Divergence in urinary 8-iso-PGF2α (iPF2α-III, 15-F2t-IsoP) levels from gas chromatography–tandem mass spectrometry quantification after thin-layer chromatography and immunoaffinity column chromatography reveals heterogeneity of 8-iso-PGF2α: Possible metho

Free radical-catalysed oxidation of arachidonic acid esterified to lipids leads to the formation of the F2-isoprostane family which may theoretically comprise up to 64 isomers. We have previously shown that the combination of TLC and GC–tandem MS (referred to as method A) allows for the accurate and highly specific quantification of 8-iso-PGF2α (iPF2α-III, 15-F2t-IsoP) in human urine. Immunoaffinity column chromatography (IAC) with immobilized antibodies raised against 8-iso-PGF2α (i.e. 15(S)-8-iso-PGF2α) has been shown by others to be highly selective and specific for this 8-iso-PGF2α isomer when quantified by GC–MS. In the present study we established IAC for urinary 8-iso-PGF2α for subsequent quantification by GC–tandem MS (referred to as method B). This method was fully validated and found to be highly accurate and precise for urinary 15(S)-8-iso-PGF2α. 8-iso-PGF2α was measured in urine of 10 young healthy humans by both methods. 8-iso-PGF2α was determined to be 291±102 pg/mg creatinine by method A and 141±41 pg/mg creatinine by method B. Analysis of the combined through and wash phases of the IAC step, i.e. of the unretained compounds, by method A showed the presence of non-immunoreactive 8-iso-PGF2α at 128±55 pg/mg creatinine. This finding suggests that urinary 8-iso-PGF2α is heterogenous, with 15(S)-8-iso-PGF2α contributing by ∼50%. PGF2α and other 8-iso-PGF2α isomers including 15(R)-8-iso-PGF2α are not IAC-immunoreactive and are chromatographically separated from 15(S)-8-iso-PGF2α. We assume that ent-15(S)-8-iso-PGF2α is also contributing by ∼50% to urinary 8-iso-PGF2α. This finding may have methodological, mechanistic and clinical implications.