Method for detecting products derived from glucuronide metabolites with the enzyme b-glucuronidase, and a reagent comprising said enzyme

Abstract

The present invention discloses a method and a highly efficient reagent to detect products derived from glucuronide metabolites presents in a sample, comprising the steps of adding to said sample an enzyme with β-glucuronidase activity originated from genus Brachyspira or any variant or mutant derived thereof; incubating the sample with the enzyme during a determined period of time; and detecting the product derived from glucuronide metabolite by means of a suitable technique. The reagent of the present invention comprises an enzyme with β-glucuronidase activity originated from genus Brachyspira or any variant or mutant derived thereof; and a suitable vehicle.

1 . A method to detect products derived from glucuronide metabolites in a sample, comprising the steps of:
 a) providing the sample in which said product is to be detected,   b) adding to said sample an enzyme with β-glucuronidase activity from genus  Brachyspira  or any variant or mutant derived thereof,   c) incubating the sample with the enzyme for a determined period of time; and   d) detecting the product derived from said glucuronide metabolite through any suitable technique.   
     
     
         2 . The method of  claim 1 , wherein products derived from glucuronide metabolites are from natural, semi-synthetic or synthetic origin. 
     
     
         3 . The method of  claim 2 , wherein products derived from glucuronide metabolites are selected from the group consisting of an opioid, an opiate, a cannabinoid, a benzodiazepine or any derivative thereof. 
     
     
         4 . The method of  claim 1 , wherein the sample is a biological sample. 
     
     
         5 . The method of  claim 4 , wherein the biological sample is selected from the group consisting of saliva, whole blood, plasma, urine, hair, skin, teeth, soft tissues, meconium and vitreous humor. 
     
     
         6 . The method of  claim 1 , wherein the enzyme with β-glucuronidase activity originates from species  Brachyspira pilosicoh.    
     
     
         7 . The method of  claim 6 , wherein the enzyme with β-glucuronidase activity is selected from the group consisting of:
 a) an amino acid sequence comprising the amino acid sequence shown in SEQ. ID. NO.1; 
 b) an amino acid sequence sharing at least a 70% of identity with the amino acid sequence defined in a). 
 
     
     
         8 . The method of  claim 1 , wherein the incubation period of the sample and the enzyme is between 1 and 30 minutes. 
     
     
         9 . The method of  claim 1 , wherein incubation of sample and enzyme is performed at a temperature range between 20 and 60 degrees Celsius. 
     
     
         10 . The method of  claim 1 , wherein incubation of sample and enzyme is performed at a range of pH between 4.0 and 9.0. 
     
     
         11 . The method of  claim 1 , wherein the suitable technique to detect products derived from glucuronide metabolites is selected from the group consisting of LC-MS, GC-MS, HPLC-MS, LC-HRMS, HPLC-HRMS, LC-MS-MS, GC-MS-MS and HPLC-MS-MS. 
     
     
         12 . A reagent to detect products derived from glucuronide metabolites in a sample, comprising the steps of:
 a) an enzyme with β-glucuronidase activity originated from genus  Brachyspira  or any variant or mutant derived thereof; and   b) a suitable vehicle.   
     
     
         13 . The reagent of  claim 12 , wherein the enzyme with β-glucuronidase activity originates from species  Brachyspira pilosicoli.    
     
     
         14 . The reagent of  claim 13 , wherein the enzyme with β-glucuronidase activity is selected from the group consisting of:
 a) an amino acid sequence comprising the amino acid sequence shown in SEQ. ID. NO.1; 
 b) an amino acid sequence sharing at least a 70% of identity with the amino acid sequence defined in a). 
 
     
     
         15 . The reagent of  claim 12 , wherein the suitable vehicle is selected from the group consisting of: water, salts, buffer solutions, Tris, HEPES, DTT, citric acid, EDTA, glycerol, sugars, amino acids or a combination thereof. 
     
     
         16 . The reagent of  claim 15 , wherein the salts are selected from the group consisting of: sodium phosphate, potassium phosphate, sodium carbonate, sodium acetate, sodium citrate, sodium chloride and potassium chloride. 
     
     
         17 . The reagent of  claim 15 , wherein sugars are selected from the group consisting of sorbitol, trehalose, sucrose, glucose, lactose, mannitol and raffinose.