Ultrahigh-pressure liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) had been previously proposed for untargeted lipidomics analysis, but this updated approach was reported by the authors to reduce run time to 4 min.
A study recently published in the journal Analytical and Bioanalytical Chemistry claims to have cut in half the time spent by conventional ultrahigh-pressure liquid chromatography (UHPLC) methods for untargeted lipidomics analysis by using high-throughput four-dimensional trapped ion-mobility mass spectrometry (HT-4D-TIMS) with a fast (4 min) reversed-phase (RP) gradient (1). With this approach, the authors aimed to address the shortcomings of UHPLC coupled to high-resolution mass spectrometry (HRMS), namely low throughput, while also allowing for large-scale screening.
Colorful 3D Illustration of the Biochemical Process of Lipid Synthesis | Image Credit: © artefacti - stock.adobe.com
This research, undertaken by a group of 10 authors at the University of Salerno in Fisciano, Italy, builds on a growing research field of interest recently discussed in detail by Anne Bendt of the Singapore Lipidomics Incubator (SLING) in a taped interview with LCGC International at the 2024 Analytica conference in Munich, Germany (2). Lipidomics was defined in a previous issue of Analytical and Bioanalytical Chemistry as the “large-scale determination of individual lipid species,” commonly identified using mass spectrometric methods, and which since its introduction in 2003 has been the subject of prior investigation as to whether a high-throughput UHPLC-HRMS determination can be feasible (3–5).
Samples used in the experimental stage of this study included human AC-16 cells, a cardiomyocyte line derived from ventricular heart tissue, as well as mouse brain, liver, sclera, and feces (1). The RP gradient, intended to bring analysis time under 5 min, allowed for implementation of TIMS that resulted in differentiation of coeluting isomeric and isobaric lipids. The authors added that the other effects of this process included correct precursor ion isolation, and an avoidance of both co-fragmentation and chimeric tandem mass spectrometry (MS/MS) spectra.
Ultimately, the HT-4D-TIMS method resulted in the annotation of more than 1900 different lipid species, according to the research team—just over 1300 at the molecular level and 600 at the sum composition level, surveying 58 different lipid subclasses. Quantitation capability covered more than three orders of magnitude, with a coefficient of variation (CV) of 0.39% of the TIMS values relative to longer LC gradients (1). These findings led the authors to conclude that the high coverage and accuracy, along with speed, afforded by their experimental platform could have valuable use in the practical world, for monitoring lipid changes in disease or drug treatment models.
(1) Merciai, F.; Basilicata, M.; La Gioia, D.; et al. Sub-5-min RP-UHPLC-TIMS for High-Throughput Untargeted Lipidomics and its Application to Multiple Matrices. Anal. Bioanal. Chem. 2024, 416, 959–970. DOI: 10.1007/s00216-023-05084-w
(2) Hroncich, C.; Lavery, P. Lipidomics Advances and Females in Mass Spec: An Analytica Interview with Anne Bendt. LCGC International. MJH Life Sciences, 2024. https://www.chromatographyonline.com/view/lipidomics-advances-females-mass-spectometry-analytica-interview-anne-bendt (accessed 2024-04-24).
(3) Züllig, T.; Trötzmüller, M.; Köfeler, H. C. Lipidomics from Sample Preparation to Data Analysis: A Primer. Anal. Bioanal. Chem. 2020, 412, 2191–2209. DOI: 10.1007/s00216-019-02241-y
(4) Cajka, T.; Fiehn, O. Comprehensive Analysis of Lipids in Biological Systems by Liquid Chromatography–Mass Spectrometry. TrAC, Trends Anal. Chem. 2014, 61, 192–206. DOI: 10.1016/j.trac.2014.04.017
(5) Xuan, Q.; Hu, C.; Yu, D.; et al. Development of a High Coverage Pseudotargeted Lipidomics Method Based on Ultra-High Performance Liquid Chromatography–Mass Spectrometry. Anal. Chem. 2018, 90 (12), 7608–7616. DOI: 10.1021/acs.analchem.8b01331
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