Immobilized sulfatase:beta-glucuronidase enzymes for the qualitative and quantitative analysis of drug conjugates

• Published in: Journal of pharmaceutical sciences Vol. 78; no. 2; p. 127
• Main Authors: Boppana, V K, Lynn, R K, Ziemniak, J A
• Format: Journal Article
• Language: English
• Published: United States 01-02-1989
• Subjects: Benzazepines - analysis; Chromatography, High Pressure Liquid; Enzymes, Immobilized; Fenoldopam; Glucuronidase; Pharmaceutical Preparations - analysis; Sulfatases - analysis
• ISSN: 0022-3549
• DOI: 10.1002/jps.2600780211

The enzymes sulfatase and beta-glucuronidase from Helix pomatia were simultaneously immobilized on aminopropyl control pore glass. Once immobilized, these enzymes retained activity under varied conditions of pH, organic solvent, and temperature. To hydrolyze the sulfate and glucuronide conjugates of xenobiotics, the immobilized enzymes were either added directly to incubation mixtures for qualitative in vitro studies or packed in a short stainless steel column and placed in an HPLC system for quantitative studies. By incorporating specific inhibitors (D-saccharic acid-1,4-lactone to inhibit beta-glucuronidase or phosphate ions to inhibit sulfatase) into the incubation mixture or into the HPLC mobile phases, selective hydrolysis of either sulfate or glucuronide conjugates was achieved. Upon removal of the inhibitors from the incubation mixtures or from the mobile phases, original enzyme activity was restored. The utility of immobilized enzymes was demonstrated for quantitative analysis of sulfate and glucuronide conjugates of fenoldopam, where the liberation of the catechol aglycone moiety was necessary for electrochemical detection.