METHOD FOR DETECTING PRODUCTS DERIVED FROM GLUCURONIDE METABOLITES WITH THE ENZYME B-GLUCURONIDASE, AND A REAGENT COMPRISING SAID ENZYME
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.