Detection of trace drugs of abuse in baby formula using solid‐phase microextraction direct analysis in real‐time mass spectrometry (SPME‐DART‐MS)

The intentional or unintentional adulteration of baby formula with drugs of abuse is one of the many increasingly complex samples forensic chemists may have to analyze. This sample type presents a challenge because of a complex matrix that can mask the detection of trace drug residues. To enable screening of baby formula for trace levels of drugs, the use of solid‐phase microextraction (SPME) coupled with direct analysis in real‐time mass spectrometry (DART‐MS) was investigated. A suite of five drugs was used as adulterants and spiked into baby formula. Samples were then extracted using SPME fibers which were analyzed by DART‐MS. Development of a proof‐of‐concept method was completed by investigating the effects of the DART gas stream temperature and the linear speed of the sample holder. Optimal values of 350°C and 0.2 mm/s were found. Once the method was established, representative responses and sensitivities for the five drugs were measured and found to be in the range of single ng/mL to hundreds ng/mL. Additional studies found that the presence of the baby formula matrix increased analyte signal (relative to methanolic solutions) by greater than 200%. Comparison of the SPME‐DART‐MS method to a traditional DART‐MS method for trace drug detection found at least a factor of 13 improvement in signal for the drugs investigated. This work demonstrates that SPME‐DART‐MS is a viable technique for the screening of complex matrices, such as baby formula, for trace drug residues and that development of a comprehensive method is warranted.     

Capturing high-resolution digital images for use in forensic document examination

In the past, pattern disciplines within forensic science have periodically faced criticism due to their subjective and qualitative nature and the perceived absence of research evaluating and supporting the foundations of their practices. Recently, however, forensic scientists and researchers in the field of pattern evidence analysis have developed and published approaches that are more quantitative, objective, and data driven. This effort includes automation, algorithms, and measurement sciences, with the end goal of enabling conclusions to be informed by quantitative models. Before employing these tools, forensic evidence must be digitized in a way that adequately balances high-quality detail and content capture with minimal background noise imparted by the selected technique. While the current work describes the process of optimizing a method to digitize physical documentary evidence for use in semi-automated trash mark examinations, it could be applied to assist other disciplines where the digitization of physical items of evidence is prevalent. For trash mark examinations specifically, it was found that high-resolution photography provided optimal digital versions of evidentiary items when compared to high-resolution scanning.     

Additional predictions for forensic DNA phenotyping of externally visible characteristics using the ForenSeq and Imagen kits

Multiplex DNA typing methods using massively parallel sequencing can be used to predict externally visible characteristics (EVCs) in forensic DNA phenotyping through the analysis of single-nucleotide polymorphisms. The focus of EVC determination has focused on hair color, eye color, and skin tone as well as visible biogeographical ancestry features. In this study, we researched off-label applications beyond what is currently marketed by the manufacturer of the Verogen ForenSeq kit primer set B and Imagen primer set E SNP loci. We investigated additional EVC predictions by examining published genome wide sequencing studies and reported allele-specific gene expression and predictive values. We have identified 15 SNPs included in the ForenSeq kit panel and Imagen kits that have additional EVC prediction capabilities beyond what is published in the Verogen manuals. The additional EVCs that can be predicted include hair graying, ephelides hyperpigmented spots, dermatoheliosis, facial pigmented spots, standing height, pattern balding, helix-rolling ear morphology, hair shape, hair thickness, facial morphology, eyebrow thickness, sarcoidosis, obesity, vitiligo, and tanning propensity. The loci can be used to augment and refine phenotype predictions with software such as MetaHuman for missing persons, cold case, and historic case investigations.     

Lipophilicity of fentalogs: Comparison of experimental and computationally derived data

Although fentanyl and a small number of derivatives used for medical or veterinary procedures are well characterized, physiochemical properties have not been determined for many of the newer fentanyl analogs. Partition coefficients (Log P) were determined for 19 fentalogs using the shake-flask method and liquid chromatography–tandem mass spectrometry (LC–MS/MS). Experimentally determined partition coefficients were compared with computationally derived data using six independent software sources (ACD/LogP, LogKOWWIN v 1.69, miLogP 2.2, OsirisP, XLOGP 3.0, ALogPS 2.1). Fentalogs with a wide variety of structural modifications were intentionally selected, yielding Log P values ranging from 1.21 to 4.90. Comparison of experimental and computationally derived Log P values were highly correlated (R² 0.854–0.967). Overall, substructure-based modeling using fragmental methods or property-based topological approaches aligned more closely with experimentally determined Log P values. LC–MS/MS was also used to estimate pK_a values for fentalogs with no previously reported data. Lipophilicity and pK_a are important considerations for analytical detection and toxicological interpretation. In silico methods allow the determination of physicochemical information prior to certified reference materials being readily available for in vitro or in vivo studies. Computationally derived data can provide insight regarding physiochemical characteristics of future fentalogs and other classes of synthetic analogs that have yet to emerge.     

The random presence of glass and paint on the clothing and footwear of members of the general population: A US baseline survey at various seasons

This study assists the interpretation of glass and paint evidence by filling an existing gap in the background occurrence that reflects the socioeconomic and demographic circumstances in the United States. The collection was performed in a college US city (Morgantown, West Virginia) to determine the effect of the type of clothing worn at different seasons on the presence of glass and paint. Tape lifts and sole scrapings (1038) were collected from 210 participants and up to six clothing and footwear areas per individual. Glass fragments were analyzed via polarized light microscopy (PLM), refractive index (RI), micro-X-Ray fluorescence (μXRF), and scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS), while paint specimens were examined by light microscopy and infrared spectroscopy (μFTIR). Higher occurrences of glass and paint were found in the winter season. The winter collection yielded 10 glass fragments and 68 paint particles, whereas the summer collection resulted in one glass fragment and 23 paint particles. The percentage of individuals with traces varied between seasons; 7% of individuals in the winter and 0.9% in the summer had glass, whereas 36% of individuals in the winter and 19% in the summer bore paint. Lastly, when considering the overall garment and footwear areas, glass was detected in 1.4% of the winter set, compared to 0.2% in the summer collection; paint was found in 9.2% of the winter collection, whereas only 4.2% was found in the summer set. There were no instances where both glass and paint were detected on the clothing and footwear of the same individual.     

Use of a commercially available hydrogel as a novel DNA surface sampling tool

A common requirement in the military, law enforcement, and forensic mission space is the need to collect trace samples from surfaces using a method that not only readily captures the sample but also retains its integrity for downstream identification and characterization. Additionally, collecting samples from three-dimensional objects (e.g., shell casings) is a challenge for which there is currently no validated standardized approach. Recently, hydrogels have been shown to have the potential for surface collection of trace bacterial spores, amino acids, and DNA. To test whether these hydrogels can serve as a viable collection medium for sampling DNA from surfaces, we carried out a series of preliminary tests examining collection efficiency and suitability of hydrogel material to recover samples of diluted, dried human DNA on a smooth polycarbonate surface. The recovery of surface DNA using a commercially available hydrogel was examined, and the efficiency compared to samples collected using a standard foam collection swab. DNA collected using the hydrogel and swab methods was then examined using quantitative polymerase chain reaction (qPCR) and short tandem repeat (STR) analysis to determine whether the collection material was compatible with these downstream processes. The hydrogel material used for this study collected the experimental DNA with comparable efficiency to standard collection swabs. In addition, qPCR and STR analyses demonstrated compatibility with the hydrogel collection and extraction process. These data suggest that hydrogels have the potential to be used as sample collection materials and deserve further characterization to elucidate their utility in collection from irregularly shaped, three-dimensional surfaces/materials.     

Detection and confirmation of fentanyls in high clay-content soil by electron ionization gas chromatography-mass spectrometry

Detection of illicit drugs in the environment, particularly in soils, often suggests the present or past location of a clandestine production center for these substances. Thus, development of efficient methods for the analysis and detection of these chemicals is of paramount importance in the field of chemical forensics. In this work, a method involving the extraction and retrospective confirmation of fentanyl, acetylfentanyl, thiofentanyl, and acetylthiofentanyl using trichloroethoxycarbonylation chemistry in a high clay-content soil is presented. The soil was spiked separately with each fentanyl at two concentrations (1 and 10 μg/g) and their extraction accomplished using ethyl acetate and aqueous NH₄OH (pH ~ 11.4) with extraction recoveries ranging from ~56% to 82% for the high-concentration (10 μg/g) samples while ranging from ~68% to 83% for the low-concentration (1 μg/g) samples. After their extraction, residues containing each fentanyl were reacted with 2,2,2-trichloroethoxycarbonyl chloride (Troc-Cl) to generate two unique and predictable products from each opioid that can be used to retrospectively confirm their presence and identity using EI-GC-MS. The method's limit of detection (MDL/LOD) for Troc-norfentanyl and Troc-noracetylfentanyl were estimated to be 29.4 and 31.8 ng/mL in the organic extracts. In addition, the method's limit of quantitation for Troc-norfentanyl and Troc-noracetylfentanyl were determined to be 88.2 and 95.5 ng/mL, respectively. Collectively, the results presented herein strengthen the use of chloroformate chemistry as an additional chemical tool to confirm the presence of these highly toxic and lethal substances in the environment.     

Understanding research methods,limitations, and applications of drug data collected by the National Forensic Laboratory Information System (NFLIS-Drug)

The National Forensic Laboratory Information System (NFLIS) is a drug surveillance program of the US Drug Enforcement Administration that systematically collects data on drugs that are seized by law enforcement and submitted to and analyzed by the Nation's forensic laboratories (NFLIS-Drug). NFLIS-Drug data are increasingly used in predictive modeling and drug surveillance to examine drug availability patterns. Given the complexity of the data and data collection, there are some common methodological pitfalls that we highlight with the aim of helping researchers avoid these concerns. The analysis done for this Technical Note is based on a review of the scientific literature that includes 428 unique, refereed article citations in 182 distinct journals published between January 1, 2005, and April 30, 2021. Each article was analyzed according to how NFLIS-Drug data were mentioned and whether NFLIS-Drug data were included. A sample of 37 articles was studied in-depth, and data issues were summarized. Using examples from the literature, this Technical Note highlights eight broad concerns that have important implications for the proper applications, interpretations, and limitations of NFLIS-Drug data with suggestions for improving research methods and accurate reporting of forensic drug data. NFLIS-Drug data are timely and provide key information to inform drug use trends across the United States; however, our present analysis shows that NFLIS-Drug data are misunderstood and represented in the literature. In addition to highlighting these issues, DEA has created several resources to assist NFLIS data users and researchers, which are summarized in the discussion.     

Identifying reliable ions for the statistical differentiation of structurally similar fentanyl analogs

Definitive identification of fentanyl analogs based on mass spectral comparison is challenging given the high degree of structural and, hence, spectral similarity. To address this, a statistical method was previously developed in which two electron-ionization (EI) mass spectra are compared using the unequal variance t-test. Normalized intensities of corresponding ions are compared, testing the null hypothesis (H₀) that the difference in intensity is equal to zero. If H₀ is accepted at all m/z values, the two spectra are statistically equivalent at the specified confidence level. If H₀ is not accepted at any m/z value, then there is a significant difference in intensity at that m/z value between the two spectra. In this work, the statistical comparison method is applied to distinguish EI spectra of valeryl fentanyl, isovaleryl fentanyl, and pivaloyl fentanyl. Spectra of the three analogs were collected over a 9-month period and at different concentrations. At the 99.9% confidence level, the spectra of corresponding isomers were statistically associated. Spectra of different isomers were statistically distinct, and ions responsible for discrimination were identified in each comparison. To account for inherent instrument variations, discriminating ions for each pairwise comparison were ranked based on the magnitude of the calculated t-statistic (t_calc) value. For a given comparison, ions with higher t_calc values are those with the greatest difference in intensity between the two spectra and, therefore, are considered more reliable for discrimination. Using these methods, objective discrimination among the spectra was achieved and ions considered most reliable for discrimination of these isomers were identified.     

An unusual blunt force trauma pattern and mechanism to the cranial vault: Investigation of an atypical infant homicide

This case report presents an unusual fracture pattern in the cranium of a four-month-old infant indicative of child abuse. Upon postmortem examination, the infant presented with numerous bilateral linear cranial fractures running perpendicular to the sagittal suture with depressed and curvilinear fractures apparent on the supra-auricular surfaces of the cranium. Histological evidence indicates multiple traumatic events to the cranium. In addition, the stair-step pattern of a parietal fracture may represent multiple contiguous fractures from repeated loading of the head at different times with variation of the focal points of compressive force. Additionally, the left humerus, left radius, and left ulna have healing metaphyseal fractures, and the left ulna also has an antemortem diaphyseal fracture which resulted in the distal metaphysis being rotated 45 degrees medially. Integration of autopsy, anthropological, and neuropathological reports for this case suggest multiple inflicted injury episodes with a repeated atypical mechanism(s) to the cranial vault of the infant. During investigative interviews, the caretaker admitted to squeezing the infant's head and neck on multiple occasions to quiet the child. This reported abusive mechanism is consistent with the pattern of symmetric cranial fractures and soft tissue injuries indicating asphyxiation. This case report provides forensic investigators with a potential trauma mechanism to explore in cases when a similar pattern of cranial trauma is observed and highlights the need for greater research on fracture propagation and fracture healing in the infant cranium.