Solid phase extraction and HPLC determination of 9-benzyladenine and isomeric 9-(nitrobenzyl)adenines and their metabolic N1-oxides present in microsomal incubates.

9-Benzyladenine, 9-(2-nitrobenzyl)adenine, 9-(3-nitrobenzyl)adenine and 9-(4-nitrobenzyl)adenine were metabolized to 9-benzyladenine-N1-oxide, 9-(2-nitrobenzyl)adenine-n1-oxide, 9-(3-nitrobenzyl)adenine-N1-oxide and 9-(4-nitrobenzyl)adenine-N1-oxide, respectively, by animal hepatic microsomes. For the quantitative determination of the substrates and metabolites present in microsomal incubates, an off-line solid phase extraction procedure, using columns paced with C18 silica bonded phase, was developed. The extraction recovery for these 9-alkyladenines and their N1-oxides was in the range of 92-101%. A reversed-phase HPLC method was established with an ODS column at a column temperature of 50 degrees C. The mobile phase consisted of H20-MeOH-diethylamine (65:35:0.5, v/v/v). pH 6.8. The above analytes were monitored at 233 nm and retention times of all analytes were within 6-14 min. The within-day coefficients of variation (CV) for the determinations were in an acceptable range. The biotransformation of BA and NBAs to N1-oxides by hamster microsomes was determined under the experimental conditions employed.