S-naproxen-ss-1-O-acyl glucuronide degradation kinetic studies by stopped-flow high-performance liquid chromatography-H-1 NMR and high-performance liquid chromatography-UV

Acyl-migrated isomers of drug beta -1-O-acyl glucuronides have been implicated in drug toxicity because they can bind to proteins. The acyl migration and hydrolysis of S-naproxen-beta -1-O-acyl glucuronide (S-nap-g) was followed by dynamic stopped-flow HPLC-H-1 NMR and HPLC methods. Nine first order rate constants in the chemical equilibrium between six species (S-nap-g, its alpha/beta -2-O-acyl, alpha/beta -3-O-acyl, alpha/beta -4-O-acyl, and alpha -1-O-acyl-migration isomers, and S-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-H-1 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-alpha/beta -2-O-acyl isomer), 0.23 (alpha/beta -2-O-acyl-alpha -1-O-acyl), 2.6 (alpha -1-O-acyl-alpha/beta -2-O-acyl), 0.12 (alpha/beta -2-O-acyl-alpha/beta -3-O-acyl), 0.048 (alpha/beta -3-O-acyl- alpha/beta -2-O-acyl), 0.059 (alpha/beta -3-O-acyl-alpha/beta -4-O-acyl), and 0.085 (alpha/beta -4-O-acyl-alpha/beta -3-O-acyl). The hydrolysis rate constants (h(-1)) were 0.025 (hydrolysis of S-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-H-1 NMR determined using HPLC-UV could predict the results obtained by the dynamic stopped-flow HPLC-H-1 NMR experiments of the individual acyl-migrated isomers. It is therefore recommended that beta -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-H-1 NMR.