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Comparison of microcapillary column length and inner diameter investigated with gradient analysis of lipids by ultrahigh-pressure liquid chromatography-mass spectrometry.

ABSTRACT: Biological samples in lipidomic studies can consist of extremely complex mixtures due to the diverse range of species and isomerism. Herein, highly efficient, in-house packed microcapillary columns introduce the potential to better separate these complex mixtures. We compared the effects of changing column length (15, 30, and 60 cm) and inner diameter (75 and 100 μm) on lipid separation efficiency by reversed-phase gradient analysis using ultrahigh-pressure liquid chromatography coupled to mass spectrometry with operating pressures ranging from 450 to 2200 bar. Seven lipid standards composed of phosphatidylcholine and triacylglycerol species were analyzed at four different gradient rates to calculate conditional peak capacity. The longest column, 60 cm, at the shallowest gradient of 2% gave the highest peak capacity of 359 with a separation window of 2 h. The intermediate column length of 30 cm with 75 μm inner diameter provided a peak capacity of 287 with a separation window of 1 h. There was no significant difference in peak capacity between 75 and 100 μm inner diameter columns. This study showed that using highly efficient microcapillary columns increased peak capacity and resolution of lipids, and thus, this technique seems promising for enhancing lipid coverage and enabling better discovery of lipid biomarkers.

SUBMITTER: Miller KE

PROVIDER: S-EPMC7727313 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Improvements in sample preparation, separation, and mass spectrometry continue to expand the coverage in LC-MS based lipidomics. While longer columns packed with smaller particles in theory give higher separation performance compared to shorter columns, the implementation of this technology above commercial limits has been sparse due to difficulties in packing long columns and successfully operating instruments at ultrahigh pressures. In this work, a liquid chromatograph that operates up to 35 kpsi was investigated for the separation and identification of lipid species from human plasma. Capillary columns between 15-50 cm long were packed with 1.7 µm BEH C18 particles and evaluated for their ability to separate lipid isomers and complex lipid extracts from human plasma. Putative lipid class identifications were assigned using accurate mass and relative retention time data of the eluting peaks. Our findings indicate that longer columns packed and operated at 35 kpsi outperform shorter columns packed and run at lower pressures in terms of peak capacity and numbers of features identified. Packing columns with relatively high concentration slurries (200 mg/mL) while sonicating the column resulted in 6-34% increase in peak capacity for 50 cm columns compared to lower slurry concentrations and no sonication. For a given analysis time, 50 cm long columns operated at 35 kpsi provided a 20-95% increase in chromatographic peak capacity compared with 15 cm columns operated at 15 kpsi. Analysis times up to 4 h were evaluated, generating peak capacities up to 410 ± 5 (n = 3, measured at 4σ) and identifying 480 ± 85 lipids (n = 2). Importantly, the results also show a correlation between the peak capacity and the number of lipids identified from a human plasma extract. This correlation indicates that ionization suppression is a limiting factor in obtaining sufficient signal for identification by mass spectrometry. The result also shows that the higher resolution obtained by shallow gradients overcomes possible signal reduction due to broader, more dilute peaks in long gradients for improving detection of lipids in LC-MS. Lastly, longer columns operated at shallow gradients allowed for the best separation of both regional and geometrical isomers. These results demonstrate a system that enables the advantages of using longer columns packed and run at ultrahigh pressure for improving lipid separations and lipidome coverage.

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Comparison of microcapillary column length and inner diameter investigated with gradient analysis of lipids by ultrahigh-pressure liquid chromatography-mass spectrometry.

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