Evaluation and classification of fentanyl-related compounds using EC-SERS and machine learning.

Multiple analytical techniques for the screening of fentanyl-related compounds exist. High discriminatory methods such as GC-MS and LC-MS are expensive, time-consuming, and less amenable to onsite analysis. Raman spectroscopy provides a rapid, inexpensive alternative. Raman variants such as electrochemical surface-enhanced Raman scattering (EC-SERS) can provide signal enhancements with 1010 magnitudes, allowing for the detection of low-concentration analytes, otherwise undetected using conventional Raman. Library search algorithms embedded in instruments utilizing SERS may suffer from accuracy when multicomponent mixtures involving fentanyl derivatives are analyzed. The complexing of machine learning techniques to Raman spectra demonstrates an improvement in the discrimination of drugs even when present in multicomponent mixtures of various ratios. Additionally, these algorithms are capable of identifying spectral features difficult to detect by manual comparisons. Therefore, the goal of this study was to evaluate fentanyl-related compounds and other drugs of abuse using EC-SERS and to process the acquired data using machine learning-convolutional neural networks (CNN). The CNN was created using Keras v 2.4.0 with Tensorflow v 2.9.1 backend. In-house binary mixtures and authentic adjudicated case samples were used to evaluate the created machine-learning models. The overall accuracy of the model was 98.4 ± 0.1% after 10-fold cross-validation. The correct identification for the in-house binary mixtures was 92%, while the authentic case samples were 85%. The high accuracies achieved in this study demonstrate the advantage of using machine learning to process spectral data when screening seized drug materials comprised of multiple components.

Comparison of portable and benchtop electrochemical instruments for detection of inorganic and organic gunshot residues in authentic shooter samples.

Analysis of gunshot residue currently lacks effective screening methods that can be implemented in real time at the crime scene. Historically, SEM-EDS has been the standard for analysis; however, advances in technology have brought portable instrumentation to the forefront of forensic science disciplines, including the screening of GSR. This study proposes electrochemical methods with disposable screen-printed carbon electrodes for GSR screening at the laboratory and points of care due to their rapid, cost-efficient, and compact platform. GSR residues were extracted from typical aluminum/carbon adhesive collection stubs and analyzed via square-wave anodic stripping voltammetry. Benchtop and portable electrochemical instruments were compared for the assessment and classification of authentic shooter samples by monitoring a panel of inorganic and organic GSR elements and compounds including lead, antimony, copper, 2,4-dinitrotoluene, diphenylamine, nitroglycerin, and ethyl centralite. The evaluation included the assessment of figures of merit and performance measures from quality controls, nonshooter, and shooter data sets. Samples collected from the hands of 200 background individuals (nonshooters), and shooters who fired leaded ammunition (100) and lead-free ammunition (50) were analyzed by the benchtop and portable systems with accuracies of 95.7% and 96.5%, respectively. The findings indicate that electrochemical methods are fast, sensitive, and specific for the identification of inorganic and organic gunshot residues. The portable potentiostat provided results comparable with the benchtop system, serving as a proof-of-concept to transition this methodology to crime scenes for a practical and inexpensive GSR screening that could reduce backlogs, improve investigative leads, and increase the impact of gunshot residues in forensic science.

Quantitation and Validation of 34 Fentanyl Analogs from Liver Tissue Using a QuEChERS Extraction and LC-MS-MS Analysis.

Since 2013, drug overdose deaths involving synthetic opioids (including fentanyl and fentanyl analogs) have increased from 3,105 to 31,335 in 2018. Postmortem toxicological analysis in fentanyl-related overdose deaths is complicated by the high potency of the drug, often resulting in low analyte concentrations and associations with toxicity, multidrug use, novelty of emerging fentanyl analogs and postmortem redistribution. Objectives for this study include the development of a quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction and subsequent liquid chromatography-mass spectrometry--mass spectrometry analysis, validation of the method following the American Academy of Forensic Sciences Standards Board (ASB) standard 036 requirements and application to authentic liver specimens for 34 analytes including fentanyl, metabolites and fentanyl analogs. The bias for all 34 fentanyl analogs did not exceed ±10% for any of the low, medium or high concentrations and the %CV did not exceed 20%. No interferences were identified. All 34 analytes were within the criteria for acceptable percent ionization suppression or enhancement with the low concentration ranging from -10.2% to 23.7% and the high concentration ranging from -7.1% to 11.0%. Liver specimens from 22 authentic postmortem cases were extracted and analyzed with all samples being positive for at least one target analyte from the 34 compounds. Of the 22 samples, 17 contained fentanyl and metabolites plus at least one fentanyl analog. The highest concentration for a fentanyl analog was 541.4 µg/kg of para-fluoroisobutyryl fentanyl (FIBF). The concentrations for fentanyl (n = 20) ranged between 3.6 and 164.9 µg/kg with a mean of 54.7 µg/kg. The fentanyl analog that was most encountered was methoxyacetyl fentanyl (n = 11) with a range of 0.2-4.6 µg/kg and a mean of 1.3 µg/kg. The QuEChERS extraction was fully validated using the ASB Standard 036 requirements for fentanyl, metabolites and fentanyl analogs in liver tissue.

Detection of organic and inorganic gunshot residues from hands using complexing agents and LC-MS/MS.

Gunshot residue (GSR) refers to a conglomerate consisting of both organic molecules (OGSR) and inorganic species (IGSR). Historically, forensic examiners have focused only on identifying the IGSR particles by their morphology and elemental composition. Nonetheless, modern ammunition formulations and challenges with the GSR transference (such as secondary and tertiary transfer) have driven research efforts for more comprehensive examinations, requiring alternative analytical techniques. This study proposes the use of LC-MS/MS for chromatographic separation and dual detection of inorganic and organic residues. The detection of both target species in the same sample increases the confidence that chemical profiles came from a gun's discharge instead of non-firearm-related sources. This strategy implements supramolecular molecules that complex with the IGSR species, allowing them to elute from the column towards the mass spectrometer while retaining isotopic ratios for quick and unambiguous identification. The macrocycle (18-crown-6-ether) complexes with lead and barium, while antimony complexes with a chelating agent (tartaric acid). The total analysis time for OGSR and IGSR in one sample is under 20 minutes. This manuscript expands from a previous proof-of-concept publication by improving figures of merit, increasing the target analytes, testing the method's feasibility through a more extensive set of authentic specimens collected from the hands of both shooters and non-shooters, and comparing performance with other analytical techniques such as ICP-MS, electrochemical methods and LIBS. The linear dynamic ranges (LDR) spread across the low ppb range for OGSR (0.3-200 ppb) and low ppm range (0.1-6.0 ppm) for IGSR. The method's accuracy increased overall when both organic and inorganic profiles were combined.

Quantitation of Fentanyl and Metabolites from Liver Tissue Using a Validated QuEChERS Extraction and LC-MS-MS Analysis.

According to the National Institute on Drug Abuse (NIDA), more than one hundred people die every day from opioid overdose. Overdose fatalities have risen as the availability of potent synthetic opioids, such as fentanyl, has increased. A forensic postmortem toxicological specimen is often in various stages of decomposition, experiencing autolysis and putrefaction, which complicates the extraction, creating a difficult challenge for toxicologists. Isolating the target drug, while creating an efficient and simplified analytical scheme, is a goal for most toxicology laboratories. The validation of a quick, easy, cheap, effective, rugged and safe extraction protocol is presented in this study as an alternative analytical method for efficient extraction and detection of fentanyl and its major metabolites: norfentanyl and despropionyl fentanyl (4-ANPP). The liquid Chromatography with tandem mass spectrometry analysis was validated following the American Academy of Forensic Sciences Standards Board (ASB) standard 036 proposed requirements. Evaluated parameters include selectivity, matrix effects (MEs), linearity, processed sample stability, bias, precision and proof of applicability using liver samples from authentic postmortem cases. MEs (represented as percent ionization suppression or enhancement) at low and high concentrations were -10.0% and 1.4% for fentanyl, -2.1% and -0.3% for 4-ANPP and 3.1% and 2.8% for norfentanyl, respectively. Bias for the three analytes ranged from -8.5% to -19.9% for the low concentrations, -3.6% to -14.7% for the medium concentrations and 1.5% to -16.1% for the high concentrations with all being within the ±20% guideline. Precision for the three analytes ranged from 2.2% to 15.1%. The linear range for the fentanyl and norfentanyl was 0.5-100 and 4-ANPP had a linear range of 0.4-80 µg/kg. The authentic postmortem liver samples ranged in fentanyl concentrations from 56.6 to 462.3 µg/kg with a mean of 149.2 µg/kg (n = 10). The range of norfentanyl concentrations were 1.9 to 50.0 µg/kg with a mean of 14.1 µg/kg (n = 10). The range of 4-ANPP concentrations were 3.2 to 23.7 µg/kg with a mean of 7.5 µg/kg (n = 7).

Rapid Determination of the 'Legal Highs' 4-MMC and 4-MEC by Spectroelectrochemistry: Simultaneous Cyclic Voltammetry and In Situ Surface-Enhanced Raman Spectroscopy.

The synthetic cathinones mephedrone (4-MMC) and 4-methylethcathinone (4-MEC) are two designer drugs that represent the rise and fall effect of this drug category within the stimulants market and are still available in several countries around the world. As a result, the qualitative and quantitative determination of 'legal highs', and their mixtures, are of great interest. This work explores for the first time the spectroelectrochemical response of these substances by coupling cyclic voltammetry (CV) with Raman spectroscopy in a portable instrument. It was found that the stimulants exhibit a voltammetric response on a gold screen-printed electrode while the surface is simultaneously electro-activated to achieve a periodic surface-enhanced Raman spectroscopy (SERS) substrate with high reproducibility. The proposed method enables a rapid and reliable determination in which both substances can be selectively analyzed through the oxidation waves of the molecules and the characteristic bands of the electrochemical SERS (EC-SERS) spectra. The feasibility and applicability of the method were assessed in simulated seized drug samples and spiked synthetic urine. This time-resolved spectroelectrochemical technique provides a cost-effective and user-friendly tool for onsite screening of synthetic stimulants in matrices with low concentration analytes for forensic applications.

Evaluation of the Simultaneous Analysis of Organic and Inorganic Gunshot Residues Within a Large Population Data Set Using Electrochemical Sensors*, †.

The increasing demand for rapid methods to identify both inorganic and organic gunshot residues (IGSR and OGSR) makes electrochemical methods, an attractive screening tool to modernize current practice. Our research group has previously demonstrated that electrochemical screening of GSR samples delivers a simple, inexpensive, and sensitive analytical solution that is capable of detecting IGSR and OGSR in less than 10 min per sample. In this study, we expand our previous work by increasing the number of GSR markers and applying machine learning classifiers to the interpretation of a larger population data set. Utilizing bare screen-printed carbon electrodes, the detection and resolution of seven markers (IGSR; lead, antimony, and copper, and OGSR; nitroglycerin, 2,4-dinitrotoluene, diphenylamine, and ethyl centralite) was achieved with limits of detection (LODs) below 1 µg/mL. A large population data set was obtained from 395 authentic shooter samples and 350 background samples. Various statistical methods and machine learning algorithms, including critical thresholds (CT), naïve Bayes (NB), logistic regression (LR), and neural networks (NN), were utilized to calculate the performance and error rates. Neural networks proved to be the best predictor when assessing the dichotomous question of detection of GSR on the hands of shooter versus nonshooter groups. Accuracies for the studied population were 81.8 % (CT), 88.1% (NB), 94.7% (LR), and 95.4% (NN), respectively. The ability to detect both IGSR and OGSR simultaneously provides a selective testing platform for gunshot residues that can provide a powerful field-testing technique and assist with decisions in case management.