Chemical derivatization involves the reaction of an analyte with a derivatization agent to modify its structure, improving the peak shape, chromatographic performance, structural analysis, ionization efficiency, and sensitivity. A novel derivatization method using 3-(chlorosulfonyl)benzoic acid is developed for the determination of monoacylglycerols, diacylglycerols, free sterols, and tocopherols using the reversed-phase ultra-high-performance liquid chromatography-tandem mass spectrometry (RP-UHPLC/MS/MS) method in the negative ion mode. The chromatographic and mass spectrometric properties of derivatized lipids are investigated by using 29 lipid standards spanning four lipid classes. The derivatization process is optimized using pooled plasma spiked by 9 internal standards, achieving an optimal yield with a reaction time of 40 min at 60 °C. The stability of the derivatives is confirmed, with short-term stability maintained for 10 h at 4 °C and long-term stability preserved for 5 days at -80 °C. The repeatability and reproducibility are verified by one/two operator(s), which underscores the simplicity and robustness of the method, and calibration curves with high linear regression coefficients illustrate the accuracy of the method. The derivatization approach, which combines RP-UHPLC/MS/MS and the use of specific fragmentation patterns, significantly reduces limits of detection, reaching 15-25 pmol/mL for free sterols in plasma. The optimized method is applied to the analysis of human plasma, leading to the identification of 92 lipid species in the targeted lipid classes. This represents a substantial improvement in sensitivity and detection capabilities compared to those of previously reported methods.

Palacký University Medical School and University Hospital Olomouc Faculty of Medicine and Dentistry Department of Oncology 1 P Pavlova 6 775 20 Olomouc Czech Republic

University of Pardubice Faculty of Chemical Technology Department of Analytical Chemistry Studentská 573 532 10 Pardubice Czech Republic

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Han X. L. Lipidomics for studying metabolism. Nat. Rev. Endocrinol. 2016, 12 (11), 668–679. 10.1038/nrendo.2016.98. PubMed DOI

Yang X. K.; Liu L.; Xi L. J.; Wu B. B.; Ku C. Y.; Wang R. Z.; Dai M.; Ping Z. G. Trends in total cholesterol control among American adults with hypercholesterolemia, 1988–2018. Nutr. Metab. Cardiovas. 2023, 33 (8), 1511–1520. 10.1016/j.numecd.2023.05.015. PubMed DOI

Zambón D.; Quintana M.; Mata P.; Alonso R.; Benavent J.; Cruz-Sánchez F.; Gich J.; Pocoví M.; Civeira F.; Capurro S.; Bachman D.; Sambamurti K.; Nicholas J.; Pappolla M. A. Higher Incidence of Mild Cognitive Impairment in Familial Hypercholesterolemia. Am. J. Med. 2010, 123 (3), 267–274. 10.1016/j.amjmed.2009.08.015. PubMed DOI PMC

Singh M.; Nam D. T.; Arseneault M.; Ramassamy C. Role of By-Products of Lipid Oxidation in Alzheimer’s Disease Brain: A Focus on Acrolein. J. Alzheimers Dis. 2010, 21 (3), 741–756. 10.3233/JAD-2010-100405. PubMed DOI

Huynh K.; Barlow C. K.; Jayawardana K. S.; Weir J. M.; Mellett N. A.; Cinel M.; Magliano D. J.; Shaw J. E.; Drew B. G.; Meikle P. J. High-Throughput Plasma Lipidomics: Detailed Mapping of the Associations with Cardiometabolic Risk Factors. Cell Chem. Biol. 2019, 26 (1), 71–84. 10.1016/j.chembiol.2018.10.008. PubMed DOI

Wolrab D.; Jirásko R.; Chocholoušková M.; Peterka O.; Holčapek M. Oncolipidomics: Mass spectrometric quantitation of lipids in cancer research. Trac-Trend Anal. Chem. 2019, 120, 115480.10.1016/j.trac.2019.04.012. DOI

Avela H. F.; Sirén H. Advances in lipidomics. Clin. Chim. Acta 2020, 510, 123–141. 10.1016/j.cca.2020.06.049. PubMed DOI

Kanehisa M.; Furumichi M.; Sato Y.; Kawashima M.; Ishiguro-Watanabe M. KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Res. 2023, 51 (1), 587–592. 10.1093/nar/gkac963. PubMed DOI PMC

Lange M.; Ni Z. X.; Criscuolo A.; Fedorova M. Liquid Chromatography Techniques in Lipidomics Research. Chromatographia. 2019, 82 (1), 77–100. 10.1007/s10337-018-3656-4. DOI

Wolrab D.; Peterka O.; Chocholoušková M.; Holčapek M. Ultrahigh-performance supercritical fluid chromatography/mass spectrometry in the lipidomic analysis. Trac-Trend Anal. Chem. 2022, 149, 116546.10.1016/j.trac.2022.116546. DOI

Holčapek M.; Liebisch G.; Ekroos K. Lipidomic Analysis. Anal. Chem. 2018, 90 (7), 4249–4257. 10.1021/acs.analchem.7b05395. PubMed DOI

Züllig T.; Trötzmüller M.; Köfeler H. C. Lipidomics from sample preparation to data analysis: a primer. Anal. Bioanal. Chem. 2020, 412 (10), 2191–2209. 10.1007/s00216-019-02241-y. PubMed DOI PMC

Murphy R. C. Challenges in mass spectrometry-based lipidomics of neutral lipids. Trac-Trend Anal. Chem. 2018, 107, 91–98. 10.1016/j.trac.2018.07.023. PubMed DOI PMC

Peterka O.; Jirásko R.; Vaňková Z.; Chocholoušková M.; Wolrab D.; Kulhánek J.; Bureš F.; Holčapek M. Simple and Reproducible Derivatization with Benzoyl Chloride: Improvement of Sensitivity for Multiple Lipid Classes in RP-UHPLC/MS. Anal. Chem. 2021, 93 (41), 13835–13843. 10.1021/acs.analchem.1c02463. PubMed DOI

Li Y. L.; Su X.; Stahl P. D.; Gross M. L. Quantification of diacylglycerol molecular species in biological samples by electrospray ionization mass spectrometry after one-step derivatization. Anal. Chem. 2007, 79 (4), 1569–1574. 10.1021/ac0615910. PubMed DOI PMC

Leiker T. J.; Barkley R. M.; Murphy R. C. Analysis of diacylglycerol molecular species in cellular lipid extracts by normal-phase LC-electrospray mass spectrometry. Int. J. Mass Spectrom. 2011, 305 (2–3), 103–108. 10.1016/j.ijms.2010.09.008. PubMed DOI PMC

Liu Y. D.; Liu H. J.; Gong G. W. Monitoring diacylglycerols in biofluids by non-isotopically paired charge derivatization combined with LC-MS/MS. Front. Chem. 2022, 10, 1062118.10.3389/fchem.2022.1062118. PubMed DOI PMC

Petrosino T.; Riccieri R.; Blasi F.; Brutti M.; D’arco G.; Bosi A.; Maurelli S.; Cossignani L.; Simonetti M. S.; Damiani P. Original normal-phase high-performance liquid chromatographic separation of monoacylglycerol classes from extra virgin olive oil Triacylglycerols for their stereospecific analysis. J. Aoac. Int. 2007, 90 (6), 1647–1654. 10.1093/jaoac/90.6.1647. PubMed DOI

Tada N.; Fujita H.; Ando Y. Synthesis of Urethane Derivatives of Mono- and Diacylglycerols for Use as HPLC Standards in the Enantiomeric Separation. J. Am. Oil Chem. Soc. 2014, 91 (7), 1131–1137. 10.1007/s11746-014-2452-z. DOI

Zhu M. L.; Lu K. G.; Jin Y. T.; Xu X. W.; Chu C. Y.; Hao H. P.; Zheng Q. L. Boronic derivatization-based strategy for monoacylglycerol identification, isomer annotation and quantification. Anal. Chim. Acta 2022, 1190, 339233.10.1016/j.aca.2021.339233. PubMed DOI

Liebisch G.; Binder M.; Schifferer R.; Langmann T.; Schulz B.; Schmitz G. High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Bba-Mol. Cell Biol. 2006, 1761 (1), 121–128. 10.1016/j.bbalip.2005.12.007. PubMed DOI

Nzekoue F. K.; Caprioli G.; Ricciutelli M.; Cortese M.; Alesi A.; Vittori S.; Sagratini G. Development of an innovative phytosterol derivatization method to improve the HPLC-DAD analysis and the ESI-MS detection of plant sterols/stanols. Food Res. Int. 2020, 131, 108998.10.1016/j.foodres.2020.108998. PubMed DOI

Woo H. K.; Go E. P.; Hoang L.; Trauger S. A.; Bowen B.; Siuzdak G.; Northen T. R. Phosphonium labeling for increasing metabolomic coverage of neutral lipids using electrospray ionization mass spectrometry. Rapid Commun. Mass Sp. 2009, 23 (12), 1849–1855. 10.1002/rcm.4076. PubMed DOI PMC

Sandhoff R.; Brügger B.; Jeckel D.; Lehmann W. D.; Wieland F. T. Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry. J. Lipid Res. 1999, 40 (1), 126–132. 10.1016/S0022-2275(20)33347-2. PubMed DOI

Ayciriex S.; Regazzetti A.; Gaudin M.; Prost E.; Dargère D.; Massicot F.; Auzeil N.; Laprévote O. Development of a novel method for quantification of sterols and oxysterols by UPLC-ESI-HRMS: application to a neuroinflammation rat model. Anal. Bioanal. Chem. 2012, 404 (10), 3049–3059. 10.1007/s00216-012-6396-6. PubMed DOI

Honda A.; Yamashita K.; Miyazaki H.; Shirai M.; Ikegami T.; Xu G. R.; Numazawa M.; Hara T.; Matsuzaki Y. Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS. J. Lipid Res. 2008, 49 (9), 2063–2073. 10.1194/jlr.D800017-JLR200. PubMed DOI

Jiang X. T.; Sidhu R.; Porter F. D.; Yanjanin N. M.; Speak A. O.; Vruchte D. T. T.; Platt F. M.; Fujiwara H.; Scherrer D. E.; Zhang J.; Dietzen D. J.; Schaffer J. E.; Ory D. S. A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma. J. Lipid Res. 2011, 52 (7), 1435–1445. 10.1194/jlr.D015735. PubMed DOI PMC

Griffiths W. J.; Crick P. J.; Wang Y. C.; Ogundare M.; Tuschl K.; Morris A. A.; Bigger B. W.; Clayton P. T.; Wang Y. Q. Analytical strategies for characterization of oxysterol lipidomes: Liver X receptor ligands in plasma. Free Radical. Bio. Med. 2013, 59, 69–84. 10.1016/j.freeradbiomed.2012.07.027. PubMed DOI

Roberg-Larsen H.; Strand M. F.; Grimsmo A.; Olsen P. A.; Dembinski J. L.; Rise F.; Lundanes E.; Greibrokk T.; Krauss S.; Wilson S. R. High sensitivity measurements of active oxysterols with automated filtration/filter backflush-solid phase extraction-liquid chromatography-mass spectrometry. J. Chromatogr. A 2012, 1255, 291–297. 10.1016/j.chroma.2012.02.002. PubMed DOI

Zhang W. P.; Zhang D. H.; Chen Q. H.; Wu J. H.; Ouyang Z.; Xia Y. Online photochemical derivatization enables comprehensive mass spectrometric analysis of unsaturated phospholipid isomers. Nat. Commun. 2019, 10 (1), 79.10.1038/s41467-018-07963-8. PubMed DOI PMC

Poad B. L. J.; Zheng X. Y.; Mitchell T. W.; Smith R. D.; Baker E. S.; Blanksby S. J. Online Ozonolysis Combined with Ion Mobility-Mass Spectrometry Provides a New Platform for Lipid Isomer Analyses. Anal. Chem. 2018, 90 (2), 1292–1300. 10.1021/acs.analchem.7b04091. PubMed DOI PMC

Deng P.; Zhong D. F.; Wang X.; Dai Y. L.; Zhou L.; Leng Y.; Chen X. Y. Analysis of diacylglycerols by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry: Double bond location and isomers separation. Anal. Chim. Acta 2016, 925, 23–33. 10.1016/j.aca.2016.04.051. PubMed DOI

Feng Y.; Chen B. M.; Yu Q. Y.; Li L. J. Identification of Double Bond Position Isomers in Unsaturated Lipids by m-CPBA Epoxidation and Mass Spectrometry Fragmentation. Anal. Chem. 2019, 91 (3), 1791–1795. 10.1021/acs.analchem.8b04905. PubMed DOI PMC

Vaňková Z.; Peterka O.; Chocholoušková M.; Wolrab D.; Jirásko R.; Holčapek M. Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS. Anal. Bioanal. Chem. 2022, 414 (1), 319–331. 10.1007/s00216-021-03492-4. PubMed DOI

Folch J.; Lees M.; Stanley G. H. S. A Simple Method for the Isolation and Purification of Total Lipides from Animal Tissues. J. Biol. Chem. 1957, 226, 497–509. 10.1016/S0021-9258(18)64849-5. PubMed DOI

Adams K. J.; Pratt B.; Bose N.; Dubois L. G.; St John-Williams L.; Perrott K. M.; Ky K.; Kapahi P.; Sharma V.; MacCoss M. J.; Moseley M. A.; Colton C. A.; MacLean B. X.; Schilling B.; Thompson J. W. Skyline for Small Molecules: A Unifying Software Package for Quantitative Metabolomics. J. Proteome Res. 2020, 19 (4), 1447–1458. 10.1021/acs.jproteome.9b00640. PubMed DOI PMC

Burla B.; Arita M.; Arita M.; Bendt A. K.; Cazenave-Gassiot A.; Dennis E. A.; Ekroos K.; Han X. L.; Ikeda K.; Liebisch G.; Lin M. K.; Loh T. P.; Meikle P. J.; Oresic M.; Quehenberger O.; Shevchenko A.; Torta F.; Wakelam M. J. O.; Wheelock C. E.; Wenk M. R. MS-based lipidomics of human blood plasma: a community-initiated position paper to develop accepted guidelines. J. Lipid Res. 2018, 59 (10), 2001–2017. 10.1194/jlr.S087163. PubMed DOI PMC

Wolrab D.; Chocholoušková M.; Jirásko R.; Peterka O.; Holčapek M. Validation of lipidomic analysis of human plasma and serum by supercritical fluid chromatography-mass spectrometry and hydrophilic interaction liquid chromatography-mass spectrometry. Anal. Bioanal. Chem. 2020, 412 (10), 2375–2388. 10.1007/s00216-020-02473-3. PubMed DOI

Holčapek M.; Jandera P.; Fischer J. Analysis of acylglycerols and methyl esters of fatty acids in vegetable oils and in biodiesel. Crit. Rev. Anal. Chem. 2001, 31 (1), 53–56. 10.1080/20014091076686. DOI

Saberi A. H.; Chin-Ping T.; Kee B. B.; Koon L. S.; Oi-Ming L. Reversed-Phase High-Performance Liquid Chromatography Analysis of 1,3-and 1,2(2,3)-Positional Isomers of Palm-Based Diacylglycerols. J. Oil Palm Res. 2013, 25 (3), 326–335.

Liebisch G.; Fahy E.; Aoki J.; Dennis E. A.; Durand T.; Ejsing C. S.; Fedorova M.; Feussner I.; Griffiths W. J.; Köfeler H.; Merrill A. H.; Murphy R. C.; O’Donnell V. B.; Oskolkova O.; Subramaniam S.; Wakelam M. J. O.; Spener F. Update on LIPID MAPS classification, nomenclature, and shorthand notation for MS-derived lipid structures. J. Lipid Res. 2020, 61 (12), 1539–1555. 10.1194/jlr.S120001025. PubMed DOI PMC

Bowden J. A.; Heckert A.; Ulmer C. Z.; Jones C. M.; Koelmel J. P.; Abdullah L.; Ahonen L.; Alnouti Y.; Armando A. M.; Asara J. M.; Bamba T.; Barr J. R.; Bergquist J.; Borchers C. H.; Brandsma J.; Breitkopf S. B.; Cajka T.; Cazenave-Gassiot A.; Checa A.; Cinel M. A.; et al. Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma. J. Lipid Res. 2017, 58 (12), 2275–2288. 10.1194/jlr.M079012. PubMed DOI PMC

Quehenberger O.; Armando A. M.; Brown A. H.; Milne S. B.; Myers D. S.; Merrill A. H.; Bandyopadhyay S.; Jones K. N.; Kelly S.; Shaner R. L.; Sullards C. M.; Wang E.; Murphy R. C.; Barkley R. M.; Leiker T. J.; Raetz C. R. H.; Guan Z. Q.; Laird G. M.; Six D. A.; Russell D. W.; et al. Lipidomics reveals a remarkable diversity of lipids in human plasma. J. Lipid Res. 2010, 51 (11), 3299–3305. 10.1194/jlr.M009449. PubMed DOI PMC

Ghorasaini M.; Mohammed Y.; Adamski J.; Bettcher L.; Bowden J. A.; Cabruja M.; Contrepois K.; Ellenberger M.; Gajera B.; Haid M.; Hornburg D.; Hunter C.; Jones C. M.; Klein T.; Mayboroda O.; Mirzaian M.; Moaddel R.; Ferrucci L.; Lovett J.; Nazir K.; et al. Cross-Laboratory Standardization of Preclinical Lipidomics Using Differential Mobility Spectrometry and Multiple Reaction Monitoring. Anal. Chem. 2021, 93 (49), 16369–16378. 10.1021/acs.analchem.1c02826. PubMed DOI PMC

Wolrab D.; Jirásko R.; Cífková E.; Höring M.; Mei D.; Chocholoušková M.; Peterka O.; Idkowiak J.; Hrnciarová T.; Kuchař L.; Ahrends R.; Brumarová R.; Friedecký D.; Vivo-Truyols G.; Skrha P.; Škrha J.; Kučera R.; Melichar B.; Liebisch G.; Burkhardt R.; et al. Lipidomic profiling of human serum enables detection of pancreatic cancer. Nat. Commun. 2022, 13 (1), 124.10.1038/s41467-021-27765-9. PubMed DOI PMC

Lerner R.; Baker D.; Schwitter C.; Neuhaus S.; Hauptmann T.; Post J. M.; Kramer S.; Bindila L. Four-dimensional trapped ion mobility spectrometry lipidomics for high throughput clinical profiling of human blood samples. Nat. Commun. 2023, 14 (1), 937.10.1038/s41467-023-36520-1. PubMed DOI PMC

Teng Y. C.; Gielen M. C.; de Gruijter N. M.; Ciurtin C.; Rosser E. C.; Karu K. Phytosterols in human serum as measured using a liquid chromatography tandem mass spectrometry. J. Steroid Biochem. 2024, 241, 106519.10.1016/j.jsbmb.2024.106519. PubMed DOI

Ceglarek U.; Dittrich J.; Leopold J.; Helmschrodt C.; Becker S.; Staab H.; Richter O.; Rohm S.; Aust G. Free cholesterol, cholesterol precursor and plant sterol levels in atherosclerotic plaques are independently associated with symptomatic advanced carotid artery stenosis. Atherosclerosis. 2020, 295, 18–24. 10.1016/j.atherosclerosis.2019.12.018. PubMed DOI

Baila-Rueda L.; Cenarro A.; Cofán M.; Orera I.; Barcelo-Batllori S.; Pocoví M.; Ros E.; Civeira F.; Nerín C.; Domeño C. Simultaneous determination of oxysterols, phytosterols and cholesterol precursors by high performance liquid chromatography tandem mass spectrometry in human serum. Anal. Methods-Uk. 2013, 5 (9), 2249–2257. 10.1039/c3ay26395a. DOI

Mendiara I.; Bentayeb K.; Nerín C.; Domeño C. Online solid-phase extraction-liquid chromatography-mass spectrometry to determine free sterols in human serum. Talanta. 2015, 132, 690–697. 10.1016/j.talanta.2014.10.029. PubMed DOI

McDonald J. G.; Smith D. D.; Stiles A. R.; Russell D. W. A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma. J. Lipid Res. 2012, 53 (7), 1399–1409. 10.1194/jlr.D022285. PubMed DOI PMC

Honda A.; Yamashita K.; Miyazaki H.; Shirai M.; Ikegami T.; Xu G. R.; Numazawa M.; Hara T.; Matsuzaki Y. Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS. J. Lipid Res. 2008, 49 (9), 2063–2073. 10.1194/jlr.D800017-JLR200. PubMed DOI