Method for detecting opioids, opiates, cannabinoids, or benzodiazepines in a sample with a b-glucuronidase enzyme

Abstract

The present invention discloses a highly efficient method for detecting opiods, opiates, cannabinoids, or benzodiazepines present in a sample, comprising the steps of adding to said sample an enzyme with β-glucuronidase activity originated from genus Brachyspira or any mutant derived thereof; incubating the sample with the enzyme; and detecting said opiods, opiates, cannabinoids, or benzodiazepines by means of a suitable technique.

1 . A method for detecting opiods, opiates, cannabinoids, or benzodiazepines in a sample, comprising the steps of:
 a) providing the sample in which said opiods, opiates, cannabinoids, or benzodiazepines are to be detected;   b) adding to said sample an enzyme with β-glucuronidase activity originated from genus  Brachyspira  or any mutant derived thereof;   c) incubating the sample with the enzyme; and   d) detecting said opiods, opiates, cannabinoids, or benzodiazepines through any suitable technique.   
     
     
         2 . The method of  claim 1 , wherein the sample is a biological sample. 
     
     
         3 . The method of  claim 2 , 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. 
     
     
         4 . The method of  claim 1 , wherein the enzyme with β-glucuronidase activity originates from species  Brachyspira pilosicoli.    
     
     
         5 . The method of  claim 4 , 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 an 80% of identity with the amino acid sequence defined in a). 
 
     
     
         6 . The method of  claim 1 , wherein the step of incubating the sample with the enzyme is performed for a period between 1 and 30 minutes. 
     
     
         7 . The method of  claim 1 , wherein the step of incubating the sample with the enzyme is performed at a temperature range between 20 and 60 degrees Celsius. 
     
     
         8 . The method of  claim 1 , wherein the step of incubating the sample with the enzyme is performed at a range of pH between 4.0 and 9.0. 
     
     
         9 . The method of  claim 1 , wherein the suitable technique for detecting opiods, opiates, cannabinoids, or benzodiazepines 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.