Changes in the frequency and pattern of drugs detected among suspected drug users during the COVID-19 pandemic in Turkey.

From March 13 until May 31, 2020, a complete lockdown in Turkey was planned and implemented by the government of Turkey. The vulnerable population with substance use disorders was affected more than others due to the social isolation measures meant to control the pandemic. This study presents detailed and broad data on drug abuse in suspected cases during the first wave of the COVID-19 pandemic in Turkey and compares the frequencies and patterns of drug abuse before and during the pandemic. The samples were screened by liquid chromatography-high resolution mass spectrometry and liquid chromatography-tandem mass spectrometry. Blood and urine samples of suspected users (n = 9669) were analyzed for drugs of abuse during the first wave of the COVID-19 pandemic and compared with their results (n = 8727) obtained just before the COVID-19 pandemic. The use of party drugs, such as MDMA and cocaine, and the classic illicit drug cannabis was significantly decreased and followed the same trend after complete lockdown was over. In contrast, methamphetamine use increased significantly during the lockdown period and continued after the lockdown. Interestingly, the number of tests that were positive for pregabalin as a misused licit drug increased, and this increase continued after the lockdown. The results showed a significant increase in drug abuse cases and changes in drug abuse trends, with an alteration toward more easily obtainable and lower-priced drugs. Using more dangerous and easily available licit and illicit drugs may cause serious health problems.

In Vitro Phase I Metabolism of the Recently Emerged Synthetic MDMB-4en-PINACA and Its Detection in Human Urine Samples.

MDMB-4en-PINACA (methyl (S)-3,3-dimethyl-2-(1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido)butanoate) is a recently emerged synthetic cannabinoid in Turkey. MDMB-4en-PINACA was detected in herbal material investigated by the Council of Forensic Medicine, Istanbul Narcotics Department in Turkey in April 2019. MDMB-4en-PINACA was added to the drug abuse list and quickly reported in biological samples after its first detection. In this study, the in vitro metabolism of MDMB-4en-PINACA was investigated by using a pooled human liver microsomes (HLMs) assay and liquid chromatography-high-resolution mass spectrometry (LC-HRMS). MDMB-4en-PINACA (5 µmol/L) was incubated with HLMs for up to 1 h, and the metabolites were identified using LC-HRMS and software-assisted data mining. The in vivo metabolism was investigated by the analysis of 22 authentic urine samples and compared to the data received from the in vitro metabolism study. Less than 7.5% of the MDMB-4en-PINACA parent compound remained after the 1 h incubation. We identified 14 metabolites, which were formed via double bond oxidation, ester hydrolysis, N-dealkylation, hydroxylation, dehydrogenation and further oxidation to N-pentanoic acid or a combination of these reactions in vitro. In 10 urine samples (total n = 22), MDMB-4en-PINACA was detected as the parent drug. Three of the identified main metabolites, double bond oxidation in combination with ester hydrolysis and hydroxylation metabolite (M3), MDMB-4en-PINACA butanoic acid (M14) and monohydroxypentyl-MDMB-4en-PINACA (M12), were suggested as suitable urinary markers. In vitro screening of 2,150 authentic urine samples for these identified MDMB-4en-PINACA metabolites resulted in 56 cases of confirmed MDMB-4en-PINACA consumption (2.6%).

The fate of acetamiprid and its degradation during long-term storage of honey.

Acetamiprid (ATP) is a neonicotinoid widely used in agriculture, which is less toxic to honeybees and is transported into the hives via the contaminated nectar and pollen as well as physical contact. Although ATP degrades in animal systems mainly through N-desmethylacetamiprid (IM-2-1), it is degraded in honey of different origins mainly through (E)-N2-carbamoyl-)-N1-[6-chloro-3-pyridyl)methyl])-N1-methylacetamidine (IM-1-2). The decomposition of ATP in honey has been followed for storage times up to 180 days at 20°C, 30°C and 40°C to represent geographical regions with different temperatures. Subsequent degradation of IM-1-2 occurred and IM-1-4 was detected as a degradation product of IM-1-2. The apparent decomposition rate constants and half-lives of ATP in honey were determined. ATP degradation to IM-1-2 was faster in honey than the rates observed in aqueous solutions of pH 3.0-5.0. In order to follow actual ATP contamination in honey, IM-1-2 analysis is proposed to be carried out instead of the analyses of ATP and IM-2-1 for safety determination to ensure application of Good Agricultural Practice and to maintain a healthy environment.

Detection and quantification of 5F-ADB and its methyl ester hydrolysis metabolite in fatal intoxication cases by liquid chromatography-high resolution mass spectrometry.

5F-ADB (methyl 2-{[1-(5-fluoropentyl)-1H-indazole-3-carbonyl]amino}-3,3-dimethylbutanoate) is a frequently abused new synthetic cannabinoid that has been sold since at least the end of 2014 on the drug market. It has been classified as an illicit drug in most European countries, and also in Turkey, Japan, and the United States. In this study, 5F-ADB and its methyl ester metabolite were determined in the blood and urine samples taken from fatal cases using liquid chromatography-highresolution mass spectrometry (LC-HRMS). The extraction of samples was performed using a solid-phase extraction method, followed by LC-HRMS analysis. The method was fully validated for linearities, limits of detection (LODs), limits of quantification (LOQs), recoveries, matrix effects, process efficiencies, accuracies, precisions, and stabilities and was applied to 70 blood and 36 urine samples from fatal cases where 5F-ADB was the only drug detected. The LODs were between 0.08 and 0.10ng/mL, and LOQs were between 0.10 and 0.12ng/mL for both blood and urine samples. 5F-ADB and its methyl ester hydrolysis metabolite were found at the blood concentrations ranging from 0.10 to 1.55ng/mL (mean=0.40ng/mL) and 0.15 to 23.4ng/mL (mean=2.69ng/mL), respectively. 5F-ADB was not detected in any urine samples. 5F-ADBmethyl ester hydrolysis metabolite was detected in 35 urine samples with a detection range of 0.28-72.2ng/mL and a mean of 9.02ng/mL. The synthetic cannabinoid 5F-ADB and its methyl ester metabolite were identified and quantified in authentic human blood and/or urine specimens obtained from 70 fatal cases. The method was successfully applied to postmortem blood and urine samples.

Metabolic profiling of synthetic cannabinoid 5F-ADB by human liver microsome incubations and urine samples using high-resolution mass spectrometry.

5F-ADB (methyl 2-{[1-(5-fluoropentyl)-1H-indazole-3-carbonyl] amino}-3,3-dimethylbutanoate) is a frequently abused new synthetic cannabinoid that has been sold since at least the end of 2014 on the drug market and has been classified as an illicit drug in most European countries, as well as Turkey, Japan, and the United States. In this study, the in vitro metabolism of 5F-ADB was investigated by using pooled human liver microsomes (HLMs) assay and liquid chromatography-high-resolution mass spectrometry (LC-HRMS). 5F-ADB (5 µmol/L) was incubated with HLMs for up to 3 hours, and the metabolites were identified using LC-HRMS and software-assisted data mining. The in vivo metabolism was investigated by the analysis of 30 authentic urine samples and was compared to the data received from the in vitro metabolism study. Less than 3.3% of the 5F-ADB parent compound remained after 1 hour of incubation, and no parent drug was detected after 3 hours. We identified 20 metabolites formed via ester hydrolysis, N-dealkylation, oxidative defluorination, hydroxylation, dehydrogenation, further oxidation to N-pentanoic acid and glucuronidation or a combination of these reactions in vitro. In 12 urine samples (n = 30), 5F-ADB was detected as the parent drug. Three of the identified main metabolites 5F-ADB carboxylic acid (M20), monohydroxypentyl-5F-ADB (M17), and carboxypentyl ADB carboxylic acid (M8) were suggested as suitable urinary markers. The screening of 8235 authentic urine samples for identified 5F-ADB metabolites in vitro resulted in 3135 cases of confirmed 5F-ADB consumption (38%).

Detection of metabolites of the new synthetic cannabinoid CUMYL-4CN-BINACA in authentic urine samples and human liver microsomes using high-resolution mass spectrometry.

CUMYL-4CN-BINACA(1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide) is a recently introduced indazole-3-carboxamide-type synthetic cannabinoid (SC) that was detected in herbal incense seized by of the Council of Forensic Medicine, Istanbul Narcotics Department, in May 2016 in Turkey. Recently introduced SCs are not detected in routine toxicological analysis; therefore, analytical methods to measure these compounds are in demand. The present study aims to identify urinary marker metabolites of CUMYL-4CN-BINACA by investigating its metabolism in human liver microsomes and to confirm the results in authentic urine samples (n = 80). In this study, 5 µM CUMYL-4CN-BINACA was incubated with human liver microsomes (HLMs) for up to 3 hours, and metabolites were identified using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Less than 21% of the CUMYL-4CN-BINACA parent compound remained after 3 hours of incubation. We identified 18 metabolites that were formed via monohydroxylation, dealkylation, oxidative decyanation to aldehyde, alcohol, and carboxylic acid formation, glucuronidation or reaction combinations. CUMYL-4CN-BINACA N-butanoic acid (M16) was found to be major metabolite in HLMs. In urine samples CUMYL-4CN-BINACA was not detected; CUMYL-4CN-BINACA N-butanoic acid (M16) was major metabolite after ß-glucuronidase hydrolysis. Based on these findings, we recommend using M16 (CUMYL-4CN-BINACA N-butanoic acid), M8 and M11 (hydroxylcumyl CUMYL-4CN-BINACA) as urinary marker metabolites to confirm CUMYL-4CN-BINACA intake.

Identification of the Synthetic Cannabinoid 1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide (CUMYL-4CN-BINACA) in Plant Material and Quantification in Post-Mortem Blood Samples.

In May 2016, a new type of synthetic indazole-3-carboxamide cannabinoid (CUMYL-4CN-BINACA) was detected in seized plant material submitted to the Istanbul Council of Forensic Medicine by the National Police Office. The major ingredient in this material was purified using preparative liquid chromatography, and its structure was identified using liquid chromatography-high-resolution mass spectrometry (LC-HR/MS), gas chromatography-electron ionization/mass spectrometry (GC-EI/MS), nuclear magnetic resonance (NMR) spectroscopy and Fourier transform-infrared spectroscopy (FT-IR). Using HR-MS, the molecular formula of the compound was determined to be C22H24N4O (MW = 360.1950). The 1H and 13C-NMR and FT-IR spectrometric data revealed that the structure of compound was 1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide (CUMYL-4CN-BINACA). After identification, it was quickly added to our generic drug list, and an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method was developed to determine its presence in blood samples. This study reports on the identification of CUMYL-4CN-BINACA in plant material using LC-HR/MS, GC-EI/MS, NMR and FT-IR as well as a validated method for quantification of CUMYL-4CN-BINACA in post-mortem blood samples by UPLC-MS-MS analysis. The quantification method has been validated in terms of linearity (0.1-50 ng/mL), selectivity, intra- and inter-assay accuracy and precision (CV < 15%), recovery (94-99%), limit of detection (0.07 ng/mL) and limit of quantification (0.1 ng/mL). Matrix effects, stability and process efficiency were also assessed. The method has been applied to 2,350 post-mortem blood samples from the autopsy cases in the Morgue Department of the Council of Forensic Medicine (Istanbul, Turkey) between 1 July 2016 and 31 December 2016.

Application of a validated LC-MS/MS method for JWH-073 and its metabolites in blood and urine in real forensic cases.

Synthetic cannabinoids, which were synthesized to improve the therapeutic effects of cannabis, have become a major issue when they are abused. They have different chemical structures from tetrahydrocannabinol (THC) but similar effects on endocannabinoid receptors. "Spice" named products have more serious side effects than cannabis and can even cause death. These mixtures are prepared by spraying chemicals onto small pieces of herbs and are being dishonestly sold as "natural" and "legal" products over the internet. Their popularity is continuously increasing. Studies on detecting synthetic cannabinoids in biological samples as well as pharmacology and toxicology studies of these chemicals are very limited. A fast, specific and robust method for the detection and quantification of JWH-073, JWH-073 N-butanoic acid, and JWH-073 N-(4-hydroxybutyl) in blood and urine has been developed that uses solid-phase extraction (SPE) followed by UPLC-MS/MS analysis. This method has been validated in terms of its linearity (0.1-50 ng/mL), selectivity, intra-assay and inter-assay accuracy and precision (CV<10%), recovery (75-95%), limits of detection (LODs) (0.08-0.13 ng/mL), and limits of quantification (LOQs) (0.11-0.17 ng/mL). Matrix effects, stability, and process efficiency parameters of this method have also been assessed. This method was applied to 2596 authentic samples received by the Department of Toxicology (Istanbul) in the Presidency of Council of Forensic Medicine (Turkey) between September 1, 2012, and February 28, 2015.

Validation of JWH-018 and its metabolites in blood and urine by UPLC-MS/MS: Monitoring in forensic cases.

The herbal products referred to as 'Spice' have been used as 'legal alternatives' to cannabis worldwide since 2004. The first synthetic cannabinoid JWH-018 was detected in 'Spice' products in 2008, and has been banned by many legal authorities since the beginning of 2009. In order to prove use of JWH cannabinoids (JWHs), specific and robust methods were needed. We have developed a specific and reliable method for the detection and quantification of JWH-018, JWH-018 N-pentanoic acid, and JWH-018 N-(5-hydroxypentyl) in blood and urine using solid-phase extraction followed by UPLC-MS/MS analysis. The method has been validated in terms of linearity (0.1-50ng/mL), selectivity, intra-assay and inter-assay accuracy and precision (CV<15%), recovery (85-98%), limits of detection (LOD) (0.08-0.14ng/mL), and quantification (LOQ) (0.10-0.21ng/mL). Matrix effects, stability, and process efficiency were also assessed. The method has been applied to 868 authentic samples received by the Department of Chemistry (Istanbul) in the Council of Forensic Medicine of the Ministry of Justice.

Determination of acetamiprid and IM-1-2 in postmortem human blood, liver, stomach contents by HPLC-DAD.

An HPLC-DAD method was developed to detect and quantify a neonicotinoid insecticide acetamiprid (ATP) and its metabolite IM-1-2 in autopsy samples of a fatal intoxication case. The postmortem blood and tissue distribution of ATP and IM-1-2 was determined for the first time. The method showed acceptable precisions and recoveries with relative standard deviations of <10% for ATP level and 1.38 % for IM-1-2. The detection and quantification limits for ATP were 0.015 µg/mL and 0.030 µg/mL for blood and were 0.035 µg/g and 0.050 µg/g for liver samples, respectively. The mean contents of ATP were 0.79 µg/g in the liver, 47.35 µg/g in the stomach contents and 2.7 µg/mL in the blood. IM-1-2 content was 17.0 µg/g in the stomach contents. ATP and IM-1-2 were not detected in the urine. The presence of ATP and IM-1-2 in the samples was confirmed by GC-MS. The method can be exploited in future forensic casework.