Opportunities in phytochemistry, ecophysiology and wood research via laser ablation direct analysis in real time imaging-mass spectrometry.

Analysis of wood transects in a manner that preserves the spatial distribution of the metabolites present is highly desirable to among other things: (1) facilitate ecophysiology studies that reveal the association between chemical make-up and environmental factors or climatic events over time; and (2) investigate the mechanisms of the synthesis and trafficking of small molecules within specialised tissues. While a variety of techniques could be applied to achieve these goals, most remain challenging and impractical. Laser ablation direct analysis in real time imaging-mass spectrometry (LADI-MS) was successfully used to survey the chemical profile of wood, while also preserving the small-molecule spatial distributions. The tree species Entandrophragma candollei Harms, Millettia laurentii DeWild., Pericopsis elata (Harms) Meeuwen, Dalbergia nigra (Vell.) Benth. and Dalbergia normandii Bosser & R.Rabev were analysed. Several compounds were associated with anatomical features. A greater diversity was detected in the vessels and parenchyma compared with the fibres. Analysis of single vessels revealed that the chemical fingerprint used for timber identification is mainly determined by vessel content. Laser ablation direct analysis in real time imaging-mass spectrometry offers unprecedented opportunities to investigate the distribution of metabolites within wood samples, while circumventing the issues associated with previous methods. This technique opens up new vistas for the discovery of small-molecule biomarkers that are linked to environmental events.

A validated method for the quantification of mitragynine in sixteen commercially available Kratom (Mitragyna speciosa) products.

The recent rise in the recreational use of plant-based "legal highs" has prompted the development of methods for the identification of the bulk material, and quantification of their psychoactive components. One of these plants is Mitragyna speciosa, commonly referred to as Kratom. While traditional use of this plant was primarily for medicinal purposes, there has been a rise in its recreational use, and as a self-prescribed medication for opioid withdrawal. Although Kratom contains many alkaloids, mitragynine and 7-hydroxymitragynine are unique psychoactive biomarkers of the species, and are responsible for its psychoactive effects. A rapid validated method for the quantification of mitragynine in Kratom plant materials by direct analysis in real time-high-resolution mass spectrometry (DART-HRMS) is presented. It has a linear range of 5-100 µg mL-1, and a lower limit of quantification of 5 µg mL-1. The protocol was applied to determination of the mitragynine content of 16 commercially available Kratom plant products purchased online. The mitragynine amounts in these materials ranged from 2.76 to 20.05 mg g-1 of dried plant material. The utilization of DART-HRMS affords a mechanism not only for the preliminary identification of bulk plant material as being M. speciosa-derived (with no sample preparation required), but also provides the opportunity to quantify its psychoactive components using the same technique.

Spatial distributions of furan and 5-hydroxymethylfurfural in unroasted and roasted Coffea arabica beans.

For the first time, the spatial distributions of the highly volatile compounds furan and 5-hydroxymethylfurfural (HMF) have been determined in cross sections of green and roasted Coffea arabica beans. The image maps were revealed by laser ablation DART imaging mass spectrometry (LADI-MS). The presence of these compounds was independently confirmed by GC-MS as well as argon DART-MS. Quantification of furan by GC-MS was completed with the final concentrations in roasted and unroasted beans determined to be 96.5 and 4.1 ng/g, respectively. Furan was observed to be distributed throughout the tissue of both green and roasted beans, while HMF was localized to the silver skin in green beans. Following roasting, the appearance of HMF was more diffuse. The implications of the broad distribution of furan on the one hand, and localization of HMF on the other, are discussed.

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.

Utilizing Direct Analysis in Real Time-High Resolution Mass Spectrometry-Derived Dark Matter Spectra to Classify and Identify Unknown Synthetic Cathinones.

Herein we describe a new method of statistical analysis processing of direct analysis in real time-high resolution mass spectrometry-derived neutral loss spectra of synthetic cathinones. The dark matter observed under collision-induced dissociation conditions is rendered as "neutral loss spectra," and these are subsequently subjected to statistical analysis processing, specifically hierarchical clustering analysis. The resulting hierarchical clustering dendrogram provides a means by which to classify an unknown as a member of a subgroup of cathinones, based on structural similarity of its backbone to that of the scaffolds of the drugs represented in the training set. The described method can be utilized for the classification and identification of a number of classes of psychoactive compounds.

Identification and classification of cathinone unknowns by statistical analysis processing of direct analysis in real time-high resolution mass spectrometry-derived "neutral loss" spectra.

An approach to the rapid determination of the structures of novel synthetic cathinone designer drugs, also known as bath salts, is reported. While cathinones fragment so extensively by electron impact mass spectrometry that their mass spectra often cannot be used to identify the structure, collision-induced dissociation (CID) direct analysis in real time-high resolution mass spectrometry (DART-HRMS) experiments furnished spectra that provided diagnostic fragmentation patterns for the analyzed cathinones. From this data, neutral loss spectra, which reflect the presence of specific chemical moieties, could be acquired. These spectra showed striking similarities between cathinones sharing structural features such as pyrrolidine rings and methylenedioxy moieties. Principle component analysis (PCA) of the neutral loss spectra of nine synthetic cathinones of various types including ethcathinones, those containing a methylenedioxy moiety appended to the benzene ring, and pyrrolidine-containing structures, illustrated that cathinones falling within the same class clustered together and could be distinguished from those of other classes. Furthermore, hierarchical clustering analysis of the neutral loss data of a model set derived from 44 synthetic cathinones, furnished a dendrogram in which structurally similar cathinones clustered together. The ability of this model system to facilitate structure determination was tested using 4-fluoroethcathinone, 3,4-methylenedioxy-α-pyrrolidinohexanophenone (MDPHP), and ethylone, which fall into the ethcathinone, pyrrolidine-containing, and methylenedioxy-containing subclasses respectively. The results showed that their neutral loss spectra correctly fell within the ethcathinone, pyrrolidine-containing and methylenedioxy-containing cathinone clades of the dendrogram, and that the neutral loss information could be used to infer the structures of these compounds. The analysis and data processing steps are rapid and samples can be analyzed in their native form without any sample processing steps. The robustness of the dendrogram dataset can be readily increased by continued addition of newly discovered structures. The approach can be broadly applied to structure determination of unknowns, and would be particularly useful for analyses where sample amounts are limited.

Development of "Laser Ablation Direct Analysis in Real Time Imaging" Mass Spectrometry: Application to Spatial Distribution Mapping of Metabolites Along the Biosynthetic Cascade Leading to Synthesis of Atropine and Scopolamine in Plant Tissue.

Methods for the accomplishment of small-molecule imaging by mass spectrometry are challenged by the need for sample pretreatment steps, such as cryo-sectioning, dehydration, chemical fixation, or application of a matrix or solvent, that must be performed to obtain interpretable spatial distribution data. Furthermore, these steps along with requirements of the mass analyzer such as high vacuum, can severely limit the range of sample types that can be analyzed by this powerful method. Here, we report the development of a laser ablation-direct analysis in real time imaging mass spectrometry approach which couples a 213 nm Nd:YAG solid state UV laser to a direct analysis in a real time ion source and high-resolution time-of-flight mass spectrometer. This platform enables facile determination of the spatial distribution of small-molecules spanning a range of polarities in a diversity of sample types and requires no matrix, vacuum, solvent, or complicated sample pretreatment steps. It furnishes high-resolution data, can be performed under ambient conditions on samples in their native form, and results in little to no fragmentation of analytes. We demonstrate its application through determination of the spatial distribution of molecules involved in the biosynthetic cascade leading to formation of the clinically relevant alkaloids atropine and scopolamine in Datura leichhardtii seed tissue.

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.