Quantification of esterified oxylipins following HILIC-fractionation of lipid classes

Created by https://lipidomicstandards.org, version v2.5.0 Contents of Report Separation Workflow 1 Overall study design ...................................................... 1 Lipid extraction ......................................................... 1 Analytical platform ....................................................... 1 Quality control ......................................................... 2 Method qualification and validation .............................................. 2 Reporting ............................................................ 2 Sample Descriptions 2 QC 1 / Human / Plasma ................................................... 2 NIST SRM 1950 / Human / Plasma ............................................. 2 Supplemenation Study / Human / Plasma .......................................... 2 Lipid Class Descriptions 2 1) Oxylipins[M-H]- / Lipid identification ........................................... 2 1) Oxylipins[M-H]- / Lipid quantification ........................................... 3 2) FA[M-H]- / Lipid identification ............................................... 4 2) FA[M-H]- / Lipid quantification .............................................. 4 Separation Workflow Overall study design Title of the study Quantification of esterified oxylipins following HILIC-fractionation of lipid classes Document creation date 10/24/2025 Principal investigator Nils Helge Schebb Institution Food Chemistry, School of Mathematics and Natural Sciences, University of Wuppertal, 42119 Wuppertal, Germany Corresponding Email [email protected] Is the workflow targeted or untargeted? Targeted Clinical No Lipid extraction Extraction method Solid-Phase Extraction pH adjustment Disodium hydrogen phosphate Solid-Phase Extraction Mixed Mode Were internal standards used? Yes Deposition method - Internal standards used d4-9-HODE, d4-13-HODE, d8-5-HETE, d8-12-HETE, d8-15-HETE Analytical platform Ionizaton additives Acetic acid Number of separation dimensions One dimension Separation type 1 LC Separation mode 1 (liquid) RP Detector Mass spectrometer MS type QQQ MS vendor SCIEX Ion source ESI MS Level MS2Mass window for precursor ion isolation (in Da total isolation window) 1 Mass resolution for detected ion at MS2 Low resolution Resolution at MS2Low Recording mode of raw data at MS2 Profile mode Was/Were additional dimension/techniques used No 1 Quality control Blanks Yes Type of Blanks Solvent blank, Internal standard blank Quality control Yes Type of QC sample Reference material Method qualification and validation Method validation Yes Lipid recovery Yes Dynamic quantification range Yes Limit of quantitation (LOQ)/Limit of detection (LOD) Yes Precision Yes Accuracy Yes Guidelines followed EMA Reporting Are reported raw data uploaded into repository? No Are metadata available? No Raw data upload No Sample Descriptions QC 1 / Human / Plasma Storage and collection conditions Available Temperature handling original sample 4-8 °C Instant sample preparation No Storage temperature -80 °C Additives None Were samples stored under inert gas? No NIST SRM 1950 / Human / Plasma Storage and collection conditions Available Temperature handling original sample 4-8 °C Instant sample preparation No Storage temperature -80 °C Additives None Supplemenation Study / Human / Plasma Storage and collection conditions Available Temperature handling original sample 4-8 °C Instant sample preparation No Storage temperature -80 °C Additives None Lipid Class Descriptions 1) Oxylipins[M-H]- / Lipid identification 2 Lipid class Oxylipins MS Level for identification MS2 Identification level Full structure MS2adduct [M-H]- Fragments for identification Fragment name alpha cleavage Isotope correction at MS2No MS2verified by standard Yes Background check at MS2Yes Did you presume assumptions for identification? No Check on: Isobaric overlap Limit of detection S/N ratio > 3 RT verified by standard Yes Separation of isobaric/isomeric interferece confirmed Yes Model for separation prediction No Lipid Identification Software manually Data manipulation Smoothing Nomenclature for intact lipid molecule No Nomenclature for fragment ions N/A 1) Oxylipins[M-H]- / Lipid quantification Quantitative Yes MS Level for quantification MS2 Internal lipid standard(s) MS2 Internal standard Fragment(s) Endogenous subclass d4-9-HODE 172.3 9-HODE d4-9-HODE 172.3 13-HODE d4-13-HODE 198.1 15-HODE d4-13-HODE 198.1 9-HOTrE d4-13-HODE 198.1 13-HOTrE d8-5-HETE 116.1 8-HETrE d8-5-HETE 116.1 12-HETrE d8-5-HETE 116.1 15-HETrE d8-5-HETE 116.1 5-HETE d8-12-HETE 184.2 8-HETE d8-5-HETE 116.1 9-HETE d8-12-HETE 184.2 11-HETE d8-12-HETE 184.2 12-HETE d8-15-HETE 226.0 15-HETE d8-15-HETE 226.0 5-HEPE d8-15-HETE 226.0 8-HEPE d8-15-HETE 226.0 9-HEPE d8-15-HETE 226.0 11-HEPE d8-15-HETE 226.0 12-HEPE d8-15-HETE 226.0 15-HEPE d8-15-HETE 226.0 18-HEPE d8-5-HETE 116.1 4-HDHA d8-5-HETE 116.1 7-HDHA d8-5-HETE 116.1 8-HDHA d8-12-HETE 184.2 10-HDHA d8-5-HETE 184.2 11-HDHA d8-12-HETE 184.2 13-HDHA d8-12-HETE 184.2 14-HDHA d8-15-HETE 226.0 16-HDHA d8-15-HETE 226.0 17-HDHA d8-15-HETE 226.0 20-HDHA 3 Type of quantification Calibration line Type of calibration line Solvent based Species calibration line 9-HODE, 13-HODE, 9-HOTrE, 13-HOTrE, 8-HETrE, 12-HETrE, 15-HETrE, 5-HETE, 8-HETE, 9-HETE, 11-HETE, 12-HETE, 15-HETE, 5-HEPE, 8-HEPE, 9-HEPE, 11-HEPE, 12-HEPE, 15-HEPE, 18-HEPE, 4-HDHA, 7-HDHA, 8-HDHA, 10-HDHA, 11-HDHA, 13-HDHA, 14-HDHA, 16-HDHA, 17-HDHA, 20-HDHA Response correction No Type I isotope correction No Limit of quantification S/N >5 and 80%<accuracy<120% Normalization to reference No Lipid Quantification Software Multiquant Batch correction No 2) FA[M-H]- / Lipid identification Lipid class FA MS Level for identification MS1 Identification level Full structure MS1adduct [M-H]- Isotope correction at MS1No MS1verified by standard No Background check at MS1Yes Did you presume assumptions for identification? No Check on: Isomeric overlap, Isobaric overlap Limit of detection S/N ratio > 3 RT verified by standard Yes Separation of isobaric/isomeric interferece confirmed Yes Model for separation prediction No Lipid Identification Software manually Data manipulation Smoothing Nomenclature for intact lipid molecule No 2) FA[M-H]- / Lipid quantification Quantitative Yes MS Level for quantification MS1 Internal lipid standard(s) MS1 Internal standard Endogenous subclass d4-C18:2 n6 C18:2 n6 d4-C20:5 n3 C18:3 n3 d8-C20:4 n6 C20:4 n6 d5-C20:5 n3 C20:5 n3 d5-C22:6 n3 C22:6 n3 Type of quantification Calibration line Type of calibration line Solvent based Species calibration line C18:2 n6, C18:3 n3, C20:4 n6, C20:5 n3, C22:6 n3 Response correction No Type I isotope correction No Limit of quantification S/N >5 and 80%<accuracy<120% Normalization to reference No Lipid Quantification Software Multiquant Batch correction No 4 5