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.     

Forensic Analysis of Stains on Fabric Using Direct Analysis in Real‐time Ionization with High‐Resolution Accurate Mass‐Mass Spectrometry

A rapid technique using direct analysis in real ‐ time (DART) ambient ionization coupled to a high ‐ resolution accurate mass‐mass spectrometer (HRAM‐MS) was employed to analyze stains on an individual's pants suspected to have been involved in a violent crime. The victim was consuming chocolate ice cream at the time of the attack, and investigators recovered the suspect's pants exhibiting splatter stains. Liquid chromatography with mass spectral detection (LC‐MS) and stereoscopic light microscopy (SLM) were also utilized in this analysis. It was determined that the stains on the pants contained theobromine and caffeine, known components of chocolate. A shard from the ceramic bowl that contained the victim's ice cream and a control chocolate ice cream sample were also found to contain caffeine and theobromine. The use of DART‐HRAM‐MS was useful in this case due to its rapid analysis capability and because of the limited amount of sample present as a stain.     

Detection and Classification of Ignitable Liquid Residues in the Presence of Matrix Interferences by Using Direct Analysis in Real Time Mass Spectrometry,

Conventional Gas Chromatography‐Mass Spectrometry (GC‐MS) methods for the analysis of ignitable liquids (ILs) are usually time‐consuming, and the data produced are difficult to interpret. A fast IL screening method using direct analysis in real time mass spectrometry (DART‐MS) is proposed in this study. GC‐MS, QuickStrip DART‐MS, and thermal desorption DART‐MS methods were used to analyze neat ILs and thermal desorption DART‐MS without extraction was used to analyze ILs on five substrates (e.g., carpet, wood, cloth, sand, and paper). Compared to GC‐MS, DART‐MS methods generated different spectral profiles for neat ILs with more peaks in the higher mass range and also provided better detection of less volatile compounds. ILs on substrates were successfully classified (98 ± 1%) using partial least squares discriminant analysis (PLS‐DA) models based on thermal desorption DART‐MS data. This study shows that DART‐MS has great potential for the high‐throughput screening of ILs on substrates.     

Identification of Eight Synthetic Cannabinoids,Including 5F‐AKB48 in Seized Herbal Products Using DART‐TOF‐MS and LC‐QTOF‐MS as Nontargeted Screening Methods

Synthetic cannabinoids are sprayed onto plant material and smoked for their marijuana‐like effects. Clandestine manufacturers modify synthetic cannabinoid structures by creating closely related analogs. Forensic laboratories are tasked with detection of these analog compounds, but targeted analytical methods are often thwarted by the structural modifications. Here, direct analysis in real time coupled to accurate mass time‐of‐flight mass spectrometry (DART‐TOF‐MS) in combination with liquid chromatography quadruple time‐of‐flight mass spectrometry (LC‐QTOF‐MS) are presented as a screening and nontargeted confirmation method, respectively. Methanol extracts of herbal material were run using both methods. Spectral data from four different herbal products were evaluated by comparing fragmentation pattern, accurate mass and retention time to available reference standards. JWH‐018, JWH‐019, AM2201, JWH‐122, 5F‐AKB48, AKB48‐N‐(4‐pentenyl) analog, UR144, and XLR11 were identified in the products. Results demonstrate that DART‐TOF‐MS affords a useful approach for rapid screening of herbal products for the presence and identification of synthetic cannabinoids.     

Detection of Brodifacoum and other Rodenticides in Drug Mixtures using Thermal Desorption Direct Analysis in Real Time Mass Spectrometry (TD‐DART‐MS)

In recent months, there has been increased reporting of seized drug and toxicology cases containing rodenticides, the active ingredient in rat poisons. Seeing as rodenticides are not scheduled substances, they are not commonly screened for in seized drug analysis. This work investigates the use of TD‐DART‐MS for the simultaneous detection of rodenticides and drugs. Six rodenticides were evaluated, an optimal method was established, and limits of detection in the tens of nanograms were calculated. Additional studies highlight that detection at less than 1% by weight in mixtures with AB‐FUBINACA, cocaine, heroin, or methamphetamine was possible. This work presents an optimized method for detection of these compounds, allowing for the simultaneous detection of drugs and rodenticides, providing drug chemists with a tool for rapid identification of these compounds for forensic or public health purposes.     

Validation of thin layer chromatography with AccuTOF-DART™ detection for forensic drug analysis

Abstract: Thin layer chromatography (TLC) is a technique that is commonly employed in the forensic drug analysis of pharmaceutical preparations. Detection is typically accomplished using various visualization spray reagents. Conventional gas chromatography–mass spectrometry (GC‐MS) analysis is typically performed to confirm the TLC results. Depending on the drugs tested and the instrument conditions required, this confirmation can take up to an hour to complete. Direct analysis in real time (DART?) is an ionization source, coupled to an accurate‐mass time‐of‐flight mass spectrometer that has the capability to ionize materials under ambient conditions. To streamline analysis, the combination of TLC with DART? detection is proposed to screen and subsequently identify drug compounds, all from the same TLC plate. DART? confirmations of TLC analyses take <10 min to complete and compare favorably to GC‐MS in sensitivity and selectivity. This study validates the use of TLC‐DART in the forensic identification of the components of several pharmaceutical preparations.     

Rapid Identification of Synthetic Cannabinoids in Herbal Incenses with DART‐MS and NMR

The usage of herbal incenses containing synthetic cannabinoids has caused an increase in medical incidents and triggered legislations to ban these products throughout the world. Law enforcement agencies are experiencing sample backlogs due to the variety of the products and the addition of new and still‐legal compounds. In our study, proton nuclear magnetic resonance (NMR) spectroscopy was employed to promptly screen the synthetic cannabinoids after their rapid, direct detection on the herbs and in the powders by direct analysis in real time mass spectrometry (DART‐MS). A simple sample preparation protocol was employed on 50 mg of herbal sample matrices for quick NMR detection. Ten synthetic cannabinoids were discovered in fifteen herbal incenses. The combined DART‐MS and NMR methods can be used to quickly screen synthetic cannabinoids in powder and herbal samples, serving as a complementary approach to conventional GC‐MS or LC‐MS methods.     

Characterization of Printing Inks Using DART‐Q‐TOF‐MS and Attenuated Total Reflectance (ATR) FTIR

The rise in improved and widely accessible printing technology has resulted in an interest to develop rapid and minimally destructive chemical analytical techniques that can characterize printing inks for forensic document analysis. Chemical characterization of printing inks allows for both discrimination of inks originating from different sources and the association of inks originating from the same source. Direct analysis in real‐time mass spectrometry (DART‐MS) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR‐FTIR) were used in tandem to analyze four different classes of printing inks: inkjets, toners, offset, and intaglio. A total of 319 samples or ~ 80 samples from each class were analyzed directly on a paper substrate using the two methods. DART‐MS was found to characterize the semi‐volatile polymeric vehicle components, while ATR‐FTIR provided chemical information associated with the bulk components of these inks. Complimentary data results in improved discrimination when both techniques are used in succession resulting in >96% discrimination for all toners, 95% for all inkjets, >92% for all offset, and >54% for all intaglio inks.     

DART‐MS as a Preliminary Screening Method for “Herbal Incense”: Chemical Analysis of Synthetic Cannabinoids

Direct analysis in real time mass spectrometry (DART‐MS) served as a method for rapid high‐throughput screening of six commercially available “Spice” products, detecting various combinations of five synthetic cannabinoids. Direct analysis in real time is an ambient ionization process that, along with high mass accuracy time‐of‐flight (TOF)‐MS to 0.0001 Da, was employed to establish the presence of cannabinoids. Mass spectra were acquired by simply suspending a small portion of sample between the ion source and the mass spectrometer inlet. The ability to test minute amounts of sample is a major advantage when very limited amounts of evidentiary material are available. In addition, reports are widespread regarding the testing backlogs that now exist because of the large influx of designer drugs. This method circumvents time‐consuming sample extraction, derivatization, chromatographic, and other sample preparative steps required for analysis by more conventional mass spectrometric methods. Accordingly, the synthetic cannabinoids AM‐2201, JWH‐122, JWH‐203, JWH‐210, and RCS‐4 were identified in commercially available herbal Spice products, singly and in tandem, at concentrations within the range of 4–141 mg/g of material. Direct analysis in real time mass spectrometry decreases the time necessary to triage analytical evidence, and therefore, it has the potential to contribute to backlog reduction and more timely criminal prosecution.     

Quantitative Analysis of Gamma‐Hydroxybutyrate at Endogenous Concentrations in Hair using Liquid Chromatography Tandem Mass Spectrometry

Abstract: A method capable of quantifying endogenous concentrations of gamma‐hydroxybutyrate (GHB) in human head hair was developed and validated using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Hair was digested under alkaline conditions, and GHB was isolated using liquid–liquid extraction. LC/MS/MS was performed using atmospheric pressure chemical ionization in the negative mode, multiple reaction monitoring, and deuterated internal standard (GHB‐D₆). Linearity was observed between 0.1 and 100 ng/mg GHB (R² = 1.000). The limits of detection and quantitation in human hair were 0.2 and 0.4 ng/mg, respectively. Accuracy at 2 ng/mg and 10 ng/mg was determined to be 97% and 94%, and intra‐assay CVs at these concentrations were 5.2% and 7.4% (n = 4). Beta‐hydroxybutyrate (BHB), alpha‐hydroxybutyrate, gamma‐butyrolactone, and 1,4‐butanediol did not produce an interference, and there was negligible ion suppression or enhancement from the matrix.     

Identification of 2‐(ethylamino)‐1‐(4‐methylphenyl)‐1‐pentanone (4‐MEAP), a New “Legal High” Sold by an Internet Vendor as 4‐Methyl Pentedrone

Online vendors are offering a new legal high, 4‐methylpentedrone (4‐MPD). Information for potential users provided by internet vendors of 4‐MPD includes incorrect structures and nonexistent CAS numbers. A sample of 4‐MPD was obtained and analyzed using GC‐MS, NMR, and LC‐EIS. The fragmentation data from the GC‐MS and LC‐EIS produced an M‐1 ion that suggested the molecular mass was 219 amu, rather than 205 amu as calculated for 4‐methylpentedrone. The difference in molecular mass corresponded to the addition of a methyl group. Based on the mass and fragmentation pattern, two standards were synthesized, 2‐(ethylamino)‐1‐(4‐methylphenyl)‐1‐pentanone and 1‐(4‐methylphenyl)‐2‐(propylamino)‐1‐butanone. The synthesis involved bromination of the appropriate ketone followed by the reaction with ethylamine or propylamine. Based on the NMR data and unique fragmentation patterns produced by these molecules, the sample was identified as 2‐(ethylamino)‐1‐(4‐methylphenyl)‐1‐pentanone, not 4‐methylpentedrone.     

Novel O‐propyl,S‐propyl,and N‐propyl substituted 1,4‐naphthoquinones and 1,4‐anthraquinones as potential fingermark development reagents for porous surfaces

Lawsone is a 2‐substituted‐1,4‐naphthoquinone derivative, which has been proposed as an alternative to the reagents currently used for fingermark detection on porous surfaces. 2‐substituted‐anthraquinones, which contain an additional conjugated benzene ring, have a similar chemical structure to that of lawsone. In this study, a new series of 2‐substituted‐1,4‐naphthoquinones and 2‐substituted‐1,4‐anthraquinones were synthesized and completely characterized by¹H NMR,¹³C NMR, IR, and HPLC‐TOF/MS analyses. All newly synthesized 2‐substituted‐1,4‐quinones were investigated for their ability to develop latent fingermarks on porous surfaces, and this ability was compared with that of lawsone. Each fingermark developed was graded using an established method; thus, quantitative data were attributed to each fingermark. It has been demonstrated that the 1,4 ‐ quinones react with amino acids present in latent fingermarks on selected paper surfaces to produce faint yellow‐orange impressions, which exhibit strong photoluminescence when illuminated with a forensic light source at 440 nm and observed through a red filter. None of the compounds caused background darkening. The results obtained were generally similar to those of lawsone, however, 8‐dibromo‐2‐(propylamino)naphthalene‐1,4‐dione and 5,8‐dibromo‐2‐(propylthio)naphthalene‐1,4‐dione yielded better results for copier paper and colored (blue) copier paper used in this analysis. To the best of our knowledge, this is the first study to examine the role of 1,4‐anthraquinone derivatives as potential fingermark development reagents. The results indicate that 1,4‐quinones have a potential to be used as reagents for enhancement of latent fingermarks.     

Detection of Cutting Agents in Drug‐Positive Seized Exhibits within the United States

The following report summarizes a study performed on seized drug exhibits collected in two U.S. states to evaluate the presence and identification of cutting agents. Aliquots of seized drug materials from Kentucky (n = 200) and Vermont (n = 315) were prepared using a dilute‐and‐shoot procedure. Initial analysis was performed using gas chromatography–mass spectrometry (GC‐MS) followed by analysis using liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC‐QTOF). Active compounds detected overall included caffeine (31.0%), quinine/quinidine (24.7%), levamisole (11.6%), acetaminophen, (8.2%) and procaine (8.2%). These compounds were found with several drugs of abuse, such as heroin, fentanyl, methamphetamine, and cocaine. This novel information about cutting agents used to dilute or alter drugs of abuse is important to criminal investigations and in the management of acute intoxications at health centers. However, common methodologies for analysis and standard reporting practices frequently do not include cutting agents, resulting in lacking or inadequate information regarding prevalence of these substances.     

The Classification of Inkjet Inks Using AccuTOF™ DART™ (Direct Analysis in Real Time) Mass Spectrometry—A Preliminary Study

A novel approach for the analysis of inkjet inks is being reported. A time‐of‐flight mass spectrometer, coupled with a Direct Analysis in Real Time (DART?) ion source (AccuTOF? DART?), was used to determine if inkjet inks from various manufacturers and models of printers could be reliably differentiated, characterized, and identified. A total of 217 ink standards were analyzed. As inkjet printing often involves the use of multiple colors (e.g., cyan, magenta, yellow, and black) to form an image or text, two different approaches to creating a library of standards and sampling methods were evaluated for implementation in a standard operating procedure. This research will show that a microscopic examination of the region of interest is requisite to identify what colors were utilized during the printing process, prior to comparing with known standards. Finally, blind testing was administered with 10 unknown samples to assess the validity and accuracy of the methodology.     

A Novel Forensic Tool for the Characterization and Comparison of Printing Ink Evidence: Development and Evaluation of a Searchable Database Using Data Fusion of Spectrochemical Methods

A searchable printing ink database was designed and validated as a tool to improve the chemical information gathered from the analysis of ink evidence. The database contains 319 samples from printing sources that represent some of the global diversity in toner, inkjet, offset, and intaglio inks. Five analytical methods were used to generate data to populate the searchable database including FTIR, SEM‐EDS, LA‐ICP‐MS, DART‐MS, and Py‐GC‐MS. The search algorithm based on partial least‐squares discriminant analysis generates a similarity “score” used for the association between similar samples. The performance of a particular analytical method to associate similar inks was found to be dependent on the ink type with LA‐ICP‐MS performing best, followed by SEM‐EDS and DART‐MS methods, while FTIR and Py‐GC‐MS were less useful in association but were still useful for classification purposes. Data fusion of data collected from two complementary methods (i.e., LA‐ICP‐MS and DART‐MS) improves the classification and association of similar inks.     

Rapid Quantitative Analysis of Multiple Explosive Compound Classes on a Single Instrument via Flow‐Injection Analysis Tandem Mass Spectrometry

A flow‐injection analysis tandem mass spectrometry (FIA MSMS) method was developed for rapid quantitative analysis of 10 different inorganic and organic explosives. Performance is optimized by tailoring the ionization method (APCI/ESI), de‐clustering potentials, and collision energies for each specific analyte. In doing so, a single instrument can be used to detect urea nitrate, potassium chlorate, 2,4,6‐trinitrotoluene, 2,4,6‐trinitrophenylmethylnitramine, triacetone triperoxide, hexamethylene triperoxide diamine, pentaerythritol tetranitrate, 1,3,5‐trinitroperhydro‐1,3,5‐triazine, nitroglycerin, and octohy‐dro‐1,3,5,7‐tetranitro‐1,3,5,7‐tetrazocine with sensitivities all in the picogram per milliliter range. In conclusion, FIA APCI/ESI MSMS is a fast (<1 min/sample), sensitive (~pg/mL LOQ), and precise (intraday RSD < 10%) method for trace explosive detection that can play an important role in criminal and attributional forensics, counterterrorism, and environmental protection areas, and has the potential to augment or replace several of the existing explosive detection methods.     

Application of LC−QTOF/MS for the validation and determination of organic explosive residues on Ionscan® swabs

The identification and confirmation of trace explosive residues along with potential precursors and degradation products require a comprehensive laboratory analysis procedure. This study presents the determination of organic explosives consisting of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 2,4,6-trinitrotoluene (TNT), 2,4,6,N-tetranitro-N-methylaniline (Tetryl), 1,3,5-trinitrobenzene (1,3,5-TNB) and pentaerythritol tetranitrate (PETN) by a high-resolution liquid chromatography quadrupole time-of-flight mass spectrometry (LC−QTOF/MS). The qualitative information including retention time, collision energy, precursor ions, and characteristic fragmentation pattern of each explosive were collected using an atmospheric pressure chemical ionization (APCI) in negative ion mode. The separation efficiency among five compounds was greatly achieved in this study. Four real explosive samples consisting of TNT, RDX, PETN and Tetryl and 12 Ionscan® quality control swabs from the Royal Thai Army were also tested to validate and verify the viability of the GC–MS method used to validate results from an Ionscan® system. The results showed that LC−QTOF/MS is a powerful technique for the identification and confirmation of thermally unstable organic explosives on Ionscan® swabs compared to a conventional GC−MS technique.     

Quantified Explosives Transfer on Surfaces for the Evaluation of Trace Detection Equipment

Trace explosive test surfaces are often required for the evaluation of trace detection equipment to determine the equipment performance. Test surfaces of C‐4, Detasheet, Semtex‐H, TNT, and HMTD were prepared by transferring trace amount of explosive deposited on polytetrafluoroethylene (PTFE) transfer strips onto different surfaces (Kraft paper, hard plastic, woven fabric, and soft vinyl). The amount of explosive transferred was deduced from the amount of explosive remaining on the PTFE strips after transfer, as quantified by direct analysis using tandem mass spectrometry with thermal desorption. From the data set of over 2000 transfers, we experienced lower transfer efficiency for Semtex‐H and Detasheet, and for soft vinyl and hard plastic. However, the rapid quantification mass spectrometric method allowed the transfer efficiency to be determined for all test surfaces used in an evaluation of trace explosive detectors, thereby permitting only the test surfaces with desired transfer to be accepted for the assessment.     

The Analysis of Black Powder Substitutes Containing Ascorbic Acid by Ion Chromatography/Mass Spectrometry*†

Abstract: Black powder substitutes containing ascorbic acid are a group of low explosives that utilize ascorbic acid as the fuel. The analysis of these powders is complicated by the degradation of ascorbic acid which occurs rapidly in solution and may also occur as the powder ages. Aqueous extracts of both intact powders and postblast residues were analyzed by an existing ion chromatography/mass spectrometry (IC/MS) method used at the Bureau of Alcohol, Tobacco, Firearms and Explosives. Results have shown that while ascorbic acid itself is not detected in this method, its diagnostic degradation products (threonic acid, monohydrated diketogulonic acid, and oxalic acid) can be identified. In addition, anions from the inorganic oxidizers (perchlorate and nitrate) and combustion products such as chloride, chlorate, and nitrite, can be identified within the same experiment. While this IC/MS method shows promise, future modifications are necessary because of limitations in identifying threonate in postblast residues, as well as coeluting compounds observed in postblast residues.     

Separation and Detection of Smokeless Powder Additives by Ultra Performance Liquid Chromatography with Tandem Mass Spectrometry (UPLC/MS/MS),

A reversed phase gradient ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) method has been developed for the analysis of smokeless powders. A total of 20 different components were separated by UPLC and detected by MS/MS in multiple reaction monitoring (MRM) mode. These compounds included diphenylamines, centralites, nitrotoluenes, nitroglycerin, and various phthalates. Simultaneous positive and negative electrospray ionization (ESI) was used along with negative atmospheric pressure chemical ionization (APCI) to detect all compounds in a single analysis. Analysis times were under 8 min with a gradient of 10–73% organic at a flow rate of 0.500 mL/min. With this method, ultraviolet and MRM limits of detection ranging from 0.08 to 2.6 ng and 0.4–64 ng injected were achieved. Commercially available smokeless powders were also extracted with methylene chloride and characterized using the developed UPLC/MS/MS method. The procedure permits the determination of compositional differences between different brands as well as lot‐to‐lot variations.