Method Development and Validation for Ultra-High Pressure Liquid Chromatography/Tandem Mass Spectrometry Determination of Multiple Prostanoids in Biological Samples

Following oxygenation of arachidonic acid by cyclooxygenase to form prostaglandin H-2 (PGH(2)), 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: PGE(2), PGD(2), 8-iso-PGF(2 alpha), PGF(2 alpha) 6-keto-PGF(1 alpha), and thromboxane B-2 (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 PGE(2), 0.027 ng/mL for PGD(2), 0.152 ng/mL for 8-iso-PGF(2 alpha), 0.179 ng/mL for PGF(2 alpha), and 6-keto-PGF(1 alpha), and 0.013 ng/mL for TXB2.