Simultaneous LC-MS/MS analysis of eicosanoids and related metabolites in human serum, sputum and BALF

Abstract

The differences among individual eicosanoids in eliciting different physiological and pathological responses are largely unknown because of the lack of valid and simple analytical methods for the quantification of individual eicosanoids and their metabolites in serum, sputum and bronchial alveolar lavage fluid (BALF). Therefore, a simple and sensitive LC-MS/MS method for the simultaneous quantification of 34 eicosanoids in human serum, sputum and BALF was developed and validated. This method is valid and sensitive with a limit of quantification ranging from 0.2 to 3 ng/mL for the various analytes, and has a large dynamic range (500 ng/mL) and a short run time (25 min). The intra- and inter-day accuracy and precision values met the acceptance criteria according to US Food and Drug Administration guidelines. Using this method, detailed eicosanoid profiles were quantified in serum, sputum and BALF from a pilot human study. In summary, a reliable and simple LC-MS/MS method to quantify major eicosanoids and their metabolites was developed and applied to quantify eicosanoids in human various fluids, demonstrating its suitability to assess eicosanoid biomarkers in human clinical trials.

Keywords: COPD; LC-MS/MS; biomarker; eicosanoids.

FIGURE 1

Representative chromatograms of all eicosanoid standards at 10 ng/mL under final chromatography and detection conditions. Peaks are labeled with analytes IDs and retention times as given in Table 1

FIGURE 2

Isobaric compounds that also share fragmentation patterns as well as retention times were distinguished via specific selected reaction monitoring (SRMs). LC–MS/MS chromatograms of various hydroxyeicosatetraenoic acids (HETEs) after the injection of a 5-HETE standard at 500 ng/mL. Isobaric HETEs including 5-HETE (13), 8-HETE (12), 11-HETE (11) and 12-HETE (10) were not resolved chromatographically, and they produced both common and selective fragments in MS/MS. The 319/301 transition was the most sensitive but was shared by all HETEs (a, c, e, g). In contrast, the less sensitive but more selective transitions of HETEs were used, including 319/114.7 for 5-HETE (b), 319/154.8 for 8-HETE (d), 319/167.2 for 11-HETE (f) and 319/179 for 12-HETE (h)

FIGURE 3

Isobaric eicosanoids that undergo in-source fragmentation were separated chromatographically. (a) The isobaric compounds PGE2 (7), PGD2 (6) and 13,14-dihydro-15-k-PGE2 (18) share the same SRM transition of 351 → 333 and had to be separated chromatographically [retention time (RT) = 11.58, 11.77, and, 12.5 min, respectively]. (b) PGJ2 (333.1 → 315.2) and (c) 15-dexoxy-delta 12,14- PGJ2 (315.2 → 271.2) have different precursors as well as fragment masses. However, PGJ2 produces an in-source fragment (315.2) with the same mass as the parent 15-dexoxy-delta 12,14-PGJ2. Therefore, the two compounds had to be separated chromatographically