Immunoaffinity purification and gas chromatography-mass spectrometric quantification of 3-alkyladenines in urine: metabolism studies and basal excretion levels in man

Abstract

Immunoaffinity gels were prepared by coupling monoclonal antibody (Mab) EM-6-47 to protein A-Sepharose, and were used to make small columns retaining 3-alkyladenines (3-alkAde) of diverse structure. An analytical procedure for determination of 3-methyladenine (3-MeAde), 3-ethyladenine (3-EtAde), 3-(2-hydroxyethyl)adenine (3-HOEtAde) and 3-benzyladenine (3-BzAde) was developed. Deuterated internal standards (d3-3-MeAde, d5-3-EtAde, d4-3-HOEtAde and d7-3-BzAde) were synthesized and added to urine samples prior to immunoaffinity purification. 3-alkAde were separated and quantitated as tert-butyl-dimethylsilyl (TBDMS) derivatives by capillary gas chromatography-low resolution mass spectrometry (GC-MS). Detection limits for 3-MeAde, 3-EtAde and 3-HOEtAde were 0.2 pmol/ml urine and for 3-BzAde, 1 pmol/ml urine. Studies in two volunteers showed that 3-MeAde and 3-HOEtAde were excreted almost quantitatively (> 90%) within 24 h, that 3-EtAde was less well excreted (67-74%) and that 3-BzAde was poorly excreted (21-25%). Studies on basal levels of 3-alkAde urinary excretion in three volunteers showed that 3-MeAde was > 90% derived from the diet as the preformed product. 3-HOEtAde was present at approximately 10 nmol/day and was reduced to approximately 1 nmol/day when the diet was standardized suggesting that it is also dietary in origin. 3-BzAde was not detected in human urine. 3-EtAde was not only excreted at low levels (< 1 nmol/day) but was also only very slightly affected by diet. This general and sensitive method will be useful in biomonitoring studies in subjects exposed to alkylating agents of diverse structure.