Accurate Quantitation of Amitriptyline and Cyclobenzaprine in Urine using IMCSzyme™ for Hydrolysis

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  • Accurate Quantitation of Amitriptyline and Cyclobenzaprine in Urine using IMCSzyme™ for Hydrolysis

Researchers with the University of South Carolina’s Departments of Chemistry and Biochemistry conducted a comparative study on enzyme hydrolysis of amitriptyline and cyclobenzaprine.  Researchers specifically used IMCSzyme, along with three other commercially available beta-glucuronidase enzymes: Haliotis rufescens (Abalone), Snail (Helix pomatia), and Limpet (Patella vulgata), to study the efficiency of hydrolysis. 

Amitriptyline and cyclobenzaprine have poor hydrolysis with beta-glucuronidase enzymes—Fisher reported a hydrolysis time of up to 18 hours for complete hydrolysis of cyclobenzaprine-N+ glucuronides.  The USC Biology Department hoped to identify a more efficient enzyme by recording pre- and post-hydrolysis concentrations for amitriptyline and cyclobenzaprine.

The comparison of the amitriptyline concentration before and after hydrolysis is important to note because any increase in concentration can be attributed to the conversion of amitriptyline glucuronide to amitriptlyine.  The difference of concentration levels, then, is the amitriptyline glucuronide concentration that was hydrolyzed by the enzyme.

IMCSzyme converted the most amitriptyline glucuronide during hydrolysis—the average amitriptyline glucuronide concentration is twenty times the concentration of the free amitriptyline in the unhydrolyzed sample.  The high glucuronide ratio from IMCSzyme is consistent with the reported amitriptyline ratios.  The other three enzymes (Helix pomatia, Patella vulgata, Abalone) had post-hydrolysis ratios ranging from 1.36 to 2.07.

The research concluded that IMCSzyme efficiently hydrolyzes amitriptyline and cyclobenzaprine glucuronides, with estimated ratios of parent compound to glucuronide consistent with the literature.  Helix pomatia, Patella vulgata and Abalone all convert less amitriptyline and cyclobenzaprine glucuronide—even under more rigorous conditions. 

The low efficiencies of the three enzymes are attributed to a combination of factors, which include impurities in the enzyme solution and differing binding affinities.  The researchers recommended that a confirmation study be conducted to monitor and quantitate purified glucuronides, which would need to be optimized on LC-MS/MS, in order to emphasize the results of this study.

For a technical note summary of this poster presented by Kaylee McDonald at the Fall 2014 SOFT Conference click here.