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User Benefits Application News OAD-MS/MS for the Structural Identification of Double- Bond Positions in Lipids Associated with Alcohol Toxicity Emily Armitage 1 , Alan Barnes 1 , Olga Deda 2 , Christina Virgiliou 2 , Neil Loftus 1 , Helen Gika 2 , Ian Wilson 3 1 Shimadzu Corporation, Manchester, UK, 2 Aristotle University of Thessaloniki, Greece 3 Imperial College, London, UK High Performance Liquid Chromatograph Mass Spectrometer Oxygen Attachment Dissociation (OAD) is a novel MS/MS fragmentation technology that induces (C=C) position-specific fragmentation. Simultaneous acquisition of OAD-MS/MS and CID-MS/MS in positive or negative ESI mode, enables lipid identification to the structural level. Automated analysis of OAD-MS/MS data is supported in MS-DIAL. OAD-MS/MS increases reporting confidence in lipid identification. Introduction In metabolomics and lipidomics studies, the structural characterisation of lipids requires the identification of carbon number as well as the number and position of double bonds. Identifying carbon-carbon double bond (C=C) position(s) presents a major challenge in unsaturated lipid characterisation, however it is vital to understand a biological mechanism, since minor structural differences between positional isomers can alter the biochemical function of a lipid. Oxygen Attachment Dissociation (OAD) MS/MS is a novel method for specific fragmentation of C=C bonds, providing additional fragmentation to CID in positive and negative ESI mode on the LCMS-9050. It involves the generation of O/OH• radicals by microwave discharge of water vapour which are introduced into the collision cell via an inductively coupled plasma through a quartz tube. Neutral radicals interact with precursor ions to specifically oxidise/dissociate the double bonds between carbons. A key advantage is that spectra can be acquired with simultaneous OAD-MS/MS and CID-MS/MS (in either positive or negative ion mode) to generate sufficient information to identify lipids to the structural level (Fig. 1). In this application, OAD-MS/MS was applied to an untargeted metabolomics study following ethanol-induced exposure in mice. Tissue extracts from gut, liver and pancreas were analysed using UHPLC-OAD-MS/MS to increase reporting confidence in identification for organ-specific changes in the lipid profiles of gut, liver and pancreas of mice in response to ethanol toxicity. Table 1 Analytical conditions UHPLC: Nexera X2 Column C18 (2.1x100 mm 1.7 µm); column maintained at 50 ˚C Flow Rate 0.4 mL/min Mobile Phase A: Water + 0.1% formic acid B: Acetonitrile + 0.1% formic acid Rinse (R0/R1/R2) IPA/MeOH/MeCN/H 2 O (1:1:1:1) + 1% formic acid Gradient 2% B (0-1 min); 40% B (2 min); 90% B (25 min, curvi-linear gradient); 100% B (27.5-31 min); 2% B (31.01-35 min) Injection 1 µL (ESI+) / 2 µL (ESI-); autosampler maintained at 4 ˚C Mass Spectrometer: LCMS-9050 with OAD Radical Source I Interface 300 ˚C, 4.0 kV (ESI+) / –3.0 kV (ESI–) Heat block, DL 400 ˚C, 250 ˚C Gas flow rates Nebulizing gas 3 L/min; Heating gas 10 L/min Drying gas 15 L/min OAD Radical Source Atomic oxygen (O) and hydroxyl radicals (OH•) are generated in a radical source. Collision gas pressure 17 kPa MS TOF-MS (m/z 60-1250) MS/MS 3 DDA-MS/MS (m/z 40-1250; 1 second cycle time; collision energy spread 6-30 V; Precursor isolation 0.8 Da (ESI+) / 3 Da (ESI–) Materials and Methods Tissue extracts (gut, liver and pancreas) were obtained from C57BL/6 mice that were either subjected to ethanol exposure or un-dosed controls. The study was conducted in accordance with EU and National ethical guidelines and approved by the Aristotle University of Thessaloniki. Phenotypic tissue samples were analysed by OAD-MS/MS to structurally characterise lipids found to be significantly differentiated in ethanol treated mice compared to healthy controls from a previous study. The analysis was performed using the analytical conditions described in Table 1. LabSolutions Insight Explore and MS-DIAL were used to process data. DDA spectra acquired in positive and negative ESI mode for each lipid were analysed to assign C=C double bond positions and matched to the OAD database in MS-DIAL. Lipids were identified according to the omega and delta nomenclature, counting carbons from the methyl end or the carboxylic end of the fatty acyl, respectively. Fig. 2 highlights an example of the identification in MS-DIAL. O O O H P O - O O N + H O n-6 n-15 n-12 n-9 n-3 356.18304 396.21468 398.19387 436.24579 476.27667 478.25651 516.30822 518.28733 552.34432 [M-H2O+H] + 570.35508 [M+H] + m/z 350 375 400 425 450 475 500 525 550 558.31930 438.22567 Fig. 1 Oxygen Attachment Dissociation (OAD) MS/MS is a novel fragmentation method, providing additional fragmentation to CID on the LCMS-9050. In this example, C=C bond position-specific fragmentation by OAD enables lipid identification to the structural level identifying this lipid as LPC 22:5(n-3,6,9,12,15) sn-1 [or LPC 22:5(7,10,13,16,19) sn-1 using the delta nomenclature]. ω LPC 22:5(n-3,6,9,12,15) sn-1 Δ LPC 22:5(7,10,13,16,19) sn-1
Application News Results For each of the lipids with C=C double bonds found to be dysregulated by ethanol toxicity in gut, liver or pancreas in a previous untargeted metabolomics study, the UHPLC-OAD- MS/MS spectral data were used to positively assign the position(s) of each double bond. This involved identification of OAD-MS/MS characteristic neutral loss fragments for each of the candidates as described for lipids 1 . Structurally characterised lipids that were increased by ethanol exposure in one or more of the tissues are given in Table 2, while those that were decreased are presented in Table 3. OAD-MS/MS enabled the structural characterisation of specific lipid isomers, indicating the exact structures affected by ethanol toxicity. As one example, four retention time separated components were annotated as LPC 22:5 in the untargeted metabolomics analysis, with only the last two components being significant. Fig. 2 Data processing and lipid identification using the OAD database in MS-DIAL. This example highlights the identification of PC 18:2(9,12)_18:2(9,12), also known as PC(18:2(n-6,9)/18:2(n-6,9)), in the gut. Fig. 3 Detection of LPC 22:5 isomers with CID-MS/MS in negative ion mode. TOF-MS extracted ion chromatogram for m/z 614.3463 [M+HCOO-H]- in pancreas tissue extract. Negative ion spectra enable the identification of the acyl chain, but retention time separated isomers (components 2 and 4) cannot be distinguished by CID- MS/MS. CID-MS/MS acquired with a gas pressure of 230 kPa and a collision energy spread of 5-55 V. From the analysis of CID-MS/MS in positive ion fragment spectra, sn-1 and sn-2 isoforms were identified given the relative fragment ion intensity at m/z 104 (a characteristic fragment ion of the sn-1 isoform known to exceed a 30-fold difference in intensity relative to the same ion in the sn-2 isoform 2 ). However, the CID-MS/MS spectra could not distinguish between each of the sn-1 components (2 and 4) or sn-2 components (1 and 3). Negative ion CID-MS/MS provided diagnostic fragments to identify the acyl chain, but the spectra were similar for retention time separated components (Fig. 3 highlights the same distribution and intensity of fragments ions in sn-1 components 2 and 4). OAD-MS/MS fragment spectra on the other hand resulted in C=C-positional information and enabled the identification of four components; LPC 22:5(n-3,6,9,12,15) sn-2 and sn-1, LPC 22:5(n-6,9,12,15,18) sn-2 and sn-1. Figure 4 highlights the different sn-1 isoforms, distinguishable by OAD-MS/MS. m/z 50 100 150 200 250 300 350 400 450 500 550 600 0 % 100 614.34542 [M+HCOO] - 554.32424 loss of CH3 329.24818 acyl chain 22:5 285.25827 loss of CO 2 from acyl chain 22:5 224.06918 glycerophosphocholine, loss of CH3 and H 2 O 168.04228 phosphocholine, loss of CH3 m/z 50 100 150 200 250 300 350 400 450 500 550 600 0 % 100 614.34553 [M+HCOO] - 554.32444 loss of CH3 329.24826 acyl chain 22:5 285.25807 loss of CO 2 from acyl chain 22:5 224.06836 glycerophosphocholine, loss of CH3 and H 2 O 168.04344 phosphocholine, loss of CH3 Component 2 MS/MS ESI- CID (CE spread 5-55V) Component 4 MS/MS ESI- CID (CE spread 5-55V) Component 1 LPC 22:5 sn-2 Component 2 LPC 22:5 sn-1 Component 3 LPC 22:5 sn-2 Component 4 LPC 22:5 sn-1 m/z 614.3463 +/-5.0ppm (ESI-) RT (min) 6.6 6.8 7.0 7.2 7.4 7.6 7.8 0.0 0.25 0.5 7.5 1.0 1.3 1.5 1.8 2.0 x10 5 Extracted ion chromatogram Isomeric lipids with m/z 782.5682 are detected in the tissue that can be specifically identified using OAD-MS/MS Map of detected precursor ion signals as components in the QTOF analysis (retention time and m/z) Measured spectrum from DDA-MS/MS analysis Reference spectrum for PC 18:2(9,12)_18:2(9,12) in the OAD database
Application News 104.10682 choline 184.07323 phosphocholine head group 356.18304 396.21468 398.19387 436.24579 476.27667 478.25651 516.30822 518.28733 552.34432 [M-H2O+H] + 570.35508 [M+H] + m/z 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 0 % 12.5 558.31930 438.22567 n-6 n-15 n-12 n-9 n-3 ω LPC 22:5(n-6,9,12,15,18) sn-1 Δ LPC 22:5(4, 7,10,13,16) sn-1 104.10687 choline 184.07330 phosphocholine head group 258.10969 sn-1 acyl loss 354.16788 396.17802 436.20944 434.23043 474.26125 476.24082 516.27175 552.34495 [M-H2O+H] + 570.35532 [M+H] + m/z 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 0 % 12.5 394.19887 356.14722 n-6 n-15 n-12 n-9 n-18 O O O H P O - O O N + H O x16 m/z 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 0 % 100 168.04356 phosphocholine, loss of CH 3 224.06972 152.99607 Glycerol-3-P, loss of H 2 O 242.07985 loss of acyl chain as ketene and CH 3 329.24806 acyl chain 22:5 614.34547 [M+HCOO] - 554.32502 loss of CH 3 483.25060 338.13726 340.11671 378.16695 380.14683 420.17836 418.19838 460.20793 458.22992 n-15 n-18 n-12 n-9 n-6 x16 m/z 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 0 % 100 614.34562 [M+HCOO] - 554.32475 loss of CH 3 168.04452 phosphocholine, loss of CH 3 224.06935 329.24811 acyl chain 22:5 382.16336 420.21569 460.24767 380.18418 422.19417 462.22361 483.25127 502.25608 500.27585 n-15 n-12 n-9 n-6 n-3 b: Positive ion OAD-MS/MS fragment assignment for positions n-3,6,9,12,15 sn-1 c: Negative ion OAD-MS/MS fragment assignment for positions n-3,6,9,12,15 sn-1 d: OAD-MS/MS specific fragmentation of C=C bonds for LPC 22:5(n-6,9,12,15,18) sn-1 e: Positive ion OAD-MS/MS fragment assignment for positions n-6,9,12,15,18 sn-1 f: Negative ion OAD-MS/MS fragment assignment for positions n-6,9,12,15,18 sn-1 ω LPC 22:5(n-3,6,9,12,15) sn-1 Δ LPC 22:5(7,10,13,16,19) sn-1 O O O H P O - O O N + H O a: OAD-MS/MS specific fragmentation of C=C bonds for LPC 22:5(n-3,6,9,12,15) sn-1 Fig. 4 LPC 22:5(n-3,6,9,12,15) sn-1 (a: molecular structure with omega and delta nomenclature, b: OAD-MS/MS in positive ion, c: OAD-MS/MS in negative ion). LPC 22:5(n-6,9,12,15,18) sn-1 (d: molecular structure with omega and delta nomenclature, e: OAD-MS/MS in positive ion, f: OAD-MS/MS in negative ion). Simultaneous CID and OAD-MS/MS spectra resulted in a specific identification for the feature which is differentially expressed following ethanol treatment in mice as identified LPC 22:5(n-6,9,12,15,18) sn-1 in a pancreatic tissue sample. CID-MS/MS specific fragments highlighted in black, OAD-MS/MS highlighted in colours as indicated.
Application News Annotation Formula RT ESI+ m/z ESI- m/z Gut Liver Pancreas Docosahexaenoic acid ethyl ester (n-3,6,9,12,15,18) C24H36O2 14.49 357.27881 355.26425 Linoleic acid ethyl ester (n-6,9) C20H36O2 15.77 309.27881 307.26425 LPC 18:1(n-9) sn-1 C26H52NO7P 7.81 522.35542 566.34634 LPC 18:1(n-9) sn-2 C26H52NO7P 7.49 522.35542 566.34634 LPC 18:2(n-6,9) sn-1 C26H50NO7P 6.78 520.33977 564.33069 LPC 18:2(n-6,9) sn-2 C26H50NO7P 6.50 520.33977 564.33069 LPC 20:3(n-6,9,12) sn-1 C28H52NO7P 7.36 546.35542 590.34634 LPC 20:3(n-6,9,12) sn-2 C28H52NO7P 7.09 546.35542 590.34634 LPC 20:4(n-6,9,12,15) sn-2 C28H50NO7P 6.53 544.33977 588.33069 LPC 20:5(n-3,6,9,12,15) sn-1 C28H48NO7P 6.01 542.32412 586.31504 LPE 18:2(n-6,9) sn-1 C23H44NO7P 6.72 478.29282 476.27826 LPE 18:2(n-6,9) sn-2 C23H44NO7P 6.44 478.29282 476.27826 LPE 20:3(n-6,9,12) sn-1 C25H46NO7P 7.31 504.30847 502.29391 LPE O-18:1(n-9) C23H48NO6P 8.19 466.32920 464.31465 LPE P-18:2(n-6,9) C23H44NO6P 7.12 462.29790 460.28335 LPI 18:2(n-6,9) sn-1 C27H49O12P 6.85 597.30344 595.28889 Oleic acid ethyl ester C20H38O2 18.04 311.29446 309.2799 PC 14:0_18:2(n-6,9) C40H76NO8P 19.74 730.53813 728.52358 PC 15:0_18:2(n-6,9) C41H78NO8P 21.40 744.55378 788.54471 PC 16:0_18:2(n-6,9) C42H80NO8P 23.14 758.56943 802.56036 PC 16:0_18:3(n-6,9,12) C42H78NO8P 21.28 756.55378 800.54471 PC 16:0_20:5(n-3,6,9,12,15) C44H78NO8P 20.31 780.55378 824.54471 PC 16:1(n-7)_18:2(n-6,9) C42H78NO8P 20.13 756.55378 800.54471 PC 18:0_18:2(n-6,9) C44H84NO8P 26.97 786.60073 830.59166 PC 18:1(n-9)_18:2(n-6,9) C44H82NO8P 23.51 784.58508 828.57601 PC 18:1(n-9)_18:2(n-6,9) C44H82NO8P 24.62 784.58508 828.57601 PC 18:2(n-6,9)/18:2(n-6,9) C44H80NO8P 20.75 782.56943 826.56036 PC 18:2(n-6,9)_20:4(n-6,9,12,15) C46H80NO8P 20.26 806.56943 850.56036 PC 36:2(n-6,9) C44H84NO8P 26.67 786.60073 830.59166 PC P-16:0/18:2(n-6,9) C42H80NO7P 24.33 742.57452 786.56544 PE 18:0_18:2(n-6,9) C41H78NO8P 27.23 744.55378 742.53923 PE 18:1(n-9)_18:2(n-6,9) C41H76NO8P 23.76 742.53813 740.52358 PE P-16:0_20:5(n-3,6,9,12,15) C41H72NO7P 21.45 722.51192 720.49736 Table 2. Identification of C=C double bond position(s) in unsaturated lipids found to be significantly increased in gut, liver and/or pancreas tissue extracts following ethanol exposure. The tissue(s) in which the lipid was found to be significant in the original study is indicated () along with the detected retention time. OAD- MS/MS data were analysed in positive and negative ion mode to confidently identify the double bond positions in each of the lipids. Annotated significant results from UHPLC-MS/MS untargeted metabolic profiling of mouse gut, liver, and pancreas following chronic ethanol exposure were considered for OAD-MS/MS analysis to increase the level of confidence in identification. Lipid annotations in the original study considered library matching between the experimental CID-MS/MS (DIA-MS/MS and DDA-MS/MS) with external databases such as LIPID MAPS, MassBank and HMDB. The level of confidence in identification as determined by the Metabolomics Standards Initiative 3 is level 2; comparison of experimental data to literature or external laboratory data. To enhance the identification of unsaturated lipids in this study, C=C bond positions were derived from specific OAD-MS/MS spectra for all unsaturated lipids that were significant. Lipids which were significantly increased or decreased following ethanol exposure are listed in Tables 2 and 3 respectively. The response to ethanol toxicity was largely tissue specific, OAD-MS/MS highlighted common constituents such as notable increases in omega-6 containing phosphatidylcholines (18:2 n- 6,9) in response to ethanol toxicity as well decreases in polyunsaturated phospholipids containing fatty acids with four or more double bonds.
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SHIMADZU Europa GmbH, www.shimadzu.eu First Edition: Jul. 2026 05-SCA-210-084-EN Annotation Formula RT ESI+ m/z ESI- m/z Gut Liver Pancreas DG 18:1(n-9)_18:2(n-6,9) C39H70O5 29.27 641.51155 N/A Linoleic acid(n-6,9) C18H32O2 11.04 281.24751 279.23295 Linolenic acid methyl ester(n-6,9) C19H32O2 12.87 293.24751 291.23295 LPC 18:3(n-6,9,12) sn-1 C26H48NO7P 6.11 518.32412 562.31504 LPC 20:4(n-6,9,12,15) sn-1 C28H50NO7P 6.77 544.33977 588.33069 LPC 20:4(n-6,9,12,15) sn-2 C28H50NO7P 6.53 544.33977 588.33069 LPC 22:4(6,9,12,15) sn-1 C30H54NO7P 8.01 572.37107 616.36199 LPC 22:4(6,9,12,15) sn-2 C30H54NO7P 7.74 572.37107 616.36199 LPC 22:5(n-6,9,12,15,18) sn-1 C30H52NO7P 7.56 570.35542 614.34634 LPC 22:5(n-6,9,12,15,18) sn-2 C30H52NO7P 7.31 568.33977 614.34634 LPC O-18:1(n-9) C26H54NO6P 8.25 508.37615 552.36708 LPC O-18:2(n-6,9) C26H52NO6P 7.14 506.36050 550.35143 LPC P-18:1(n-9) C26H52NO6P 8.26 506.36050 550.35143 LPE 22:5(n-6,9,12,15,18) sn-1 C27H46NO7P 7.51 528.30847 526.29391 LPE 22:5(n-6,9,12,15,18) sn-2 C27H46NO7P 7.26 528.30847 526.29391 LPE O-18:1(n-9) C23H48NO6P 8.19 466.32920 464.31465 LPE P-18:1(n-9) C23H46NO6P 8.21 464.31355 462.29900 LPE P-18:2(n-6,9) C23H44NO6P 7.12 462.29790 460.28335 LPE P-20:1(n-9) C25H50NO6P 10.01 492.34485 490.33030 LPE P-22:1(n-9) C27H54NO6P 12.13 520.37615 518.36160 LPI 20:4(n-6,9,12,15) sn-2 C29H49O12P 6.56 621.30344 619.28889 Oleic acid (n-9) C18H34O2 12.73 283.26316 281.24860 Oleic acid methyl ester (n-9) C19H36O2 16.66 297.27881 295.26425 Palmitoleic acid methyl ester (n-7) C17H32O2 13.88 269.24751 267.23295 PC 16:0_22:4(n-6,9,12,15) C46H84NO8P 25.25 810.60073 854.59166 PC 16:0_22:5(n-3,6,9,12,15) C46H82NO8P 23.97 808.58508 852.57601 PC 16:1(n-7)_20:4(n-6,9,12,15) C44H78NO8P 19.66 780.55378 824.54471 PC 16:1(n-7)_22:6(n-3,6,9,12,15,18) C46H78NO8P 18.89 804.55378 848.54471 PC 18:1(n-9)_20:4(n-6,9,12,15) C46H82NO8P 22.88 808.58508 852.57601 PC 20:4(n-6,9,12,15)_22:6(n-3,6,9,12,15,18) C50H80NO8P 18.89 854.56943 898.56036 PE 16:0_22:5(n-6,9,12,15,18) C43H76NO8P 24.21 766.53813 764.52358 PE 18:0_22:6(n-3,6,9,12,15,18) C45H78NO8P 24.56 792.55378 790.53923 Table 3. Identification of C=C double bond position(s) in unsaturated lipids found to be significantly decreased in gut, liver and/or pancreas tissue extracts following ethanol exposure. The tissue(s) in which the lipid was found to be significant in the original study is indicated () along with the detected retention time. OAD-MS/MS data were analysed in positive and negative ion mode to confidently identify the double bond positions in each of the lipids. References 1. Uchino, H., Tsugawa, H., Takahashi, H., & Arita, M. (2022). Computational mass spectrometry accelerates C = C position-resolved untargeted lipidomics using oxygen attachment dissociation. Communications Chemistry, 5(1). 2. Han, X., & Gross, R. W. (1996). Structural Determination of Lysophospholipid Regioisomers by Electrospray Ionization Tandem Mass Spectrometry. Journal of the American Chemical Society, 118(2), 451–457. 3. Spicer, R., Salek, R. & Steinbeck, C. (2017) A decade after the metabolomics standards initiative it's time for a revision. Sci Data 4, 170138. Conclusion OAD-MS/MS is a novel fragmentation method providing complementary structural information to CID in positive and negative ion mode. It is less hazardous than other methods and provides higher efficiency fragmentation, especially for singly charged positive and negative ion precursors. OAD-MS/MS enables C=C positional assignments in lipids and has been applied to structurally characterise lipids found to be potential markers of ethanol toxicity in a previous study using high resolution metabolomics analysis by UHPLC-CID-MS/MS. The enhanced level of structural identification provided by OAD- MS/MS allows the potential to improve our understanding of the biological roles of lipids. LabSolutions Insight Explore and Nexera are trademarks of Shimadzu Corporation or its affiliated companies in Japan and/or other countries.
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