Benzoyl chloride derivatization improves selectivity and sensitivity of lipidomic quantitation in human serum of pancreatic cancer patients using RP-UHPLC/MS/MS

Sep 30, 2026

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Chemical derivatization is used to enhance lipid chromatography and mass spectrometric sensitivity, supporting biomarker research. A targeted, validated workflow combines benzoyl chloride derivatization with reversed-phase ultrahigh-performance liquid chromatography tandem mass spectrometry (RP-UHPLC/MS/MS) for quantitative analysis of human serum lipidomes. Across 450 lipid species from 19 lipid subclasses, identification relies on multiple reaction monitoring transitions, retention dependencies, dilution series, and derivatization tags. Sensitivity increases versus conventional methods, most strongly for monoacylglycerols, diacylglycerols, sphingoid bases, and free sterols. Accuracy is validated with NIST SRM 1950 and confirmed by ring-trial consensus values. Clinical profiling shows pancreatic cancer–associated dysregulation of lipid metabolism, with upregulated monoacylglycerols and sphingosine and pronounced downregulation of specific sphingolipids and phospholipids. The approach yields new, structurally informative insights consistent with trends from other lipidomic methods.

LanguageEnglish

CategoryHealthcare

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Pages13

Reading Time33 min

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Abstract

Introduction

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Analytical and Bioanalytical Chemistry (2025) 418:733–745 [https://doi.org/10.1007/s00216-025-06151-0](https://doi.org/10.1007/s00216-025-06151-0) Benzoyl chloride derivatization improves selectivity and sensitivity of lipidomic quantitation in human serum of pancreatic cancer patients using RP-UHPLC/MS/MS Ondřej Peterka1 · Zuzana Lásko1 · Robert Jirásko1 · Petra Peroutková1 · Anna Taylor1 · Beatrice Mohelníková‑Duchoňová2 · Irena Kozubíková2 · Martin Loveček3 · Bohuslav Melichar2 · Michal Holčapek1 Received: 29 July 2025 / Revised: 19 September 2025 / Accepted: 22 September 2025 / Published online: 6 October 2025 © The Author(s) 2025 Abstract Chemical derivatization is a powerful strategy for enhancing the chromatographic behavior and mass spectrometric sensitivity of lipids, which play an essential role in cellular processes and show high potential in cancer biomarker research. In this study, we describe a targeted and validated method that combines benzoyl chloride derivatization with reversed-phase ultrahigh-performance liquid chromatography tandem mass spectrometry (RP-UHPLC/MS/MS) for the quantitative analysis of the human serum lipidome. In total, 450 lipid species from 19 lipid subclasses were identified based on a combination of multiple reaction monitoring transitions, retention dependencies, dilution series, and derivatization tags. The developed methodology increases the sensitivity for most investigated lipid classes in comparison to conventional methods, but the highest improvement was observed for monoacylglycerols, diacylglycerols, sphingoid bases, and free sterols. The method’s accuracy was confirmed using NIST SRM 1950, as the determined concentrations were in agreement with the consensus values from ring trials. Lipidomic profiling of clinical samples revealed a significant dysregulation of lipid metabolism in pancreatic cancer patients compared to healthy controls. Key findings included the upregulation of most monoacylglycerolsand sphingosine, and a pronounced downregulation of sphingolipids with very long saturated N-acyl chains and phospholipids containing fatty acyl compositions 18:2 and 20:4 . This targeted approach is consistent with the trends previously seen with other methods and also provides new findings and more detailed structural insights into metabolic alterations in pancreatic cancer. Keywords Derivatization · Lipidomic quantitation · Liquid chromatography · Mass spectrometry · Human serum · Pancreatic cancer Published in the topical collection featuring Promising EarlyCareer (Bio)Analytical Researchers in 2026 with guest editors Antje J. Baeumner, Soledad Cárdenas, and Alberto Cavazzini. * Michal Holčapek [Michal.Holcapek@upce.cz](Michal.Holcapek@upce.cz) 1 Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic 2 Department of Oncology, Faculty of Medicine and Dentistry, Palacký University Olomouc and University Hospital, Zdravotníků 248, 775 20 Olomouc, Czech Republic 3 Department of Surgery, Faculty of Medicine and Dentistry, Palacký University Olomouc and University Hospital, Hněvotínská 3, 779 00 Olomouc, Czech Republic Abbreviations ACN BMI Bz-Cl CE Cer DE DG DP DW FA FC Hex2Cer HexCer HR IPA IS Acetonitrile Body mass index Benzoyl chloride Cholesterol ester Ceramide Desmosterol ester Diacylglycerol Declustering potential Dwell weights Fatty acid Fold change Dihexosylceramide Hexosylceramide High-resolution 2-Propanol Internal standard IS-Mix LOQLPCLPELPI LRMDAMeOH MGMRM MSN NLS ns -OOPLS-DA -PPCAPC PDACPE PIPIS PR Pyr QC QqQ QTOF QTRAP RP-UHPLC/MS/MS RSDSESHexCer SMSPBSRMST T TGUPHLC/MS VIP Internal standard mixture Limit of quantitation Lysophosphatidylcholine Lysophosphatidylethanolamine Lysophosphatidylinositol Low-resolution Multivariate data analysis Methanol Monoacylglycerol Multiple reaction monitori

FAQs

What is the main analytical strategy described in the study?

The study uses benzoyl chloride derivatization combined with reversed-phase ultrahigh-performance liquid chromatography tandem mass spectrometry (RP-UHPLC/MS/MS) to quantify lipids in human serum.

How many lipid species and lipid subclasses were identified?

A total of 450 lipid species from 19 lipid subclasses were identified using multiple reaction monitoring transitions, retention dependencies, dilution series, and derivatization tags.

Which lipid classes showed the largest sensitivity improvement?

The highest improvement was observed for monoacylglycerols, diacylglycerols, sphingoid bases, and free sterols compared with conventional methods.

Sep 30, 2026

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