Anna Catharina Suhr, Barbara Maier, Mathias Brügel, Alisa Kleinhempel, Daniel Teupser, Michael Vogeser
1Institute of Laboratory Medicine, Hospital of the University of Munich, Marchioninistr. 15, 81377 Munich, Germany
We used ferromagnetic particles as a novel technique to deproteinize plasma samples prior to UHPLC-MS/MS analysis for the quantification of important lipid mediators. A combination of "ferromagnetic particle enhanced deproteination" and subsequent "on-line solid phase extraction" realises quick and convenient sample preparation as well as it provides high sensitivity and robustness. We were able to show that this approach allows accurate and precise quantification of seven exemplary eicosanoids (TXB2, PGE2, PGD2, 5-HETE, 11-HETE, 12-HETE, and arachidonic acid). The use of this semi-automated sample preparation facilitates the screening of large study cohorts. In general ferromagnetic particles might smooth the trail to automation in UHPLC-MS/MS.
Introduction: Eicosanoids play an important role in the human body as lipid mediators, e.g. in inflammation and coagulation. Our aim was the development of an UHPLC-MS/MS method to quantify seven eicosanoids of particular interest (TXB2, PGE2, PGD2, 5-HETE, 11-HETE, 12-HETE, and arachidonic acid). Additionally, sample preparation ought to be simple and quick enough to test large study cohorts.
Method: We employed stable-isotope dilution analysis with a corresponding deuterated internal standard compound for all analytes (except for 11-HETE for which no isotopic labelled analogue was commercially available so 12-HETE-d8 was used). Since no analyte free human plasma was available, eight multi-calibrators and three levels of quality controls (QC) were prepared in ethanol. As additional QC materials based on authentic matrix, two human plasma pools with low and high concentrations of eicosanoids were used. We employed the innovative approach of “ferromagnetic particle enhanced deproteination”: In brief, our protocol started with mixing 100 µL of human plasma with 25 µL of the internal standard solution and in a second step 40 µL magnetic particle suspension (Magna Medics, Geleen, The Netherlands) were added. To denaturate the proteins 300 µl acetonitrile were used. The ferromagnetic particles were used to capture the denaturated proteins and - when being placed on a magnetic separator - pull them to the wall of the sample tube leaving a clear supernatant. 200 µL of the supernatants were evaporated to dryness under nitrogen and resolved in 100 µL of methanol/water (50/50, v/v) to concentrate the analytes. A further clean-up was performed using “on-line solid phase extraction” with a trapping column (Oasis HLB, Waters, Milford, USA)) included in a Waters Acquity UPLC system. The chromatographic separation was achieved on an Acquity BEH Shield RP18 column with a total run time of 7.5 min. The mass spectrometer (Waters Xevo TQ-S) was equipped with an electrospray ionization source operating in negative mode. Multiple reaction monitoring was employed to quantify the analytes.
Results: The method was evaluated thoroughly with a protocol based on the EMA guideline for bioanalytical method validation. The results were very satisfying: Linearity was proven for a calibration range of 0.1 - 80 µg/L for TXB2, PGE2, PGD2, 0.05 - 40 µg/L for 11-HETE and 5-HETE, 0.5 - 400 µg/L for 12-HETE, and 25 - 10000 µg/L for arachidonic acid, respectively. Imprecision ranged between 3-14% for ethanolic QC and 6-15% for authentic matrix controls (depending on the analyte). Accuracy was 105-110% (only determined for ethanolic QCs). Several experiments, including e.g. dilution, change of column lots or work-up by several operators demonstrated the robustness of the method.
Conclusion: We were able to show the feasibility of "ferromagnetic particle enhanced deproteination" as a rapid and robust way of sample preparation for quantitative LC-MS/MS analyses of plasma samples. This approach was successfully validated as part of an UHPLC-MS/MS method for the quantification of seven exemplary eicosanoids. The “ferromagnetic particle enhanced deproteination” might be an attractive and generic tool to advance automation in LC-MS/MS since there is no need for centrifugation or application of vacuum or pressure.