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

a technology of b-glucuronidase and enzyme, applied in the field of biotechnology, can solve the problems of corroding vulnerable equipment, stricter security conditions, and degrading sensitive compounds

Inactive Publication Date: 2020-04-16
LA PIEDRA BIOTECNOLOGIA SPA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is a method for detecting opioids, opiates, cannabinoids, or benzodiazepines in a sample through the use of an enzyme with β-glucuronidase activity. The method involves adding the enzyme to the sample and then incubating it with the enzyme for a certain period of time. The sample can be a biological sample such as saliva, whole blood, plasma, urine, hair, skin, teeth, soft tissues, meconium, vitreous humor, water or food. The detection is then carried out through any suitable analytical technique such as liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), mass spectrometry (LC-MS, GC-MS, or HRMS) or tandem mass spectrometry (LC-MS-MS, GC-MS-MS). The technical effect of the invention is an improved method for detecting opioids, opiates, cannabinoids, or benzodiazepines in samples, which provides faster, more reliable, and accurate detection.

Problems solved by technology

However, the acid conditions and temperature required in this process degradate sensitive compounds, for example, benzodiazepines.
Moreover, working with acids in the laboratory requires stricter security conditions, producing corrosion of vulnerable equipment.
Generally, preparations of enzymes obtained from molluscs tend to have more contaminant substances that may interfere with further analysis of glucuronide metabolites.
These detection times are excessive for testing laboratories which analyze drugs in biological samples, and the situation is even more dramatic in cases such as analysis of natural opioids (codeine and morphine) and semi-synthetic opioids (hydromorphone, oxycodone and oxymorphone).

Method used

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

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Effect test

example 1

and Expression of β-glucuronidase Enzyme from B. pilosicoli

[0045]To express the β-glucuronidase enzyme, nucleotide sequence from said protein was used, originated from Brachyspira pilosicoli strain B2904 described in Genbank (access code CP003490), which is shown in SEC.ID.NO.2, and codon optimization was performed using the algorithm OptimumGene™ (GenScript) to efficiently express said sequence in E. coli. To do this, codon adaptation index (CAI), understood as distribution of frequency of codon usage through the sequence, was adjusted from 0.66 to 0.88, in which a CAI value of 1.0 is considered to be perfect in the desired expression organism; and a CAI value greater than 0.8 is regarded as good in terms of high gene expression levels. Guanine-cytosine content was also optimized to extend mRNA half-life and stem-loop structures were eliminated, since they disrupt ribosome binding and mRNA stability. Optimized nucleotide sequence obtained is shown in SEC.ID.NO.3. Amino acid sequen...

example 2

n Time of a Sample with β-glucuronidase from B. pilosicoli

[0050]The required time to hydrolyze morphine-3-glucuronide and codeine-6-glucuronide with the enzyme β-glucuronidase in excess (named BGTurbo™) from B. pilosicoli was evaluated with the following protocol: 10 μL of glucuronide metabolites at a concentration of 100 μg / mL, free standards (free codeine and morphine calibrators at concentrations of 312.5, 625, 1,000, 1,250 and 1,875 ng / mL), and internal standards (40 μL of deuterated free drug at a concentration of 1 μg / mL to each sample) were added to a sample of 0.4 mL of blank urine and then submitted to vortex agitation. Then, 360 μL of sodium phosphate (pH 7; 140 mM) solution was added to each sample. Then, 240 μL of β-glucuronidase (BGTurbo™) were added to each sample and then agitated by vortex. Samples were incubated at 55 degrees Celsius in an oven during 0, 10, 20, 30, 40, 50 and 60 minutes. A total of 21 samples were used: 7 points of measure at different times, each...

example 3

on of Specific Activity of β-glucuronidase from B. pilosicoli

[0060]Specific activity of the β-glucuronidase enzyme from B. pilosicoli (BGTurbo™) was calculated and compared with a mean value of three calculations of the specific activity of β-glucuronidase from E. coli, using the following protocol: 350 μL of sodium phosphate buffer solution (NaH2PO4) 0.1 M, pH 6.8 were mixed with 350 μL of phenolphthalein β-D-glucuronide sodium salt (0.64 mg / mL) and the mixture was incubated at 37 degrees Celsius. Then, 50 μL of solution containing the enzyme were added to the mixture and incubated at 37 degrees Celsius during 30 minutes. After the first 15 minutes the mixture was gently agitated and stopped at 30 minutes from the beginning of the reaction with 2.5 mL of a glycine solution 0.2 M, pH 10.4. Absorbance was read at 540 nm and the amount of enzyme used was calculated (U / mL) when adjusting the values in a calibration curve. Protein concentration was measured using Bradford method, as wi...

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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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional of U.S. patent application Ser. No. 15 / 442,607, filed on Feb. 24, 2017, entitled “METHOD FOR DETECTING PRODUCTS DERIVED FROM GLUCURONIDE METABOLITES WITH THE ENZYME B-GLUCURONIDASE, AND A REAGENT COMPRISING SAID ENZYME,” which claims priority under 35 USC § 119 to GB Patent Application No. 1614546.8, filed on Aug. 26, 2016, entitled “METHOD FOR DETECTING PRODUCTS DERIVED FROM GLUCURONIDE METABOLITES WITH THE ENZYME BETA-GLUCURONIDASE, AND A REAGENT COMPRISING SAID ENZYME,” the contents of both of which are incorporated herein by reference in their entireties.TECHNICAL FIELD[0002]The present invention is related with the technical field of biotechnology, and particularly provides a method for detecting products derived from glucuronide metabolites such as opioids, opiates, cannabinoids, or benzodiazepines present in a sample, using for this purpose a β-glucuronidase enzyme from bacterial origin.BACKGROUND O...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/569C12Q1/34G01N33/94
CPCG01N30/72C12Q1/34G01N2333/924G01N33/56916G01N33/9486G01N33/948C12Y302/01031
Inventor ROZAS ANDREU, MANUELALMONACID CORONADO, DANIEL E.
Owner LA PIEDRA BIOTECNOLOGIA SPA
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