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LC-MS/MS profiling of colon oxysterols and cholesterol precursors in mouse model of ulcerative colitis
Jiachun Lin1, Xue Yang1, Anhui Wang2
1China Pharmaceutical University Nanjing Drum Tower Hospital, Nanjing, 210009, China; Key Laboratory of Drug Quality Control & Pharmacovigilance (China Pharmaceutical University), Ministry of Educational, 24 Tongjia Lane, Nanjing 210009, China.
Abstract:
Oxysterols and cholesterol precursors are being increasingly investigated in humans and laboratory animals as markers for various diseases in addition to their important functions. However, the quantitative analysis of these bioactive molecules is obstructed by high structural similarity, poor ionization efficiency and low abundance. The current assay methods are still cumbersome to be of practical use, and their applicability in different bio-samples needs to be evaluated and optimized as necessary. In the present work, chromatographic separation conditions were carefully studied to achieve baseline separation of difficult-to-isolate compound pairs. On the other hand, an efficient sample purification method was established for colon tissue samples with good recoveries of sterols, demonstrating negligible autoxidation of cholesterol into oxysterols. The developed UPLC-APCI-MS/MS method was thoroughly validated and applied to measure oxysterols and cholesterol precursors in colon tissue of dextran sulfate sodium (DSS)-induced mouse colitis models, and it is expected to be successfully applied to the quantitative determination of such components in other tissue samples.
Insights
This study developed a new method to accurately measure oxysterols and cholesterol precursors, crucial disease markers. The technique improves upon existing assays for better disease research and diagnosis.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Disease Biomarkers
Background:
- Oxysterols and cholesterol precursors are vital molecules investigated as disease markers.
- Quantitative analysis is challenging due to structural similarity, low abundance, and poor ionization.
- Current assay methods are cumbersome and require optimization for diverse bio-samples.
Purpose of the Study:
- To develop and validate a robust analytical method for quantifying oxysterols and cholesterol precursors.
- To optimize chromatographic separation and sample purification for complex biological matrices.
- To apply the method to study these sterols in a mouse model of colitis.
Main Methods:
- Careful optimization of chromatographic separation conditions for difficult-to-isolate sterol pairs.
- Establishment of an efficient sample purification protocol for colon tissue with high sterol recovery.
- Development and validation of an ultra-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (UPLC-APCI-MS/MS) method.
Main Results:
- Achieved baseline separation of structurally similar sterol compounds.
- Demonstrated high sterol recoveries and negligible autoxidation during colon tissue sample purification.
- Successfully applied the validated UPLC-APCI-MS/MS method to quantify sterols in a DSS-induced mouse colitis model.
Conclusions:
- The developed UPLC-APCI-MS/MS method provides accurate and efficient quantification of oxysterols and cholesterol precursors.
- The optimized sample preparation and chromatographic conditions overcome previous analytical limitations.
- This method is expected to be broadly applicable for sterol analysis in various tissue types and disease models.
