mode and between 20, 24 and 28 for the negative ion mode. The MS/MS spectra obtained were those combining the information obtained with the different collision energies applied to each ionization mode. The MS/MS spectra were obtained with a resolution of 17,500; an AGC target of 1x105; an isolation window of 1 m/z; and a maximum injection time of 100 ms. The cycles consisted of one full scan mass spectrum and ten data-dependent MS/MS scans, which were repeated continuously throughout the experiments, with the dynamic exclusion of 30 s and an intensity threshold of 8x104. Data acquisition was carried out using the Xcalibur data system (V3.3, Thermo Fisher Scientific, USA).LC-MS data were identified using MS-DIAL v4.716 and integrated using MZmine v2.42 software17. Identification of the PL species was performed as previously described18,19. Briefly, to correctly identify the PL species, all peaks with raw intensity less than 1x104 and mass error greater than 5 ppm were excluded. The assignment of each PL species was confirmed by analysis and interpretation of the MS/MS spectra with the aid of MS-DIAL. PC, LPC and SM were analysed in the LC-MS spectra in the positive ion mode, as [M+H]+ ions. The presence of the fragmention at m/z 184, corresponding to the phosphocholine polar head group, in the MS/MS of [M+H]+ ions allows identifying PL molecular species belonging to the PC, LPC and SM classes, which were further differentiated by the characteristic retention times. The identification of PC, LPC and SM classes was confirmed in the LC-MS spectra in the negative ion mode, as formate adducts ([M+HCOO]− ions). MS/MS spectra of [M+HCOO]− ions of these three PL classes should display the typical fragment ion at m/z 168 (phosphocholine polar head group minus a methyl moiety). Carboxylate anions of fatty acyl chains can also be seen for PC and LPC. PE and LPE classes were analysed in negative ion mode ([M−H]− ions). The fragment ion at m/z 140 (phosphoethanolamine polar head group) and the carboxylate anions of fatty acyl chains can be found in the MS/MS data from negative ion mode. PI and PS species were analysed in negative ion mode, as [M−H]− ions. The presence of the fragment ion at m/z 241, corresponding to the phosphoinositol polar head group, in the MS/MS of [M-H]- ions, allows the identification of PI molecular species. PS species were identified in the MS/MS of [M-H]- ions by the neutral loss of -87Da from the molecular ion. The identification of the remaining lipid species belonging to the classes of carnitines (CAR), cholesteryl esters (CE, fragment ion at m/z 369) and triacylglycerols (TG), was made in LC-MS spectra in the positive ion mode, as [M+H]+ ions, [M+NH4]+ ions and [M+NH4]+ ions respectively. Diacylglycerols (DG) were identified by both [M+Na]+ and [M+NH4]+ ions.Relative quantification was performed by exporting the peak area values to a computer spreadsheet. For data normalization, the peak areas of the extracted ion chromatograms (XIC) of the lipid precursors of each class were divided by the peak areas of the internal standards selected for the class. Missing values were replaced by 1/5 of the minimum positive values detected in the data set. The data sets composed of the XIC areas obtained by the RP-LC-MS analysis were normalized to the internal standard, generalized log2 and normalized with EigenMS. Principal component analysis (PCA) was performed using the R libraries FactoMineR and factoextra, and ellipses were drawn with a level of 0.90. Univariate statistical analysis was performed using the ANOVA test following Tukey`s post hoc test, after testing for normality (Shapiro–Wilk test) and homogeneity of variance (Levene test). If normality was rejected, Kruskal-Wallis followed by Dunn tests were applied. P values were adjusted for multiple comparisons by the false discovery rate (FDR) correction (q-value). Heatmaps were created using the R package pheatmap using “Euclidean” as the clustering distance and “ward.D” as the clustering method. Univariate and multivariate statistical analyses were performed using R version 3.5.1 in Rstudio version 1.1.4. All graphics and boxplots were created using the R package ggplot2.