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 implica ted 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 follo wed 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, i ts alpha/beta -2-O-acyl, alpha/beta -3-O-acyl, alpha/beta -4-O-acyl, and al pha -1-O-acyl-migration isomers, and S-naproxen aglycone) were determined b y HPLC-UV studies in 25 mM potassium phosphate buffer, pH 7.40, 25 mM potas sium 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 pot assium 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-acy l-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/b eta -2-O-acyl), 0.059 (alpha/beta -3-O-acyl-alpha/beta -4-O-acyl), and 0.08 5 (alpha/beta -4-O-acyl-alpha/beta -3-O-acyl). The hydrolysis rate constant s (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 ki netic rate constants by up to 80%. The kinetic rate constants for the degra dation of S-nap-g in the mobile phase used for HPLC-H-1 NMR determined usin g HPLC-UV could predict the results obtained by the dynamic stopped-flow HP LC-H-1 NMR experiments of the individual acyl-migrated isomers. It is there fore recommended that beta -1-O-acyl glucuronide degradation kinetics be in vestigated by HPLC-UV methods once the identification and elution order of the isomers have been established by HPLC-H-1 NMR.