title: Variations in enzymatic hydrolysis efficiencies for amitriptyline and cyclobenzaprine in urine.
abstract: A collaborative study was conducted to investigate discrepancies in recoveries of two commonly prescribed compounds, amitriptyline and cyclobenzaprine, in patient urine samples when hydrolyzed with different enzymes from different sources. A 2- to 10-fold increase in analyte recoveries was seen for patient samples hydrolyzed using a recombinant β-glucuronidase (IMCSzyme™) over samples hydrolyzed with β-glucuronidase from Haliotis rufescens We report outcomes from four commercially available β-glucuronidase enzymes (IMCSzyme™, Patella vulgata, Helix pomatia and H. rufescens) on patient samples that tested positive for amitriptyline and cyclobenzaprine. Our results confirm reduced hydrolysis of glucuronides by β-glucuronidases isolated from mollusks, but near complete conversion when using the recombinant enzyme. Our premise is that systematic differences in hydrolysis efficiencies due to varying substrate affinity among enzyme subtypes potentially impacts accuracy and reliability of measurements.
authors.affiliation: Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, USA.
authors.forename: Kaylee R
authors.initials: KR
authors.lastname: Mastrianni
authors.affiliation: Integrated MicroChromatography Systems, LLC, 541 Main Street, Suite 117, Columbia, SC 29208, USA.
authors.forename: L Andrew
authors.initials: LA
authors.lastname: Lee
authors.affiliation: Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, USA.
authors.forename: William E
authors.initials: WE
authors.lastname: Brewer
authors.affiliation: PSO Laboratory, LLC, 2250 South Washington Ave, Suite 202, Lansing, MI 48910, USA.
authors.forename: Nagaraju
authors.initials: N
authors.lastname: Dongari
authors.affiliation: PSO Laboratory, LLC, 2250 South Washington Ave, Suite 202, Lansing, MI 48910, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Barna
authors.affiliation: Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, USA morgansl@mailbox.sc.edu.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Morgan
mesh_terms: D000639:Amitriptyline / Q000031:analogs & derivatives* / Q000032:analysis* / Q000652:urine; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D005966:Glucuronidase / Q000737:chemistry* / Q000652:urine; D020719:Glucuronides / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: D020719:Glucuronides; D000639:Amitriptyline; C004704:cyclobenzaprine; D005966:Glucuronidase