Consumer expectations, drug effects, price and purity of heroin and cocaine purchased at drug consumption rooms.

BACKGROUND: Drug consumption rooms offer heroin and cocaine consumers a secure and hygienic environment including medical and social guidance. Despite the support and mentoring, only sparse information is available about how drug quality, drug prices and user expectations match at these locations. The present study reports analysis of these three parameters in two drug consumption rooms in Luxembourg. METHODS: Drug users were invited to participate in the project by handing in a few milligrams of the product they planned to consume for chemical analysis and filling out a short questionnaire about the price and their expectations. After consumption, they were asked to report the experienced effects. Drug quality was accessed using LC-Q-ToF and HPLC-UV, and a statistical analysis was carried out of the questionnaires that were correctly filled out. RESULTS: A total of 513 drug samples have been analyzed. Most consumers were looking for the relaxing/calming effects of heroin and the stimulating effects of cocaine, but they generally overestimated heroin potency and underestimated cocaine potency. No strong correlation based on Spearman's ρ between drug user estimations, drug prices and drug quality was found. CONCLUSION: To the best of our knowledge, this study is the first to combine drug analysis with heroin and cocaine user feedback about expectation, drug prices and drug effects. The analytical results were of great interest for users and the staff working at the drug consumption rooms. They may be a strong supplementary communication tool for health care workers when discussing effects and risks of highly toxic substance consumption.

Direct In Vivo Analysis of CBD- and THC-Acid Cannabinoids and Classification of Cannabis Cultivars Using SpiderMass.

In recent years, cannabis and hemp-based products have become increasingly popular for recreational use, edibles, beverages, health care products, and medicines. The rapid detection and differentiation of phytocannabinoids is, therefore, essential to assess the potency and the therapeutic and nutritional values of cannabis cultivars. Here, we implemented SpiderMass technology for in vivo detection of cannabidiolic acid (CBDA) and ∆9-tetrahydrocannabinolicacid (∆9-THCA), and other endogenous organic plant compounds, to access distribution gradients within the plants and differentiate between cultivars. The SpiderMass system is composed of an IR-laser handheld microsampling probe connected to a mass spectrometer through a transfer tube. The analysis was performed on different plant organs from freshly cultivated cannabis plants in only a few seconds. SpiderMass analysis easily discriminated the two acid phytocannabinoid isomers via MS/MS, and the built statistical models differentiated between four cannabis cultivars. Different abundancies of the two acid phytocannabinoids were found along the plant as well as between different cultivars. Overall, these results introduce direct analysis by SpiderMass as a compelling analytical alternative for rapid hemp analysis.

Investigation on heroin and cocaine quality in Luxembourg.

BACKGROUND: Heroin and cocaine are among the most dangerous illicit drugs available and their presence on the market is increasing. These facts have led to the investigation of the quality of heroin and cocaine samples seized in Luxembourg by police and customs but also collected at the national supervised drug consumption facilities. METHODS: Samples obtained from 2019 to 2020 were analyzed to determine their composition and content using GC-MS, HPLC-UV and LC-Q-ToF. The statistical evaluation of concentration changes depending on the source of collection is based on an ANOVA single factor test and a two-tailed t test. RESULTS: Results showed important differences between seizure and collection sources. For both drugs, customs samples had significantly higher concentrations than police samples and the latter had significantly higher concentrations than samples from drug consumption facilities, whereas for heroin two cutting steps were identified, for cocaine samples only one appears to occur on the local market. Indeed, cocaine samples seized by police consisted of a mixture of low and high concentration samples. CONCLUSION: The results show that extensive adulteration with pharmacological active and inactive compounds takes place at local levels, which, however, are different for heroin and cocaine. This knowledge on variability of quality of drugs should be considered in the elaboration of drug and harm prevention strategies.

Cooking heroin the Turkish way: chemical investigation on an unusual heroin preparation method.

BACKGROUND: Reports from experienced heroin users about an alternative and appreciated but harmful so-called "Turkish" heroin preparation technic led to the chemical investigation of the compounds produced during this process and investigation of the presence of other psychoactive contaminants. METHODS: Comparison of diacetylmorphine, 6-monoacetylmorphine, morphine, paracetamol and caffeine concentrations were performed in the non-processed material, after processing according to the standard and to the alternative preparation methods using liquid chromatography coupled to quadrupole time of flight mass spectrometry followed by statistical evaluation of the results. RESULTS: The two preparation methods had in common a diminution of diacetylmorphine as compared to the starting material but significantly more 6-monoacetylmorphine was produced using the "Turkish" preparation method as compared to the standard method. CONCLUSION: The high amount of psychoactive 6-monoacetylmorphine may have an impact on the reported effects of heroin using the "Turkish" preparation procedure.

Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p.

The ATP binding cassette (ABC) transporters Pdr11p and its paralog Aus1p are expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and are required for sterol uptake. However, the precise mechanism by which these ABC transporters facilitate sterol movement is unknown. In this study, an overexpression and purification procedure was developed with the aim to characterise the Pdr11p transporter. Engineering of Pdr11p variants fused at the C terminus with green fluorescent protein (Pdr11p-GFP) and containing a FLAG tag at the N terminus facilitated expression analysis and one-step purification, respectively. The detergent-solubilised and purified protein displayed a stable ATPase activity with a broad pH optimum near 7.4. Mutagenesis of the conserved lysine to methionine (K788M) in the Walker A motif abolished ATP hydrolysis. Remarkably, and in contrast to Aus1p, ATPase activity of Pdr11p was insensitive to orthovanadate and not specifically stimulated by phosphatidylserine upon reconstitution into liposomes. Our results highlight distinct differences between Pdr11p and Aus1p and create an experimental basis for further biochemical studies of both ABC transporters to elucidate their function.

Parallel reaction monitoring using quadrupole-Orbitrap mass spectrometer: Principle and applications.

Targeted mass spectrometry-based approaches are nowadays widely used for quantitative proteomics studies and more recently have been implemented on high resolution/accurate mass (HRAM) instruments resulting in a considerable performance improvement. More specifically, the parallel reaction monitoring technique (PRM) performed on quadrupole-Orbitrap mass spectrometers, leveraging the high resolution and trapping capabilities of the instrument, offers a clear advantage over the conventional selected reaction monitoring (SRM) measurements executed on triple quadrupole instruments. Analyses performed in HRAM mode allow for an improved discrimination between signals derived from analytes and those resulting from matrix interferences translating in the reliable quantification of low abundance components. The purpose of the study defines various implementation schemes of PRM, namely: (i) exploratory experiments assessing the detectability of very large sets of peptides (100-1000), (ii) wide-screen analyses using (crude) internal standards to obtain statistically meaningful (relative) quantitative analyses, and (iii) precise/accurate quantification of a limited number of analytes using calibrated internal standards. Each of the three implementation schemes requires specific acquisition methods with defined parameters to appropriately control the acquisition during the actual peptide elution. This tutorial describes the different PRM approaches and discusses their benefits and limitations in terms of quantification performance and confidence in analyte identification.

A simple protocol to routinely assess the uniformity of proteomics analyses.

Mass-spectrometry-based proteomic approaches are increasingly applied to biological and clinical studies. Initially used by specialized laboratories, the technology has matured and gained acceptance by the community, using various analytical processes and platforms. To facilitate data comparison and integration across laboratories, there is a need to harmonize analytical processes to ensure the generation of reliable proteomic data sets. This is especially critical in the context of large initiatives, such as the Human Proteome Project promoted by the Human Proteome Organization (HUPO). Quality control is a first step toward the harmonization of proteomics data sets. We have developed a procedure to routinely assess the uniformity of proteomics analyses. It relies on a simple protocol based on three proteins and two sets of isotopically labeled peptides, one being added prior to tryptic digestion and the second one prior to liquid chromatography-mass spectrometry (LC-MS) analysis. The proposed method evaluates in a single step both the sample preparation, by measuring the relative amounts of endogenous peptides and their isotopically labeled counterparts, and the LC-MS platform performance, by monitoring the main LC-MS attributes for reference peptides. The procedure is simple and easy to implement into routine workflows typically employed by the proteomics community.

Technical considerations for large-scale parallel reaction monitoring analysis.

Targeted methods have gained acceptance among proteomics community to perform quantitative experiments. However, the current reference to conduct such experiments relies on selected reaction monitoring (SRM) analyses performed on triple quadrupole mass spectrometers, although it suffers from some limitations. First, the low resolution quadrupole mass analyzers do not present enough selectivity to discriminate the analytes from interferences commonly encountered in biological samples. Second, the number of peptides monitored in one single experiment often remains limited. The introduction of high resolution/accurate mass instruments with fast sequencing capabilities has enabled the development of novel quantitative methods. More specifically, the new quadrupole-orbitrap mass spectrometer operated in parallel reaction monitoring (PRM) mode showed detection and quantification performances similar or better than those obtained in SRM, due to the increased selectivity of the high-resolution orbitrap mass analyzer. The versatility of the instrument, with its ability to multiplex the selection of precursor ions and to operate with varying quadrupole isolation windows, has enabled the design of large-scale experiments, which require the optimization of several acquisition parameters to maintain high performance. It includes the adjustments of the fill time of the trapping device and the tight scheduling of elution times of the peptides, ideally adjusted on-the-fly. BIOLOGICAL SIGNIFICANCE: The present study constitutes a valuable baseline for the proteomics community to better control the trade-off between sensitivity and number of analyzed peptides in large-scale parallel reaction monitoring (PRM) experiments performed on a quadrupole-orbitrap instrument. A standard acquisition method requires careful setting of the parameters, namely the fill time in accordance with the control of the resolving power and the degree of multiplexing on one hand, and the quadrupole selection window on the other hand. This study helps in establishing acquisition parameters for large-scale PRM experiments, while maintaining sufficient sensitivity. In addition, the real-time correction of the scheduled peptide monitoring windows, to compensate for possible elution time drift, was explored. This approach supports the use of narrowed monitoring windows in PRM analyses, which greatly scale up the number of peptides targeted in a single LC-MS experiment. The broad application of the presented approaches by the community is likely to allow the establishment of an unprecedented scale for targeted proteomics, thus matching the pressing demand of systems biology or biomarker evaluation studies. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes?

Rational design of a low molecular weight, stable, potent, and long-lasting GPR103 aza-ß3-pseudopeptide agonist.

26RFa, a novel RFamide neuropeptide, is the endogenous ligand of the former orphan receptor GPR103. Intracerebroventricular injection of 26RFa and its C-terminal heptapeptide, 26RFa((20-26)), stimulates food intake in rodents. To develop potent, stable ligands of GPR103 with low molecular weight, we have designed a series of aza-ß(3)-containing 26RFa((20-26)) analogues for their propensity to establish intramolecular hydrogen bonds, and we have evaluated their ability to increase [Ca(2+)](i) in GPR103-transfected cells. We have identified a compound, [Cmpi(21),aza-ß(3)-Hht(23)]26RFa((21-26)), which was 8-fold more potent than 26RFa((20-26)) in mobilizing [Ca(2+)](i). This pseudopeptide was more stable in serum than 26RFa((20-26)) and exerted a longer lasting orexigenic effect in mice. This study constitutes an important step toward the development of 26RFa analogues that could prove useful for the treatment of feeding disorders.