Development and validation of an extraction method for the determination of pro-inflammatory eicosanoids in human plasma using liquid chromatography–tandem mass spectrometry

• Simple “supernatant-and-shoot” methodology.
• Low sample volume (50 μL plasma).
• Efficient experimental design for validation purposes.

A simple method coupled to liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and validated for the extraction of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) from human plasma. The extraction protocol consisted of adding formic acid (10 μL) and acetonitrile (140 μL) to human plasma (50 μL) and further injection of the supernatant (25 μL) into the LC–MS/MS. The method was selective for PGE2 and LTB4 and the regression models, based on deuterated internal standards (30 ng mL−1 PGE2-d4 and 40 ng mL−1 LTB4-d4), were linear over the concentration range 1.0–50.0 ng mL−1 with limits of detection (3 × σblank) and quantification (6 × σblank) of 0.5 ng mL−1 and 1 ng mL−1 for both eicosanoids. The recovery ranges were 95.1–104.7% for PGE2 and 86.4–103.2% for LTB4. The developed method was successfully implemented on plasma samples from patients before and after exposure to certain anti-inflammatory treatment.