Quantitative analysis of 6-keto-prostaglandin F1 alpha using immunoaffinity purification and gas chromatography-mass...

This paper describes an immunoaffinity purification technique for 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) prior to quantitative analysis by high-resolution gas chromatography-negative-ion chemical ionization mass spectrometry (HRGC-NICIMS). Polyclonal antibodies to 6KPGF1 alpha were partially purified using Staphylococcus aureus Protein A immobilized on Sepharose CL-4B. This partially purified fraction was covalently bound to silica gel using N-hydroxysuccinimidyl-functionalized silica. Columns constructed using this gel quantitatively bound 6KPGF1 alpha which could be eluted quantitatively with acetonitrile-water (19:1). Binding capacity was reconstituted by washing with 0.01 M phosphate buffer (pH 7.4). Human urinary and canine plasma 6KPGF1 alpha was sufficiently purified using these columns that HRGC-NICIMS analysis of the methoxime-pentafluorobenzyl-tris-trimethylsilyl derivative was interference-free.