Free radical-catalysed oxidation of arachidonic acid esterified to lipids leads to the formation of the F 2 -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-PGF 2 α (iPF 2 α -III, 15-F 2 t -IsoP) in human urine. Immunoaffinity column chromatography (IAC) with immobilized antibodies raised against 8-iso-PGF 2 α (i.e. 15(S)-8-iso-PGF 2 α ) has been shown by others to be highly selective and specific for this 8-iso-PGF 2 α isomer when quantified by GC-MS. In the present study we established IAC for urinary 8-iso-PGF 2 α 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-PGF 2 α . 8-iso-PGF 2 α was measured in urine of 10 young healthy humans by both methods. 8-iso-PGF 2 α 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-PGF 2 α at 128+/-55 pg/mg creatinine. This finding suggests that urinary 8-iso-PGF 2 α is heterogenous, with 15(S)-8-iso-PGF 2 α contributing by ~50%. PGF 2 α and other 8-iso-PGF 2 α isomers including 15(R)-8-iso-PGF 2 α are not IAC-immunoreactive and are chromatographically separated from 15(S)-8-iso-PGF 2 α . We assume that ent-15(S)-8-iso-PGF 2 α is also contributing by ~50% to urinary 8-iso-PGF 2 α . This finding may have methodological, mechanistic and clinical implications.