Continued selection on cryptic SARS-CoV-2 observed in Missouri wastewater.

Deep sequencing of wastewater to detect SARS-CoV-2 has been used during the COVID-19 pandemic to monitor viral variants as they appear and circulate in communities. SARS-CoV-2 lineages of an unknown source that have not been detected in clinical samples, referred to as cryptic lineages, are sometimes repeatedly detected from specific locations. We have continued to detect one such lineage previously seen in a Missouri site. This cryptic lineage has continued to evolve, indicating continued selective pressure similar to that observed in Omicron lineages.

Computational Insights into the Mechanism of Nitric Oxide Generation from S-Nitrosoglutathione Catalyzed by a Copper Metal-Organic Framework.

The controlled generation of nitric oxide (NO) from endogenous sources, such as S-nitrosoglutathione (GSNO), has significant implications for biomedical implants due to the vasodilatory and other beneficial properties of NO. The water-stable metal-organic framework (MOF) Cu-1,3,5-tris[1H-1,2,3-triazol-5-yl]benzene has been shown to catalyze the production of NO and glutathione disulfide (GSSG) from GSNO in aqueous solution as well as in blood. Previous experimental work provided kinetic data for the catalysis of the 2GSNO → 2NO + GSSG reaction, leading to various proposed mechanisms. Herein, this catalytic process is examined using density functional theory. Minimal functional models of the Cu-MOF cluster and glutathione moieties are established, and three distinct catalytic mechanisms are explored. The most thermodynamically favorable mechanism studied is consistent with prior experimental findings. This mechanism involves coordination of GSNO to copper via sulfur rather than nitrogen and requires a reductive elimination that produces a Cu(I) intermediate, implicating a redox-active copper site. The experimentally observed inhibition of reactivity at high pH values is explained in terms of deprotonation of a triazole linker, which decreases the structural stability of the Cu(I) intermediate. These fundamental mechanistic insights may be generally applicable to other MOF catalysts for NO generation.

Enzymatic Degradation of Glycosaminoglycans and Proteoglycan-Mimetic Materials in Solution and on Polyelectrolyte Multilayer Surfaces.

Proteoglycans (PGs) play many important roles in biology, contributing to the mechanical properties of tissues, helping to organize extracellular matrix components, and participating in signaling mechanisms related to mechanotransduction, cell differentiation, immune responses, and wound healing. Our lab has designed two different types of PG mimics: polyelectrolyte complex nanoparticles (PCNs) and PG-mimetic graft copolymers (GCs), both of which are prepared using naturally occurring glycosaminoglycans. This work evaluates the enzymatic stability of these PG mimics using hyaluronidases (I-S, IV-S, and II), chondroitinase ABC, and lysozyme, for PG mimics suspended in solution and adsorbed onto surfaces. Hyaluronan (HA)- and chondroitin sulfate (CS)-containing PG mimics are degraded by the hyaluronidases. PCNs prepared with CS and GCs prepared with heparin are the only CS- and HA-containing PG mimics protected from chondroitinase ABC. None of the materials are measurably degraded by lysozyme. Adsorption to polyelectrolyte multilayer surfaces protects PG mimics from degradation, compared to when PG mimics are combined with enzymes in solution; all surfaces are still intact after 21 days of enzyme exposure. This work reveals how the stability of PG mimics is controlled by both the composition and macromolecular assembly of the PG mimic and also by the size and specificity of the enzyme. Understanding and tuning these degradation susceptibilities are essential for advancing their applications in cardiovascular materials, orthopedic materials, and growth factor delivery applications.

Detection of glucosamine as a marker for Aspergillus niger: a potential screening method for fungal infections.

Several species of fungus from the genus Aspergillus are implicated in pulmonary infections in immunocompromised patients. Broad screening methods for fungal infections are desirable, as cultures require a considerable amount of time to provide results. Herein, we developed degradation and detection methods to produce and detect D-glucosamine (GlcN) from Aspergillus niger, a species of filamentous fungus. Ultimately, these techniques hold the potential to contribute to the diagnosis of pulmonary fungal infections in immunocompromised patients. In the following studies, we produced GlcN from fungal-derived chitin to serve as a marker for Aspergillus niger. To accomplish this, A. niger cells were lysed and subjected to a hydrochloric acid degradation protocol. Products were isolated, reconstituted in aqueous solutions, and analyzed using hydrophilic interaction liquid chromatography (HILIC) in tandem with electrospray ionization time-of-flight mass spectrometry. Our results indicated that GlcN was produced from A. niger. To validate these results, products obtained via fungal degradation were compared to products obtained from the degradation of two chitin polymers. The observed retention times and mass spectral extractions provided a two-step validation confirming that GlcN was produced from fungal-derived chitin. Our studies qualitatively illustrate that GlcN can be produced from A. niger; applying these methods to a more diverse range of fungi offers the potential to render a broad screening method for fungal detection pertinent to diagnosis of fungal infections.

Thermogravimetric Analysis and Mass Spectrometry Allow for Determination of Chemisorbed Reaction Products on Metal Organic Frameworks.

Thermogravimetric analysis (TGA) is a technique which can probe chemisorption of substrates onto metal organic frameworks. A TGA method was developed to examine the catalytic oxidation of S-nitrosoglutathione (GSNO) by the MOF H3[(Cu4Cl)3(BTTri)8] (abbr. Cu-BTTri; H3BTTri = 1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene), yielding glutathione disulfide (GSSG) and nitric oxide (NO). Thermal analysis of reduced glutathione (GSH), GSSG, GSNO, and Cu-BTTri revealed thermal resolution of all four analytes through different thermal onset temperatures and weight percent changes. Two reaction systems were probed: an aerobic column flow reaction and an anaerobic solution batch reaction with gas agitation. In both systems, Cu-BTTri was reacted with a 1 mM GSH, GSSG, or GSNO solution, copiously rinsed with distilled-deionized water (dd-H2O), dried (25 °C, < 1 Torr), and assessed by TGA. Additionally, stock, effluent or supernatant, and rinse solutions for each glutathione derivative within each reaction system were assessed by mass spectrometry (MS) to inform on chemical transformations promoted by Cu-BTTri as well as relative analyte concentrations. Both reaction systems exhibited chemisorption of glutathione derivatives to the MOF by TGA. Mass spectrometry analyses revealed that in both systems, GSH was oxidized to GSSG, which chemisorbed to the MOF whereas GSSG remained unchanged during chemisorption. For GSNO, chemisorption to the MOF without reaction was observed in the aerobic column setup, whereas conversion to GSSG and subsequent chemisorption was observed in the anaerobic batch setup. These findings suggest that within this reaction system, GSSG is the primary adsorbent of concern with regards to strong binding to Cu-BTTri. Development of similar thermal methods could allow for the probing of MOF reactivity for a wide range of systems, informing on important considerations such as reduced catalytic efficiency from poisoning, recyclability, and loading capacities of contaminants or toxins with MOFs.

Homochiral Metal-Organic Frameworks for Enantioselective Separations in Liquid Chromatography.

Selective separation of enantiomers is a substantial challenge for the pharmaceutical industry. Chromatography on chiral stationary phases is the standard method, but at a very high cost for industrial-scale purification due to the high cost of the chiral stationary phases. Typically, these materials are poorly robust, expensive to manufacture, and often too specific for a single desired substrate, lacking desirable versatility across different chiral analytes. Here, we disclose a porous, robust homochiral metal-organic framework (MOF), TAMOF-1, built from copper(II) and an affordable linker prepared from natural l-histidine. TAMOF-1 has shown to be able to separate a variety of model racemic mixtures, including drugs, in a wide range of solvents of different polarity, outperforming several commercial chiral columns for HPLC separations. Although not exploited in the present article, it is worthy to mention that the preparation of this new material is scalable to the multikilogram scale, opening unprecedented possibilities for low-energy chiral separation at the industrial scale.

S-Nitrosoglutathione exhibits greater stability than S-nitroso-N-acetylpenicillamine under common laboratory conditions: A comparative stability study.

S-Nitrosothiols (RSNOs) such as S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) are susceptible to decomposition by stimuli including heat, light, and trace metal ions. Using stepwise isothermal thermogravimetric analysis (TGA), we observed that NO-forming homolytic cleavage of the S-N bond occurs at 134.7 ±â€¯0.8 °C in GSNO and 132.8 ±â€¯0.9 °C in SNAP, contrasting with the value of 150 °C that has been previously reported for both RSNOs. Using mass spectrometry (MS), nuclear magnetic resonance (NMR), and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), we analyzed the decomposition products from TGA experiments. The organic product of GSNO decomposition was glutathione disulfide, while SNAP decomposed to form N-acetylpenicillamine disulfide as well as other products, including tri- and tetrasulfides. In addition, we assessed the relative solution stabilities of GSNO and SNAP under common laboratory conditions, which include variable temperature, pH, and light exposure with rigorous exclusion of trace metal ions by chelation. GSNO exhibited greater stability than SNAP over a 7-day period except in one instance. Both RSNOs demonstrated an inverse relationship between solution stability and temperature, with refrigeration considerably extending shelf life. A decrease in pH from 7.4 to 5.0 also enhanced the stability of both RSNOs. A further decrease in pH from 5.0 to 3.0 resulted in decreased stability for both RSNOs, and is notably the only occasion in which SNAP proved more stable than GSNO. After 1 h of exposure to overhead fluorescent lighting, both RSNOs displayed high susceptibility to light-induced decomposition. After 7 h, GSNO and SNAP decomposed 19.3 ±â€¯0.5% and 30 ±â€¯2%, respectively.

Nitric oxide generation from S-nitrosoglutathione: New activity of indium and a survey of metal ion effects.

S-Nitrosothiols (RSNOs) such as S-nitrosoglutathione (GSNO) are known to produce nitric oxide (NO) through thermal, photolytic, and metal ion-promoted pathways, which has led to their increasing use as exogenous sources of therapeutic NO. Despite the burgeoning NO release applications for RSNOs, their susceptibility to metal-promoted decomposition has rarely been examined in a uniform manner through the specific measurement of NO release. In this study, the ability of various transition and post-transition metal ions to promote NO release from GSNO was surveyed by chemiluminescence-based NO detection. Substantial NO formation (>10-fold increase relative to GSNO baseline) was detected after the addition of Cu2+, Au3+, Pd2+, Pt2+, and V3+. Modest increases were observed in the cases of Co2+, Hf4+, Fe2+, Fe3+, Mn2+, Hg2+, Ni2+, Ag+, Sn2+, and Zr4+, while no effect was evident for Al3+, Cr3+, Pb2+, Sc3+, and Zn2+. It was further observed that In+ compounds initiate the apparent NO-forming decomposition of GSNO, while In0 and In3+ are inactive, indicating that In+ exerts a previously unknown effect on GSNO.

Amino-Incorporated Tricarboxylate Metal-Organic Framework for the Sensitive Fluorescence Detection of Heavy Metal Ions with Insights into the Origin of Photoluminescence Response.

Metal-organic frameworks (MOFs) are powerful chemosensors when designed to undergo a detectable change in optical properties upon interacting with target analytes. This work contributes to the overall understanding of metal-ion interactions with MOFs to elicit changes in fluorescence emission, a necessary step en route to developing more sensitive and selective systems for metal-ion sensing. Toward this goal, the photophysical properties of an amino-containing MOF, Cu3(NH2BTC)2, were investigated. The MOF was highly sensitive for the detection of Fe2+ and Fe3+ exhibiting the most intense fluorescence quenching with the lowest detectable change in signal occurring at 1.55 ppm (27.8 µM) at room temperature in minutes. Other metal ions, including Pb2+, Cu2+, Mn2+, Ni2+, and Co2+, were also detected at 5.7, 12, 3.0, 1.6, and 0.2 ppm, respectively, demonstrating the range of sensing capabilities. Additional experiments were performed to elucidate the pathway of metal-ion detection, including the investigation of photoluminescent changes upon the introduction of acids (HCl, ZrCl4, and AlCl3) and several anions (CO32-, OAc-, and Cr2O72-). To determine the influence of the amino functional group on interactions with the analytes, isoreticular Cu3BTC2 and postsynthetically modified Cu3(NH2BTC)2 (to hinder access to the free-amine moiety) were also investigated, revealing that the free amine is essential for the detection of the anions and is likely involved in the detection of several divalent metal ions. On the other hand, divalent metal ion Fe2+ likely induces an emission intensity change by interaction with the carboxylate of the MOF ligand or the open Cu2+ sites. Taken together, this report demonstrates that Cu3(NH2BTC)2 has unique photoluminescent properties and likely elicits detection of ions (ppm, µM) via multiple pathways. As such, future development of MOF-based sensors should include MOFs with free accessible functional handles (such as an amine) to be strategically modified to selectively detect specific metal ions.

Synthesis of novel nitroreductase enzyme-activated nitric oxide prodrugs to site-specifically kill bacteria.

The developing antibiotic resistance crisis creates a serious need for new antimicrobial agents. In this work, novel nitroaromatic-protected piperazine diazeniumdiolate (nitric oxide donor) prodrugs are synthesized to release nitric oxide upon enzyme activation to kill bacteria. Antibacterial prodrugs could help reduce side effects due to antibiotics, only releasing the therapeutic where infections are concentrated. The nitroreductase enzyme, which is found almost exclusively in bacteria, reduces the nitroaromatic-protecting group of the synthesized compounds and catalyzes the release of nitric oxide. This paper shows that nitric oxide release from the synthesized compounds only occurs in the presence of a bacteria-derived nitroreductase enzyme, demonstrating the possibility of site-specific delivery of an antibacterial therapeutic. The amount of nitric oxide release is measured at concentrations of 0.01, 0.1, and 1 mM, and is well within antibacterial levels at concentrations of 0.1 and 1 mM. The antibacterial activity of the compounds is demonstrated after exposure of the compounds to Escherichia coli, a nitroreductase-producing bacterial species, leading to up to a 94% reduction in the number of viable bacteria after 24 h at 1 mM concentrations of the prodrug. This study is the first example of an antibacterial diazeniumdiolate prodrug activated by a nitroreductase enzyme, and further demonstrates the possibilities of antibacterial prodrugs.

Small Molecule Interferences in Resazurin and MTT-Based Metabolic Assays in the Absence of Cells.

In vitro assays (such as resazurin and MTT) provide an opportunity to determine the cytotoxicity of novel therapeutics before moving forward with expensive and resource-intensive in vivo studies. A concern with using these assays, however, is the production of false responses in the presence of particular chemical functionalities. To better understand this phenomenon, 19 small molecules at 6 concentrations (1 µM-100 mM) were tested in the presence of resazurin and MTT reagents to highlight potential interfering species. Through the use of absorbance measurements (using well-plate assays and UV-vis spectroscopy) with parallel MS analysis, we have shown that significant conversion of the assay reagents readily occurs in the presence of many tested interfering species without the need for any cellular activity. The most attributable sources of interference seem to arise from the presence of thiol and carboxylic acid moieties. Interestingly, the detectable interferences were more prevalent and larger in the presence of MTT (19 species with some deviations >3000%) compared to resazurin (16 species with largest deviation of ∼150%). Additionally, those deviations in the presence of resazurin were only substantial at high concentrations, while MTT showed deviations across the tested concentrations. This comprehensive study gives insight into chemical functional groups (thiols, amines, amides, carboxylic acids) that may interfere with resazurin and MTT assays in the absence of metabolic activity and indicates that proper control studies must be performed to obtain accurate data from these in vitro assays.

Functionalization of Metal-Organic Frameworks To Achieve Controllable Wettability.

The overall versatility of a material can be immensely expanded by the ability to controllably tune its hydrophobicity. Herein we took advantage of steric bias to demonstrate that tricarboxylate metal-organic frameworks (MOFs) can undergo covalent postsynthetic modification to confer various degrees of hydrophobicity. MOF copper 2-aminobenzene-1,3,5-tricarboxylate was modified with varying-length aliphatic carbon chains. Unmodified Cu3(NH2BTC)2 degrades in minutes upon contact with water, whereas modification as low as 14% results in powders that show significantly enhanced hydrophobic character with contact angles up to 147°. The modified material is capable of withstanding direct contact with water for 30 min with no visual evidence of altered surface characteristics. A linear relationship was observed between the length of the tethered chain and the water contact angle. These results reveal a predictable method for achieving a range of desirable sorption rates and highly controllable hydrophobic character. This work thereby expands the possibilities of rationally modifying MOFs for a plethora of target-specific applications.

Nitric oxide mediates activity-dependent plasticity of retinal bipolar cell output via S-nitrosylation.

Coding a wide range of light intensities in natural scenes poses a challenge for the retina: adaptation to bright light should not compromise sensitivity to dim light. Here we report a novel form of activity-dependent synaptic plasticity, specifically, a "weighted potentiation" that selectively increases output of Mb-type bipolar cells in the goldfish retina in response to weak inputs but leaves the input-output ratio for strong stimuli unaffected. In retinal slice preparation, strong depolarization of bipolar terminals significantly lowered the threshold for calcium spike initiation, which originated from a shift in activation of voltage-gated calcium currents (ICa) to more negative potentials. The process depended upon glutamate-evoked retrograde nitric oxide (NO) signaling as it was eliminated by pretreatment with an NO synthase blocker, TRIM. The NO-dependent ICa modulation was cGMP independent but could be blocked by N-ethylmaleimide (NEM), indicating that NO acted via an S-nitrosylation mechanism. Importantly, the NO action resulted in a weighted potentiation of Mb output in response to small (≤-30 mV) depolarizations. Coincidentally, light flashes with intensity ≥ 2.4 × 10(8) photons/cm(2)/s lowered the latency of scotopic (≤ 2.4 × 10(8) photons/cm(2)/s) light-evoked calcium spikes in Mb axon terminals in an NEM-sensitive manner, but light responses above cone threshold (≥ 3.5 × 10(9) photons/cm(2)/s) were unaltered. Under bright scotopic/mesopic conditions, this novel form of Mb output potentiation selectively amplifies dim retinal inputs at Mb → ganglion cell synapses. We propose that this process might counteract decreases in retinal sensitivity during light adaptation by preventing the loss of visual information carried by dim scotopic signals.

Accurate nitric oxide measurements from donors in cell media: identification of scavenging agents.

Nitric oxide (NO) is an essential messenger in human physiology, mediating cellular processes ranging from proliferation to apoptosis. The effects of NO are concentration dependent, and control over the instantaneous amount of NO available to cells is essential for determining the therapeutic NO dosages for various applications. As such, the development of NO therapeutic materials relies on accurate quantitative NO measurements that provide both total NO release from the NO donor as well as instantaneous NO concentrations. On the basis of the complexity of the cell media environment, inaccurate NO reporting often occurs for in vitro studies. These inaccuracies result from using inert media such as phosphate buffer saline (PBS), failing to account for the reactivity of media components. In this work, we describe a method for directly quantifying the instantaneous and total amounts of NO from commonly used NO donors in commercially available cell media routinely used for endothelial and neural cell lines. A riboflavin-tryptophan complex found in the media was identified as the major scavenger of NO in the cell media and likely reacts with NO via a radical-radical reaction. This finding significantly impacts the amount of available NO. The scavenging effects are concentration dependent on the riboflavin-tryptophan complex and the NO release rate from the NO donor. The results of this study provide insights on the exogenous amounts of NO that are present in cell media and may provide an explanation for differences in NO dosages between buffer experiments and in vitro and in vivo studies.

Achieving Biomedically Desirable Nitric Oxide Release from Glucose Monitor Surfaces Via a Cu-Based Catalyst Coating.

We report the design of a blood-contacting glucose monitor with a nitric oxide (NO)-releasing metal-organic framework (MOF) embedded within the outer polymer layer of a glucose sensor to promote the release of NO from endogenous NO donors. The sensors were tested by using amperometry across a range of glucose concentrations to assess whether the presence of either the MOF or NO decreased the performance of the glucose monitor. Even though signal response was diminished, the sensors maintained a good regression fit (R2 = 0.9944) and a similar dynamic range and reproducibility in the presence of S-nitrosoglutathione.

Copper(II) Ions Originating from CuBTC MOF Act as a Soluble Catalyst in the Friedländer Synthesis.

The copper-based metal-organic framework (MOF), CuBTC (where H3BTC = benzene-1,3,5-tricarboxylate), has been reported as a reusable heterogeneous catalyst for the Friedländer synthesis of substituted quinolines, which are desirable targets in the pharmaceutical industry. Because of this application, we further investigated the CuBTC-catalyzed Friedländer synthesis of 3-acetyl-2-methyl-4-phenylquinoline. CuBTC was synthesized in-house and used as a catalyst for the Friedländer synthesis. Fresh and used CuBTC were analyzed using scanning electron microscopy (SEM), powder X-ray diffraction (pXRD), and X-ray photoelectron spectroscopy (XPS). The used CuBTC shows structural breakdown in pXRD patterns and SEM images. Despite the structural breakdown, the desired product, 3-acetyl-2-methyl-4-phenylquinoline, is still produced in a moderate yield (76.3% ± 0.2), as confirmed via time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy. Inductively coupled plasma atomic emission spectroscopy of the recovered supernatant solution indicates the presence of copper(II) ions in solution. Thus, we hypothesized that the standard Friedländer conditions may degrade the CuBTC framework, resulting in copper(II) ions in solution. Control experiments with copper(II) from Cu(NO3)2·3H2O catalyzes the Friedländer reaction in yields (75.6% ± 0.1) equal to that of the CuBTC MOF. Overall, our findings suggest that CuBTC acts as a copper(II) source, and the copper(II) ions originating from the CuBTC MOF are responsible for the observed catalysis.

The different adsorption-degradation behaviors of SARS-CoV-2 by bioactive chemicals in wastewater: The suppression kinetics and their implications for wastewater-based epidemiology.

Wastewater-Based Epidemiology (WBE) is widely used to monitor the progression of SARS-CoV-2 pandemic. While there is a clear correlation between the number of COVID patients in a sewershed and the viral load in the wastewater, there is notable variability across different treatment plants. In particular, some facilities consistently exhibit higher viral content per diagnosed patient, implying a potential underestimation of the number of COVID patients, while others show a low viral load per diagnosed case, indicating potential attenuation of genetic material from the sewershed. In this study, we investigated the impact of nonylphenol ethoxylate (NPHE), linear alkylbenzene sulfonic acid (LABS), bisoctyl dimethyl ammonium chloride (BDAC), and didecyldimethylammonium chloride (DDAC), the surfactants that have been commonly used as detergents, emulsifiers, wetting agents on the stability of SARS-CoV-2 in wastewater. The results showed multiple and dynamic mechanisms, including degradation and desorption, can occur simultaneously during the interaction between SARS-CoV-2 and different chemicals depending on the physicochemical properties of each chemical. Through the elucidation of the dynamic interactions, the findings from this study could help the state health organizations and scientific community to optimize the SARS-CoV-2 wastewater-based epidemiology strategies.

Applications for nitric oxide in halting proliferation of tumor cells.

Tumor resistance to cytotoxic therapeutics coupled with dose-limiting toxicity is a serious hurdle in the field of medical oncology. In the face of this obstacle, nitric oxide has emerged as a powerful adjuvant for the hypersensitization of tumors to more traditional chemo- and radio-therapeutics. Furthermore, emerging evidence indicates that nitric oxide donors have the potential to function independently in the clinical management of cancer. Herein, we discuss the role of nitric oxide in cancer and the potential for nitric oxide donors to support conventional therapeutics.

Continuous flow catalysis with CuBTC improves reaction time for synthesis of xanthene derivatives.

The copper-based metal-organic framework (MOF) CuBTC (where H3BTC = benzene-1,3,5-tricarboxylate) has been shown to be an efficient heterogeneous catalyst for the generation of 1,8-dioxo-octa-hydro xanthene derivatives, which are valuable synthetic targets for the pharmaceutical industry. We have applied this catalytic capability of CuBTC to a continuous flow system to produce the open chain form of 3,3,6,6-tetramethyl-9-phenyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione, a xanthene derivative from benzaldehyde and dimedone. An acid work-up after producing the open chain form of the xanthene derivative was used to achieve ring closure and form the final xanthene product. The CuBTC used to catalyze the reaction under continuous flow was confirmed to be stable throughout this process via analysis by SEM, pXRD, and FT-IR spectroscopy, elemental analysis, and XPS. The reaction to produce the open-chain form of the xanthene derivative produced an average yield of 33% ± 14% under the continuous flow (compared to 33% ± 0.12% of performing it under batch conditions). Based on the data obtained from this work, the continuous flow system required 22.5x less time to produce the desired xanthene derivative at comparable yields to batch reaction conditions. These results would allow for the xanthene derivative to be produced much faster, at a lower cost, and require less personal time while also removing the need to perform catalyst remove post reaction.

Modeling a pesticide remediation strategy for preparative liquid chromatography using high-performance liquid chromatography.

BACKGROUND: Cannabis sativa L. also known as industrial hemp, is primarily cultivated as source material for cannabinoids cannabidiol (CBD) and ∆9-tetrahydrocannabinol (∆9-THC). Pesticide contamination during plant growth is a common issue in the cannabis industry which can render plant biomass and products made from contaminated material unusable. Remediation strategies to ensure safety compliance are vital to the industry, and special consideration should be given to methods that are non-destructive to concomitant cannabinoids. Preparative liquid chromatography (PLC) is an attractive strategy for remediating pesticide contaminants while also facilitating targeted isolation cannabinoids in cannabis biomass. METHODS: The present study evaluated the benchtop-scale suitability of pesticide remediation by liquid chromatographic eluent fractionation, by comparing retention times of 11 pesticides relative to 26 cannabinoids. The ten pesticides evaluated for retention times are clothianidin, imidacloprid, piperonyl butoxide, pyrethrins (I/II mixture), diuron, permethrin, boscalid, carbaryl, spinosyn A, and myclobutanil. Analytes were separated prior to quantification on an Agilent Infinity II 1260 high performance liquid chromatography with diode array detection (HPLC-DAD). The detection wavelengths used were 208, 220, 230, and 240 nm. Primary studies were performed using an Agilent InfinityLab Poroshell 120 EC-C18 3.0 × 50 mm column with 2.7 µm particle diameter, using a binary gradient. Preliminary studies on Phenomenex Luna 10 µm C18 PREP stationary phase were performed using a 150 × 4.6 mm column. RESULTS: The retention times of standards and cannabis matrices were evaluated. The matrices used were raw cannabis flower, ethanol crude extract, CO2 crude extract, distillate, distillation mother liquors, and distillation bottoms. The pesticides clothianidin, imidacloprid, carbaryl, diuron, spinosyn A, and myclobutanil eluted in the first 3.6 min, and all cannabinoids (except for 7-OH-CBD) eluted in the final 12.6 min of the 19-minute gradient for all matrices evaluated. The elution times of 7-OH-CBD and boscalid were 3.44 and 3.55 min, respectively. DISCUSSION: 7-OH-CBD is a metabolite of CBD and was not observed in the cannabis matrices evaluated. Thus, the present method is suitable for separating 7/11 pesticides and 25/26 cannabinoids tested in the six cannabis matrices tested. 7-OH-CBD, pyrethrins I and II (RTA: 6.8 min, RTB: 10.5 min), permethrin (RTA: 11.9 min, RTB: 12.2 min), and piperonyl butoxide (RTA: 8.3 min, RTB: 11.7 min), will require additional fractionation or purification steps. CONCLUSIONS: The benchtop method was demonstrated have congruent elution profiles using preparative-scale stationary phase. The resolution of pesticides from cannabinoids in this method indicates that eluent fractionation is a highly attractive industrial solution for pesticide remediation of contaminated cannabis materials and targeted isolation of cannabinoids.