ENZYMATIC HYDROLYSIS OF GLUCURONIDE CONJUGATED DRUGS IN THE PRESENCE OF WATER MISCIBLE ORGANIC MEDIA
The present disclosure provides methods of hydrolyzing a drug-glucuronide conjugate, and methods of determining a drug concentration comprising hydrolyzing a drug-glucuronide conjugate in the presence of a water miscible organic solvent that can simultaneously prevent bacterial growth in the enzyme and prevent analyte adsorption.
1 . A method of determining a concentration of a drug in a urine sample, the method comprising:
obtaining a urine sample containing a drug-glucuronide conjugate;
contacting the urine sample with an enzyme configured to cleave the drug-glucuronide conjugate to form a solution comprising the drug; and
determining a concentration of the drug in the solution.
2 . The method of claim 1 further comprising extrapolating the concentration of the drug in the solution to determine a concentration of the drug in the urine sample.
3 . The method of claim 1 further comprising diluting the urine sample before determining the concentration of the drug in the solution.
4 . The method of claim 1 further comprising adding a water miscible organic solvent to the urine sample.
5 . The method of claim 1 , wherein the step of adding the solvent further includes adding water to the urine sample.
6 . The method of claim 1 , wherein the solvent is present in an amount of at least about 20% (vol/vol %).
7 . The method of claim 1 , wherein the solvent is present in an amount of at least about 40% (vol/vol %).
8 . The method of claim 1 , wherein the organic solvent is present in an amount of at least about 60% (vol/vol %).
9 . The method of claim 1 , wherein the step of contacting the drug-glucuronide conjugate with the enzyme occurs at a temperature of no more than about 65° C.
10 . The method of claim 1 , wherein the step of contacting the drug-glucuronide conjugate with the enzyme occurs at a temperature of no more than about 55° C.
11 . The method of claim 1 , wherein the step of contacting the drug-glucuronide conjugate with the enzyme occurs at a temperature of about 19° C. to about 25° C.
12 . The method of claim 1 , wherein the solvent comprises methanol.
13 . The method of claim 1 , wherein the solvent comprises ethanol.
14 . The method of claim 1 , wherein the solvent comprises acetonitrile.
15 . The method of claim 1 , wherein the solvent comprises dimethyl formamide (DMF).
16 . The method of claim 1 , wherein the solvent comprises dimethyl sulfoxide (DMSO).
17 . The method of claim 1 , wherein the solvent is inert and polar.
18 . The method of claim 1 , wherein the “solvent” is inert and polar and is a combination of 2 or more of methanol, ethanol, acetonitrile, dimethylformamide (DMF), dimethyl sulfoxide (DMSO).
19 . The method of claim 1 , wherein the enzyme comprises a recombinant glucuronidase.
20 . The method of claim 19 , wherein the recombinant glucuronidase is IMCSzyme™.
21 . The method of claim 1 , wherein the enzyme comprises a naturally derived glucuronidase.
22 . The method of claim 21 , wherein the naturally derived glucuronidase is Red Abalone beta-glucuronidase.
23 . The method of claim 1 , wherein the water miscible organic solvent prevents bacterial growth in a solution of the enzyme.
24 . The method of claim 1 , wherein the contacting occurs in a test container, and wherein the water miscible organic solvent is present in an amount sufficient to substantially prevent the drug-glucuronide conjugate and/or the drug from adhering to a wall of the test container.
25 . The method of claim 1 , wherein the enzyme is provided as a solution comprising at least about 10% (vol/vol or wt/vol) of the water miscible organic solvent.