Formation of the Quaternary Ammonium-Linked Glucuronide of Nicotine in Human Liver Microsomes: Identification and Stereoselectivity in the Kinetics

The formation of the N 1-glucuronide metabolite of each nicotine enantiomer was studied in pooled human liver microsomes ( n = 6). The metabolite formed from natural S (−)-nicotine was identified by comparison of the high-pressure liquid chromatography (HPLC) retention time and positive ion electrospray ionization-mass spectral characteristics with a synthetic reference standard. A radiometric HPLC method was used to quantify the metabolite. The specificity of the assay method was demonstrated by experiments in which β-glucuronidase treatment of incubated assay samples resulted in elimination of the peak due to the N 1-glucuronide metabolite. The glucuronides of S (−)- and R (+)-nicotine were formed by one-enzyme kinetics, with K m values of 0.11 and 0.23 mM and V max values of 132 and 70 pmol/min/mg of protein, respectively. There is marked stereoselectivity in the apparent intrinsic clearance values ( V max / K m ) in that the value for S (−)-nicotine is 4 times greater than for the R (+)-isomer (1.2 versus 0.31 μl/min/mg of protein).