Target and suspect screening of (new) psychoactive substances in South Korean wastewater by LC-HRMS.

New psychoactive substances (NPS) are a type of abused drug designed to mimic the effects of the currently known illicit drugs, whose structures are constantly changing to escape surveillance. The quick identification of NPS use in the community therefore demands immediate action. This study aimed to develop a target and suspect screening method using LC-HRMS to identify NPS in wastewater samples. An in-house database of 95 traditional and NPS was built using the reference standards, and an analytical method was developed. Wastewater samples were collected from 29 wastewater treatment plants (WWTP) across South Korea, representing 50 % of the total population. The psychoactive substances in waste water samples were screened using in-house database and developed analytical methods. A total of 14 substances were detected in the target analysis, including three NPS (N-methyl-2-AI, 25E-NBOMe, and 25D-NBOMe) and 11 traditional psychoactive substances and their metabolites (zolpidem phenyl-4-COOH, ephedrine, ritalinic acid, tramadol, phenmetrazine, phendimetrazine, phentermine, methamphetamine, codeine, morphine, and ketamine). Out of these, N-methyl-2-AI, zolpidem phenyl-4-COOH, ephedrine, ritalinic acid, tramadol, phenmetrazine, and phendimetrazine were detected with a detection frequency of over 50 %. Primarily, N-methyl-2-Al was detected in all the wastewater samples. Additionally, four NPSs (amphetamine-N-propyl, benzydamine, isoethcathinone, methoxyphenamine) were tentatively identified at level 2b in a suspect screening analysis. This is the most comprehensive study to investigate NPS using target and suspect analysis methods at the national level. This study raises a need for continuous monitoring of NPS in South Korea.

Organ distribution of diclazepam, pyrazolam and 3-fluorophenmetrazine.

The organ distribution of 3-fluorophenmetrazine (3-FPM), pyrazolam, diclazepam as well as its main metabolites delorazepam, lormetazepam and lorazepam, was investigated. A solid phase extraction (SPE) and a QuEChERS (acronym for quick, easy, cheap, effective, rugged and safe) - approach were used for the extraction of the analytes from human tissues, body fluids and stomach contents. The detection was performed on a liquid chromatography-tandem mass spectrometry system (LCMS/MS). The analytes of interest were detected in all body fluids and tissues. Results showed femoral blood concentrations of 10 µg/L for 3-FPM, 28 µg/L for pyrazolam, 1 µg/L for diclazepam, 100 µg/L for delorazepam, 6 µg/L for lormetazepam, and 22 µg/L for lorazepam. Tissues (muscle, kidney and liver) and bile exhibited higher concentrations of the mentioned analytes than in blood. Additional positive findings in femoral blood were for 2-fluoroamphetamine (2-FA, approx. 89 µg/L), 2-flourometamphetamine (2-FMA, hint), methiopropamine (approx. 2.2 µg/L), amphetamine (approx. 21 µg/L) and caffeine (positive). Delorazepam showed the highest ratio of heart (C) and femoral blood (P) concentration (C/P ratio = 2.5), supported by the concentrations detected in psoas muscle (430 µg/kg) and stomach content (approx. 210 µg/L, absolute 84 µg). The C/P ratio indicates that delorazepam displays susceptibility for post-mortem redistribution (PMR), supported by the findings in muscle tissue. 3-FPM, pyrazolam, diclazepam, lorazepam and lormetazepam did apparently not exhibit any PMR. The cause of death, in conjunction with autopsy findings was concluded as a positional asphyxia promoted by poly-drug intoxication by arising from designer benzodiazepines and the presence of synthetic stimulants.

Validation of an LC-MS/MS method for the quantitative analysis of 1P-LSD and its tentative metabolite LSD in fortified urine and serum samples including stability tests for 1P-LSD under different storage conditions.

A variety of hallucinogens of the lysergamide type has emerged on the drug market in recent years and one such uncontrolled derivative of lysergic acid diethylamide (LSD) is 1-propionyl-LSD (1P-LSD). Due to the high potency of LSD and some of its derivatives (common doses: 50-200 µg), sensitive methods are required for the analysis of biological samples such as serum and urine. The occurrence of an intoxication case required the development of a fully validated, highly sensitive method for the quantification of 1P-LSD and LSD in urine and serum using LC-MS/MS. Given that LSD is unstable in biological samples when exposed to light or elevated temperatures, we also conducted stability tests for 1P-LSD in urine and serum under different storage conditions. The validation results revealed that the analysis method was accurate and precise with good linearity over a wide calibration range (0.015-0.4 ng mL-1). The limit of detection (LOD) and the lower limit of quantification (LLOQ) of 1P-LSD and LSD in serum and urine were 0.005 ng mL-1 and 0.015 ng mL-1, respectively. The stability tests showed no major degradation of 1P-LSD in urine and serum stored at -20 °C, 5 °C or at room temperature for up to five days, regardless of protection from light. However, LSD was detected in all samples stored at room temperature showing a temperature-dependent hydrolysis of 1P-LSD to LSD to some extent (up to 21% in serum). Serum samples were particularly prone to hydrolysis possibly due to enzymatically catalyzed reactions. The addition of sodium fluoride prevented the enzymatic formation of LSD. The method was applied to samples obtained from the intoxication case involving 1P-LSD. The analysis uncovered 0.51 ng mL-1 LSD in urine and 3.4 ng mL-1 LSD in serum, whereas 1P-LSD remained undetected. So far pharmacokinetic data of 1P-LSD is missing, but with respect to the results of our stability tests and the investigated case rapid hydrolysis to LSD in-vivo seems more likely than instabilities of 1P-LSD in urine and serum samples.

Synthesis, analytical characterization, and monoamine transporter activity of the new psychoactive substance 4-methylphenmetrazine (4-MPM), with differentiation from its ortho- and meta- positional isomers.

The availability of new psychoactive substances (NPS) on the recreational drug market continues to create challenges for scientists in the forensic, clinical and toxicology fields. Phenmetrazine (3-methyl-2-phenylmorpholine) and an array of its analogs form a class of psychostimulants that are well documented in the patent and scientific literature. The present study reports on two phenmetrazine analogs that have been encountered on the NPS market following the introduction of 3-fluorophenmetrazine (3-FPM), namely 4-methylphenmetrazine (4-MPM), and 3-methylphenmetrazine (3-MPM). This study describes the syntheses, analytical characterization, and pharmacological evaluation of the positional isomers of MPM. Analytical characterizations employed various chromatographic, spectroscopic, and mass spectrometric platforms. Pharmacological studies were conducted to assess whether MPM isomers might display stimulant-like effects similar to the parent compound phenmetrazine. The isomers were tested for their ability to inhibit uptake or stimulate release of tritiated substrates at dopamine, norepinephrine and serotonin transporters using in vitro transporter assays in rat brain synaptosomes. The analytical characterization of three vendor samples revealed the presence of 4-MPM in two of the samples and 3-MPM in the third sample, which agreed with the product label. The pharmacological findings suggest that 2-MPM and 3-MPM will exhibit stimulant properties similar to the parent compound phenmetrazine, whereas 4-MPM may display entactogen properties more similar to 3,4-methylenedioxymethamphetamine (MDMA). The combination of test purchases, analytical characterization, targeted organic synthesis, and pharmacological evaluation of NPS and their isomers is an effective approach for the provision of data on these substances as they emerge in the marketplace.

Determination of stimulants using gas chromatography/high-resolution time-of-flight mass spectrometry and a soft ionization source.

RATIONALE: The aim of this study was to investigate the mass spectral fragmentation of a small set of stimulants in a high-resolution time-of-flight mass spectrometer equipped with a soft ionization source using vacuum ultraviolet (VUV) photons emitted from different plasma gases. It was postulated that the use of a plasma gas such as Xe, which emits photons at a lower energy than Kr or Ar, would lead to softer ionization of the test compounds, and thus to less fragmentation. METHODS: A set of nine stimulants: cocaine, codeine, nicotine, methadone, phenmetrazine, pentylenetetrazole, niketamide, fencamfamine, and caffeine, was analyzed by gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) in positive ion mode with this soft ionization source, using either Xe, Kr, or Ar as plasma gases. Working solutions of the test compounds at 0.1 to 100 ng/µL were used to establish instrument sensitivity and linearity. RESULTS: All test compounds, except methadone and pentylenetetrazole, exhibited strong molecular ions and no fragmentation with Xe-microplasma photoionization (MPPI). Methadone exhibited significant fragmentation not only with Xe, but also with Kr and Ar, and pentylenetetrazole could not be ionized with Xe, probably because its ionization energy is above 8.44 eV. The Kr- and Ar-MPPI mass spectra of the test compounds showed that the relative intensity of the molecular ion decreased as the photon energy increased. CONCLUSIONS: When coupled to a TOF mass spectrometer this soft ionization source has demonstrated signal-to-noise (S/N) ratios from 7 to 730 at 100 pg per injection (depending on the compound), and a dynamic range of three orders of magnitude (100 pg to 100 ng) for some of the test compounds.

Simultaneous analysis of psychotropic phenylalkylamines in oral fluid by GC-MS with automated SPE and its application to legal cases.

Phenylalkylamine derivatives, such as methamphetamine (MA), amphetamine (AM), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), phentermine (PT), fenfluramine (FFA) and phenmetrazine (PM), and ketamine (KT) are widely abused recreational or anorectic drugs in Korea and are regulated under the Controlled Substance Act in Korea. Phenylalkylamines and ketamine analysis is normally performed using both urine and hair samples but there is no established method for the simultaneous analysis of all these phenylalkylamines and ketamine in oral fluids. Oral fluid is easy to collect/handle and can provide an indication of recent drug abuse. In this study, to confirm the presence of phenylalkylamine derivatives and ketamine in oral fluid after screening with an immunoassay, an analytical method using automated solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) was developed and fully validated according to international guidelines. The applicability of the assay was demonstrated by analyzing of authentic oral fluid samples and the results of oral fluid analysis were compared with those in urine and hair to to evaluate the feasibility of oral fluid in forensic cases. The recovery of phenylalkylamines and ketamine from oral fluid collection devices was also assessed. Oral fluid specimens from 23 drug abuse suspects submitted by the police were collected using Salivette (Sarstedt, Nümbrecht, Germany), Quantisal (Immunalysis, Pomona, CA) or direct expectoration. The samples were screened using a biochip array analyzer (Evidence Investigator, Randox, Antrim, UK). For confirmation, the samples were analyzed by GC-MS in selected-ion monitoring (SIM) mode after extraction using automated SPE (RapidTrace, Zymark, MA, USA) with a mixed-mode cation exchange cartridge (CLEAN SCREEN, 130 mg/3 ml, UCT, PA, USA) and derivatization with trifluoroacetic anhydride (TFA). The results from the immunoassay were consistent with those from GC-MS. Twenty oral fluid samples gave positive results for MA, AM, PT and/or PM among the 23 cases, which gave positive results in urine and/or hair. Although large variations in the MA, AM, PT and PM concentrations were observed in three different specimens, the oral fluid specimen was useful for demonstrating phenylalkylamines and ketamine abuse as an alternative specimen for urine.

Determination of Phenmetrazine in urine by gas chromatography-mass spectrometry.

A sensitive and simple gas chromatographic--mass spectrometric method is described for the determination of the central nervous system (CNS) stimulant phenmetrazine in urine. The extraction and derivatization were combined into a single step with isooctane and methyl chloroformate. The limit of quantitation was 0.05 micrograms/mliters urine, and the method was linear up to 100 micrograms/mliters. The coefficients of variation (CV) for within-day runs were 1.2% and 2.4% (n = 5) for two controls containing 1.0 micrograms/mliters and 50 micrograms/mliters, respectively. During a six-month period, the same controls showed CVs of 9.1% and 8.7%, respectively (n = 40), indicating a somewhat lower between-run precision. Phenmetrazine was present in 83 out of 3000 urine samples that were screened for CNS stimulants during this period, and the concentrations ranged from 0.5-370 micrograms/mliters.

Negative-ion mass spectrometry of amphetamine congeners.

Negative-ion chemical ionization mass spectrometry with a conventional combined gas chromatograph-mass spectrometer has been used for the analysis of amphetamine-like compounds. Ammonia was used as the reagent gas, which gives rise to few but specific sample-fragment ions, such as (M--1)- as the base peak, m/z 91, and a smaller peak corresponding to the end part of the side chain. Five reference compounds and a urine sample from an overdose case were analyzed. Comparative positive-ion chemical ionization reference spectra were also recorded.

Cocaine-related death.

Cocaine use and abuse, an ancient custom, is once again commonplace. While severe toxicity appears to be rare, overt poisoning including death can occur. This report documents nine cases of death associated with cocaine use; in three of these cocaine appears to be causative. Toxicologic analysis of body fluids and tissues was affirmative and levels are reported. Cocaine should be considered in serious drug overdose-reactions, especially after illicit injection.

Comparative gas chromatographic analysis of narcotics. III. Phenmetrazine hydrochloride.

Chemical signatures of phenmetrazine hydrochloride were studied by gas chromatography. The inter-batch variations of the signatures were found to be large whereas the intra-batch variations were usually small. The method is used for the tracing of seized phenmetrazine hydrochloride samples to common sources, which in turn may permit further tracing back to chains of illicit distribution of this drug. The applicability of the method to other narcotics is also discussed.