Abstract

Acyl-migrated isomers of drug β-1-O-acyl glucuronides have been implicated in drug toxicity because they can bind to proteins. The acyl migration and hydrolysis ofS-naproxen-β-1-O-acyl glucuronide (S-nap-g) was followed by dynamic stopped-flow HPLC-¹H NMR and HPLC methods. Nine first order rate constants in the chemical equilibrium between six species (S-nap-g, its α/β-2-O-acyl, α/β-3-O-acyl, α/β-4-O-acyl, and α-1-O-acyl-migration isomers, andS-naproxen aglycone) were determined by HPLC-UV studies in 25 mM potassium phosphate buffer, pH 7.40, 25 mM potassium phosphate buffer in D₂O pD 7.40, and 25 mM potassium phosphate buffer in D₂O pD 7.40/MeCN 80:20 v/v (HPLC-¹H NMR mobile phase). In the 25 mM potassium phosphate buffer (pH 7.40) the acyl-migration rate constants (h⁻¹) were 0.18 (S-nap-g–α/β-2-O-acyl isomer), 0.23 (α/β-2-O-acyl–α-1-O-acyl), 2.6 (α-1-O-acyl–α/β-2-O-acyl), 0.12 (α/β-2-O-acyl–α/β-3-O-acyl), 0.048 (α/β-3-O-acyl–α/β-2-O-acyl), 0.059 (α/β-3-O-acyl–α/β-4-O-acyl), and 0.085 (α/β-4-O-acyl–α/β-3-O-acyl). The hydrolysis rate constants (h⁻¹) were 0.025 (hydrolysis ofS-nap-g) and 0.0058 (hydrolysis of all acyl-migrated isomers). D₂O and MeCN decreased the magnitude of all nine kinetic rate constants by up to 80%. The kinetic rate constants for the degradation of S-nap-g in the mobile phase used for HPLC-¹H NMR determined using HPLC-UV could predict the results obtained by the dynamic stopped-flow HPLC-¹H NMR experiments of the individual acyl-migrated isomers. It is therefore recommended that β-1-O-acyl glucuronide degradation kinetics be investigated by HPLC-UV methods once the identification and elution order of the isomers have been established by HPLC-¹H NMR.