Method development and validation for ultra-high pressure liquid chromatography/tandem mass spectrometry determination of multiple prostanoids in biological samples

Abstract

Following oxygenation of arachidonic acid by cyclooxygenase to form prostaglandin H2 (PGH2), a variety of prostanoids can be generated with diverse physiologic effects on pain, inflammation, allergy, cardiovascular system, cancer, etc. To facilitate the quantitative analysis of prostanoids in human serum of cell culture, an ultra-high pressure LC (UHPLC)/MS/MS method was developed and validated for the measurement of six eicosanoids belonging to the cyclooxygenase pathway: PGE2, PGD2, 8-iso-PGF2alpha, PGF2alpha, 6-keto-PGF1alpha, and thromboxane B2 (TXB2). Selectivity, matrix effects, calibration model, precision, and accuracy (intraday and interday), lower limit of quantitation (LLOQ), recovery, stability, and sample dilution were evaluated. Fast UHPLC separation was carried out in only 0.5 min with isocratic elution, and each prostanoid was measured using negative electrospray ionization MS with collision-induced dissociation and selected reaction monitoring. UHPLC/MS/MS provided high throughput with peak widths of approximately 3 s and an LLOQ of 0.020 ng/mL for PGE2, 0.027 ng/mL for PGD2, 0.152 ng/mL for 8-iso-PGF2alpha, 0.179 ng/mL for PGF2alpha and 6-keto-PGF1alpha, and 0.013 ng/mL for TXB2.

Figure 1

Chemical structures of PGE₂, PGD₂, PGF_2α, 8-iso-PGF_2α, 6-keto-PGF_1α, and TXB₂.

Figure 2

Negative ESI UHPLC/MS/MS SRM chromatograms of PGE₂, PGD₂, 8-iso-PGF_2α, PGF_2α, 6-keto-PGF_1α, and TXB₂ (1 ng/mL each) and internal standards d₄-PGE₂, and d₄-PGD₂ (20 ng/mL each) extracted from DMEM containing 10% pooled blank human serum.

Figure 3

Negative ESI UHPLC/MS/MS SRM chromatograms of (A) PGE₂ and PGD₂ (0.05 ng/mL), (B) 8-iso-PGF_2α and PGF_2α (0.5 ng/mL), (C) 6-keto-PGF_1α (0.5 ng/mL), and (D) TXB₂ (0.05 ng/mL) extracted from DMEM containing 10% pooled blank human serum.

Figure 4

Negative ESI UHPLC/MS/MS SRM chromatograms of PGE₂, PGD₂, 8-iso-PGF_2α, PGF_2α, 6-keto-PGF_1α, and TXB₂, d₄-PGE₂, and d₄-PGD₂ extracted from BMDM cell culture. Note that 8-iso-PGF_2α, PGF_2α, or 6-keto-PGF_1α was not detected in these cells (see chromatograms for the SRM transitions of m/z 353 to m/z 193 and m/z 369 to m/z 163, respectively).