Detection of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, and PV8 in blood in a forensic case using liquid chromatography-electrospray ionization linear ion trap mass spectrometry.

We present a case of fatal poisoning by 4-F-methcathinone (4-FMC; also called flephedrone), 4-methoxy-α-pyrrolidinopentiophenone (4-MeO-α-PVP), 4-fluoro-α-pyrrolidinopentiophenone (4-F-α-PVP), and α-pyrrolidinohepatanophenone (PV8). In this study, we compared the mass spectra of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, PV8, and α-pyrrolidinohexanophenone between LC-ESI-LIT-MS and GC-EI-MS analyses. Subsequently, we applied LC-ESI-LIT-MS for detection and quantification analyses of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, and PV8 in human authentic whole blood samples. More specific mass spectra for the target compounds were obtained with the LC-ESI-LIT-MS qualitative analyses than with the GC-EI-MS analyses, indicating that LC-ESI-LIT-MS was more suitable for the qualitative analysis of cathinones. The LC-ESI-LIT-MS validation data showed moderately good linearity and reproducibility for the compounds in the quantitative analyses at the range of 1-500 ng/mL. The detection limits of four cathinones ranged from 0.1 to 1 ng/mL. The concentrations of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, and PV8 in heart whole blood samples were 365, 449, 145, and 218 ng/mL, respectively. Those of the 4 cathinones in femoral vein whole blood samples were 397, 383, 127, and 167 ng/mL, respectively. We can then assume that the cause of death was acute poisoning by a combination of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, and PV8. In this article, we present a detailed LC-ESI-LIT-MS procedure for detection and quantification analyses of 4-FMC, 4-MeO-α-PVP, 4-F-α-PVP, and PV8 in authentic human whole blood samples.

Determination of New Psychoactive Substances in Whole Blood Using Microwave Fast Derivatization and Gas Chromatography/Mass Spectrometry.

The production and consumption of new psychoactive substances (NPSs) has been raising a major concern worldwide. Due to easy access and available information, many NPSs continue to be synthesized with an alarming increase of those available to purchase, despite all the control efforts created. A new analytical method was developed and validated to determine a group of phenethylamines and synthetic cathinones: cathinone, flephedrone, buphedrone, 4-MTA, α-PVP, methylone, 2C-P, ethylone, pentylone, MDPV and bromo-dragonFLY in whole blood. A mixed-mode solid phase extraction was applied to 250 µL of sample, and the extracts were derivatized with fast microwave technique before being analyzed by gas chromatography-mass spectrometry (GC-MS). The validation procedure followed the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines with parameters that included selectivity, linearity, limits of detection and quantification, intra- and inter-day precision and accuracy, recoveries and stability. The method presented linearity between 5 and 500 ng/mL for cathinone, buphedrone, 4-MTA, methylone, 2C-P and bromo-dragonFLY, 10-500 ng/mL for flephedrone, ethylone, pentylone and MDPV, and 40-500 ng/mL for α-PVP, with determination coefficients above 0.99 for all analytes. Recoveries ranged between 70.3% and 116.6%, and regarding intra- and inter-day precision, the relative mean errors were typically lower than 8.6%. The method was successfully applied to over 100 authentic samples from the Laboratory of Chemistry and Forensic Toxicology, Centre Branch, of the National Institute of Legal Medicine and Forensic Sciences, Portugal.

A case of intoxication with a mixture of synthetic cannabinoids EAM-2201, AB-PINACA and AB-FUBINACA, and a synthetic cathinone α-PVP.

We report a case of intoxication with a mixture of three synthetic cannabinoids and a synthetic cathinone, which have been disclosed by a highly sensitive progressing technology. A man was found dead, and his forensic autopsy was performed at our department. After further examinations of his specimens, EAM-2201 and α-PVP have been newly found in his lung. The concentrations of EAM-2201 have not been reported yet in any authentic human specimens although its existence (not quantified) in blood was reported in 2015. Therefore, a sensitive quantitation method of these compounds in blood and solid tissues has been devised using the sensitive instrument. The limits of detection of these compounds were in the range of 3-10 pg/ml with their quantification range of 10-1000 pg/ml in blood. The femoral vein blood levels of EAM-2201 and AB-PINACA were 56.6 ±â€¯4.2 and 12.6 ±â€¯0.1 pg/ml, respectively, and AB-FUBINACA could be detected but not quantifiable in the blood specimens; α-PVP could not be detected. The standard addition method was employed for the quantification of these compounds in the lung, liver and kidney specimens. The lung levels of EAM-2201, AB-PINACA, AB-FUBINACA and α-PVP were 348 ±â€¯34, 355 ±â€¯30, 124 ±â€¯12 and 59.0 ±â€¯7.4 pg/g, respectively. In conclusion, in this study, the concentrations of EAM-2201 in authentic human specimens including blood and solid tissues and those of AB-PINACA and AB-FUBINACA in solid tissue specimens were quantified for the first time to our knowledge.

New challenges in toxicology of new psychoactive substances exemplified by fatal cases after UR-144 and UR-144 with pentedrone administration determined by LC-ESI-MS-MS in blood samples.

The topic of this paper relates to the study of cases involving the use of new psychoactive substances (NPS) from the classes of synthetic cannabinoids and cathinones, analyzed from multiple viewpoints including clinical and medico-legal perspectives. The paper investigates three fatal cases in which UR-144 and UR-144 with pentedrone identified in the bodies of victims during post-mortem examinations were responsible for the tragic consequences and proved to be the indirect cause of death. The victims were men aged 16, 22 and 40 years who used drugs, for example they smoked marijuana or its substitutes in the form of synthetic cannabinoids. In addition, all of them had behavioural problems. On account of emotional imbalance attributable probably to the presence of UR-144 (in one case) and a mixture of UR-144 and pentedrone (in the other two cases), two men committed suicide by jumping from a height and hanging, and one man had fatal accidental poisoning with pentedrone which was used to enhance the effect of previously used UR-144. The presence of UR-144 and pentedrone in the post-mortem material was analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS). The results of toxicological tests were analyzed with a focus on possible tragic side effects caused by the presence of UR-144 and UR-144 with pentedrone in the body.

Active vaccination attenuates the psychostimulant effects of α-PVP and MDPV in rats.

Recreational use of substituted cathinones continues to be an emerging public health problem in the United States; cathinone derivatives α-pyrrolidinopentiophenone (α-PVP) and 3,4-methylenedioxypyrovalerone (MDPV), which have been linked to human fatalities and show high potential for abuse liability in animal models, are of particular concern. The objective of this study was to develop an immunotherapeutic strategy for attenuating the effects of α-PVP and MDPV in rats, using drug-conjugate vaccines created to generate antibodies with neutralizing capacity. Immunoconjugates (α-PVP-KLH and MDPV-KLH) or the control carrier protein, keyhole limpet hemocyanin (KLH), were administered to groups (N = 12) of male Sprague-Dawley rats on Weeks 0, 2 and 4. Groups were administered α-PVP or MDPV (0.0, 0.25, 0.5, 1.0, 5.0 mg/kg, i.p.) in acute drug challenges and tested for changes in wheel activity. Increased wheel activity produced by α-PVP or MDPV in the controls was attenuated in the α-PVP-KLH and MDPV-KLH vaccinated groups, respectively. Rectal temperature decreases produced by MDPV in the controls were reduced in duration in the MDPV-KLH vaccine group. A separate group (N = 19) was trained to intravenously self-administer α-PVP (0.05, 0.1 mg/kg/inf) and vaccinated with KLH or α-PVP-KLH, post-acquisition. Self-administration in α-PVP-KLH rats was initially higher than in the KLH rats but then significantly decreased following a final vaccine booster, unlike the stable intake of KLH rats. The data demonstrate that active vaccination provides functional protection against the effects of α-PVP and MDPV, in vivo, and recommend additional development of vaccines as potential therapeutics for mitigating the effects of designer cathinone derivatives.

A fatal intoxication related to MDPV and pentedrone combined with antipsychotic and antidepressant substances in Cyprus.

This is a case report of a fatal intoxication in Cyprus related to 3,4-methylenedioxypyrovalerone (MDPV) and 2-(methylamino)-1-phenylpentan-1-one (pentedrone) intake combined with antipsychotic and antidepressant substances. A 42- year old man with a history of serious psychiatric illness was found unresponsive in his bed. Seized materials were also found close to his body. The forensic autopsy reported myocardial infarction due to multidrug intoxication. Toxicology screening in blood and urine was applied. Biological specimens were analysed by enzyme immunoassay procedure and GC/MS. MDPV, pentedrone and etizolam detected and quantitated in blood and urine. Other drugs quantitated in blood were also olanzapine, mirtazapine, and ephedrine. This was the first fatal case reported in Cyprus associated with new psychoactive substances. Additionally, this was the first case reported to Early Warning System of the European Monitoring Center of Drugs and Drug Abuse (EMCDDA), related to multidrug intoxication, attributed to the consumption of cathinones, designer benzodiazepines, and other drugs.

[Fatal poisoning of pentedron ­ cases reports].

The issue of sudden deaths due to acute pentedron poisoning is presented in the report. The analysis included three cases autopsied. Biological material were delivered to the Toxicological Laboratory ToxLab placed in Katowice, during the autopsy were subjected to chemical-toxicological analysis. Analysis of blood samples in the first case present concentration of the pentedrone were 385 ng/ml, and present the concentration of delta- 9-tetrahydrocannabinol were 2.6 ng/ml. Analysis of blood and urine samples in the next case present pentedrone concentrations were 280 ng/ml i 255 ng/ml. Analysis of blood samples in the third case present concentration of the pentedrone were 340 ng/ml.

Characterization of a Hemoglobin Adduct from Ethyl Vinyl Ketone Detected in Human Blood Samples.

Electrophiles have the ability to form adducts to nucleophilic sites in proteins and DNA. Internal exposure to such compounds thus constitutes a risk for toxic effects. Screening of adducts using mass spectrometric methods by adductomic approaches offers possibilities to detect unknown electrophiles present in tissues. Previously, we employed untargeted adductomics to detect 19 unknown adducts to N-terminal valine in hemoglobin (Hb) in human blood. This article describes the characterization of one of these adducts, which was identified as the adduct from ethyl vinyl ketone (EVK). The mean adduct level was 40 ± 12 pmol/g Hb in 12 human blood samples; adduct levels from acrylamide (AA) and methyl vinyl ketone (MVK) were quantified for comparison. Using l-valine p-nitroanilide (Val-pNA), introduced as a model of the N-terminal valine, the rate of formation of the EVK adduct was studied, and the rate constant determined to 200 M(-1)h(-1) at 37 °C. In blood, the reaction rate was too fast to be feasibly measured, EVK showing a half-life <1 min. Parallel experiments with AA and MVK showed that the two vinyl ketones react approximately 2 × 10(3) times faster than AA. The EVK-Hb adduct was found to be unstable, with a half-life of 7.6 h. From the mean adduct level measured in human blood, a daily dose (area under the concentration-time-curve, AUC) of 7 nMh EVK was estimated. The AUC of AA from intake via food is about 20 times higher. EVK is naturally present in a wide range of foods and is also used as a food additive. Most probably, naturally formed EVK is a major source to observed adducts. Evaluation of available toxicological data and information on occurrence of EVK indicate that further studies of EVK are motivated. This study illustrates a quantitative strategy in the initial evaluation of the significance of an adduct detected through adduct screening.

Identification of major metabolites of the catechol-O-methyltransferase inhibitor nitecapone in the rat and dog.

Metabolites of nitecapone [3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione], a potent catechol-O-methyltransferase inhibitor with gastroprotective and antiulcerogenic effects, were isolated by extraction and HPLC from dog and rat urine after enzymatic hydrolysis and as glucuronic acid and sulfate conjugates. Eight and 10 nonconjugated metabolites and unchanged nitecapone were found in hydrolyzed dog and rat urine, respectively, and identified by HPLC with diode-array UV detection, electron ionization mass spectrometry, and IR spectroscopy. In both species the main phase I metabolic pathways were: 1) reduction of the side chain carbon-carbon double bond and carbonyl groups and 2) cleavage of the side-chain double bond, giving an aromatic aldehyde that was partly oxidized to the corresponding carboxylic acid. These phase I metabolites and unchanged nitecapone were excreted in urine mainly as their glucuronides and sulfates in both species. Additionally, the 3-O-methylated metabolite, not found in urine, was identified in rat plasma.

Determination of nitecapone and (13C6)nitecapone in human plasma by gas chromatography/mass spectrometry.

A gas chromatographic/mass spectrometric method is developed and validated for simultaneous determination of nitecapone and its 13C6-labelled analogue in human plasma using (2H6,13C6)nitecapone as internal standard. The method involves extraction of the analytes from plasma to ethyl acetate-hexane mixture (20:80) and conversion to bis(trimethylsilyl) ethers prior to determination by gas chromatography/mass spectrometry using selected ion monitoring. The quantification range is 0.5-2000 ng ml-1. Precision ranges from 11.3% (coefficient of variation) at low levels to 2.4% at high levels. Recovery is about 50% in the whole range. The method is applied to a pharmacokinetic study where nitecapone diluted with 14C-labelled nitecapone is given intravenously concomitantly with an oral dose of (13C6)nitecapone.

Determination of a catechol-O-methyltransferase inhibitor, nitecapone, in human plasma and urine by liquid chromatography.

Methods based on reversed-phase liquid chromatography with amperometric detection have been developed for determination of nitecapone, 3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione, a COMT inhibitor, in human plasma and urine. Nitecapone was extracted with ethyl acetate-hexane mixtures from plasma after acidification with hydrochloric acid and from urine as the tetrabutylammonium ion-pair of its diphenylborate derivative. The recoveries of both methods exceeded 70% and the relative standard deviations for within-day precision were less than 4% and 8% at 50 ng ml-1 and at the quantitation limits, respectively. The methods are selective, sensitive and precise enough for determination of 4-5 ng ml-1 of nitecapone in plasma and urine and are thus suitable for the kind of pharmacokinetic studies exemplified in this paper.