New Extraction Technique Tested on Polar Metabolites in Human Plasma

Scientists from the University of Oslo in Norway have combined electromembrane extraction (EME) and flow injection-tandem mass spectrometry (FI–MS/MS) to determine polar endogeneous metabolites in human plasma. Their findings were published in the Journal of Chromatography A (1).

Bags of yellow fresh frpzen plasma ready for transfusion. | Image Credit: © pirke - stock.adobe.com

High-throughput methodology has become a popular means of bioanalysis when dealing with complex samples or analytes. The general meaning for “high-throughput” is an analytical technique’s ability to determine hundreds of samples on a daily basis, or a technique’s ability to generate valid results for single or multiple analytes in less than a minute. That said, when determining polar, endogenous metabolites in complex samples (such as biological fluids), this can be difficult to achieve. In these scenarios, one must typically compromise between simple sample preparation, followed by long chromatographic separation, or vice versa, in order to limit matrix effects. Metabolomics is also becoming more popular within bioanalysis, being a powerful tool for gaining information about an individual’s health state, since endogenous metabolites can be biomarkers for predicting diseases, such as cancer or cardiovascular disease.

Electromembrane extraction (EME) is based on electrokinetic migration of target analytes from an aqueous donor solution, across a thin supported liquid membrane (SLM) composed of organic solvent, and into an aqueous acceptor solution. For high-throughput applications, electrically driven transport allows for faster extractions compared to liquid-phase microextraction, where transport is solely based on passive diffusion. To quote the scientists, “EME provides a simple, one-step extraction of analytes into a fully LC–MS compatible clean and aqueous solution, thus avoiding any time-consuming steps such as centrifugation, evaporation, and reconstitution” (1).

To address the issues with high-throughput techniques, scientists coupled a one-step 96-well (parallel extraction) electromembrane extraction (EME) method with flow injection-MS/MS (FI–MS/MS) of 0.7 min per sample. This allowed for very high-throughput analysis of 12 polar and endogenous metabolites from unprecipitated plasma that had limited dilution. First, EME parameters were first optimized to ensure the highest extraction recoveries, all while maintaining low and stable currents. Extracts were analyzed using three methods: a rapid gradient separation, a rapid isocratic separation, and a FI-method (without LC column), all followed by MS-detection, to compare their performance against the throughput. The FI-method had a total run time of 43 s per sample (including injection), matching EME throughput. This theoretically allowed over 2000 samples to be analyzed in 24 h.

The throughput of the EME method matched subsequent analyses, with recoveries ranging from 6–93% and both repeatability and linearity being 2–15% and R² ≥ 0.9949, respectively, for all but two compounds. Matrix effects reached approximately 50% after EME and varied less than 11% between six plasma donors; this represented a major improvement relative to simple protein precipitation, where signals were entirely suppressed.

According to the scientists, this is supposedly the first paper to report on EME coupled to FI–MS. Compared to other studies that deal with using EME on polar bases, the method developed in this study showed EME capable of analyzing plasma samples of limited dilution while having good stability of the EME-system. Though this study was only limited to polar bases, the performances exhibited in this study show that EME coupled to FI–MS could also be feasible for other substance classes, such as basic and acidic hydrophobic drugs. EME-FI–MS/MS, if handled properly, could potentially be a future platform for very-high throughput applications in bioanalysis.

References

(1) Hay, A. O.; Hansen, F. A. Exploring Electromembrane Extraction Coupled to Fast LC-MS/MS as a High-Throughput Platform for Determination of 12 Polar Endogenous Metabolites in Human Plasma. J. Chromatogr. A 2024, 1737, 465451. DOI: 10.1016/j.chroma.2024.465451