Metabolic profiling by reversed-phase/ion-exchange mass spectrometry

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Chemistry

Background:

• Metabolic profiling commonly uses mass spectrometry (MS) with reversed-phase (RP) and hydrophilic interaction chromatography (HILIC).
• Independent or coupled chromatography setups often lead to data overlap or require multiple liquid chromatography (LC) systems.
• Existing methods can be complex, time-consuming, and may not efficiently separate all clinically relevant metabolites.

Purpose of the Study:

• To develop a streamlined, single LC system for comprehensive metabolic profiling.
• To enable simultaneous separation of both polar and non-polar metabolites without derivatization.
• To improve the efficiency and accuracy of metabolite identification and quantification in biological samples.

Main Methods:

• Development of a single 20-minute chromatographic method utilizing an in-line reversed-phase (RP) and ion-exchange (IEX) column arrangement.
• Creation of an in-house library with 397 authentic metabolite standards.
• Analysis of pooled plasma and urine samples using the developed LC-MS method.

Main Results:

• Successful separation of clinically significant polar and non-polar compounds in a single run.
• Detection of 5445 and 4111 ion features in plasma and urine, respectively, leading to 88 and 82 confirmed metabolite identifications.
• High precision and clinically relevant concentration detection of metabolites, with excellent separation of critical isomers (e.g., methylmalonic acid, succinic acid, alloisoleucine, isoleucine/leucine).

Conclusions:

• The in-line RP-IEX configuration provides an efficient and robust method for metabolic profiling.
• This single LC system approach overcomes limitations of traditional methods, simplifying analysis and improving metabolite coverage.
• The method demonstrates high analytical quality and is suitable for detecting metabolites at clinically relevant levels.