Direct monitoring of enzyme reactions using micellar electrokinetic capillary chromatography. Optimisation of drug glucuronide and sulfate conjugate hydrolysis.
This paper demonstrates the use of micellar electrokinetic capillary chromatography (MECC) to monitor enzyme reaction conditions. The hydrolysis reactions of model conjugated substrates (morphine and reduced flunixin glucuronides, napthyl sulfate), by proprietary beta-glucuronidase preparations, were studied under varied experimental conditions. Reactions were carried out in autosampler vials with incubation in a thermostatted CE autosampler tray. MECC was performed using borax buffer (17.5 mM, pH 9.3) modified with sodium dodecyl sulfate (70 mM). Repetitive injections were made from the sample vial throughout the course of the reactions at a frequency of up to 10 h-1. MECC provided a rapid and reproducible assay for the model substrates. Baseline interference from the enzymes prevented measurement of product increase, therefore substrate decrease was measured from the peak areas. Monitoring of reactions in this way has proved valuable in the optimisation of hydrolysis conditions used in sample preparation for drug analysis. beta-Glucuronidase preparations from Helix pomatia were found to give the best performance of those evaluated in terms of deconjugation efficiency.
Taylor
M R
MR
Horseracing Forensic Laboratory, Suffolk, UK.
Westwood
S A
SA
Perrett
D
D
D000818:Animals; D003002:Clonixin / Q000031:analogs & derivatives / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D005965:Glucuronates / Q000032:analysis* / Q000378:metabolism; D005966:Glucuronidase / Q000378:metabolism*; D006372:Helix, Snails / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009281:Naphthalenes / Q000032:analysis; D013431:Sulfates / Q000032:analysis* / Q000378:metabolism; D013463:Sulfuric Acid Esters / Q000378:metabolism; D013696:Temperature
D005965:Glucuronates; D009281:Naphthalenes; D013431:Sulfates; D013463:Sulfuric Acid Esters; C029092:naphthyl sulfate; C014557:flunixin; D005966:Glucuronidase; D003002:Clonixin