ABSTRACT
The stereochemical stability of the popular drugs of abuse 2-, 3- and 4-chloromethcathinone was studied in the mobile phase used for the isolation of their enantiomers by high-performance liquid chromatography, as well as in various biological matrixes such as whole blood, saliva and urine. For 2-, 3-, and 4-chloromethcathinones the rate constants and half-lives of their first order racemization reaction was assessed. It was found that at 25 °C the racemization rate constant decreases in the order 2-CMC > 3-CMC > 4-CMC while their stereochemical stability in biological matrixes decreases in the order urine > saliva > whole blood. This information must be considered for the adequate storage of purified enantiomers in the collected fractions, as well as in the studies focused on their enantioselective transformation in the human body.
Subject(s)
Drug Stability , Stereoisomerism , Humans , Chromatography, High Pressure Liquid/methods , Saliva/chemistry , Propiophenones/chemistry , Propiophenones/blood , Half-LifeABSTRACT
Recently, hexahydrocannabinol (HHC) was posed under strict control in Europe due to the increasing HHC-containing material seizures. The lack of analytical methods in clinical laboratories to detect HHC and its metabolites in biological matrices may result in related intoxication underreporting. We developed and validated a comprehensive GC-MS/MS method to quantify 9(R)-HHC, 9(S)-HHC, 9αOH-HHC, 9ßOH-HHC, 8(R)OH-9(R)-HHC, 8(S)OH-9(S)HHC, 11OH-9(R)HHC, 11OH-9(S)HHC, 11nor-carboxy-9(R)-HHC, and 11nor-carboxy-9(S)-HHC in whole blood, urine, and oral fluid. A novel QuEChERS extraction protocol was optimized selecting the best extraction conditions suitable for all the three matrices. Urine and blood were incubated with ß-glucuronidase at 60 °C for 2 h. QuEChERS extraction was developed assessing different ratios of Na2SO4:NaCl (4:1, 2:1, 1:1, w/w) to be added to 200 µL of any matrix added with acetonitrile. The chromatographic separation was achieved on a 7890B GC with an HP-5ms column, (30 m, 0.25 mm × 0.25 µm) in 12.50 min. The analytes were detected with a triple-quadrupole mass spectrometer in the MRM mode. The method was fully validated following OSAC guidelines. The method showed good validation parameters in all the matrices. The method was applied to ten real samples of whole blood (n = 4), urine (n = 3), and oral fluid (n = 3). 9(R)-HHC was the prevalent epimer in all the samples (9(R)/9(S) = 2.26). As reported, hydroxylated metabolites are proposed as urinary biomarkers, while carboxylated metabolites are hematic biomarkers. Furthermore, 8(R)OH-9(R)HHC was confirmed as the most abundant metabolite in all urine samples.
Subject(s)
Dronabinol , Gas Chromatography-Mass Spectrometry , Tandem Mass Spectrometry , Humans , Gas Chromatography-Mass Spectrometry/methods , Tandem Mass Spectrometry/methods , Dronabinol/urine , Dronabinol/blood , Dronabinol/analogs & derivatives , Saliva/chemistry , Saliva/metabolism , Reproducibility of ResultsABSTRACT
In this study we report on efforts to develop an enantioselective method for the detection of the drug of abuse clephedrone (1-(4-chlorophenyl)-2-(methylamino)-1-propanone (4-chloromethcathinone, also known as 4-CMC or para-chloro-methcathinone)) and its phase-1 metabolites in human biological fluids. The major goal is not to only report results, but primarily to emphasize the various challenges encountered when developing a reliable analytical method for the detection and quantification of novel psychoactive substances (NPS) and their metabolites in the matrix of interest. Such challenges start with the lack of chemical stability of some NPS in biological matrices. Additionally, most often metabolites are unavailable in pure form to serve as analytical standards, just as deuterated standards for native drugs and metabolites are frequently not commercially available. Furthermore, if the NPS is chiral, enantiomerically pure standards with known absolute stereochemistry are required, as well as a stereochemical stability of a drug and its metabolites becomes an issue. In addition, the chirality of a NPS significantly increases the number of species to be detected in the sample and thus challenges the development of an adequate separation method. These issues are shortly addressed, and some solutions offered in this manuscript.
Subject(s)
Psychotropic Drugs , Stereoisomerism , Psychotropic Drugs/analysis , Psychotropic Drugs/chemistry , Humans , Propiophenones/chemistry , Propiophenones/analysis , Illicit Drugs/analysis , Illicit Drugs/chemistry , Substance Abuse Detection/methodsABSTRACT
Ostarine (enobasarm) is a selective androgen receptor modulator with great therapeutic potential. However, it is also used by athletes to promote muscle growth and enhance performances without the typical adverse effects of anabolic steroids. Ostarine popularity increased in recent years, and it is currently the most abused "other anabolic agent" (subclass S1.2. of the "anabolic agents" class S1) from the World Anti-Doping Agency's (WADA) prohibited list. Several cases of liver toxicity were recently reported in regular users. Detecting ostarine or markers of intake in biological matrices is essential to document ostarine use in doping. Therefore, we sought to investigate ostarine metabolism to identify optimal markers of consumption. The substance was incubated with human hepatocytes, and urine samples from six ostarine-positive cases were screened. Analyses were performed via liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) and software-assisted data mining, with in silico metabolite predictions. Ten metabolites were identified with hydroxylation, ether cleavage, dealkylation, O-glucuronidation, and/or sulfation. The production of cyanophenol-sulfate might participate in the mechanism of ostarine liver toxicity. We suggest ostarine-glucuronide (C25H22O9N3F3, diagnostic fragments at m/z 118, 185, and 269) and hydroxybenzonitrile-ostarine-glucuronide (C25H22O10N3F3, diagnostic fragments at m/z 134, 185, and 269) in non-hydrolyzed urine and ostarine and hydroxybenzonitrile-ostarine (C19H14O4N3F3, diagnostic fragments at m/z 134, 185, and 269) in hydrolyzed urine as markers to document ostarine intake in doping.
Subject(s)
Anabolic Agents , Doping in Sports , Humans , Male , Anabolic Agents/metabolism , Anabolic Agents/urine , Hepatocytes/metabolism , Hepatocytes/drug effects , Tandem Mass Spectrometry , Receptors, Androgen/metabolism , Substance Abuse Detection/methods , Chromatography, Liquid , Adult , AnilidesABSTRACT
Hydrogen/deuterium (H/D) isotope effects are not unusual in chromatography and such phenomena have been observed in both gas- and liquid-phase separations. Despite the numerous reports on this topic, the understanding of mechanisms and the underlying noncovalent interactions at play remains rather challenging. In our recent study, we reported baseline separation of isotopologoues of some amphetamine (AMP) derivatives on achiral and polysaccharide-based chiral columns, as well as some correlations between the degree of separation of enantiomers and isotopologues on (the same) polysaccharide-based chiral column(s). Following our previous findings on isotope effects in high-performance liquid chromatography, we report herein a comparative study on the isotope effects observed with AMP and methamphetamine (MET). The impact of some pivotal factors such as the number of deuterium atoms part of AMP isotopologues, the structure of its isotopomers, the chemical structure of the achiral and chiral stationary phases used in this study, and the use of methanol- vs acetonitrile-containing mobile phases on the isotope effects was examined and discussed. Quantitative correlations between the observed isotope effects and the enantioselectivity of the chiral columns used are also shortly discussed. Furthermore, considering the chromatographic results as benchmark experimental data, we attempted to elucidate the molecular bases of the observed phenomena using quantum mechanics calculations.
Subject(s)
Amphetamine , Deuterium , Polysaccharides , Chromatography, High Pressure Liquid/methods , Stereoisomerism , Deuterium/chemistry , Amphetamine/chemistry , Amphetamine/isolation & purification , Polysaccharides/chemistry , Polysaccharides/isolation & purification , Methamphetamine/chemistry , Methamphetamine/isolation & purification , Acetonitriles/chemistry , Methanol/chemistryABSTRACT
A method of analysis was developed for the simultaneous chemo- and enantioseparation of 2-, 3-, and 4-chloromethcathinones by high-performance liquid chromatography tandem mass-spectrometry. The fast method enables the reliable identification of positional isomers of chloromethcathinones in biological samples. In addition, the same method can be used for the enantioselective quantitative determination of one of these compounds and its major phase-1 metabolites in biological fluids. The developed method was applied to oral fluid samples collected by police during routine random traffic control in Belgium from January to November, 2023. It was found that 3-CMC was more frequently abused compared to 4-CMC. Although some differences were observed between the concentrations of enantiomers in OF, most likely the drugs were abused in the racemic form. No abuse of 2-CMC was detected at the timepoint of sample collection.
Subject(s)
Saliva , Tandem Mass Spectrometry , Tandem Mass Spectrometry/methods , Chromatography, High Pressure Liquid/methods , Humans , Saliva/chemistry , Stereoisomerism , Propiophenones/chemistry , Propiophenones/analysis , Substance Abuse Detection/methods , BelgiumABSTRACT
Recently we published in this journal an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 3,4-methylenedioxymethamphetamine (MDMA) and its major phase-1 metabolites, 4-hydroxy-3-methoxyamphetamine (HMA), 4-hydroxy-3-methoxymethamphetamine (HMMA) and 3,4-methylenedioxyamphetamine (MDA) in human plasma, sweat, oral fluid and urine. Since we did not achieve simultaneous enantioseparation of all 4 compounds with a single chiral column, two amylose-based chiral columns were used alternatively. Further optimization of the mobile phase in the present study enabled baseline separation of all four pairs of enantiomers on a single Lux AMP column. In addition, by optimization of the column dimension and applied flow-rate it became possible to complete the separation within 6â¯minutes. These new methods were applied to the analysis of human plasma, oral fluid and urine. While results on the concentration of MDMA and its metabolites in various biological fluids were reported in our recent publication, in the present study an attempt was made to hydrolyze glucuronides in urine samples by using alternatively, hydrochloric acid or glucuronidase and to evaluate the effect of hydrolysis on the concentration and enantiomeric distribution of hydroxy metabolites of MDMA such as HMA and HMMA.
Subject(s)
3,4-Methylenedioxyamphetamine , Lactates , Methamphetamine , N-Methyl-3,4-methylenedioxyamphetamine , Humans , N-Methyl-3,4-methylenedioxyamphetamine/urine , Chromatography, High Pressure Liquid , Tandem Mass Spectrometry , Chromatography, Liquid , Stereoisomerism , 3,4-Methylenedioxyamphetamine/urineABSTRACT
BACKGROUND: Approximately 30 million people worldwide consume new psychoactive substances (NPS), creating a serious public health issue due to their toxicity and potency. Drug-induced liver injury is the leading cause of liver disease, responsible for 4% of global deaths each year. CONTENT: A systematic literature search revealed 64 case reports, in vitro and in vivo studies on NPS hepatotoxicity. Maximum elevated concentrations of aspartate aminotransferase (136 to 15 632â U/L), alanine transaminase (121.5 to 9162â U/L), total bilirubin (0.7 to 702â mg/dL; 0.04 to 39.03â mmol/L), direct (0.2-15.1â mg/dL; 0.01-0.84â mmol/L) and indirect (5.3â mg/dL; 0.29â mmol/L) bilirubin, alkaline phosphatase (79-260â U/L), and gamma-glutamyltransferase (260â U/L) were observed as biochemical markers of liver damage, with acute and fulminant liver failure the major toxic effects described in the NPS case reports. In vitro laboratory studies and subsequent in vivo NPS exposure studies on rats and mice provide data on potential mechanisms of toxicity. Oxidative stress, plasma membrane stability, and cellular energy changes led to apoptosis and cell death. Experimental studies of human liver microsome incubation with synthetic NPS, with and without specific cytochrome P450 inhibitors, highlighted specific enzyme inhibitions and potential drug-drug interactions leading to hepatotoxicity. SUMMARY: Mild to severe hepatotoxic effects following synthetic NPS exposure were described in case reports. In diagnosing the etiology of liver damage, synthetic NPS exposure should be considered as part of the differential diagnosis. Identification of NPS toxicity is important for educating patients on the dangers of NPS consumption and to suggest promising treatments for observed hepatotoxicity.
Subject(s)
Chemical and Drug Induced Liver Injury , Liver Diseases , Humans , Rats , Mice , Animals , Liver/metabolism , Liver Diseases/diagnosis , Chemical and Drug Induced Liver Injury/diagnosis , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/metabolism , Alkaline Phosphatase , Alanine Transaminase , BilirubinABSTRACT
In 2023, hexahydrocannabinol (HHC) attracted the attention of international agencies due to its rapid spread in the illegal market. Although it was discovered in 1940, less is known about the pharmacology of its two naturally occurring epimers, 9(R)-HHC and 9(S)-HHC. Thus, we aimed to investigate the disposition of hexahydrocannabinol epimers and their metabolites in whole blood, urine and oral fluid following a single controlled administration of a 50:50 mixture of 9(R)-HHC and 9(S)-HHC smoked with tobacco. To this end, six non-user volunteers smoked 25 mg of the HHC mixture in 500 mg of tobacco. Blood and oral fluid were sampled at different time points up to 3 h after the intake, while urine was collected between 0 and 2 h and between 2 and 6 h. The samples were analyzed with a validated HPLC-MS/MS method to quantify 9(R)-HHC, 9(S)-HHC and eight metabolites. 9(R)-HHC showed the highest Cmax and AUC0-3h in all the investigated matrices, with an average concentration 3-fold higher than that of 9(S)-HHC. In oral fluid, no metabolites were detected, while they were observed as glucuronides in urine and blood, but with different profiles. Indeed, 11nor-9(R)-HHC was the most abundant metabolite in blood, while 8(R)OH-9(R) HHC was the most prevalent in urine. Interestingly, 11nor 9(S) COOH HHC was detected only in blood, whereas 8(S)OH-9(S) HHC was detected only in urine.
ABSTRACT
The different behavior of enantiomers of chiral compounds in non-isotropic environments (among them in living organism) is well known. On the other hand, the importance of a kinetic isotope effect in the biomedical field has become evident during past few decades. Thus, separation of both, enantiomers and isotopologues is now critical. Only very few published studies have attempted the simultaneous separation of enantioisotopologues. In this article we report baseline separation of partially deuterated isotopologues of a few amphetamine derivatives in high-performance liquid chromatography (HPLC) using achiral columns. In addition, the simultaneous separations of enantiomers and isotopologues (i.e. enantioisotopologues) were attempted on polysaccharide-based chiral columns. For several compounds the isotope effect was tunable and could be switched from a "normal" to "inverse" by making changes to the mobile-phase composition. A stronger isotope effect was observed in acetonitrile-containing mobile phases compared to methanol-containing ones with both chiral and achiral columns. In a separation system where both "normal" and "inverse" isotope effects were observed the "normal" isotope effect was favored in polar organic solvents while increasing content of the aqueous component in the reversed-phase (RP) mobile phase favored an "inverse" isotope effect. This observation indicates that polar, hydrogen bonding-type noncovalent interactions are involved in the "normal" isotope effect, while apolar hydrophobic-type interactions are mostly responsible for the "inverse" isotope effect.
Subject(s)
Amphetamine , Polysaccharides , Chromatography, High Pressure Liquid/methods , Polysaccharides/chemistry , Solvents/chemistry , Isotopes , StereoisomerismABSTRACT
The New Psychoactive Substances (NPS) phenomenon represents an ever-changing global issue, with a number of new molecules entering the illicit market every year in response to international banning laws [...].
Subject(s)
Psychotropic Drugs , Psychotropic Drugs/adverse effectsABSTRACT
In the present study an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the first time for quantitative determination of the recreational drug of abuse methylone and its major metabolites in oral fluid. The simultaneous chemo- and enantioseparation of methylone and its major metabolites was performed on a polysaccharide-based chiral column based on amylose tris(5-chloro-3-methylphenylcarbamate) as chiral selector (Lux i-Amylose-3) with methanol containing 0.4 % (v/v) aqueous ammonium hydroxide as mobile phase. The time required for enantioselective analysis of methylone and its 2 major metabolites was 15 min. This method was fully validated following the Organization of Scientific Area Committees (OSAC) for Forensic Science guidelines. This method was applied for the enantioselective determination of methylone and its metabolites in oral fluid and enantioselectivity in metabolism and pharmacokinetic of the parent compound and metabolites was observed. While the first enantiomer of methylone was found at higher concentration, both metabolites shown greater concentration for the second enantiomer. The results revealed that MET undergoes an enantioselective biotransformation to its metabolites HMMC and MDC, with S-(-)-MET more rapidly metabolized and eliminated from the body.
Subject(s)
Amylose , Tandem Mass Spectrometry , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Amylose/chemistry , StereoisomerismABSTRACT
The aim of this study was to investigate methylone and its metabolites concentration in oral fluid following controlled increasing doses, focusing on the effect of oral fluid pH. Samples were obtained from a clinical trial where twelve healthy volunteers participated after ingestion of 50, 100, 150 and 200 mg of methylone. Concentration of methylone and its metabolites 4-hydroxy-3-methoxy-N-methylcathinone (HMMC) and 3,4-methylenedioxycathinone in oral fluid were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pharmacokinetic parameters were estimated, and the oral fluid-to-plasma ratio (OF/P) at each time interval was calculated and correlated with the oral fluid pH using data from our previous study in plasma. Methylone was detected at all time intervals after each dose; MDC and HMMC were not detectable after the lowest dose. Oral fluid concentrations of methylone ranged between 88.3-503.8, 85.5-5002.3, 182.8-13,201.8 and 214.6-22,684.6 ng/mL following 50, 100, 150 and 200 mg doses, respectively, peaked between 1.5 and 2.0 h, and were followed by a progressive decrease. Oral fluid pH was demonstrated to be affected by methylone administration. Oral fluid is a valid alternative to plasma for methylone determination for clinical and toxicological studies, allowing for a simple, easy and non-invasive sample collection.
ABSTRACT
According to the EU Early Warning System (EWS), synthetic cathinones (SCs) are the second largest new psychoactive substances (NPS) class, with 162 synthetic cathinones monitored by the EU EWS. They have a similar structure to cathinone, principally found in Catha Edulis; they have a phenethylamine related structure but also exhibit amphetamine-like stimulant effects. Illegal laboratories regularly develop new substances and place them on the market. For this reason, during the last decade this class of substances has presented a great challenge for public health and forensic toxicologists. Acting on different systems and with various mechanisms of action, the spectrum of side effects caused by the intake of these drugs of abuse is very broad. To date, most studies have focused on the substances' cardiac effects, and very few on their associated neurotoxicity. Specifically, synthetic cathinones appear to be involved in different neurological events, including increased alertness, mild agitation, severe psychosis, hyperthermia and death. A systematic literature search in PubMed and Scopus databases according to PRISMA guidelines was performed. A total of 515 studies published from 2005 to 2022 (350 articles from PubMed and 165 from Scopus) were initially screened for eligibility. The papers excluded, according to the criteria described in the Method Section (n = 401) and after full text analyses (n = 82), were 483 in total. The remaining 76 were included in the present review, as they met fully the inclusion criteria. The present work provides a comprehensive review on neurotoxic mechanisms of synthetic cathinones highlighting intoxication cases and fatalities in humans, as well as the toxic effects on animals (in particular rats, mice and zebrafish larvae). The reviewed studies showed brain-related adverse effects, including encephalopathy, coma and convulsions, and sympathomimetic and hallucinogenic toxidromes, together with the risk of developing excited/agitated delirium syndrome and serotonin syndrome.
Subject(s)
Central Nervous System Stimulants , Neurotoxicity Syndromes , Mice , Rats , Humans , Animals , Synthetic Cathinone , Zebrafish , Central Nervous System Stimulants/toxicity , Fever , Amphetamine , Neurotoxicity Syndromes/etiology , Psychotropic Drugs/toxicityABSTRACT
In the present work an isocratic enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the separation and quantitative determination of dextro - and levo -methorphan and their pharmacologically relevant metabolites, dextrorphan and levorphanol, respectively, in human blood samples. The separation of enantiomers of methorphan and metabolites was performed on the polysaccharide-based chiral column Lux AMP in combination with acetonitrile and 5 mM aqueous ammonium bicarbonate pH 11 in the ratio 50:50 (%, v/v) as mobile phase with the flow rate 1 mL/min. The mass spectrometer was operated in scheduled multiple reaction monitoring (MRM) mode, with four transitions for each dextromethorpan, levomethorphan, dextrorphan and dextromethorphan-d3 and two transitions for each levorphanol, levorphanol-d3 and dextrorphan-d3. Application of this method to human post-mortem blood samples confirmed cases of severe overdosing with dextromethorphan, levomethorphan, and less commonly with both.
Subject(s)
Dextromethorphan , Dextrorphan , Humans , Dextromethorphan/chemistry , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid , Tandem Mass Spectrometry/methods , Stereoisomerism , LevorphanolABSTRACT
The aim of this study was to determine the excretion of methylone and its metabolites in sweat following the ingestion of increasing controlled doses of 50, 100, 150 and 200 mg of methylone to twelve healthy volunteers involved in a clinical trial. Methylone and its metabolites 4-hydroxy-3-methoxy-N-methylcathinone (HMMC) and 3,4-methylenedioxycathinone (MDC) were analyzed in sweat patches by liquid chromatography-tandem mass spectrometry. Methylone and MDC were detected in sweat at 2 h and reached their highest accumulation (Cmax) at 24 h after the administration of 50, 100, 150 and 200 mg doses. In contrast, HMMC was not detectable at any time interval after each dose. Sweat proved to be a suitable matrix for methylone and its metabolites' determination in clinical and toxicological studies, providing a concentration that reveals recent drug consumption.
Subject(s)
Methamphetamine , Sweat , Humans , Chromatography, Liquid/methods , Mass Spectrometry , Methamphetamine/metabolism , Sweat/chemistryABSTRACT
In January 2020, the World Health Organization (WHO) issued a Public Health Emergency of International Concern, declaring the COVID-19 outbreak a pandemic in March 2020. Stringent measures decreased consumption of some drugs, moving the illicit market to alternative substances, such as New Psychoactive Substances (NPS). A systematic literature search was performed, using scientific databases such as PubMed, Scopus, Web of Science and institutional and government websites, to identify reported intoxications and fatalities from NPS during the COVID-19 pandemic. The search terms were: COVID-19, SARS-CoV-2, severe acute respiratory syndrome coronavirus 2, coronavirus disease 2019, intox*, fatal*, new psychoactive substance, novel psychoactive substance, smart drugs, new psychoactive substance, novel synthetic opioid, synthetic opioid, synthetic cathinone, bath salts, legal highs, nitazene, bath salt, legal high, synthetic cannabinoid, phenethylamine, phencyclidine, piperazine, novel benzodiazepine, benzodiazepine analogue, designer benzodiazepines, tryptamine and psychostimulant. From January 2020 to March 2022, 215 NPS exposures were reported in Europe, UK, Japan and USA. Single NPS class intoxications accounted for 25, while mixed NPS class intoxications represented only 3 cases. A total of 130 NPS single class fatalities and 56 fatalities involving mixed NPS classes were published during the pandemic. Synthetic opioids were the NPS class most abused, followed by synthetic cathinones and synthetic cannabinoids. Notably, designer benzodiazepines were frequently found in combination with fentalogues. Considering the stress to communities and healthcare systems generated by the pandemic, NPS-related information may be underestimated. However, we could not define the exact impacts of COVID-19 on processing of toxicological data, autopsy and death investigations.
ABSTRACT
"Light cannabis" is a product legally sold in Europe with Δ9-tetrahydrocannabinol (THC) concentration <0.2% and variable cannabidiol (CBD) content. In this study, we aimed to assess the time courses of THC and metabolites (11-nor-9-carboxy-THC and 11-hydroxy-THC) and CBD and metabolites (CBD-7-oic acid, 7-hydroxy-CBD, 6α-hydroxy-CBD and 6ß-hydroxy-CBD) in whole blood of 10 healthy participants after smoking one or four light cannabis cigarettes (0.16% THC and 5.8% CBD). Blood samples were collected 0.5-4 h after administration. Blood analysis was performed by reversed-phase ultra-performance liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode after glucuronide hydrolysis and liquid-liquid extraction in basic and acidic conditions. The method was validated following the most recent guidelines in toxicology: the method was linear, accurate, precise and sensitive (lower limits of quantification ranged from 0.005 to 0.01 ng/mL); carryover, matrix effect, recovery, process efficiency and dilution integrity were also assessed. As previously reported, the main metabolites of THC were THC-COOH and then 11-OH-THC, and the main metabolites of CBD were 7-OH-CBD and then 7-COOH-CBD. The time of the first collection, which likely occurred after the maximal concentration of most of the analytes, and the short monitoring time, up to 4 h after smoking, limited the evaluation of the pharmacokinetic parameters.
Subject(s)
Cannabidiol , Cannabis , Hallucinogens , Humans , Tandem Mass Spectrometry/methods , Chromatography, High Pressure Liquid/methods , Smokers , Dronabinol/analysisABSTRACT
The aim of this study is to define, for the first time, human methylone and HMMC plasma pharmacokinetics following controlled administration of 50-200 mg methylone to 12 male volunteers. A new LC-MS/MS method was validated to quantify methylone, MDMA, and their metabolites in plasma. The study was a randomized, cross-over, double-blinded and placebo-controlled study, with a total of 468 plasma samples collected. First, 10 µL of MDMA-d5, MDA-d5 and methylone-d3 internal standards were added to 100 µL of plasma. Two mL of chloroform and ethyl acetate 9:1 (v/v) were then added, mixed well and centrifuged. The supernatant was fortified with 0.1 mL acidified methanol and evaporated under nitrogen. Samples were reconstituted with a mobile phase and injected into the LC-MS/MS instrument. The method was fully validated according to OSAC guidelines (USA). Methylone plasma concentrations increased in a dose-proportional manner, as demonstrated by the increasing maximum concentration (Cmax) and area under the curve of concentrations (AUC). Methylone Cmax values were reported as 153, 304, 355 and 604 ng/mL, AUC0-24 values were reported as 1042.8, 2441.2, 3524.4 and 5067.9 h·ng/mL and T1/2 values as 5.8, 6.4, 6.9 and 6.4 h following the 50, 100, 150 and 200 mg doses, respectively. Methylone exhibited rapid kinetics with a Tmax of 1.5 h for the 50 mg dose and 2 h approximately after all the other doses. HMMC exhibited faster kinetics compared to methylone, with a Cmax value that was 10-14-fold lower and an AUC0-24 value that was 21-29-fold lower. Methylone pharmacokinetics was linear across 50-200 mg oral doses in humans, unlike the previously described non-linear oral MDMA pharmacokinetics. An LC-MS/MS method for the quantification of methylone, MDMA and their metabolites in human plasma was achieved. Methylone exhibited linear pharmacokinetics in humans with oral doses of 50-200 mg.
Subject(s)
Methamphetamine , Tandem Mass Spectrometry , Humans , Male , Chromatography, Liquid , Tandem Mass Spectrometry/methods , Methamphetamine/metabolism , Area Under Curve , Administration, OralABSTRACT
Cannabidiol (CBD) exhibits anti-inflammatory, anxiolytic, antiseizure, and neuroprotective proprieties without addictive or psychotropic side effects, as opposed to Δ9-tetrahydrocannabinol (THC). While recreational cannabis contains higher THC and lower CBD concentrations, medical cannabis contains THC and CBD in different ratios, along with minor phytocannabinoids, terpenes, flavonoids and other chemicals. A volumetric absorptive microsampling (VAMS) method combined with ultra-high-performance liquid chromatography coupled with mass spectrometry in tandem for quantification of CBD, THC and their respective metabolites: cannabidiol-7-oic acid (7-COOH-CBD); 7-hydroxy-cannabidiol (7-OH-CBD); 6-alpha-hydroxy-cannabidiol (6-α-OH-CBD); and 6-beta-hydroxycannabidiol (6-ß-OH-CBD); 11- Hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH). After overnight enzymatic glucuronide hydrolysis at 37°C, samples underwent acidic along with basic liquid-liquid extraction with hexane: ethyl acetate (9:1, v/v). Chromatographic separation was carried out on a C18 column, with the mass spectrometer operated in multiple reaction monitoring mode and negative electrospray ionization. Seven patients with intractable epilepsy were dosed with various CBD-containing formulations and blood collected just before their daily morning administration. The method was validated following international guidelines in toxicology. Linear ranges were (ng/ml) 0.5-25 THC, 11-OH-THC, THCCOOH, 6-α-OH-CBD and 6-ß-OH-CBD; 10-500 CBD and 7-OH-CBD; and 20-5000 7-COOH-CBD. 7-COOH-CBD was present in the highest concentrations, followed by 7-OH-CBD and CBD. This analytical method is useful for investigating CBD, THC and their major metabolites in epilepsy patients treated with CBD preparations employing a minimally invasive microsampling technique requiring only 30 µL blood.