Analysis of new synthetic cannabinoid in human urine by LC-MS/MS

Synthetic cannabinoids (SCs) are a group of novel psychoactive substances, which bind to cannabinoid receptors (CB1 and CB2). One of the largest groups of SCs are smoking mixtures, which are intended as legal replacements of cannabis and distributed on the illicit drug market. In 2016, 11 new SCs were reported by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). The most commonly used techniques for quantitation of SCs in urine are high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this study we analysed in 2016-2017 released 6 new SCs such as 5F-MN18, AMB-CHMINACA, AMB-FUBINACA, APP-CHMINACA, CUMYL-4CNBINACA and THJ-2201. The LC-MS/MS system was optimised and the mass of identified parent ions, daughter ions and related retention time were determined. Matrix effect, extraction recovery and process efficiency were evaluated by the method proposed by Matuszewski et al

Identification of the main metabolites of three synthetic cannabinoids using LC-MS/MS technique

The consumption of designer drugs today is a serious problem, especially among young people involvement. ‘Herbal mixtures’ containing synthetic cannabinoids (SCs) that mimic the effect of marijuana and there are easily available via the Internet. For analysis of urine samples, knowledge of the main metabolites is necessary as the mother compounds are usually not found in urine after using, due to their fast metabolism. The aims of this study were the in vitro identification of metabolites of ADB-FUBINACA, 5F-MDMB-PICA and CUMYL-PEGACLONE and to determine which analytical targets are excreted into urine. Metabolites identified after incubation of SCs with pooled human liver microsomes (HLM). The authentic urine samples were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for investigation of the major in vivo metabolites. The main metabolites were the mono-hydroxylation of ADB-FUBINACA and CUMYL-PEGACLONE in positive urine specimens. We didn’t have positive sample of 5F-MDMB-PICA

Laboratory challenges of detecting synthetic cannabinoids in urine samples - a new sample preparation method

Synthetic cannabinoids (SCs) put the spotlight on the designer drugs’ market due to their dangerous – in some cases lethal – biological effects and easy accessibility. They are more potent than the well-known Δ⁹-tetrahydrocannabinol (THC) thanks to their special pharmacodynamic properties. The number of novel SCs on the market and of their users is growing which urges the forensic laboratories to use precise SCs detection methods routinely. Our aim was to develop a new sample preparation method for the newest 24 SCs analysis in urine samples achieving high recovery of SCs. Ethyl acetate was used instead of the traditional acetonitrile, and the targeted analysis of SCs was performed by use of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) method. The related matrix effect and process efficiency of sample preparation method were taken in consideration as well in our study

Heart-cutting two-dimensional liquid chromatography coupled to quadrupole-orbitrap high resolution mass spectrometry for determination of N,N-dimethyltryptamine in rat plasma and brain; Method development and application

The orthogonal heart-cutting liquid chromatography (LC) modes coupled to high-resolution tandem mass spectrometry (HRMS/MS) provide a number of possibilities to enhance selectivity and sensitivity for the determination of targeted compounds in complex biological matricies. Here we report the development of a new fast 2D-LC-(HRMS/MS) method and its successful application for quantitative determination of the level of plasma and brain N,N-dimethyltriptamine (DMT) using α-methyltryptamine (AMT) as internal standard in an experimental model of cerebral ischemia/reperfusion using DMT administration. The 2D-LC separation was carried out by a combination of hydrophilic interaction liquid chromatography (HILIC) in the first dimension followed by second-dimensional reversed-phase (RP) chromatography within a total run time of 10 min. The enrichment of HILIC effluent of interest containing DMT was performed using a C18 trapping column. During method development several parameters of sample preparation procedures, chromatographic separation and mass spectrometric detection were optimised to achieve high DMT recovery (plasma: 90 %, brain: 88 %) and sensitivity (plasma: 0.108 ng/mL of LOD, brain: 0.212 ng/g of LOD) applying targeted analytical method with strict LC and HRMSMS confirmatory criteria. Concerning rat plasma sample, the concentration of DMT before hypoxia (49.3–114.3 ng/mL plasma) was generally higher than that after hypoxia (10.6–96.1 ng/mL plasma). After treatment, the concentration of DMT in brain was elevated up to the range of 2–6.1 ng/g. Overall, our analytical approach is suitable to detect and confirm the presence of DMT administered to experimental animals with therapeutic purpose in a reliable manner

Development of targeted LC-MS/MS method for analysis of diclofenac and its main metabolites in rat liver perfusion solution obtained by new type of ex vivo perfusion system

Isolated perfused rat liver (IPRL) is used as ex vivo simulation for more accurate evaluation of pharmacokinetic parameters of target drugs, production of their metabolites and their excretion in the bile. Compared to in vivo systems, IPRL avoids the clearance of any other organ and permits the control over the factors that may affect the hepatic metabolism, for example, the flow rate of the perfusate, pH, pressure, and concentration of chemicals. This simple experimental technique, unlike the cultured cells, keeps the intactness of the liver, in other words, cells other than hepatocytes, drug transporters' distribution, as well as enzymes, are still affecting the metabolism which gives a realistic picture [1-2]. The portal vein of the anesthetized rat was cannulated and the perfusion solution saturated with carbogen (95% O2 and 5% CO2) was pumped through it. The solution was directed through the transected abdominal vein to the waste. A cannula was inserted into the inferior vena cava through the right heart atrium through which the perfusate leaved the liver after the ligation of the abdominal vein. The isolated liver was then placed into the buffer vapour chamber of the recirculated perfusion system and the perfusate samples were collected during different time intervals. The main goal of our work was the development of targeted reversed-phase LC-MS/MS analytical method for the analysis of diclofenac (DF) and its main metabolites namely diclofenac-O-acyl glucuronide (Glu-DF) and 4-hydroxydiclofenac (4´OH-DF) in the perfusion solution of rat liver. We are planning to use diclofenac as a reference compound in the future investigation of metabolism of designer drugs. DF and its metabolites were extracted by the optimized liquid-liquid extraction procedure. LC separation was achieved by gradient elution of the mobile phase consisted of 0.1% formic acid in water and 0.1% of formic acid in acetonitrile on a Luna Phenyl-Hexyl column at 50˚ C with a run time of 10 minutes. The Agilent 1100 LC system was coupled to the triple quadrupole TSQ 7000 mass spectrometer. The appropriate transitions were monitored during the region of elution of each analyte. The linearity range of DF was between 100 ng/mL and 40 μg/mL. The related matrix effect, recovery and process efficiency were successfully evaluated