Determination of double bond positions in unsaturated fatty acids by pre-column derivatization with dimethyl and dipyridyl disulfide followed by LC-SWATH-MS analysis

Abstract

Comprehensive in-depth structural characterization of free mono-unsaturated and polyunsaturated fatty acids often requires the determination of carbon-carbon double bond positions due to their impact on physiological properties and relevance in biological samples or during impurity profiling of pharmaceuticals. In this research, we report on the evaluation of disulfides as suitable derivatization reagents for the determination of carbon-carbon double bond positions of unsaturated free fatty acids by UHPLC-ESI-QTOF-MS/MS analysis and SWATH (sequential windowed acquisition of all theoretical mass spectra) acquisition. Iodine-catalyzed derivatization of C = C double bonds with dimethyl disulfide (DMDS) enabled detection of characteristic carboxy-terminal MS2 fragments for various fatty acids in ESI negative mode. The determination of double bond positions of fatty acids with up to three double bonds, the transfer of the method to plasma samples, and its limitations have been shown. To achieve charge-switching for positive ion mode MS-detection, derivatization with 2,2'-dipyridyldisulfide (DPDS) was investigated. It enabled detection of both corresponding characteristic omega-end- and carboxy-end-fragments for fatty acids with up to two double bonds in positive ion mode. It provides a straightforward strategy for designing MRM transitions for targeted LC-MS/MS assays. Both derivatization techniques represent a simple and inexpensive way for the determination of double bond positions in fatty acids with low number of double bonds. No adaptation of MS hardware is required and the specific isotopic pattern of resulting sulfur-containing products provides additional structural confirmation. This reaction scheme opens up the avenue of structural tuning of disulfide reagents beyond DMDS and DPDS using reagents like cystine and analogs to achieve enhanced performance and sensitivity.

Keywords: 2,2′-Dipyridyldisulfide (DPDS); Collision-induced dissociation (CID); Data-independent acquisition (DIA); Dimethyl disulfide (DMDS); Isomer; Lipidomics.

Fig. 1

DMDS-derivatized mono-unsaturated fatty acids: a MS1-EIC of derivatized oleic acid (blue; m/z 375.240 ± 0.01) and cis-vaccenic acid (red; m/z 375.240 ± 0.01) from TOF–MS scans, MS2-EIC of characteristic fragments (m/z 215.111 ± 0.01, m/z 187.080 ± 0.01; intensity multiplied with 20 for better visibility) from PIS (MRM-HR) of respective mono-derivatized products; b derivatization kinetics of mono-derivatized oleic acid (fitted, pseudo 1st order, BoxLucas1); c MS2 spectrum (CE: 45 V) of derivatized oleic acid with tentative fragmentation pattern leading to fragment with m/z 187.080; d MS2 spectrum (CE: 45 V) of derivatized cis-vaccenic acid with tentative fragmentation pattern leading to fragment m/z 215.111

Fig. 2

DPDS-derivatized mono-unsaturated fatty acids, detected as [M + H]⁺: a MS1-EIC of derivatized oleic acid (blue; m/z 503.276 ± 0.01) and cis-vaccenic acid (red; m/z 503.276 ± 0.01) from TOF–MS scans, MS2-EICs of characteristic fragments (40-fold multiplied for visibility; derived from OA: m/z 248.110, 236.147, 266.121; derived from VA: m/z 208.115, 276.142, 294.152) from PIS (MRM-HR) of respective mono-derivatized products; b derivatization kinetics of mono-derivatized oleic acid (fitted, pseudo 1st order, BoxLucas1); c MS2 spectrum (CE: 45 V) of derivatized oleic acid; d tentative fragmentation patterns of oleic acid; e MS2 spectrum (CE: 45 V) of derivatized cis-vaccenic acid; f tentative fragmentation patterns of cis-vaccenic acid

Fig. 3

DMDS-derivatized linoleic acid (C18:2n-6,9): a MS1-EIC of derivatized linoleic acid (blue; m/z 373.224 ± 0.01), cyclic products of linoleic acid (red; m/z 405.197 ± 0.01) from TOF–MS scan and MS2-EIC of fragmentation products (40-fold increased for visibility) from PIS (MRM-HR) of mono-derivatized linoleic acid; b derivatization kinetics of linoleic acid (fitted, pseudo 1st order, BoxLucas1); c MS2 spectrum (CE: 45 V) of derivatized linoleic acid; d tentative fragmentation patterns leading to characteristic fragments (position 9: m/z 187.080, 215.111; position 12: m/z 195.139, 239.111, 225.095)

Fig. 4

DMDS-derivatized α-linolenic acid (C18:3n-3,6,9): a MS1-EIC of mono-derivatized α-linolenic acid (blue; m/z 371.208 ± 0.01), bis-derivatized α-linolenic acid (red; m/z 465.200 ± 0.01), and cyclic products (purple, m/z 403.178 ± 0.01) from TOF–MS scan, MS2-EICs (CE: 45 V) of fragments (300-fold increased: m/z 227.111, 235.170, 255.142, 267.142; 100-fold multiplied: m/z 195.139, 215.111, 187.080, 207.139) from PIS (MRM-HR) of mono-derivatized α-linolenic acid; b derivatization kinetics (fitted, mono-derivatized: ExpGrowDec, di-derivatized: BoxLucas1, cyclic product: BoxLucas1); c proposed fragmentation patterns leading to characteristic fragments

Fig. 5

SWATH-MS measurements of DMDS-derivatized PUFA-mix (a), underivatized plasma extract (b), and DMDS-derivatized plasma extract (c). a MS1-EIC of mono-derivatized oleic acid (OA, m/z 375.239 ± 0.01), linoleic acid (LA, m/z 373.224 ± 0.01), α-/γ-linolenic acid (ALA/GLA, m/z 371.208 ± 0.01), stearidonic acid (STEA, m/z 369.193 ± 0.01), dihomo-γ-linolenic acid (DGLA, m/z 399.240 ± 0.01), arachidonic acid (AA, m/z 397.224 ± 0.01), eicosapentaenoic acid (EPA, m/z 395.208 ± 0.01, multiplied with 10 for better visibility), docosatetraenoic acid (DTA, m/z 425.255 ± 0.01, multiplied with 2 for better visibility), and docosahexaenoic acid (DHA, m/z 421.224 ± 0.01, multiplied with 2 for better visibility); MS2-EICs (CE: 45 V) of characteristic fragments from SWATH window m/z 349–380 (20-fold multiplied: m/z 187.080, 215.111, 195.139, 225.095; 50-fold multiplied: m/z 239.111, 227.111, 255.142, 207.139, 267.142, 235.170, 197.064, 183.048, 193.123); b underivatized plasma extract: MS1-EICs of underivatized C16:1, C18:1, C18:2, C18:3, C20:4, C22:4, C22:5, and C22:6 (assigned with MS-DIAL, but does not allow a statement about double bond positions); c derivatized plasma extract: MS1-EIC of derivatized FAs with assigned double bond positions: palmitoleic acid (C16:1), oleic acid (C18:1), and linoleic acid (C18:2) and corresponding MS2-EICs (CE: 45 V) of their characteristic fragments (60-fold multiplied (SWATH window m/z 319–350): m/z 153.092, 187.080; 40-fold multiplied (SWATH window m/z 349–380): m/z 227.111, 225.095, 239.111, 195.139, 215.111; tenfold multiplied (SWATH window m/z 349–380): m/z 187.080). All MS1-EICs are extracted from the TOF–MS scans and MS2-EIC from respective SWATH-MS windows