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Off-line mixed-mode liquid chromatography coupled with reversed phase high performance liquid chromatography-high resolution mass spectrometry to improve coverage in lipidomics analysis.

ABSTRACT: The confident identification and in-depth profiling of molecular lipid species remain to be a challenge in lipidomics analysis. In this work, an off-line two-dimensional mixed-mode and reversed-phase liquid chromatography (RPLC) method combined with high-field quadrupole orbitrap mass spectrometer (Q Exactive HF) was developed to profile lipids from complex biological samples. In the first dimension, 22 different lipid classes were separated on a monolithic silica column with elution order from neutral to polar lipids. A total of 13 fractions were collected and run on a RPLC C30 column in the second dimension for further separation of the lipid molecular species based on their hydrophobicity, with the elution order being determined by both the length and degree of unsaturation in the fatty-acyl chain. The method was applied to analyze lipids extracted from rat plasma and rat liver. Fatty acid methyl ester analysis by gas chromatography-mass spectrometry was used to identify the fatty acyls from total lipid extracts, which provided a more confident identification of the lipid species present in these samples. More than 800 lipids were identified in each sample and their molecular structures were confidentially confirmed using tandem mass spectrometry (MS/MS). The number of lipid molecular species identified in both rat plasma and rat liver by this off-line two-dimensional method is approximately twice of that by one-dimensional RPLC-MS/MS employing a C30 column. This off-line two-dimensional mixed-mode LC-RPLC-MS/MS method is a promising technique for comprehensive lipid profiling in complex biological matrices.

SUBMITTER: Narvaez-Rivas M

PROVIDER: S-EPMC5260842 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Off-line mixed-mode liquid chromatography coupled with reversed phase high performance liquid chromatography-high resolution mass spectrometry to improve coverage in lipidomics analysis.

Narváez-Rivas Mónica M Vu Ngoc N Chen Guan-Yuan GY Zhang Qibin Q

Analytica chimica acta 20161210

The confident identification and in-depth profiling of molecular lipid species remain to be a challenge in lipidomics analysis. In this work, an off-line two-dimensional mixed-mode and reversed-phase liquid chromatography (RPLC) method combined with high-field quadrupole orbitrap mass spectrometer (Q Exactive HF) was developed to profile lipids from complex biological samples. In the first dimension, 22 different lipid classes were separated on a monolithic silica column with elution order from ...[more]

PMID: 28081809

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Project description:Bile acids (BAs) are cholesterol metabolites with important biological functions. They undergo extensive host-gut microbial co-metabolisms during the enterohepatic circulation, creating a vast structural diversity and resulting in great challenges to separate and detect them. Based on the bioanalytical reports in the past decade, this work developed three chromatographic gradient methods to separate a total of 48 BA standards on an ethylene-bridged hybrid (BEH) C18 column and high-strength silica (HSS) T3 column and accordingly unraveled the factors affecting the separation and detection of them by liquid chromatography coupled with mass spectrometry (LC-MS). It was shown that both the acidity and ammonium levels in mobile phases reduced the electrospray ionization (ESI) of BAs as anions of [M-H]-, especially for those unconjugated ones without 12-hydroxylation. It was also found that the retention of taurine conjugates on the BEH C18 column was sensitive to the strength of formic acid and ammonium in mobile phases. By using the volatile buffers with an equivalent ammonium level as mobile phases, we comprehensively demonstrated the effects of the elution pH value on the retention behaviors of BAs on both the BEH C18 column and HSS T3 column. Based on the retention data acquired on a C18 column, we presented the ionization constants (pK a) of various BAs with the widest coverage beyond those of previous reports. When we made attempts to establish the structure-retention relationships (SRRs) of BAs, the lack of discriminative structural descriptors for BA stereoisomers emerged as the bottleneck problem. The methods and results presented in this work are especially useful for the development of reliable, sensitive, high-throughput, and robust LC-MS bioanalytical protocols for the quantitative metabolomic studies. Graphical Abstract Nonlinear curve fitting of capacity factors and elution pH value for the separation of common unconjugated bile acids.

Dataset Information

Off-line mixed-mode liquid chromatography coupled with reversed phase high performance liquid chromatography-high resolution mass spectrometry to improve coverage in lipidomics analysis.

Publications

Off-line mixed-mode liquid chromatography coupled with reversed phase high performance liquid chromatography-high resolution mass spectrometry to improve coverage in lipidomics analysis.

Similar Datasets

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