Micro-flow LC- drift tube IMS-MS with high-resolution demultiplexing for enhanced lipidome coverage of volume-limited biological samples.
Abstract
The analysis of volume-limited biological specimens remains a major challenge in lipidomics, requiring analytical platforms with improved sensitivity and structural characterization capabilities. Here, a microflow liquid chromatography ion mobility mass spectrometry (microLC-IMS-MS) platform incorporating high-resolution demultiplexing (HRdm) was developed and benchmarked against conventional ultraperformance liquid chromatography (UPLC) for untargeted lipidomics. Following optimization of the LC hardware and ion source conditions, microLC provided median 2- to 14-fold increases in peak area, up to 10-fold increases in peak height, and approximately 3-fold higher signal-to-noise ratios relative to UPLC. Calibration with 34 isotopically labeled standards spanning eight lipid classes gave detection limits of 0.22-14.3 μg/L (median 1.6 μg/L; 0.80 pg on column) and quantitative ranges covering a median of 2.2 orders of magnitude. Analysis of NIST SRM 1950 plasma enabled the annotation of 130 lipid species compared with 74 by UPLC-MS, representing a 76% increase in lipid coverage. When combined with HRdm, the platform resolved five lipid isomer pairs that remained unresolved under conventional UPLC conditions. The method was further applied to volume-limited biological specimens, enabling the identification of 62 lipids from as few as 1x103 human Müller glia cells and lipid profiling of human vitreous humor samples from a pilot cohort of glaucoma patients and matched controls. Variations in lipid features were observed in an exploratory pilot clinical study, supporting the applicability of the platform to clinically relevant ocular biofluids; given the small cohort, these observations should be regarded as hypothesis-generating. These results establish microLC-HRdm-MS as a sensitive and reproducible platform for lipidomics of volume-limited and structurally complex specimens.
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