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

Abstract 9-Benzyladenine, 9-(2-nitrobenzyl)adenine, 9-(3-nitrobenzyl)adenine and 9-(4-nitrobenzyl)adenine were metabolized to 9-benzyladenine-N 1 -oxide, 9-(2-nitrobenzyl)adenine-N 1 -oxide, 9-(3-nitrobenzyl)adenine-N 1 -oxide and 9-(4-nitrobenzyl)adenine-N 1 -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 packed with C18 silica bonded phase, was developed. The extraction recovery for these 9-alkyladenines and their N 1 -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°C. The mobile phase consisted of H 2 O-MeOH-diethylamine (65:35:0.5, v/v/v), pH 6.8. The above analytes were monitored at 233 nm and the 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 N 1 -oxides by hamster microsomes was determined under the experimental conditions employed.