Characterization of the designer drug deschloroketamine (2-methylamino-2-phenylcyclohexanone) by gas chromatography/mass spectrometry, liquid chromatography/high-resolution mass spectrometry, multistage mass spectrometry, and nuclear magnetic resonance.

RATIONALE: Clinical and forensic toxicology laboratories are challenged every day by the analytical aspects of the new psychoactive substances phenomenon. In this study we describe the analytical characterization of a new ketamine derivative, deschloroketamine (2-methylamino-2-phenylcyclohexanone), contained in seized powders. METHODS: The analytical techniques employed include gas chromatography/mass spectrometry (GC/MS), liquid chromatography/electrospray ionization coupled with Orbitrap high-resolution/MS (LC/ESI-HRMS), multistage MS (ESI-MS(n)), and NMR. The LC/ESI-HRMS analyses consisted of accurate mass measurements of MH(+) ions in full-scan mode; comparison of experimental and calculated MH(+) isotopic patterns; and examination of the isotopic fine structure (IFS) of the M + 1, M + 2, M + 3 isotopic peaks relative to the monoisotopic M + 0 peak. The collision-induced product ions of the MH(+) ions were studied by both HRMS and MS(n). (1)H and (13)C NMR measurements were carried out to confirm the chemical structure of the analyte. RESULTS: The EI mass spectra obtained by GC/MS analysis showed the presence of molecular ions at m/z 203, and main fragment ions at m/z 175, 174, 160, 147, 146, and 132. The application of LC/ESI-HRMS allowed us to obtain: the accurate mass of deschloroketamine MH(+) ions with a mass accuracy of 1.47 ppm; fully superimposable experimental and calculated MH(+) isotopic patterns, with a relative isotopic abundance value of 3.69 %; and the IFS of the M + 1, M + 2, M + 3 isotopic peaks completely in accordance with theoretical values. Examination of the product ions of MH(+), as well as the study of both (1)H and (13)C NMR spectra, enabled the full characterization of the molecular structure of deschloroketamine. CONCLUSIONS: The combination of the employed analytical techniques allowed the characterization of the seized psychoactive substance, in spite of the lack of a reference standard. Deschloroketamine is a ketamine analogue considered to be more potent and longer lasting than ketamine, and this paper is probably the first to report on its analytical characterization.

Determination of cannabinoids in 50 µL whole blood samples by online extraction using turbulent flow chromatography and LC-HRAM-Orbitrap-MS: Application on driving under the influence of drugs cases.

The analysis of cannabinoids in whole blood is usually done by traditional mass spectrometry (MS) techniques, after offline cleanup or derivatization steps which can be lengthy, laborious, and expensive. We present a simple, fast, highly specific, and sensitive method for the determination of Δ9 -tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), 11-hydroxy-Δ9 -tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THC-COOH) in 50 µL whole blood samples. After the addition of deuterated internal standards (IS) and a simple protein precipitation step, an online extraction of sample supernatants using turbulent flow chromatography (TurboFlow-Thermo Scientific) was carried out. Analytes were separated on a C18 analytical column and detected by LC-HRAM-Orbitrap-MS using a Thermo Scientific Q Exactive Focus MS system. MS detection was performed in polarity switching and selected ion monitoring (SIM) modes using five specific acquisition windows, at a resolution of 70,000 (FWHM). Total run time was about 10 min including preanalytical steps. Method validation was carried out by determining limit of detection (LOD), lower limit of quantitation (LLOQ), linearity range, analytical accuracy, intra-assay and interassay precision, carry-over, matrix effect, extraction recovery, and selectivity, for all analytes. Measurement uncertainties were also evaluated, and a decision rule was set with confidence for forensic purposes. The method may become suitable for clinical and forensic toxicology applications, taking advantage of the small matrix volume required, the simple and cost-effective sample preparation procedure, and the fast analytical run time. Performances were monitored over a long-term period and tested on 7620 driving under the influence of drugs (DUID) samples, including 641 positive samples.

Analytical Characterization of 3-MeO-PCP and 3-MMC in Seized Products and Biosamples: The Role of LC-HRAM-Orbitrap-MS and Solid Deposition GC-FTIR.

Among the phencyclidine (PCP) and synthetic cathinone analogs present on the street market, 3-methoxyphencyclidine (3-MeO-PCP) is one of the most popular dissociative hallucinogen drugs, while 3-methylmethcathinone (3-MMC) is a commonly encountered psychostimulant. Numerous 3-MeO-PCP- and 3-MMC-related intoxication cases have been reported worldwide. Identification of the positional isomers of MeO-PCP and MMC families are particularly challenging for clinical and forensic laboratories; this is mostly due to their difficult chromatographic separation (particularly when using liquid chromatography-LC) and similar mass spectrometric behaviors. 3-MeO-PCP and 3-MMC were identified in two powders, detained by two subjects and seized by the police, by different analytical techniques, including liquid chromatography-high-resolution accurate-mass Orbitrap mass spectrometry (LC-HRAM-Orbitrap-MS), and solid deposition gas chromatography-Fourier transform infrared spectroscopy (sd-GC-FTIR). LC-HRAM-Orbitrap-MS allowed us to assign the elemental formulae C18H27NO (MeO-PCP) and C11H15NO (MMC) through accurate mass measurement of the two MH+ ions, and the comparison of experimental and calculated MH+ isotopic patterns. However, MH+ collision-induced product ions spectra were not conclusive in discriminating between the positional isomers [(3-MeO-PCP vs. 4-MeO-PCP) and (3-MMC vs. 4-MMC and 2-MMC)]. Likewise, sd-GC-FTIR easily allowed us to differentiate between the MeO-PCP and MMC positional isomers unambiguously, confirming the presence of 3-MeO-PCP and 3-MMC, due to the high-quality match factor of the experimental FTIR spectra against the target FTIR spectra of MeO-PCP and MMC isomers in a dedicated library. 3-MeO-PCP (in contrast to 3-MMC) was also detected in blood and urine samples of both subjects and analyzed in the context of routine forensic casework by LC-HRAM-Orbitrap-MS following a simple deproteinization step. In addition, this untargeted approach allowed us to detect dozens of phase I and phase II 3-MeO-PCP metabolites in all biological specimens. Analysis of the extracted samples by sd-GC-FTIR revealed the presence of 3-MeO-PCP, thus confirming the intake of such specific methoxy-PCP isomer in both cases. These results highlight the effectiveness of LC-HRAM-Orbitrap-MS and sd-GC-FTIR data in attaining full structural characterization of the psychoactive drugs, even in absence of reference standards, in both non-biological and biological specimens.

Analytically Confirmed Intoxication by 4-Fluoromethylphenidate, an Analog of Methylphenidate.

4-Fluoromethylphenidate (4F-MPH) is an halogenated derivative of methylphenidate (MPH), a re-uptake inhibitor for dopamine and norepinephrine used for the treatment of attention deficit hyperactivity disorders. In the last few years, several compounds structurally related to MPH have been marked as new psychoactive substances (NPS) with stimulating and euphoric effects similar to the parent drug, but with more dopaminergic activity. This report represents the first case of an analytically confirmed non-fatal intoxication by 4F-MPH. A 26-year-old female was admitted to the emergency department with neuropsychiatric and cardiologic symptoms that lasted for a week, during which she sniffed a powder named 4F-MPH acquired as entactogen on the Internet. The patient required sedation with intravenous diazepam and was discharged two days later with a prescription of promazine and quetiapine. The seized product was analytically characterized by gas chromatography-mass spectrometry, liquid chromatography high-resolution mass spectrometry and nuclear magnetic resonance. These analyses confirmed the composition of the product as a 4F-MPH diastereomeric (±)-threo and (±)-erythro mixture, with a large preponderance of the active (±)-threo isomer. A minimal validation, intended for rare analytes, was performed for the quantification of 4F-MPH in the biological samples by liquid chromatography-tandem mass spectrometry. Accuracy (bias) and precision were within ±15% for both blood and urine. The blood and urine concentration of (±)-threo 4F-MPH were 32 ng/mL and 827 ng/mL, respectively. Analyses for classic drugs (opiates, methadone, cocaine, cannabis metabolites, amphetamines, ecstasy and LSD), ethanol, qualitative full screen by gas chromatography-mass spectrometry and targeted analysis for 50 NPS by liquid chromatography-tandem mass spectrometry tested negative; comorbidities were excluded, too. Based on these data, it can be assumed that the clinical manifestations were due to 4F-MPH only.

Detection of 3-methylmethcathinone and its metabolites 3-methylephedrine and 3-methylnorephedrine in pubic hair samples by liquid chromatography-high resolution/high accuracy Orbitrap mass spectrometry.

Hair testing is considered to be one of the most efficient tool to investigate drug-related histories, particularly when the period of use needs to be tested back to many days or even months before sampling. High-resolution mass spectrometry represents today one of the most specific and sensitive analytical techniques to detect psychoactive substances in hair samples following single or multiple drug exposures. In this study pubic hair testing, by means of liquid chromatography-high resolution/high accuracy Orbitrap mass spectrometry, was employed to document the potential intake of five new psychoactive substances by a drug dealer. Pubic hair samples were decontaminated and pulverized with a ball mill, and, after the addition of the internal standard 3,4-methylenedioxypropylamphetamine, extracted with methanol:trifluoroacetic acid 9:1 at 45°C for one night. The obtained extracts were analyzed on a Thermo Fisher Scientific Accela 1250 liquid chromatography system coupled to a Thermo Fisher Scientific single-stage Exactive HCD mass spectrometry system. 3-methylmethcathinone (3-MMC) was found to be present at a concentration of 25.8ng/mg in the pubic hair sample, whereas the other four designer drugs were found to be absent. 3-methylephedrines and 3-methylnorephedrines, metabolites of 3-MMC, were identified in the same sample, thereby proving the 3-MMC intake by the drug dealer.

UHPLC-MS/MS and UHPLC-HRMS identification of zolpidem and zopiclone main urinary metabolites and method development for their toxicological determination.

Zolpidem and zopiclone (Z-compounds) are non-benzodiazepine hypnotics of new generation that can be used in drug-facilitated sexual assault (DFSA). Their determination in biological fluids, mainly urine, is of primary importance; nevertheless, although they are excreted almost entirely as metabolites, available methods deal mainly with the determination of the unmetabolized drug. This paper describes a method for the determination in urine of Z-compounds and their metabolites by ultra-high-pressure liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) and UHPLC coupled with high resolution/high accuracy Orbitrap® mass spectrometry (UHPLC-HRMS). The metabolic profile was studied on real samples collected from subjects in therapy with zolpidem or zopiclone; the main urinary metabolites were identified and their MS behaviour studied by MS/MS and HRMS. Two carboxy- and three hydroxy- metabolites, that could be also detected by gas chromatography/mass spectrometry (GC-MS) as trimethylsylyl derivatives, have been identified for zolpidem. Also, at least one dihydroxilated metabolite was detected. As for zopiclone, the two main metabolites detected were N-demethyl and N-oxide zopiclone. For both substances, the unmetabolized compounds were excreted in low amounts in urine. In consideration of these data, a UHPLC-MS/MS method for the determination of Z-compounds and their main metabolites after isotopic dilution with deuterated analogues of zolpidem and zopiclone and direct injection of urine samples was set up. The proposed UHPLC-MS/MS method appears to be practically applicable for the analysis of urine samples in analytical and forensic toxicology cases, as well as in cases of suspected DFSA.

Variability of blood alcohol content (BAC) determinations: the role of measurement uncertainty, significant figures, and decision rules for compliance assessment in the frame of a multiple BAC threshold law.

The measurement of blood-alcohol content (BAC) is a crucial analytical determination required to assess if an offence (e.g. driving under the influence of alcohol) has been committed. For various reasons, results of forensic alcohol analysis are often challenged by the defence. As a consequence, measurement uncertainty becomes a critical topic when assessing compliance with specification limits for forensic purposes. The aims of this study were: (1) to investigate major sources of variability for BAC determinations; (2) to estimate measurement uncertainty for routine BAC determinations; (3) to discuss the role of measurement uncertainty in compliance assessment; (4) to set decision rules for a multiple BAC threshold law, as provided in the Italian Highway Code; (5) to address the topic of the zero-alcohol limit from the forensic toxicology point of view; and (6) to discuss the role of significant figures and rounding errors on measurement uncertainty and compliance assessment. Measurement variability was investigated by the analysis of data collected from real cases and internal quality control. The contribution of both pre-analytical and analytical processes to measurement variability was considered. The resulting expanded measurement uncertainty was 8.0%. Decision rules for the multiple BAC threshold Italian law were set by adopting a guard-banding approach. 0.1 g/L was chosen as cut-off level to assess compliance with the zero-alcohol limit. The role of significant figures and rounding errors in compliance assessment was discussed by providing examples which stressed the importance of these topics for forensic purposes.

Pneumatically assisted desorption/ionization: 1. Some thoughts on the possible ionization mechanism(s).

We have observed that spraying solvent droplets on a zopiclone tablet produced MH(+) ions also in the absence of any electrical field and without the addition of organic acids to the sprayed solvent. The choice of a drug tablet as test bench has been done for the signal stability, higher than that observed when the drug is directly placed on a stainless steel surface. This behavior indicates that the formation of MH(+) ions is mainly due to pneumatical effects and the results are discussed with respect to those obtained by other research groups. Different mechanisms contributing to MH(+) production under these conditions are proposed and discussed. The local heating of the solvent thin layer present on the surface has been calculated and the small temperature increase (and the consequent small decrease of pK(a) value) suggests that this effect can play only a minor role. However, different solvents have been employed for studying this aspect and, quite surprisingly, the best results have been obtained with acetonitrile (ACN). Experiments performed by spraying CD(3)CN showed again the formation of MH(+) and not MD(+), and this excludes the role of ACN as protonating medium. A further thought was stimulated by the behavior observed by varying the sheath gas (N(2)) flow, showing that the MH(+) ion intensity increases by increasing the flow. Side effects related to the highest kinetic energy of the spraying droplets can be considered, but an active role of N(2) in the MH(+) formation could be taken into account, by considering the possible ionization of N(2) by collisional phenomena. The N(2)(+*) ions could undergo a charge-exchange reaction with analyte molecules leading to a short-lived odd electron ion which behaves as protonating media for neutral molecules. The above-described mechanism does not require either the presence of electrical fields nor the addition of organic acid to the sprayer solvent and can give a rationale for what was observed when only pneumatical conditions are employed.

A case of beta-carboline alkaloid intoxication following ingestion of Peganum harmala seed extract.

Beta-carboline alkaloids harmine, harmaline, and tetrahydroharmine can stimulate the central nervous system by inhibiting the metabolism of amine neurotransmitters, or by direct interaction with specific receptors; they are found in numerous plants, including Peganum harmala, Passiflora incarnata and Banisteriopsis caapi, and in the entheogen preparation Ayahuasca, which is traditionally brewed using B. caapi to enhance the activity of amine hallucinogenic drugs. The ingestion of plant preparations containing beta-carboline alkaloids may result in toxic effects, namely visual and auditory hallucinations, locomotor ataxia, nausea, vomiting, confusion and agitation. We report a case of intoxication following intentional ingestion of P. harmala seed infusion; P. harmala seeds were bought over the Internet. The harmala alkaloids were identified by gas chromatography-mass spectrometry in the seed extract and the patient's urine. This is, to our knowledge, the first case of P. harmala intoxication corroborated by toxicological findings.

An improved method for cyanide determination in blood using solid-phase microextraction and gas chromatography/mass spectrometry.

A new method is described for the qualitative and quantitative analysis of cyanide, a very short-acting and powerful toxic agent, in human whole blood. It involves the conversion of cyanide into hydrogen cyanide and its subsequent headspace solid-phase microextraction (HS-SPME) and detection by gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring (SIM) mode. Optimizing the conditions for the GC/MS (type of column, injection conditions, temperature program) and SPME (choice of SPME fiber, effect of salts, adsorption and desorption times, adsorption temperature) led to the choice of a 75-microm carboxen/polydimethylsiloxane SPME fiber, with D3-acetonitrile as internal standard, and a capillary GC column with a polar stationary phase. Method validation was carried out in terms of linearity, precision and accuracy in both aqueous solutions and blood. The limit of detection (LOD) and limit of quantitation (LOQ) were determined only in aqueous solutions. The assay is linear over three orders of magnitude (water 0.01-10, blood 0.05-10 microg/mL); and the LOD and LOQ in water were 0.006 and 0.01 microg/mL, respectively. Good intra- and inter-assay precision was obtained, always <8%. The method is simple, fast and sensitive enough for the rapid diagnosis of cyanide intoxication in clinical and forensic toxicology.