A rapid, simple and sensitive method was developed for the determination of 8-iso-PGF2α in urine using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS); 8-iso-PGF2α-d4 was used as the internal standard (IS). Chromatographic separation was performed using an Acquity BEH C18 column with a mobile phase composition of A: 0.1% acetic acid in methanol:acetonitrile (1:1, v:v) and B: 0.1% acetic acid in water (A:B, 32.5:67.5, v:v). Detection was performed on a triple-quadrupole tandem mass spectrometer using atmospheric pressure chemical ionization (APCI) in negative mode and using multiple reaction monitoring (MRM). The MS/MS ion transitions monitored were m/z 353→193 and 357→197 for 8-iso-PGF2α and IS, respectively. The calibration curve was prepared in PBS buffer because of the presence of endogenous concentrations of analyte in the control matrix; the internal standard successfully correcting for matrix effects. Good linearity was observed over the concentration range of 0.025-20 ng/mL; the method proving to be accurate and reliable was successfully used in support of a pharmacodynamic study in humans.
Keywords: 8-Iso-PGF(2α); Analytical validation; Biomarker; Pharmacodynamics; UPLC–MS/MS; Urine.
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