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

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-PGF2small alpha, Greek (iPF2small alpha, Greek-III, 15-F2t-IsoP) in human urine. Immunoaffinity column chromatography (IAC) with immobilized antibodies raised against 8-iso-PGF2small alpha, Greek (i.e. 15(S)-8-iso-PGF2small alpha, Greek) has been shown by others to be highly selective and specific for this 8-iso-PGF2small alpha, Greek isomer when quantified by GC-MS. In the present study we established IAC for urinary 8-iso-PGF2small alpha, Greek 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-PGF2small alpha, Greek. 8-iso-PGF2small alpha, Greek was measured in urine of 10 young healthy humans by both methods. 8-iso-PGF2small alpha, Greek 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-PGF2small alpha, Greek at 128±55 pg/mg creatinine. This finding suggests that urinary 8-iso-PGF2small alpha, Greek is heterogenous, with 15(S)-8-iso-PGF2small alpha, Greek contributing by ~50%. PGF2small alpha, Greek and other 8-iso-PGF2small alpha, Greek isomers including 15(R)-8-iso-PGF2small alpha, Greek are not IAC-immunoreactive and are chromatographically separated from 15(S)-8-iso-PGF2small alpha, Greek. We assume that ent-15(S)-8-iso-PGF2small alpha, Greek is also contributing by ~50% to urinary 8-iso-PGF2small alpha, Greek. This finding may have methodological, mechanistic and clinical implications.