A Rapid, High-Throughput Validated Method for the Quantification of Atropine in Datura stramonium Seeds Using Direct Analysis in Real Time-High Resolution Mass Spectrometry (DART-HRMS).

The utility of direct analysis in real time-high resolution mass spectrometry (DART-HRMS) for quantification of a variety of compounds has been explored, but the number of reports of validated methods using this technique is limited. Furthermore, despite the increasing use in crime labs of DART-HRMS for the detection and identification of drugs of abuse, very few published reports have appeared describing how the method can be exploited for the analysis of small molecules of interest within complex matrices such as plant tissues. Herein we describe the steps to be taken to establish a validated quantification method for psychoactive compounds within complex plant matrices through its application to the detection and quantification of atropine in Datura stramonium seeds. Six calibration standard series are analyzed eight times over a period of several days to create a calibration curve. The resulting calibration curve is tested using six quality control samples and finally utilized to determine the concentration of atropine in a D. stramonium seed extract. The linear range for quantification of atropine in this study was found to be comparable to that reported previously using GC, LC, HPLC, and UHPLC-MS methods. Furthermore, the method can be applied to the quantification of other biomarkers in plant materials, despite the complexity of the plant matrix. The speed of the analysis (<10 min for duplicate analysis of 20 samples) and the ability to integrate peaks using accurate masses for specificity are advantages of the DART-HRMS quantification approach.

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, ß-pinene, ß-thujone, ß-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 © 2016 John Wiley & Sons, Ltd.

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry.

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

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.

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.

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

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.

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.

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.

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.

Rapid detection by direct analysis in real time-mass spectrometry (DART-MS) of psychoactive plant drugs of abuse: the case of Mitragyna speciosa aka "Kratom".

Mitragyna speciosa, also known commonly as "Kratom" or "Ketum", is a plant with psychoactive properties that have been attributed to the presence of various indole alkaloids such as mitragynine and 7-hydroxymitragynine. M. speciosa use is gaining popularity internationally as a natural and legal alternative to narcotics. As a drug of abuse, its detection and identification are not straightforward, since M. speciosa plant material is not particularly distinctive. Here, we show that direct analysis in real time-mass spectrometry (DART-MS) can be used not only to rapidly identify M. speciosa plant material and distinguish it from other plants, but also to distinguish between M. speciosa plant varieties, based on differences between their chemical profiles. The method is rapid and the analysis expeditious. Plant material such as that found at a crime scene can be analyzed directly with no sample pre-preparation steps. Furthermore, we show that the basis set of principal components that permit characterization of the plant material can be used to positively identify M. speciosa.

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.

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.

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.

Direct analysis in real time mass spectrometry (DART-MS) of "bath salt" cathinone drug mixtures.

Rapid and versatile direct analysis in real time mass spectrometry (DART-MS) methods were developed for detection and characterization of synthetic cathinone designer drugs, also known as "bath salts". The speed and efficiency associated with DART-MS testing of such highly unpredictable samples demonstrate the technique as an attractive alternative to conventional GC-MS and LC-MS methods. A series of isobaric and closely related synthetic cathinones, alone and in mixtures, were differentiated using high mass accuracy and in-source collision induced dissociation (CID). Crime laboratories have observed a dramatic rise in the use of these substances, which has caused sample testing backlogs, particularly since the myriad of structurally related compounds are challenging to efficiently differentiate. This challenge is compounded by the perpetual emergence of new structural variants as soon as older generation derivatives become scheduled. Because of the numerous chemical substances that fall into these categories, along with the varying composition and complexity of mixtures of these drugs, DART-MS CID has the potential to dramatically streamline sample analysis, minimize the number of sample preparation steps, and enable rapid characterization of emerging structural analogs.

Direct analysis in real time mass spectrometry with collision-induced dissociation for structural analysis of synthetic cannabinoids.

RATIONALE: The emergence of numerous cannabinoid designer drugs has been tied to large spikes in emergency room visits and overdoses. Identifying these substances is difficult for the following reasons: (1) the compounds are novel, closely structurally related, and do not usually test positive in drug screens; (2) novel analogs rapidly appear on the market; (3) no standard protocols exist for their identification; and (4) customized and extensive sample preparation/extraction and analysis procedures are required to demonstrate their presence. METHODS: Direct analysis in real time mass spectrometry (DART-MS) employing collision-induced dissociation (CID) provided confirmatory structural information that was useful in characterizing the various cannabinoid analogs, including those contained in mixtures. CID analysis illustrated that, although closely related compounds fragment in a similar fashion, their structural differences still resulted in multiple diagnostic peaks that provided additional confidence towards structural identification. RESULTS: DART-MS spectra were acquired under CID conditions to rapidly differentiate among five synthetic cannabinoids contained within 'herbal' products purchased locally in New York State (USA). The spectra exhibited [M+H](+) ions and product ions unique to each cannabinoid that corresponded to major structural features. Five different cannabinoid analogs, alone and as mixtures of at least two cannabinoids, were identified in six herbal products and differentiated by their CID product ion patterns. CONCLUSIONS: Illicit synthetic cannabinoid products continue to be readily available despite national and international restrictions. These products contain a wide range of active components, and, in many cases, multiple active ingredients. DART-MS allows rapid analyses of these synthetic cannabinoids based on the exact masses of their [M+H](+) ions and product ion peaks generated using CID.