Variations in enzymatic hydrolysis efficiencies for amitriptyline and cyclobenzaprine in urine

• Published in: Journal of analytical toxicology Vol. 40; no. 9; pp. 732 - 737
• Main Authors: Mastrianni, Kaylee R, Lee, L Andrew, Brewer, William E, Dongari, Nagaraju, Barna, Michael, Morgan, Stephen L
• Format: Journal Article
• Language: English
• Published: England 01.11.2016
• Subjects: Amitriptyline - analogs & derivatives; Amitriptyline - analysis; Amitriptyline - urine; Calibration; Chromatography, High Pressure Liquid; Enzyme Activation; Glucuronidase - chemistry; Glucuronidase - urine; Glucuronides - chemistry; Humans; Hydrolysis; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization - methods
• ISSN: 0146-4760; 1945-2403
• DOI: 10.1093/jat/bkw062

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.