Update on Urine Adulterants and Synthetic Urine Samples to Subvert Urine Drug Testing.

To avoid a positive urine drug test, donors might try to subvert the test, either by adulterating the specimen with a product designed to interfere with testing or by substituting the specimen for a synthetic urine. A market search conducted in December of 2020 identified 3 adulterants and 32 synthetic urines, and a selection was procured based on specific criteria. Samples prepared with the 3 adulterants and 10 synthetic urines were submitted for testing at five forensic drug testing laboratories to perform immunoassay screening, chromatographic confirmation analysis and specimen validity testing (SVT). One adulterant determined to contain iodate reduced THC-COOH concentrations by 65% and the concentrations of 6-acetylmorphine, morphine, oxycodone, oxymorphone, hydrocodone and hydromorphone by 6-27%. Another adulterant determined to contain nitrite reduced THC-COOH concentrations by 22%, while the third did not affect drug screening or confirmatory testing. Both active adulterants could be identified through positive oxidant screens as well as through signal suppression in cloned enzyme donor immunoassay (CEDIA). The synthetic urines could not be identified either through traditional SVT or by the AdultaCheck10 dipstick. The Synthetic UrineCheck dipstick produced a difference in response between the authentic urine specimen and the synthetic urine samples, but the difference was small and difficult to observe. While most synthetic urines now contain uric acid, magnesium and caffeine, the results indicated that a biomarker panel including endogenous and exogenous markers of authentic urine performed well and clearly demonstrated the absence of biomarkers in the synthetic urines. The SVT assay also offers potential targets for future screening assays.

Confined DART-MS for Rapid Chemical Analysis of Electronic Cigarette Aerosols and Spiked Drugs.

A confined direct analysis in a real time mass spectrometry (DART-MS) system and method were developed for coupling directly with commercial electronic cigarettes for rapid analysis without sample preparation. The system consisted of a confining heated glass T-junction, DART ionization source, and Vapur interface to assist aerodynamic transport. Suction generated by positioning the electronic cigarette at the junction inlet allowed for direct chemical analysis of aerosolized electronic liquids from both automatic devices powered by drag and manual button-operated devices, which is unachievable with traditional DART-MS. Parametric analyses for the system investigated Vapur suction flow rate, junction heating, puff duration, and coil power levels. Using this method, rapid chemical analyses of electronic cigarette aerosols from electronic liquids, spiked illicit drugs, and polymeric or plasticizer contaminants were performed in <30 s. The confined DART-MS method provides a streamlined tool for rapid screening of illicit and hazardous chemical profiles emitting from electronic cigarettes.

Inorganic oxidizer detection from propellants, pyrotechnics, and homemade explosive powders using gradient elution moving boundary electrophoresis.

Advancement in rapid targeted chemical analysis of homemade and improvised explosive devices is critical for the identification of explosives-based hazards and threats. Gradient elution moving boundary electrophoresis (GEMBE), a robust electrokinetic separation technique, was employed for the separation and detection of common inorganic oxidizers from frequently encountered fuel-oxidizer mixtures. The GEMBE system incorporated sample and run buffer reservoirs, a short capillary (5 cm), an applied electric field, and a pressure-driven counterflow. GEMBE provided a separation format that allowed for continuous injection of sample, selectivity of analytes, and no sample cleanup or filtration prior to analysis. Nitrate, chlorate, and perchlorate oxidizers were successfully detected from low explosive propellants (e.g., black powders and black powder substitutes), pyrotechnics (e.g., flash powder), and tertiary explosive mixtures (e.g., ammonium nitrate- and potassium chlorate-based fuel-oxidizer mixtures). Separation of these mixtures exhibited detection without interference from a plethora of additional organic and inorganic fuels, enabled single particle analysis, and demonstrated semiquantitative capabilities. The bulk counterflow successfully excluded difficult components from fouling the capillary, yielding estimated limits of detection down to approximately 10 µmol/L. Finally, nitrate was separated and detected from postblast debris collected and directly analyzed from two nitrate-based charges.

Detection of fuel-oxidizer explosives utilizing portable capillary electrophoresis with wipe-based sampling.

Portable analytical instrumentation that can provide an alarm indication for the presence of explosives and related components is critical for the identification of explosives-based hazards and threats. Many explosives incident reports involve an inorganic oxidizer-fuel mixture which can include pyrotechnics, fireworks, flash powders, black powders, black powder substitutes, and improvised or homemade explosives. A portable CE instrument with targeted analysis of common inorganic oxidizer ions, for example, chlorate, perchlorate, and nitrate, was used here as a rapid detection platform. Unlike frequently used gas-phase separation and detection instrumentation such as ion mobility spectrometry (IMS), an automated liquid extraction mechanism is required for CE separation using acetate paper sample collection wipes. Target inorganic oxidizers were inkjet-printed onto sample wipes to investigate instrument response relative to the collected analyte spatial distribution. Overall, analyte signal intensities increased with off-center sample deposition due to improved sample extraction from wipes and no change in response was observed for varied array distributions across wipes. The system demonstrated sub 200 ng detection limits for all target analytes, with further improvement when normalizing to an internal standard.

Emerging techniques for the detection of pyrotechnic residues from seized postal packages containing fireworks.

High volume screening of parcels with the aim to trace the illegal distribution and selling of fireworks using postal services is challenging. Inspection services have limited manpower and means to perform extensive visual inspection. In this study, the presence of solid pyrotechnic residues collected from cardboard shipping parcels containing fireworks was investigated for direct in-field chemical detection. Two emerging trace detection techniques, i.e., capillary electrophoresis (CE)-based inorganic oxidizer detector and infrared thermal desorption (IRTD) coupled with direct analysis in real time mass spectrometry (DART-MS), were investigated for their potential as screening tools. Detection of non-visible pyrotechnic trace residues from real-case seized parcels was demonstrated using both screening techniques. However, the high nitrate background in the commercial CE system complicated its screening for black powder traces. IRTD-DART-MS allowed differentiation between flash and black powder by identification of the molecular inorganic ions. Compared to the portable CE instrument, rapid screening using IRTD-DART-MS is currently limited to laboratory settings. The capabilities of these emerging techniques established solid particle and trace residue chemical detection as interesting options for parcel screening in a logistic setting.

Trace Detection and Chemical Analysis of Homemade Fuel-Oxidizer Mixture Explosives: Emerging Challenges and Perspectives.

The chemical analysis of homemade explosives (HMEs) and improvised explosive devices (IEDs) remains challenging for fieldable analytical instrumentation and sensors. Complex explosive fuel-oxidizer mixtures, black and smokeless powders, flash powders, and pyrotechnics often include an array of potential organic and inorganic components that present unique interference and matrix effect difficulties. The widely varying physicochemical properties of these components as well as external environmental interferents and background challenge many sampling and sensing modalities. This review provides perspective on these emerging challenges, critically discusses developments in sampling, sensors, and instrumentation, and showcases advancements for the trace detection of inorganic-based explosives.

Separation and Detection of Trace Fentanyl from Complex Mixtures Using Gradient Elution Moving Boundary Electrophoresis.

The current opioid epidemic remains an ongoing challenge, exacerbated by the extreme potency of synthetic opioids (e.g., fentanyl and fentanyl analogues), leading to an increase in adulterated heroin-related deaths. The increasing prevalence of fentanyl and fentanyl analogues in mixtures with heroin and other adulterants, excipients, and bulking agents has placed an emphasis on trace analysis methods for their detection from complex drug mixtures. Here, gradient elution moving boundary electrophoresis (GEMBE), a robust and miniaturized electrophoretic separation technique, was employed for the separation and detection of fentanyl and nine (9) fentanyl analogues from mixtures. GEMBE incorporated a short capillary (5 cm × 15 µm i.d.) for the electrophoretic separation of analytes with an opposing bulk counterflow. As the velocity of the counterflow was varied, analytes with differing electrophoretic mobilities entered the separation channel at different times and were analyzed as moving boundaries by contactless conductivity detection. The continuous injection of sample, driven by a controlled and variable pressure, both provided selectivity of the analytes and prevented contaminants or particulate within the sample from entering the separation capillary. Fentanyl was successfully separated and detected down to 2.5 µmol/L and demonstrated only 50% to 60% signal suppression in dilute binary mixtures with heroin and other common adulterants and excipients at 30:1 (compound/fentanyl) concentration ratios. In addition, GEMBE was successfully applied to a few adjudicated case samples of fentanyl-related mixtures exhibiting dyes and visible particulate. The short capillaries, contactless detection format utilized here, and continuous injection of sample allow for a small footprint platform that is easy-to-use for forensic analyses.

Color manipulation through microchip tinting for colorimetric detection using hue image analysis.

Colorimetry with microfluidic devices has been proven to be an advantageous method for in situ analyses where limited resources and rapid response for untrained users are desired. Image analysis using a small camera or cell phone can be easily incorporated for an objective readout, eliminating variations from normal differences in color perception and environmental factors during analysis. The image analysis using the parameter hue, for example, has been utilized as a highly effective, objective analysis method that correlates with the psychological way color is perceived. Hue analysis, however, is best used for colorimetric reactions that result in distinct changes from one color to a markedly different color and can be inadequate to distinguish between subtle or monotonal (colorless-to-colored) color changes. We address this with three unique color manipulation (i.e., tinting) techniques that provide greater discrimination with such color changes, thus yielding improved limits of detection for various colorimetric reactions that may have previously been limited. Tinting is invoked through dyeing the reagent substrate, colored printing the device, or colored lighting during image capture, and is shown to effectively shift the background color of the reaction detection area. Hydrogen peroxide, a constituent of peroxide-based explosives, is associated with a monochromatic color change upon reaction, and this is used to demonstrate the effectiveness of the tinting methods in improving the limit of detection from an undetectable color change to 0.1 mg mL-1.

Objective Method for Presumptive Field-Testing of Illicit Drug Possession Using Centrifugal Microdevices and Smartphone Analysis.

Current colorimetric presumptive identification of illicit drugs for determining illegal possession of controlled substances by law enforcement relies solely on the subjective interpretation of color change using drug- or class-specific reactions. Here, we describe the use of inexpensive polyester-toner, rotation-driven microfluidic devices with a smartphone as a potential alternative for current presumptive colorimetric field-testing of illicit drugs, allowing for an objective and user-friendly image analysis technique for detection. The centrifugal microfluidic platform accommodates simultaneous presumptive testing of material from a single input to multiple reaction chambers, enabling rapid screening. Hue and saturation image analysis parameters are used to define threshold values for the detection of cocaine and methamphetamine as proof-of-principle with 0.25 and 0.75 mg/mL limits of detection, respectively, with nonvolatile reagents stored on-board and smartphone for detection. Reported LODs are lower than those concentrations used in the field. Additionally, the developed objective detection method addresses the testing of drugs with various common cutting agents, including those known to produce false negative and positive results. We demonstrate the effectiveness of the method by successfully identifying the composition of 30 unknown samples.

Microfluidic enzymatic DNA extraction on a hybrid polyester-toner-PMMA device.

To date, the forensic community regards solid phase extraction (SPE) as the most effective methodology for the purification of DNA for use in short tandem repeat (STR) polymerase chain reaction (PCR) amplification. While a dominant methodology, SPE protocols generally necessitate the use of PCR inhibitors (guanidine, IPA) and, in addition, can demand timescales of up to 30 min due to the necessary load, wash and elution steps. The recent discovery and characterization of the EA1 protease has allowed the user to enzymatically extract (not purify) DNA, dramatically simplifying the task of producing a PCR-ready template. Despite this, this procedure has yet to make a significant impact on microfluidic technologies. Here, we describe a microfluidic device that implements the EA1 enzyme for DNA extraction by incorporating it into a hybrid microdevice comprising laminated polyester (Pe) and PMMA layers. The PMMA layer provides a macro-to-micro interface for introducing the biological sample into the microfluidic architecture, whilst also possessing the necessary dimensions to function as the swab acceptor. Pre-loaded reagents are then introduced to the swab chamber centrifugally, initiating DNA extraction at 75 °C. The extraction of DNA occurs in timescales of less than 3 min and any external hardware associated with the transportation of reagents by pneumatic pumping is eliminated. Finally, multiplexing is demonstrated with a circular device containing eight separate chambers for the simultaneous processing of eight buccal swab samples. The studies here provide DNA concentrations up to 10 ng µL(-1) with a 100% success rate in less than 3 minutes. The STR profiles generated using these extracted samples demonstrate that the DNA is of PCR forensic-quality and adequate for human identification.

Inexpensive, rapid prototyping of microfluidic devices using overhead transparencies and a laser print, cut and laminate fabrication method.

We describe a technique for fabricating microfluidic devices with complex multilayer architectures using a laser printer, a CO2 laser cutter, an office laminator and common overhead transparencies as a printable substrate via a laser print, cut and laminate (PCL) methodology. The printer toner serves three functions: (i) it defines the microfluidic architecture, which is printed on the overhead transparencies; (ii) it acts as the adhesive agent for the bonding of multiple transparency layers; and (iii) it provides, in its unmodified state, printable, hydrophobic 'valves' for fluidic flow control. By using common graphics software, e.g., CorelDRAW or AutoCAD, the protocol produces microfluidic devices with a design-to-device time of ∼40 min. Devices of any shape can be generated for an array of multistep assays, with colorimetric detection of molecular species ranging from small molecules to proteins. Channels with varying depths can be formed using multiple transparency layers in which a CO2 laser is used to remove the polyester from the channel sections of the internal layers. The simplicity of the protocol, availability of the equipment and substrate and cost-effective nature of the process make microfluidic devices available to those who might benefit most from expedited, microscale chemistry.

Capillary electrophoresis separation of the desamino degradation products of oxytocin.

Oxytocin (OT) is an endogenous and therapeutic hormone necessary for maternal health. It is also the subject of fast growing research in the field of behavioral science. This article describes a rapid CE method using UV detection at 214 nm for the determination of the deamidation products of OT. Deamidation is the most common degradation pathway of peptides and proteins and can lead to reduced therapeutic efficiency of biopharmaceuticals. To achieve a separation of the seven structurally similar desamino peptides from OT, 11 mM sulfobutyl ether ß-CD and 10% v/v MeOH were added to a BGE of 50 mM phosphate buffer at pH 6.0. The assay is linear within ≤5-100 µM for all species with a total analysis time of 12 min. The method was then applied to monitor the heat-stress degradation of OT at 70°C, where all seven desamino species were observed over a 96 h period.

2-[1-(2-Hy-droxy-6-meth-oxy-phen-yl)ethyl-idene]-N-methyl-hydrazinecarbothio-amide acetonitrile monosolvate.

In the title compound, C11H15N3O2S·C2H3N, the dihedral angle between the benzene ring and the mean plane of the hydrazinecarbothio-amide group is 75.1 (2)°. In the crystal, the main mol-ecule is linked to the solvent mol-ecule by a weak N-H⋯N hydrogen bond while O-H⋯S hydrogen bonds link the mol-ecules into columns along [100].

6-(3-Chloro-phen-yl)imidazo[2,1-b][1,3,4]thia-diazole.

In the title compound, C10H8ClN3S, the dihedral angle between the mean planes of the benzene and imidazo[2,1-b][1,3,4]thia-diazole rings is 6.0 (9)°. In the crystal, mol-ecules are assembled by the formation of centrosymmetric dimers by π-stacking of the thia-diazole and benzene rings of neighboring mol-ecules [centroid-centroid distance = 3.6938 (11) Å] along [010].

2-(4-Methyl-phen-yl)acetohydrazide.

In the title compound, C9H12N2O, the dihedral angle between the benzene ring and the mean plane of the acetohydrazide group is 88.2 (7)°. In the crystal, N-H⋯O hydrogen bonds and weak C-H⋯O inter-actions link the mol-ecules into infinite ribbons along [001].

4-Methyl-phenyl quinoline-2-carboxyl-ate.

In the title compound, C(17)H(13)NO(2), two mol-ecules crystallize in the asymmetric unit. The dihedral angle between the mean planes of the quinoline and benzene rings are 78.3 (4) and 88.2 (3)°. The carboxyl-ate group is twisted slightly from the quinoline ring by 7.1 (2) and 13.3 (4)°, respectively. In the crystal, weak C-H⋯O inter-actions are observed. Further stabilization is provided by weak π-π stacking inter-actions, with centroid-centroid distances of 3.564 (9)/3.689 (2) and 3.830 (1)/3.896 (5)Å, respectively.