Immunoaffinity purification and gas chromatography-mass spectrometric quantification of 3-alkyladenines in urine: metabolism studies and basal excretion levels in man.
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.
Prevost
V
V
International Agency for Research on Cancer, Unit of Environmental Carcinogens and Host Factors, Lyon, France.
Shuker
D E
DE
Friesen
M D
MD
Eberle
G
G
Rajewsky
M F
MF
Bartsch
H
H
D000225:Adenine / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism / Q000652:urine*; D000328:Adult; D000911:Antibodies, Monoclonal; D003903:Deuterium; D004032:Diet; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D007163:Immunosorbent Techniques; D008297:Male; D012685:Sepharose / Q000031:analogs & derivatives; D013205:Staphylococcal Protein A
D000911:Antibodies, Monoclonal; D013205:Staphylococcal Protein A; C022595:3-ethyladenine; C079840:3-(2-hydroxyethyl)adenine; C025946:3-methyladenine; C035771:Sepharose CL 4B; C079839:3-benzyladenine; D012685:Sepharose; D003903:Deuterium; D000225:Adenine