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While state sequence analysis (SSA) has been long used in social sciences, its use in pharmacoepidemiology is still in its infancy. Indeed, this technique is relatively easy to use, and its intrinsic visual nature may help investigators to untangle the latent information within prescription data, facilitating the individuation of specific patterns and possible inappropriate use of medications. In this paper, we provide an educational primer of the most important learning concepts and methods of SSA, including measurement of dissimilarities between sequences, the application of clustering methods to identify sequence patterns, the use of complexity measures for sequence patterns, the graphical visualization of sequences, and the use of SSA in predictive models. As a worked example, we present an application of SSA to opioid prescription patterns in patients with non-cancer pain, using real-world data from Italy. We show how SSA allows the identification of patterns in prescriptions in these data that might not be evident using standard statistical approaches and how these patterns are associated with future discontinuation of opioid therapy.

Interest in research into bioactive peptides (BPs) is growing because of their health-promoting ability. Several bioactivities have been ascribed to peptides, including antioxidant, antihypertensive and antimicrobial properties. As they can be produced from precursor proteins, the investigation of BPs in foods is becoming increasingly popular. For the same reason, production of BPs from by-products has also emerged as a possible means of reducing waste and recovering value-added compounds suitable for functional food production and supplements. Milk, meat and fish are the most investigated sources of BPs, but vegetable-derived peptides are also of interest. Vegetables are commonly consumed, and agro-industrial wastes constitute a cheap, large and lower environmental impact source of proteins. The use of advanced analytical techniques for separation and identification of peptides would greatly benefit the discovery of new BPs. In this context, this review provides an overview of the most recent applications in BP investigations for vegetable food and by-products. The most important issues regarding peptide isolation and separation, by single or multiple chromatographic techniques, are discussed. Additionally, problems connected with peptide identification in plants and non-model plants are discussed regarding the particular case of BP identification. Finally, the issue of peptide validation to confirm sequence and bioactivity is presented.

To the Editor: We previously reported on an alternative activity-based screening method to detect (synthetic) opioids/opiates in blood samples (1) in which ^-opioid receptor activation could be monitored via a splitluciferase system. Because it may be difficult to determine whether a person with a (potential) opioid overdose received an antidote, our bioassay should also be capable of assessing whether an antagonist is present in the sample because these may conceal the presence of opioid agonists. Author Contributions: Allauthors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation ofdata; (b) drafting or revising the article for intellectual content; (c) final approval ofthe published article; and (d) agreement to be accountable for all aspects ofthe article thus ensuringthatquestions related to the accuracy or integrity ofany part ofthe article are appropriately investigated and resolved.

Dried matrix spot (DMS) techniques have gained increasing attention in bioanalytical and forensic toxicology for the detection of opiates and opioids, offering minimally invasive sampling, enhanced sample stability, and simplified storage and transport. This review provides a critical overview of recent methodological advances and applications of DMS across multiple biological matrices, including blood, plasma, urine, and oral fluid. Particular focus is given to sample preparation protocols, extraction strategies, analytical instrumentation, and method performance. Dried blood spots (DBS) remain the most established format; however, alternative matrices such as dried plasma, urine, and saliva spots (DPS, DUS, DSS) are expanding the scope of DMS, particularly in decentralised and point-of-care contexts. Despite clear advantages, such as reduced biohazard risk and compatibility with high-throughput workflows, several limitations persist, including low sample volumes, matrix-specific recovery issues, and lack of standardised procedures. Future efforts should aim to optimise paper substrates, improve solvent–matrix compatibility, and integrate DMS workflows with automated or miniaturised mass spectrometry platforms. Overall, DMS techniques represent a versatile and evolving analytical platform with strong potential for reliable opioid monitoring in both clinical and forensic settings.

Magnetic graphene nanoparticles coated with a new deep eutectic solvent (Fe 3 O 4 @GO-DES) were developed for efficient preconcentration of methadone. The extracted methadone was then analyzed by gas chromatography–flame ionization detection (GC–FID) or gas chromatography–mass spectrometry (GC–MS). Fe 3 O 4 @GO-DES were characterized by Fourier transform IR and X-ray diffraction techniques. Ultrasound was used to enhance the dispersion of the sorbent, with a high extraction recovery. Some parameters affecting the extraction recovery, such as pH, type of deep eutectic solvent, sample volume, amount of sorbent, extraction time, and type of eluent, were investigated. Under optimum conditions, the method developed was linear in the concentration range from 3 to 45,000 μg L -1 for GC–FID and from 0.1 to 500 μg L -1 for GC–MS, with a detection limit of 0.8 μg L -1 for GC–FID and 0.03 μg L -1 for GC–MS. The relative standard deviations ( n  = 6) as the intraday and interday precisions of the methadone spike at a concentration of 100 μg L -1 were 5.8% and 8.4% respectively for GC–FID. The preconcentration factor was 250. Relative recoveries from spiked plasma, urine, and water samples ranged from 95.1% to 101.5%.

Novel synthetic opioids (NSOs) represent an emerging group of novel psychoactive substances, acting as agonists at the opioid receptors. NSOs include fentanyl-related compounds, e.g. methoxyacetylfentanyl (MeACF), and non-fentanyl analogs, e.g. “U compounds” including U-47700. Here we present three cases of death involving MeACF and U-47700, with particular reference to preliminary data on pharmacokinetics and tissue distribution.After a complete post-mortem examination, general unknown screenings and analysis of drugs of abuse were performed on postmortem samples by immunoassays, gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry. To quantify the analytes of interest in post-mortem blood and tissues, the standard addition method was used. A toxicological significance score (TSS), weighing the role of the NSO in each death case, was assigned.Case 1 died at the hospital after consumption of U-47700, methadone (serum levels: 2,600 ng/ml and 37 ng/ml), tilidine and benzodiazepines. In case 2, U-47700 (204 ng/ml) together with methadone (290 ng/ml), flubromazepam (480 ng/ml) and diazepam (300 ng/ml) were detected in peripheral blood. In case 3, methoxyacetylfentanyl (266 ng/ml), furanylfentanyl (4.3 ng/ml) 4-ANPP (15 ng/ml) and alprazolam (69 ng/ml) were quantified in femoral blood. In all cases, the NSO likely contributed to the death (TSS = 3).NSOs appear to be often consumed in the setting of polydrug intoxications, especially in combination with other opioids and benzodiazepines, which often exert synergistic effects. The standard addition method remains the most reliable in post-mortem analysis and toxicological results should always be evaluated together with circumstantial and autopsy data.

Hair analysis for drugs has become extensively used for forensic investigation in recent years. To best interpret hair drug content in post-mortem conditions, the extent of external contamination by biological fluids, such as blood, must be taken into account to avoid false positive results. The present study evaluated opiates and opioids incorporation into hair from blood containing different concentrations of morphine (MOR), 6-mono-acetyl morphine (6-AM), codeine (COD), dihydrocodeine (DHC), tramadol (TRA), oxycodone (OXY), methadone (MET), 2-ethylidene-1,5-dimethyl-3,3- diphenyl pyrrolidine (EDDP), buprenorphine (BUP) and norbuprenorphine (NBUP). The hair strands contaminated by brief soaking into blood were stored at room temperature (RT) or at 4°C during 6 hours, 1, 3, 7 or 14 days. After decontamination by extensive washing, we show that all opiates and opioids were incorporated into hair within a few hours at RT and 4°C, without significant changes over time. The concentrations of opiates and opioids in hair reached the cut-off levels established by the Society of Hair Testing (SoHT) for therapeutic (MET, COD), or toxic or lethal (all other molecules) blood concentrations. The metabolite to parent drug concentration ratios were determined for NBUP/BUP, MOR/6-AM and EDDP/MET and could be helpful as indicators of blood external contamination. •Opiates and opioids incorporate deeply into hair after short exposure to blood.•No influence of temperature of storage on opiates and opioids integration into hair.•Avoid analyzing hair samples contaminated with blood containing opiates and opioids.•Metabolite to parent drug ratio could be helpful as indicator of hair contamination.

Scaling Up Point-of-Care Fentanyl TestingAlthough fentanyl is now the dominant driver of the U.S. opioid epidemic, our health care system has struggled to adapt toxicology screening practices to this reality.

State prescription drug monitoring programs are common tools intended to reduce prescription drug abuse and diversion, or the nonmedical use of a prescribed drug. The success of these programs depends largely upon physicians' awareness and use of them. We conducted a nationally representative mail survey of 1,000 practicing primary care physicians in 2014 to characterize their attitudes toward and awareness and use of prescription drug monitoring programs. A total of 420 eligible physicians (adjusted response rate: 58 percent) returned completed surveys. Among all physicians surveyed, 72 percent were aware of their state's prescription drug monitoring program, and 53 percent reported using one of the programs. We identified several barriers that may prevent greater use of the programs, including the time-consuming nature of information retrieval and the lack of an intuitive format for data provided by the programs. These results suggest that the majority of US primary care physicians are aware of and use prescription drug monitoring programs at least on occasion, although many did not access these programs routinely. To increase the use of the programs in clinical practice, states should consider implementing legal mandates, investing in prescriber education and outreach, and taking measures to enhance ease of access to and use of the programs.

•The lateral flow immunoassays detected fentanyl in both urine and saliva.•The lateral flow immunoassays detected fentanyl following an in vivo exposure.•These lateral flow immunoassays cross-reacted with several fentanyl analogs.•Fentanyl was detected in case samples by the lateral flow immunoassays. In 2017, 47,600 overdose deaths were reported to be associated with the abuse of opioids, including prescription painkillers (e.g. oxycodone), opiates (e.g. heroin), or synthetic opioids (e.g. fentanyl) within the United States. The recent spike in the presence of synthetic opioids in lots of heroin distributed on the street present specific and significant challenges to law enforcement. Synthetic opioids are extremely toxic substances, which can easily be inhaled. This type of exposure can lead to accidental overdoses by law enforcement and other first responders answering calls involving illicit drugs containing these substances. Due to this extreme toxicity, it is important for these individuals to have tools that can be easily deployed for accurate presumptive field tests. Currently, there are only a limited number of presumptive tests available for fentanyl detection. In this study, we addressed this technology gap by evaluating newly developed lateral flow immunoassays (LFIs) designed for the detection of fentanyl and its derivatives. These LFIs were evaluated for effectiveness in different biofluid matrices, following an in vivo exposure, cross-reactivity with fentanyl analogs, and in case samples. This study demonstrates that LFIs have the potential to be used by law enforcement for the detection of synthetic opioids.