Assessment of recombinant beta-Glucuronidase vs native beta-Glucuronidase and alkaline conditions for the hydrolysis of THC-glucuronide

Abstract

The use of enzymes plays an important role in the hydrolysis of conjugated molecules during the preparation of biological samples, especially urine. Several clinical and forensic applications, including Therapeutic Drug Monitoring (TDM) and toxicological analyses, can take advantage of an effective, easy, time-saving procedure. In this perspective, we aimed to assess the yield of two different beta-glucuronidase enzymes, one recombinant and one native, in comparison with the alkaline hydrolysis routinely used in our laboratory for the analysis of cannabinoids in urine samples. A total of 82 anonymous urine samples were tested with an ISO 17025 accredited UHPLC-MS/MS method for the detection of THC-carboxylated metabolite. During the sample preparation, three different pre-analytical hydrolysis steps were performed: (A) 100 μL of urine were added with 4 μL of NaOH 10 N, then incubated at 55° for 15 minutes; (B) 100 μL of urine were added with 200 μL of recombinant beta-glucuronidase (rec-β-gluc), then incubated at room temperature for 5 minutes; (C) 100 μL of urine were added with 10 μL of native beta-glucuronidase from Helix Pomatia (nat-β-gluc), then incubated at 55° for 15 minutes. In a second experiment, further 37 anonymous urine samples were tested with different volumes (respectively, 20, 50 and 100 μL) of rec-β-gluc, to optimize the working conditions. According to the concentrations obtained with the routine procedure, the 82 samples were divided into “high concentration” (HC, above 55 ng/mL) and “low concentration” (LC, below 55 ng/mL) samples. For the HC samples, the yield obtained using rec-β-gluc was on average 13.7% higher than alkaline hydrolysis and 23.4% higher than nat-β-gluc procedure. For the LC samples, the yield with rec-β-gluc was on average 12.4% higher than alkaline hydrolysis and 15.5% higher than the nat-β-gluc procedure. A Wilcoxon test was run, and all procedures proved significantly different. The second experiment showed that the THC-carboxylated metabolite concentrations were not significantly different using the three different volumes of rec-β-gluc, proving that a minimum amount of 20 μL is enough to obtain the highest hydrolysis efficiency. The recombinant beta-glucuronidase provided the most efficient hydrolytic activity to cleave the glucuronide of THC-carboxylated metabolite. Further experiments are needed to verify that similar results are obtained for other glucuronate metabolites, including pharmaceuticals and other cannabinoids, primarily cannabidiol. By achieving a high yield in only 5 minutes without a heating step, the laboratories, especially in clinical, forensic, TDM, and workplace drug testing, will be enabled to increase the number of processed urine samples, with no detriment of the analytical performances.