MetaboQuan-R for Acylcarnitines in Human Serum: A Rapid, Targeted UPLC-MS/MS Method for Metabolomic Research Studies

Significance of the topic

The role of acylcarnitines as key intermediates in fatty acid transport and metabolism underpins their importance as biomarkers in clinical and research settings. Rapid, targeted quantification of these species aids in understanding metabolic dysregulation and supports studies in metabolomics, lipidomics, and related fields.

Objectives and Study Overview

This application note presents the development of a high-throughput UPLC-MS/MS method for simultaneous analysis of 20 acylcarnitine species in human serum. The aim was to eliminate derivatization steps common to flow injection analysis and to achieve clear separation of isobaric and isomeric compounds within a three-minute run time.

Methodology and Instrumentation

Sample preparation involved protein precipitation of human serum with methanol, dilution of the supernatant, and micro-injection (0.5 µL) onto the UPLC-MS/MS system. Chromatographic separation used an ACQUITY UPLC I-Class system with a CORTECS T3 2.7 µm, 2.1×30 mm column at 60 °C. A gradient of aqueous 0.01% formic acid with 0.2 mM ammonium formate to 50% isopropanol in acetonitrile (0.01% formic acid, 0.2 mM ammonium formate) carried analytes at 1.3 mL/min over 0.8 minutes, followed by a wash and re-equilibration.

• Mass spectrometry employed a Xevo TQ-S micro in positive ESI mode with MRM detection.
• Source parameters included 150 °C source temperature, 650 °C desolvation, 2.0 kV capillary voltage, 50 L/hr cone gas flow.
• Data acquisition and processing utilized MassLynx, TargetLynx and the Quanpedia database as part of MetaboQuan-R.

Results and Discussion

The method achieved baseline separation and detection of 20 acylcarnitine analytes with retention times ranging from 0.07 to 0.69 minutes. Saturated and unsaturated species, including C5:1, C8:1, C10:1, and C12:1, exhibited multiple peaks consistent with isomeric forms. Chromatograms demonstrated clear resolution of both classes within the rapid gradient. Quantitative performance remained robust across physiological concentration ranges, confirming suitability for serum analysis.

Benefits and Practical Application

The presented workflow offers:

• Sub-three-minute run times enabling high throughput processing of large sample cohorts.
• Elimination of derivatization steps, simplifying sample handling.
• Flexibility of a generic LC-MS configuration for seamless transition to other compound classes.

This approach streamlines metabolomic studies and can integrate into targeted multi-omics pipelines for research and quality control.

Future Trends and Potential Applications

Advances may include further miniaturization of flow paths, integration with automated sample preparation platforms, and expansion to broader acylcarnitine panels or alternative biological matrices. Data analysis enhancements through machine learning could enable deeper biomarker discovery and personalized metabolic profiling.

Conclusion

A rapid, robust UPLC-MS/MS method has been established for quantitative profiling of acylcarnitines in human serum. By combining high throughput, simplified workflow, and precise isomer separation, this protocol supports versatile applications in metabolomic research without the need for derivatization.