Virginia Department of Forensic Science

Novel psychoactive substances pose a significant analytical challenge for forensic laboratories. Gas Chromatography with Infrared Spectroscopy (GC-IR) is typically presented as producing visually distinct spectra. However, this is not the case for all compounds. In this study, we showcase three synthetic cathinone analogs (methylone, N-ethylpentylone, and pentylone) which produce extremely visually similar solid state IR spectra.A primary dataset of these analogs was generated at the Amsterdam Police Laboratory. Libraries were created using two different sample preparation methods. The same compounds were also analyzed at the Virginia Department of Forensic Science (DFS). It was observed that changes in either instrument or sample preparation were enough to pose challenges to both the visual assessment and a library matching algorithm. Additionally, week-to-week variation was observed within the primary dataset. Principal Component Analysis (PCA) in combination with mahalanobis distances for objective comparison was assessed. A leave-one-sample-out cross validation accurately identified 100% of the samples in the primary dataset.This study shows how the application of chemometrics to spectral GC-IR data can substantiate the instrument’s differentiation capabilities and provide valuable objective support for a compound identification. In addition, limitations in the consistency of GC-IR spectra over time and across instruments and sample preparation methods were observed which could affect how the forensic community utilizes these techniques. Specifically, this study shows that shared GC-IR libraries might pose selectivity limitations. Therefore, laboratories must exercise caution if shared/generic GC-IR libraries are utilized for casework, and it is recommended that instrument and solvent specific libraries are generated.

The development of probabilistic genotyping (PG) systems to quantitatively analyze DNA mixture samples has been transformative in forensic science. TrueAllele® Casework (TA) and STRmix™ (STRmix) are the two most widely used PG systems in the United States. The two systems were challenged with 48 two-, three-, and four-person mock casework samples, for a total of 152 likelihood ratio (LR) comparisons. TA and STRmix converged on the same result (supportive, non-supportive, or inconclusive) for ~91% of contributor-specific comparisons. Where moderate or substantial differences in log(LR) values were observed, 9% affected the conclusion of the reference association to the mixture. The PG systems exhibited high correlations for estimated contributor-specific template quantities (~92%) and log(LR)s produced (>88%). When the log(LR)s for only low-template contributors (<100 pg) were compared, the R2 value dropped to ~68% and the difference became statistically significant. Of the 14 contributor comparisons where the conclusion differed, two were contradictory (supportive vs. non-supportive) and 12 were either inconclusive versus non-supportive or inconclusive versus supportive. The differing results were likely due to dissimilarities in the mixture input file as STRmix uses a lab-defined analytical threshold (AT) and TA models to 10 RFUs for each electropherogram. When 7 of the 14 mixtures were reanalyzed by STRmix using a 10 RFU AT, the log(LR)s for the low-template contributors became more similar to TAs. This study shows that while both systems may produce accurate and calibrated LRs, their results can deviate, especially for low-template, degraded contributors, and the deviation is generally predictable.

Synthetic opioids such as fentanyl account for over 71,000 of the approximately 107,000 overdose deaths reported in the United States in 2021. Fentanyl remains the fourth most identified drug by state and local forensic laboratories, and the second most identified drug by federal laboratories. The unambiguous identification of fentanyl-related substances (FRS) is challenging due to the absence or low abundance of a molecular ion in a typical gas chromatography-mass spectrometry (GC-MS) analysis and due to a low number of fragment ions that are similar among the many potential isomers of FRS. This study describes the utility of a previously reported gas chromatography-infrared (GC-IR) library for the identification of FRS within a blind, interlaboratory study (ILS) involving seven forensic laboratories. Twenty FRS reference materials, including those with isomer pairs in the library, were selected based on either their presence in the NIST library and/or some similarity of the mass spectra information produced. The ILS participants were requested to use the Florida International University (FIU) GC-MS and GC-IR libraries supplied by FIU to search for matches to their unknown spectra generated from in-house GC-MS and GC-IR analysis. The laboratories reported improvement in the positive identification of unknown FRS from ~75% using GC-MS alone to 100% correct identification using GC-IR analysis. One laboratory participant used solid phase IR analysis, which produced spectra incompatible with the vapor phase GC-IR library to generate a good comparison spectrum. However, this improved when searched against a solid phase IR library.

The reliability of forensic inorganic gunshot residue (iGSR) analysis depends strongly on the performance of the scanning electron microscope with energy dispersive X-ray spectrometer (SEM/EDS) instrument and the software that automates the data acquisition. Without a record of the baseline performance of the instrument, it is often hard to know when performance has deteriorated. This paper provides a set of tests which can be performed when the instrument is commissioned, or when significant modifications are made, to establish the baseline performance and a smaller set of tests that can be performed regularly to document the current performance of the instrument. Together, these represent a powerful way to establish, document and evaluate the performance of the instrumentation used for iGSR analysis. Furthermore, most of these tests are suitable for characterizing SEM/EDS instruments used for other forensic or non-forensic uses.

Gabapentin, a GABA analogue, is a nonopioid prescribed for seizure control and neuralgic pain. Its abuse for recreational purposes has been increasing in recent years as the number of prescriptions also increases. In our series, we review 104 cases of decedents who tested positive for gabapentin in postmortem blood samples and an additional 53 nonfatal cases of motor vehicle drivers suspected of driving under the influence. In 47.1% of the fatality cases, gabapentin was directly involved in death. Most gabapentin fatalities had several other intoxicants present (opioids, over-the-counter medications, antianxiety, and antidepressant medications). In cases in which gabapentin was determined to be a cause of death, the blood concentrations ranged from 1.1 to 134.0mg/L. Persons who died of a gabapentin-related drug death were prescribed the drug legitimately 91.4% of the time, with 84.2% of those also having a known prior history of abuse or misuse of prescription medications.

A direct analysis in real time ion source coupled with a time-of-flight mass spectrometer (DART-TOF-MS) is a suitable confirmatory technique for the analysis of pharmaceutical preparations, with accompanying reference sources for preparation markings. The DART-TOF-MS instrument allows for simple sample preparation and decreased analysis time, both crucial in a forensic laboratory setting. Differentiation can be made between active drug ingredients with the same molecular weight, such as hydrocodone and codeine, as well as pharmaceutical preparation mixtures, such as oxycodone and acetaminophen, using exact masses of the protonated molecules and fragment peaks compared to a standard.

The goal of this study was to survey optical and biochemical variation in cell populations deposited onto a surface through touch or contact and identify specific features that may be used to distinguish and then sort cell populations from separate contributors in a trace biological mixture. Although we were not able to detect meaningful biochemical variation in touch samples deposited by different contributors through preliminary antibody surveys, we did observe distinct differences in red autofluorescence emissions (650–670 nm), with as much as a tenfold difference in mean fluorescence intensities observed between certain pairs of donors. Results indicate that the level of red autofluorescence in touch samples can be influenced by a donor’s contact with specific material prior to handling the substrate from which cells were collected. In particular, we observed increased red autofluorescence in cells deposited subsequent to handling laboratory gloves, plant material, and certain types of marker ink, which could be easily visualized microscopically or using flow cytometry, and persisted after hand washing. To test whether these observed optical differences could potentially be used as the basis for a cell separation workflow, a controlled two-person touch mixture was separated into two fractions via fluorescence-activated cell sorting (FACS) using gating criteria based on intensity of 650–670 nm emissions and then subjected to DNA analysis. Genetic analysis of the sorted fractions provided partial DNA profiles that were consistent with separation of individual contributors from the mixture suggesting that variation in autofluorescence signatures, even if driven by extrinsic factors, may nonetheless be a useful means of isolating contributors to some touch mixtures.Graphical Conceptual workflow diagram. Trace biological mixtures containing cells from multiple individuals are analyzed by flow cytometry. Cells are then physically separated into two populations based on intensity of red autofluorescence using Fluorescence Activated Cell Sorting. Each isolated cell fraction is subjected to DNA analysis resulting in a DNA profile for each contributor.

Microfluidics has the potential to significantly improve the speed, throughput, and cost performance of electrophoretic short tandem repeat (STR) analysis by translating the process into a miniaturized and integrated format. Current STR analysis bypasses the post-PCR sample cleanup step in order to save time and cost, resulting in poor injection efficiency, bias against larger loci, and delicate injection timing controls. Here we describe the operation of an integrated high-throughput sample cleanup and capillary array electrophoresis microsystem that employs a streptavidin capture gel chemistry coupled to a simple direct-injection geometry for simultaneously analyzing 12 STR samples in less than 30 min with >10-fold improved sensitivity.