Isolation of Urinary 3-Methyladenine Using Immunoaffinity Columns Prior to Determination by Low-Resolution Gas Chromatography-Mass Spectrometry

An ammonium sulfate precipated immunoglobin G (IgG) fraction from rabbit antiserum, prepared by use of novel haptenic derivatives, was used to make immunoaffinity columns for purification of 3-methyladenine (3-MeAde) from human urine. IgG was covalently bound to protein A-Sepharose, and the resulting affinity gel columns were sufficiently stable for multiple reuse. 3-MeAde (up to 200 ng) was adsorbed at pH 7.4 and, after extensive washing, eluted with 1 M acetic acid. Recovery of 3-MeAde was typically >90%. For gas chromatography-mass spectrometry analysis, deuterium-labeled (d₃) 3-MeAde (50 ng per sample) was used as an internal standard. 3-MeAde was determined as the mono-tert-butyldimethylsilyl derivative and quantitated by measurement of ions at m/z 206 (3-MeAde-d₀) and m/z 209 (3-MeAde-d₃). Repeated analyses of a human urine sample show excellent reproducibility of the method.