Ashton D. Lesiak

DART-MS in-source collision induced dissociation and high mass accuracy for new psychoactive substance determinations

The influx of new psychoactive substances is a problem that is challenging the analytical capabilities of enforcement agencies. Cathinone designer drugs are less likely to be included in routine drug screens and typical drug formulations are commonly mixtures with continually shifting components. Ambient ionization mass spectrometry employs relatively mild conditions to desorb and ionize solid samples, imparting much less energy than that associated with conventional mass spectrometry methods. Direct analysis in real time mass spectrometry (DART-MS) is an ambient ionization method that was employed to rapidly screen cathinones, alone and in mixtures, readily enabling differentiation of the active drug(s) from various cutting agents. Accurate mass determinations provided preliminary identification of the various components of drug mixtures. The data generated in forensic mass spectrometry can be used for both elemental composition formulations and isotope abundance calculations for determination of unknown psychoactive substances, and we demonstrate how this data could be applied to the presence of new drugs as the active components shift in response to regulations. Isotope abundance calculations were used to develop a candidate pool of possible molecular formulas associated with cathinones as a specific class of designer drugs. Together, the combination of a time-of-flight (TOF) mass analyzer along with in-source collision-induced dissociation (CID) spectra were used to drastically narrow the pool of candidates to a single molecular formula. The [M+H](+) and product ion peaks provided data for presumptive analysis of various substituted synthetic cathinones in a manner that is complementary to conventional GC-MS analysis of new psychoactive substances.

DART-MS for rapid, preliminary screening of urine for DMAA

Dimethylamylamine (DMAA) is a sympathomimetic amine found in weight-loss/workout supplements or used as an appetite suppressant. DMAA is a stimulant that is banned by the World Anti-Doping Agency (WADA). Adverse health effects as well as fatalities have been implicated with its use. Direct analysis in real time mass spectrometry (DART-MS) is an ambient ionization method that was employed to rapidly identify the presence of DMAA in various samples without any extraction or preparations whatsoever. DMAA was first identified in supplements, sampled directly in their solid forms. Furthermore, DMAA was detected directly in urine over 48 h as a means of indicating recent abuse of the substance. DART-MS analysis is instantaneous, and coupled with the high mass accuracy associated with the time-of-flight mass analyzer, results in unequivocal identification of the presence of DMAA. These features demonstrate DART-MS as an attractive potential alternative screening method for the presence of drugs and medications or for toxicological investigations.

Plant Seed Species Identification from Chemical Fingerprints: A High-Throughput Application of Direct Analysis in Real Time Mass Spectrometry

Plant species identification based on the morphological features of plant parts is a well-established science in botany. However, species identification from seeds has largely been unexplored, despite the fact that the seeds contain all of the genetic information that distinguishes one plant from another. Using seeds of genus Datura plants, we show here that the mass spectrum-derived chemical fingerprints for seeds of the same species are similar. On the other hand, seeds from different species within the same genus display distinct chemical signatures, even though they may contain similar characteristic biomarkers. The intraspecies chemical signature similarities on the one hand, and interspecies fingerprint differences on the other, can be processed by multivariate statistical analysis methods to enable rapid species-level identification and differentiation. The chemical fingerprints can be acquired rapidly and in a high-throughput manner by direct analysis in real time mass spectrometry (DART-MS) analysis of the seeds in their native form, without use of a solvent extract. Importantly, knowledge of the identity of the detected molecules is not required for species level identification. However, confirmation of the presence within the seeds of various characteristic tropane and other alkaloids, including atropine, scopolamine, scopoline, tropine, tropinone, and tyramine, was accomplished by comparison of the in-source collision-induced dissociation (CID) fragmentation patterns of authentic standards, to the fragmentation patterns observed in the seeds when analyzed under similar in-source CID conditions. The advantages, applications, and implications of the chemometric processing of DART-MS derived seed chemical signatures for species level identification and differentiation are discussed.

Recent advances in forensic drug analysis by DART-MS

Mass spectrometry methods play a major role in many forensic applications. While gas chromatography-mass spectrometry methods are commonly used in crime laboratories and enforcement agencies, a variety of advanced techniques are now available that can improve upon standard methods and address emerging issues in forensic science. New mass spectrometry technologies include more versatile ionization sources, allowing the next generation of instrumentation to be more multipurpose and adaptable to the needs of the discipline. Direct analysis in real-time mass spectrometry is an ambient ionization method that allows direct testing of gas, liquid and solid samples without the need for any preparation or extraction, based on thermal desorption and ionization directly from the sample surface. This Review will provide an in-depth description of direct analysis in real-time time-of-flight mass spectrometry as applied to samples relevant to forensic science, with a focus on analysis and characterization related to forensic drug chemistry.

A High Throughput Ambient Mass Spectrometric Approach to Species Identification and Classification from Chemical Fingerprint Signatures

A high throughput method for species identification and classification through chemometric processing of direct analysis in real time (DART) mass spectrometry-derived fingerprint signatures has been developed. The method entails introduction of samples to the open air space between the DART ion source and the mass spectrometer inlet, with the entire observed mass spectral fingerprint subjected to unsupervised hierarchical clustering processing. A range of both polar and non-polar chemotypes are instantaneously detected. The result is identification and species level classification based on the entire DART-MS spectrum. Here, we illustrate how the method can be used to: (1) distinguish between endangered woods regulated by the Convention for the International Trade of Endangered Flora and Fauna (CITES) treaty; (2) assess the origin and by extension the properties of biodiesel feedstocks; (3) determine insect species from analysis of puparial casings; (4) distinguish between psychoactive plants products; and (5) differentiate between Eucalyptus species. An advantage of the hierarchical clustering approach to processing of the DART-MS derived fingerprint is that it shows both similarities and differences between species based on their chemotypes. Furthermore, full knowledge of the identities of the constituents contained within the small molecule profile of analyzed samples is not required.

Direct analysis in real time high resolution mass spectrometry as a tool for rapid characterization of mind-altering plant materials and revelation of supplement adulteration--The case of Kanna.

We demonstrate the utility of direct analysis in real time ionization coupled with high resolution time-of-flight mass spectrometry (DART-HRTOFMS) in revealing the adulteration of commercially available Sceletium tortuosum, a mind-altering plant-based drug commonly known as Kanna. Accurate masses consistent with alkaloids previously isolated from S. tortuosum plant material enabled identification of the products as Kanna, and in-source collision-induced dissociation (CID) confirmed the presence of one of these alkaloids, hordenine, while simultaneously revealing the presence of an adulterant. The stimulant ephedrine, which has been banned in herbal products and supplements, was confirmed to be present in a sample through the use of in-source CID. High-throughput DART-HRTOFMS was shown to be a powerful tool to not only screen plant-based drugs of abuse for psychotropic alkaloids, but also to reveal the presence of scheduled substances and adulterants.

Application of ambient ionization high resolution mass spectrometry to determination of the botanical provenance of the constituents of psychoactive drug mixtures

A continuing challenge in analytical chemistry is species-level determination of the constituents of mixtures that are made of a combination of plant species. There is an added urgency to identify components in botanical mixtures that have mind altering properties, due to the increasing global abuse of combinations of such plants. Here we demonstrate the proof of principle that ambient ionization mass spectrometry, namely direct analysis in real time-high resolution mass spectrometry (DART-HRMS), and statistical analysis tools can be used to rapidly determine the individual components within a psychoactive brew (Ayahuasca) made from a mixture of botanicals. Five plant species used in Ayahuasca preparations were subjected to DART-HRMS analysis. The chemical fingerprint of each was reproducible but unique, thus enabling discrimination between them. The presence of important biomarkers, including N,N-dimethyltryptamine, harmaline and harmine, was confirmed using in-source collision-induced dissociation (CID). Six Ayahuasca brews made from combinations of various plant species were shown to possess a high level of similarity, despite having been made from different constituents. Nevertheless, the application of principal component analysis (PCA) was useful in distinguishing between each of the brews based on the botanical species used in the preparations. From a training set based on 900 individual analyses, three principal components covered 86.38% of the variance, and the leave-one-out cross validation was 98.88%. This is the first report of ambient ionization MS being successfully used for determination of the individual components of plant mixtures.

Mechanosensitivity below Ground: Touch-Sensitive Smell-Producing Roots in the Shy Plant Mimosa pudica

Plant roots can exhibit a type of mechanosensitivity whereby they emit noxious organosulfur compounds in response to touch. The roots of the shy plant Mimosa pudica emit a cocktail of small organic and inorganic sulfur compounds and reactive intermediates into the environment, including SO2, methanesulfinic acid, pyruvic acid, lactic acid, ethanesulfinic acid, propanesulfenic acid, 2-aminothiophenol, S-propyl propane 1-thiosulfinate, phenothiazine, and thioformaldehyde, an elusive and highly unstable compound that, to our knowledge, has never before been reported to be emitted by a plant. When soil around the roots is dislodged or when seedling roots are touched, an odor is detected. The perceived odor corresponds to the emission of higher amounts of propanesulfenic acid, 2-aminothiophenol, S-propyl propane 1-thiosulfinate, and phenothiazine. The mechanosensitivity response is selective. Whereas touching the roots with soil or human skin resulted in odor detection, agitating the roots with other materials such as glass did not induce a similar response. Light and electron microscopy studies of the roots revealed the presence of microscopic sac-like root protuberances. Elemental analysis of these projections by energy-dispersive x-ray spectroscopy revealed them to contain higher levels of K+ and Cl− compared with the surrounding tissue. Exposing the protuberances to stimuli that caused odor emission resulted in reductions in the levels of K+ and Cl− in the touched area. The mechanistic implications of the variety of sulfur compounds observed vis-à-vis the pathways for their formation are discussed.

More than just heat: ambient ionization mass spectrometry for determination of the species of origin of processed commercial products—application to psychoactive pepper supplements

The application of direct analysis in real time high resolution mass spectrometry (DART-HRMS) to the determination of the originating species of the plants from which processed commercially available plant-based products are derived is described. As a proof of principle, the method was employed for determination of the provenance of psychoactive pepper species, namely Piper methysticum (aka kava) and P. betle (aka betel). In addition to being of agricultural importance, these species are also of relevance in a forensics context. DART-HRMS spectra showed that extractions, heat treatment and other steps associated with the manufacture of these products result in significant differences in the mass spectral fingerprints observed. Nevertheless, the presence of key species-specific biomarkers such as kavalactones and chalcones in P. methysticum, and a variety of terpenes in P. betle, were retained. Chemometric processing by principal component analysis using a selection of feature masses that represented both compounds common to each of the species, and others that distinguished them, showed that the two Piper spp. could be readily identified, regardless of the manufacturing process used to create the product, with a leave-one-out cross validation result of 100%. Furthermore, unsupervised statistical analysis processing by hierarchical clustering not only enabled P. methysticum and P. betle products to be distinguished from one another, but also permitted further discrimination that was based on the processing method used to produce them. Advantages of the method over commonly used conventional protocols include minimal methods development; the capability of analyzing material in its native form without resorting to solvent extraction, derivatization or other sample preparation steps; speed; and the ability to detect and definitively identify biomarkers characteristic of a species. The method has wide applicability and is particularly useful for analysis of products from plants whose genes have not been mapped, and which, as a consequence, cannot be subjected to DNA analysis to determine the originating plant species.

Rapid Species‐level Identification of Salvias by Chemometric Processing of Ambient Ionisation Mass Spectrometry‐derived Chemical Profiles

INTRODUCTION The Salvia genus contains numerous economically important plants that have horticultural, culinary and nutraceutical uses. They are often similar in appearance, making species determination difficult. Species identification of dried Salvia products is also challenging since distinguishing plant morphological features are no longer present. OBJECTIVE The development of a simple high-throughput method of analysis of fresh and dried Salvia leaves that would permit rapid species-level identification and detection of diagnostic biomarkers. METHODOLOGY Plant leaves were analysed in their native form by DART-MS without the need for any sample preparation steps. This furnished chemical fingerprints characteristic of each species. In the same experiment, in-source collision-induced dissociation was used to identify biomarkers. Biomarker presence was also independently confirmed by GC-MS. Chemometric processing of DART-MS profiles was performed by kernel discriminant analysis (KDA) and soft independent modelling of class analogy (SIMCA) to classify the fingerprints according to species. RESULTS The approach was successful despite the occurrence of diurnal cycle and plant-age related chemical profile variations within species. In a single rapid experiment, the presence of essential oil biomarkers such as 3-carene, α-pinene, β-pinene, β-thujone, β-caryophyllene, camphor and borneol could be confirmed. The method was applied to rapid identification and differentiation of Salvia apiana, S. dominica, S. elegans, S. officinalis, S. farinacea and S. patens. CONCLUSION Species-level identification of Salvia plant material could be accomplished by chemometric processing of DART-HRMS-derived chemical profiles of both fresh and dried Salvia material. Copyright