Isolation of urinary 3-methyladenine using immunoaffinity columns prior to determination by low-resolution gas chromatography-mass spectrometry.
An ammonium sulfate precipitated 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 greater than 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.
Friesen
M D
MD
International Agency for Research on Cancer, Lyon, France.
Garren
L
L
Prevost
V
V
Shuker
D E
DE
D000225:Adenine / Q000031:analogs & derivatives* / Q000652:urine; D000818:Animals; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002846:Chromatography, Affinity; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007158:Immunologic Techniques; D011817:Rabbits
C025946:3-methyladenine; D000225:Adenine