Two sensitive and selective quantitative methods for 6‐oxo‐PGF1α, the stable hydrolysis product of prostacyclin are described and compared. Prostaglandins were extracted from biological fluids with organic solvents. Samples for gas chromatographic mass spectrometric analysis required additional thin‐layer chromatographic separation prior to conversion to the O‐methyloxime, methyl ester, tri‐trimethylsilyl ether. Ion fragments at m/z 418 and 508 (protium) and m/z 422 and 512 (deuterium) were monitored to prepare 6‐oxo‐PGF1α standard curves. The gas chromatographic mass spectrometric detection limit was 500 pg injected on column with a method coefficient of variation of 11.6% at this level. At 590 pg ml−1 the coefficient of variation for reproducibility of measurement was 1.77%. Antisera raised against a 6‐oxo‐PGF1α‐bovine serum albumin conjugate in sheep had a higher titre and greater selectivity than those raised in rabbits. Cross‐reaction of sheep antisera with all prostaglandins and fatty acids tested was less than 0.5%. The radioimmunoassay limit of detection was 60 pg ml−1 (6 pg per tube) with a coefficient of variation of 10.4% (intra‐assay) and 10.75% (inter‐assay). The double blind comparison of gas chromatographic mass spectrometric and radioimmunoassay quantitation of the same samples gave a correlation coefficient of 0.97. Both methods offer sensitivity, selectivity and reproducibility. Gas chromatography mass spectrometry is necessary to validate the radioimmunoassay method which offers advantages of time, sample capacity and volume, expenditure and sensitivity.
ASJC Scopus subject areas
- Molecular Medicine