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Poster: Hydrolysis optimization for eight cannabinoids in human urine employing recombinant E. coli β-glucuronidase

Distinguishing recent cannabis intake during treatment, workplace, clinical or forensic urine testing is confounded by prolonged excretion in chronic cannabis users. Previous studies for Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) in urine after tandem alkaline/glucuronidase hydrolysis were unable to differentiate recent cannabis intake in a single specimen. The objective of this research was to develop an optimized hydrolysis approach for THC, 11-OH-THC, THCCOOH, cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), tetrahydrocannabivarin (THCV) and 11-nor-9-carboxy-THCV (THCVCOOH) in urine employing recombinant β-glucuronidase (EBG; 50,000 U/mL, Kura Biotec, Puerto-Vara, Chile) to evaluate these analytes as indicators of recent cannabis intake.. View it here.

Poster: Streamlining Sample Preparation with Second Generation Enzymes

Even though enzymatic hydrolysis is the optimum approach used for sample preparation in analytical toxicology (Fu et al., 2010), some drawbacks of this technique include long incubation times, poor analyte recovery and short column lifetime due to high concentration of proteins loaded onto the HPLC column. We tested a first generation beta-glucuronidase currently in use in our laboratory (DR2102 from Campbell Scientific) against a beta-glucuronidase also isolated from Haliotis rufescens (BG100® from Kura Biotec) and a second generation enzyme (BGTurbo™ also from Kura Biotec), assessing final analyte recovery and total protein concentration loaded onto HPLC column. The aim of this work was to compare hydrolysis efficiency and potential column lifetime of first and second generation beta-glucuronidase enzymes used for analyte quantification.. View it here.

Research Article: Rapid screening for drugs of abuse in biological fluids by ultra high performance liquid chromatography/Orbitrap mass spectrometry. E. Jagerdeo, 2016

We present a UPLC®-High Resolution Mass Spectrometric method to simultaneously screen for nineteen benzodiazepines, twelve opiates, cocaine and three metabolites, and three “Z-drug” hypnotic sedatives in both blood and urine specimens. Sample processing consists of a high-speed, high-temperature enzymatic hydrolysis (BG100) for urine samples followed by a rapid supported liquid extraction (SLE). The combination of ultra-high resolution chromatography with high resolution mass spectrometry allows all 38 analytes to be uniquely detected with a ten minute analytical run. Limits of detection for all target analytes are 3 ng/mL or better, with only 0.3 mL of specimen used for analysis. The combination of low sample volume with fast processing and analysis makes this method a suitable replacement for immunoassay screening of the targeted drug classes, while providing far superior specificity and better limits of detection than can routinely be obtained by immunoassay. Read it here.

Article Review: BG100® Powers detection of emerging Synthetic Cannabinoids

Chemists at the US National Institute on Drug Abuse (NIDA) have  developed a non-targeted LC-QTOF acquisition method for confirming 47 synthetic cannabinoid metabolites in human urine. This review compares two sample preparations. The optimization was performed with Kura BG100® hydrolysis followed by ISOLUTE® SLE+ in order to “reduce costs and minimize instrument downtime”. With the upgrade a high-throughput screening method was made possible. Scheidweiler et al. have developed an assay that is “…designed to be easily updated for the latest emerging NPS” (NPS=New Psychoactive Substance). View it here.

Poster: Rapid and Efficient Enzymatic Hydrolysis of Codeine and Morphine Glucuronides in Urine, Kumi Ellis 2015.

LCMS procedures for the confirmation of drugs of abuse in urine are now used in most laboratories, because multiple drugs can be assayed at the same time and sample processing is quick and easy, resulting in faster turn around times. For opiate analysis, hydrolysis of the glucuronide bonds is required and enzymatic hydrolysis is the preferred option. However, up to date the yields of codeine from the enzymes used have been relatively low even with long incubation times, as the codeine 6 glucuronide bond is difficult to cleave. This study examines the Kura BG 100® enzyme and Patella vulgata to breakdown the glucuronide bonds of codeine and morphine under various incubation conditions. View it here.

Application Note: Flash and Room Temperature Hydrolysis of Benzodiazepines with BG100® and EBG™

Fast sample turnaround and workflow simplification are critical to the success of new analytical processes. The focus of this study was to develop rapid and simplified processes for the hydrolysis of the benzodiazepines found in urine using BG100® (Flash Hydrolysis) and EBG™ (Room Temperature (RT) Hydrolysis), respectively.. See it here.

Application Note: Rapid and Efficient Opiate Hydrolysis with BG100®

An efficient opiate hydrolysis protocol is important for every TDM, forensic and WPDT lab in the world. Hydrolysis with acid and generic enzymes is not optimal because they either destroy 6-acetylmorphine (6-AM), an analyte of interest, or they do not hydrolyze codeine efficiently in a short time. Here we summarize our findings on the key parameters of Opiates hydrolysis . See it here.

Webinar: Approaches for Optimizing Hydrolysis of Cannabinoids, Cannabidiol and Synthetic Cannabinoids.

Hydrolysis Optimization Webinar on April 29th | Karl B. Scheidweiler PhD revealed how to drive hydrolysis for more efficient and reliable drug-testing. Recent findings on cannabis and synthetic cannabinoids showed the importance of this critical step in sample preparation. The substance abuse expert focuses on analytical method validation and has co-authored over 30 researches with Dr. Marylin Huestis at NIDA’s Chemistry and Drug Metabolism Section. See it here.

Technical Info: Optimization of the Working Conditions for BAM Mix. Temperature and pH Effect in Recovery Results.

Conjugation reactions of xenobiotics represent probably the most important biotransformation reaction in humans and animals. Glucuronidation and sulfonation are by far the most important conjugation reaction in this process. Traditionally beta-glucuronidase and aryl-sulfatase preparations (Helix pomatia, P. vulgata, bovine liver) present different optimum pH. This obliges users to choose which metabolite to hydrolyze and recover, or to run separate hydrolysis steps, resulting in a problematic and complex process. This study reveals the capacity of BAM, a mix of beta-glucuronidase and aryl-sulfatase enzymes mix from Haliotis rufescens, to simultaneously and efficiently deconjugate glucuronide and sulfate conjugates with a unique set of incubation conditions. Optimum pH efficiency ranges of both enzymes – beta-glucuronidase and aryl-sulfatase – overlap defining a simultaneous efficiency range between pH 4.5 – 5.2. In this pH range both enzymes are working at least at 80% of their maximum hydrolysis rate. Besides an optimum ideal pH of 4.8 is identified where both enzyme work at 98% of their hydrolysis rate. An optimal temperature for BAM that was dependent on the incubation time was also found. Protocols for different incubation times with BAM are provided. Read it here.

Poster: A New Enzyme for the Hydrolysis of Codeine 6 Glucuronide, Ellis, 2013.

Codeine 6 glucuronide is the major metabolite of codeine and hydrolysis is required to break the glucuronide bond when quantifying codeine in urine samples. Previously enzymes derived from sources such as Helix pomatia, Escherichia coli and Patella vulgata have been used but recoveries of codeine achieved have only been up to 60% when the incubation was carried out overnight. Acid hydrolysis has been the method of choice when quantitative yields of codeine are required. The advantages for using enzymatic hydrolysis include cleaner extracts and the ability to assay for numerous drugs in the same run. Acid hydrolysis compromises measurement of 6 acetyl morphine by converting it to morphine, is more labour intensive and poses a safety risk to staff. We have observed a significant variation of inter laboratory results for codeine concentrations in non spiked urine samples in the Austox quality assurance program, because of varying recoveries of codeine from the different methods of hydrolysis used. The aim of this study was to find an enzyme that would give satisfactory recoveries of codeine using a short incubation time. View it here.

Poster: Optimization of Glucuronidase Hydrolysis of Drug Facilitated Assault Urine Samples, Rogers, 2014.

For toxicological analysis in drug facilitated sexual assault (DFSA) cases, urine is usually the preferred sample type over blood. This is mainly because drugs have a longer detection window in urine. However, extensive phase II metabolism of many DFSA related drugs, for example opiates and benzodiazepines, can mean that very little free drug is detectable in the urine. Glucuronidation is a principal phase II metabolic route for many drugs. Enzymatic hydrolysis using commercially available glucuronidase enzymes is a recommended method for improving the detection of highly conjugated drugs in DFSA urine samples. This work examined the efficacy of 3 commercially available beta glucuronidase enzyme products for the hydrolysis of glucuronidated metabolites, and was conducted as part of a Flinders University Honours project in conjunction with Forensic Science SA. View it here.

Poster: Enzymatic Hydrolysis Efficiency of Glucuronide Conjugates in Human Urine, Tann, 2014.

The purpose of this research was to study and optimize the hydrolysis efficiency of various β-glucuronidase enzymes used for hydrolyzing glucuronide conjugates in urine samples. Results indicate significant differences in enzyme efficiencies between the different types of enzyme as well as differences between sources of the same type of enzyme. The differences were most pronounced with codeine-6- β-D-glucuronide, the most difficult test compound to hydrolyze. View it here.

Research Article: 3,4 Methylenedioxypyrovalerone (MDPV) and metabolites quantification in human and rat plasma by liquid chromatography high resolution mass spectrometry. Anizan, 2014

Synthetic cathinones are recreational drugs that mimic the effects of illicit stimulants like cocaine, amphetamine or Ecstasy. Among the available synthetic cathinones in the United States, 3,4-methylenedioxypyrovalerone (MDPV) is commonly abused and associated with dangerous side effects. MDPV is a dopamine transporter blocker 10-fold more potent than cocaine as a locomotor stimulant in rats.

We developed and validated a liquid chromatography–high resolution mass spectrometry method to quantify MDPV and its primary metabolites in 100 mL human and rat plasma. Plasma hydrolysis was followed by protein precipitation before analysis. Limits of detection were 0.1 mg L1, with linear ranges from 0.25 to 1000 mg L1.

As proof of this method, rat plasma specimens were analyzed after intraperitoneal and subcutaneous dosing with MDPV (0.5 mg kg1). MDPV, 3,4-catechol-PV and 4-OH-3-MeO-PV concentrations ranged from not detected to 107.5 mg L1. Read it here.

Research Article: Impact of enzymatic and alkaline hydrolysis on CBD concentration in urine, Bergamaschi, 2013.

A sensitive and specific analytical method for cannabidiol (CBD) in urine was needed to define urinary CBD pharmacokinetics after controlled CBD administration, and to confirm compliance with CBD medications including Sativex—a cannabis plant extract containing 1:1 Δ9-tetrahydrocannabinol (THC) and CBD. Non-psychoactive CBD has a wide range of therapeutic applications and may also influence
psychotropic smoked cannabis effects. Few methods exist for the quantification of CBD excretion in urine, and no data are available for phase II metabolism of CBD to CBD-glucuronide or CBD-sulfate. We optimized the hydrolysis of CBD-glucuronide and/or -sulfate, and developed and validated a GC-MS method for urinary CBD quantification. Solid-phase extraction isolated and concentrated analytes prior to GC-MS.
Method validation included overnight hydrolysis (16 h) at 37 °C with 2,500 units of BG100. Enzyme hydrolyzed urine specimens exhibited more than a 250-fold CBD concentration increase compared to alkaline and non-hydrolyzed specimens. This method can be applied for urinary CBD quantification and further pharmacokinetics characterization following controlled CBD administration. Full text here.

Research Article: Quantification of six cannabinoids and metabolites in oral fluid by liquid chromatography-tandem mass spectrometry, Desrosiers, 2014.

Δ9-Tetrahydrocannabinol (THC) is the most commonly analyzed cannabinoid in oral fluid (OF); however, its metabolite 11-nor-9-carboxy-THC (THCCOOH) offers the advantage of documenting active consumption, as it is not detected in cannabis smoke. Analytical
challenges such as low (ng/L) THCCOOH OF concentrations hampered routine OF THCCOOH monitoring. Presence of minor cannabinoids like cannabidiol and cannabinol offer the advantage of identifying recent cannabis intake. Published OF cannabinoids methods have limitations, including few analytes and lengthy derivatization. We developed and validated a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for THC, its metabolites, 11-hydroxy-THC and THCCOOH quantification, and other natural cannabinoids including tetrahydrocannabivarin (THCV), cannabidiol (CBD), and cannabigerol (CBG) in 1mL OF collected with the Quantisal device.

This method will be highly useful for workplace, criminal justice, drug treatment and driving under the influence of cannabis OF testing. Read the article here.

Trends in Beta-glucuronidase enzymatic hydrolysis