Method development of high-throughput eicosanoid profiling for micro-sampling plasma

Importance of the topic

Fatty acid metabolites such as eicosanoids are central mediators of inflammation and other pathophysiological processes. High throughput profiling of these compounds in plasma supports biomarker discovery and mechanistic studies while minimizing sample volume requirements in clinical and animal research.

Objectives and overview of the study

The study aimed to develop a rapid and sensitive LC MS MS method for comprehensive profiling of eicosanoids and related fatty acid metabolites from minimal plasma volumes. A micro sampling device was employed to collect exactly 5.6 micro liter of plasma for high throughput analysis using 200 multiple reaction monitoring transitions covering 114 target compounds and 18 internal standards.

Methods and instrumentation

Sample collection used a micro sampling device to isolate 5.6 micro liter plasma from whole blood. Protein precipitation was performed by adding methanol with formic acid and deuterated internal standards followed by centrifugation. Solid phase extraction on STRATA X cartridges concentrated analytes which were dried and reconstituted in methanol with formic acid. Chromatographic separation was achieved on a Shim Pack GIST HP C18 column 2.1x100 mm 1.9 micro m particle size with a 10 minute gradient at 0.4 mL per min flow. Detection was carried out on a Shimadzu LCMS 8060 triple quadrupole mass spectrometer with ESI polarity switching and up to 200 MRM transitions at 5 ms switch time.

Used instrumentation

Shimadzu Nexera LC 40 UHPLC system
Shimadzu LCMS 8060 Ultra Fast Triple Quadrupole Mass Spectrometer
STRATA X 10 mg SPE cartridges
Shim Pack GIST HP C18 column 2.1x100 mm 1.9 micro m
Micro sampling device MSW2

Main results and discussion

The method enabled detection of over 30 eicosanoids and more than 100 fatty acid metabolites from 5.6 micro liter plasma. Key arachidonic acid derivatives including various HETEs and DHETs were quantified with reproducibility CV below 20 percent and micro sampling accuracy around 3 percent. The approach achieved quantitation of 109 targets in serum and plasma samples with high peak resolution and sensitivity. Reproducibility of the micro sampling and analytical workflow supported reliable profiling in triplicate analyses.

Benefits and practical applications of the method

The minimal sample volume requirement facilitates studies in small animals or limited clinical samples and allows serial sampling. The 10 minute analysis time supports high throughput screening in pharmacology toxicology and clinical biomarker research. The use of micro sampling reduces animal usage and enables repeated sampling over time.

Future trends and possibilities of use

Further automation of sample preparation and integration with on line SPE could increase throughput. Expansion to cover additional lipid mediators and polar metabolites may broaden clinical and research applications. Coupling with advanced data processing and machine learning can accelerate biomarker discovery. Single cell and microsampling platforms may enable spatial metabolomics in tissues.

Conclusion

A novel high throughput LC MS MS method for eicosanoid profiling from micro sampled plasma was established. The workflow combines precise micro sampling with rapid chromatographic separation and sensitive MRM detection to quantify over 100 fatty acid metabolites from 6 micro liter samples. This approach offers robust reproducibility and supports diverse applications in biomedical research.

Reference

• M Yamada T Nakamura A Murata T Hattori MP 546 67th ASMS 2019
• M Yamada Y Kita T Kohira et al J Chromatography B 995 74 84 2015