Development of SFC-MS Method for Quantification of Eicosanoids Biosynthesized in Primary Human Blood Cells

Abstract

Eicosanoids are lipid mediators generated from arachidonic acid with pro- and anti-inflammatory properties. Despite these lipid mediators being known for decades, quantitative determination in biological samples is still challenging due to low abundance, instability, the existence of regio- and stereoisomers, and a wide polarity range that hampers chromatographic separation. In this study, we developed a supercritical fluid chromatography mass spectrometry (SFC-MS) platform for the quantification of relevant eicosanoids. Application of a chiral amylose-based column and modifier combination of 2-propanol/acetonitrile offered separation and sufficient resolution of 11 eicosanoids (5-, 12-, 15-HETE, PGB1, LTB4, t-LTB4, 20-OH-LTB4, PGE2, PGD2, PGF2α, TxB2) with baseline separation of isobaric analytes within 12 min. The method was validated in terms of range (78–2500 ng/mL), linearity, accuracy, precision, and recovery according to EMA guidelines. Finally, we confirmed the method’s applicability by quantifying eicosanoid levels in human primary blood cells. In conclusion, we present a validated SFC-MS method for the determination of relevant eicosanoids in biological samples with a wide range of polarity while maintaining baseline separation of isobars, which allows coupling to a single quadrupole mass detector.

Identification and Imaging of Prostaglandin Isomers Utilizing MS3 Product Ions and Silver Cationization

“…Several analytical methods have been developed for the analysis of PGs, including enzyme immunoassays, − liquid chromatography–mass spectrometry (LC-MS), − supercritical fluid chromatography (SFC) MS, , and gas chromatography–mass spectrometry (GC-MS). − However, pinpointing PGs with isomeric resolution is not straightforward due to the presence of several isomers. For example, PGD 2 , PGE 2 , PGI 2 , Δ12-PGD 2 , TXA 2 , lipoxin A 4 (LXA 4 ), and LXB 4 are all isomers with the same chemical formula; C 20 H 32 O 5 .…”