Isolation of Caribbean Ciguatoxin-5 (C-CTX5) and confirmation of its structure by NMR spectroscopy.

Ciguatera poisoning occurs throughout subtropical and tropical regions globally. The Virgin Islands in the Caribbean Sea is a known hyperendemic region for ciguatera and has been associated with Caribbean ciguatoxin (C-CTX) contamination in fish. An algal C-CTX (C-CTX5) was identified in Gambierdiscus silvae and G. caribeaus isolated from benthic algal samples collected in waters south St. Thomas, US Virgin Islands. The highest CTX-producing isolate, G. silvae 1602 SH-6, was grown at large-scale to isolate sufficient C-CTX5 for structural confirmation by NMR spectroscopy. A series of orthogonal extraction and fractionation procedures resulted in purification of approximately 40 µg of C-CTX5, as estimated by quantitative NMR. A suite of 1D and 2D NMR experiments were acquired that verified the structure originally proposed for C-CTX5. The structural confirmation and successful isolation of C-CTX5 opens the way for work on the stability, toxicology and biotransformation of C-CTXs, as well as for the production of quantitative reference materials for analytical method development and validation. The strategies developed for purification of C-CTX5 may also apply to isolation and purification of CTXs from the Pacific Ocean and other regions.

Targeted Metabolite Fingerprints of Thirteen Gambierdiscus, Five Coolia and Two Fukuyoa Species.

The genus Gambierdiscus produces an array of bioactive hydrophilic and lipophilic secondary metabolites that range in mode of action and toxicity. In this study, the metabolite fingerprint was mapped for thirteen Gambierdiscus, five Coolia and two Fukuyoa species (34 isolates) by assessing the production of 56 characterised secondary metabolites. Gambierdiscus polynesiensis was the only species to produce Pacific-ciguatoxin-3B (P-CTX3B), P-CTX3C, iso-P-CTX3B/C, P-CTX4A, P-CTX4B and iso-P-CTX4A/B. G. australes produced maitotoxin-1 (MTX-1) and MTX-5, G. cheloniae produced MTX-6 and G. honu produced MTX-7. Ubiquitous production of 44-methylgambierone was observed amongst all the Gambierdiscus isolates, with nine species also producing gambierone. Additional gambierone analogues, including anhydrogambierone (tentatively described herein), were also detected in all Gambierdiscus species, two Coolia and two Fukuyoa species. Gambieroxide was detected in G. lewisii and G. pacificus and gambieric acid A was detected in ten Gambierdiscus species, with G. australes (CAWD381) being the only isolate to produce gambieric acids A-D. This study has demonstrated that the isolates tested to date produce the known CTXs or MTXs, but not both, and highlighted several species that produced 'unknown' compounds displaying characteristics of cyclic polyethers, which will be the focus of future compound discovery efforts.

Gambierone and Sodium Channel Specific Bioactivity Are Associated with the Extracellular Metabolite Pool of the Marine Dinoflagellate Coolia palmyrensis.

Tropical epibenthic dinoflagellate communities produce a plethora of bioactive secondary metabolites, including the toxins ciguatoxins (CTXs) and potentially gambierones, that can contaminate fishes, leading to ciguatera poisoning (CP) when consumed by humans. Many studies have assessed the cellular toxicity of causative dinoflagellate species to better understand the dynamics of CP outbreaks. However, few studies have explored extracellular toxin pools which may also enter the food web, including through alternative and unanticipated routes of exposure. Additionally, the extracellular exhibition of toxins would suggest an ecological function and may prove important to the ecology of the CP-associated dinoflagellate species. In this study, semi-purified extracts obtained from the media of a Coolia palmyrensis strain (DISL57) isolated from the U.S. Virgin Islands were assessed for bioactivity via a sodium channel specific mouse neuroblastoma cell viability assay and associated metabolites evaluated by targeted and non-targeted liquid chromatography tandem and high-resolution mass spectrometry. We found that extracts of C. palmyrensis media exhibit both veratrine enhancing bioactivity and non-specific bioactivity. LC-HR-MS analysis of the same extract fractions identified gambierone and multiple undescribed peaks with mass spectral characteristics suggestive of structural similarities to polyether compounds. These findings implicate C. palmyrensis as a potential contributor to CP and highlight extracellular toxin pools as a potentially significant source of toxins that may enter the food web through multiple exposure pathways.

Structural Characterization of Maitotoxins Produced by Toxic Gambierdiscus Species.

Identifying compounds responsible for the observed toxicity of the Gambierdiscus species is a critical step to ascertaining whether they contribute to ciguatera poisoning. Macroalgae samples were collected during research expeditions to Rarotonga (Cook Islands) and North Meyer Island (Kermadec Islands), from which two new Gambierdiscus species were characterized, G. cheloniae CAWD232 and G. honu CAWD242. Previous chemical and toxicological investigations of these species demonstrated that they did not produce the routinely monitored Pacific ciguatoxins nor maitotoxin-1 (MTX-1), yet were highly toxic to mice via intraperitoneal (i.p.) injection. Bioassay-guided fractionation of methanolic extracts, incorporating wet chemistry and chromatographic techniques, was used to isolate two new MTX analogs; MTX-6 from G. cheloniae CAWD232 and MTX-7 from G. honu CAWD242. Structural characterization of the new MTX analogs used a combination of analytical chemistry techniques, including LC-MS, LC-MS/MS, HR-MS, oxidative cleavage and reduction, and NMR spectroscopy. A substantial portion of the MTX-7 structure was elucidated, and (to a lesser extent) that of MTX-6. Key differences from MTX-1 included monosulfation, additional hydroxyl groups, an extra double bond, and in the case of MTX-7, an additional methyl group. To date, this is the most extensive structural characterization performed on an MTX analog since the complete structure of MTX-1 was published in 1993. MTX-7 was extremely toxic to mice via i.p. injection (LD50 of 0.235 µg/kg), although no toxicity was observed at the highest dose rate via oral administration (155.8 µg/kg). Future research is required to investigate the bioaccumulation and likely biotransformation of the MTX analogs in the marine food web.

LC-HRMS and Chemical Derivatization Strategies for the Structure Elucidation of Caribbean Ciguatoxins: Identification of C-CTX-3 and -4.

Ciguatera poisoning is linked to the ingestion of seafood that is contaminated with ciguatoxins (CTXs). The structural variability of these polyether toxins in nature remains poorly understood due to the low concentrations present even in highly toxic fish, which makes isolation and chemical characterization difficult. We studied the mass spectrometric fragmentation of Caribbean CTXs, i.e., the epimers C-CTX-1 and -2 (1 and 2), using a sensitive UHPLC-HRMS/MS approach in order to identify product ions of diagnostic value. We found that the fragmentation of the ladder-frame backbone follows a characteristic pattern and propose a generalized nomenclature for the ions formed. These data were applied to the structural characterization of a pair of so far poorly characterized isomers, C-CTX-3 and -4 (3 and 4), which we found to be reduced at C-56 relative to 1 and 2. Furthermore, we tested and applied reduction and oxidation reactions, monitored by LC-HRMS, in order to confirm the structures of 3 and 4. Reduction of 1 and 2 with NaBH4 afforded 3 and 4, thereby unambiguously confirming the identities of 3 and 4. In summary, this work provides a foundation for mass spectrometry-based characterization of new C-CTXs, including a suite of simple chemical reactions to assist the examination of structural modifications.

The Terroir of Cannabis: Terpene Metabolomics as a Tool to Understand Cannabis sativa Selections.

The phytochemical diversity of Cannabis chemovars is not well understood, and many chemovars were created in informal breeding programs without records of parentage or the criteria for selection. Key criteria for selection sometimes included aroma notes and visual cues, which some breeders associated with pharmacological activity. We hypothesized that the process of selection for scents believed to be related to specific tetrahydrocannabinol levels has resulted in modified terpene biosynthesis in these chemovars. Thirty-two cannabinoids, 29 monoterpenes and 38 sesquiterpenes were measured in 33 chemovars from 5 licensed producers. A classification system based on cannabinoid content was used with targeted metabolomic tools to determine relationships in the phytochemistry. Three monoterpenes, limonene, ß-myrcene, and α-pinene, and two sesquiterpenes, caryophyllene and humulene, were abundant in the majority of chemovars. Nine terpenes were present in tetrahydrocannabinol-dominant chemovars. Three monoterpenes and four sesquiterpenes were predominantly found in cannabidiol-containing chemovars. Low abundance terpenes may have been the aromatic cues identified by breeders. The medicinal activity of some of the terpenes is likely to contribute to the pharmacological effect of specific chemovars. Together, these data demonstrate the synergy of compounds in Cannabis chemovars and point to the need for additional research to understand the phytochemical complexity.

Leaner and greener analysis of cannabinoids.

There is an explosion in the number of labs analyzing cannabinoids in marijuana (Cannabis sativa L., Cannabaceae) but existing methods are inefficient, require expert analysts, and use large volumes of potentially environmentally damaging solvents. The objective of this work was to develop and validate an accurate method for analyzing cannabinoids in cannabis raw materials and finished products that is more efficient and uses fewer toxic solvents. An HPLC-DAD method was developed for eight cannabinoids in cannabis flowers and oils using a statistically guided optimization plan based on the principles of green chemistry. A single-laboratory validation determined the linearity, selectivity, accuracy, repeatability, intermediate precision, limit of detection, and limit of quantitation of the method. Amounts of individual cannabinoids above the limit of quantitation in the flowers ranged from 0.02 to 14.9% w/w, with repeatability ranging from 0.78 to 10.08% relative standard deviation. The intermediate precision determined using HorRat ratios ranged from 0.3 to 2.0. The LOQs for individual cannabinoids in flowers ranged from 0.02 to 0.17% w/w. This is a significant improvement over previous methods and is suitable for a wide range of applications including regulatory compliance, clinical studies, direct patient medical services, and commercial suppliers.

Single-Laboratory Validation for the Determination of Flavonoids in Hawthorn Leaves and Finished Products by LC-UV.

Suitably validated analytical methods that can be used to quantify medicinally active phytochemicals in natural health products are required by regulators, manufacturers, and consumers. Hawthorn (Crataegus) is a botanical ingredient in natural health products used for the treatment of cardiovascular disorders. A method for the quantitation of vitexin-2″-O- rhamnoside, vitexin, isovitexin, rutin, and hyperoside in hawthorn leaf and flower raw materials and finished products was optimized and validated according to AOAC International guidelines. A two-level partial factorial study was used to guide the optimization of the sample preparation. The optimal conditions were found to be a 60-minute extraction using 50 : 48 : 2 methanol : water : acetic acid followed by a 25-minute separation using a reversed-phased liquid chromatography column with ultraviolet absorbance detection. The single-laboratory validation study evaluated method selectivity, accuracy, repeatability, linearity, limit of quantitation, and limit of detection. Individual flavonoid content ranged from 0.05 mg/g to 17.5 mg/g in solid dosage forms and raw materials. Repeatability ranged from 0.7 to 11.7 % relative standard deviation corresponding to HorRat ranges from 0.2 to 1.6. Calibration curves for each flavonoid were linear within the analytical ranges with correlation coefficients greater than 99.9 %. Herein is the first report of a validated method that is fit for the purpose of quantifying five major phytochemical marker compounds in both raw materials and finished products made from North American (Crataegus douglasii) and European (Crataegus monogyna and Crataegus laevigata) hawthorn species. The method includes optimized extraction of samples without a prolonged drying process and reduced liquid chromatography separation time.

3-Epimers of Caribbean ciguatoxins in fish and algae.

Ciguatera poisoning (CP) is endemic to several subtropical and tropical regions and is caused by the consumption of fish contaminated with ciguatoxins (CTXs). The recent discovery of Caribbean CTXs (C-CTXs) in Gambierdiscus spp. isolated from the Caribbean resulted in the identification of a precursor analogue, C-CTX5, that is reduced into C-CTX1. C-CTX5 has two reducible sites, a ketone at C-3 and hemiketal at C-56. Chemical reductions of C-CTX5 into C-CTX3/4 resulted in two peaks in the LC-HRMS chromatograms with a ratio that differed markedly from that observed in fish extracts and the reduction of C-CTX1 isolated from fish. Reduction of C-CTX5 should have produced four diastereoisomers of C-CTX3/4, prompting a more detailed study of the reduction products. LC-HRMS with a slow gradient was used to separate and detect the four stereoisomers of C-CTX3/4, and to determine the distribution of these analogues in naturally contaminated fish tissues and following chemical reduction of isolated analogues. The results showed that in naturally contaminated fish tissues C-CTX1/2 is a mixture of two diastereoisomers at C-3 and that C-CTX3/4 is a mixture of two pairs of diastereoisomers at C-3 and C-56. The data suggests that there is variability in the enzymatic reduction at C-3 and C-56 of C-CTXs in reef fish, leading to variations in the ratios of the four stereoisomers. Based on these findings, a naming convention for C-CTXs is proposed which aligns with that used for Pacific CTX congeners and will aid in the identification of the structure and stereochemistry of the different CTX analogues.

Azaspiracid-59 accumulation and transformation in mussels (Mytilus edulis) after feeding with Azadinium poporum (Dinophyceae).

Azaspiracid-59 (AZA-59) was detected in plankton in coastal waters of the Pacific Northwest USA. Given that bivalves metabolize and transform accumulated phycotoxins, a strain of Azadinium poporum isolated from the coast of Washington State that is a known producer of AZA-59 was used in a controlled feeding experiment with mussels (Mytilus edulis) to assess AZA-59 accumulation rates and transformation into shellfish metabolites. Mussels started feeding immediately after the addition of A. poporum. Mussels were generally healthy during the entire experimental exposure of 18 days with prevailingly high rates of clearance (ca 100 mL per mussel and hour) and ingestion. Mussel were extracted after different exposure times and were analyzed by liquid chromatography coupled with low- and high-resolution mass spectrometry. In the course of the experiment a number of putative AZA-59 metabolites were detected including hydroxyl and carboxy analogs that corresponded with previously reported mussel metabolites of AZA-1. A significant formation of 3-OH fatty acid acyl esters relative to free AZAs was observed through the time course of the study, with numerous fatty acid ester variants of AZA-59 confirmed. These results illustrate the potential for metabolism of AZA59 in shellfish and provide important information for local AZA monitoring and toxicity testing along the Northern Pacific US coast.

Investigation of 44-Methylgambierone Reactivity with Periodate: Structural Reassignment, Solvent Instability and Formation of a Furanoid Analogue.

Gambierones are sulfated polyethers produced by benthic dinoflagellates in the genera Gambierdiscus, Coolia and Fukuyoa. While relative toxicity data for gambierones suggests they are low compared with ciguatoxin analogues, gambierones have been suggested for use as marker compounds for environmental monitoring programs for the presence of Gambierdiscus in marine waters. The published structure of gambierone and analogues of it, including 44-methylgambierone (44-MeGAM), have been reported to possess 1,2- and 4,5-cis diols, while only the 1,2- diol unit has been shown to undergo periodate oxidation. An in-depth analysis of previously reported NMR data for 44-MeGAM in CD3OD showed that the C-4 stereochemistry of 44-MeGAM and other gamberiones was mis-assigned, that the 4-CH2-CHOH-CH2OH and OH groups are equatorially and axially oriented, respectively, rather than vice versa as previously reported. This re-examination of existing 44-MeGAM NMR data also showed that its C-12 and C-13 assignments (and those for other gambierones) should be reversed. In an effort to better understand the C-4 stereochemical and periodate reaction characteristics of gambierones (C-4 is an epimerizable hemiacetal carbon), additional NMR data was acquired in D6-DMSO. Unexpectedly, progressive conversion of 44-MeGAM to a long-term stable ring-A furanoid analogue was observed. A subsequent series of microscale stability trials identified several solvents that affected the solution-stability of 44-MeGAM, and these findings should be taken into consideration during isolation, handling, storage and bioassay evaluations of gambierones in future studies.

Algal ciguatoxin identified as source of ciguatera poisoning in the Caribbean.

Ciguatera poisoning (CP) is a severe seafood-borne disease, caused by the consumption of reef fish contaminated with Caribbean ciguatoxins (C-CTXs) in the Caribbean and tropical Atlantic. However, C-CTXs have not been identified from their presumed algal source, so the relationship to the CTXs in fish causing illness remains unknown. This has hindered the development of detection methods, diagnostics, monitoring programs, and limited fundamental knowledge on the environmental factors that regulate C-CTX production. In this study, in vitro and chemical techniques were applied to unambiguously identify a novel C-CTX analogue, C-CTX5, from Gambierdiscus silvae and Gambierdiscus caribaeus strains from the Caribbean. Metabolism in vitro by fish liver microsomes converted algal C-CTX5 into C-CTX1/2, the dominant CTX in ciguatoxic fish from the Caribbean. Furthermore, C-CTX5 from G. silvae was confirmed to have voltage-gated sodium-channel-specific activity. This finding is crucial for risk assessment, understanding the fate of C-CTXs in food webs, and is a prerequisite for development of effective analytical methods and monitoring programs. The identification of an algal precursor produced by two Gambierdiscus species is a major breakthrough for ciguatera research that will foster major advances in this important seafood safety issue.

Selective and Efficient Capture and Release of vic-Diol-Containing Pacific and Caribbean Ciguatoxins from Fish Extracts with a Boronate Affinity Polymer.

Ciguatera poisoning can occur following the consumption of fish contaminated with trace levels of ciguatoxins (CTXs). These trace levels represent an analytical challenge for confirmation by LC-MS due to matrix interferences and the high instrument sensitivity required. Sample preparation procedures are laborious and require extensive cleanup procedures to address these issues. The application of a selective isolation technique employing boronate affinity polymers was therefore investigated for the capture of vic-diol-containing Caribbean and Pacific CTXs from fish extracts. A dispersive SPE procedure was developed where nearly complete binding of CTXs in fish extracts occurred with boric acid gel in less than 1 h. Release of the bound CTXs resulted in >95% recovery of C-CTX1/2, C-CTX3/4, CTX1B, 54-deoxyCTX1B, and 52-epi-54-deoxyCTX1B from the extracts. This selective extraction tool has the potential to greatly simplify both analytical sample preparation and preparative extraction and isolation of CTXs for structure elucidation and production of standards.

Selective extraction of gambierone and related metabolites in Gambierdiscus silvae using m-aminophenylboronic acid-agarose gel and liquid chromatography-high-resolution mass spectrometric detection.

Gambierdiscus spp. are epi-benthic dinoflagellates that have been associated with ciguatera poisoning. These microalgae can have complex secondary metabolite profiles including ciguatoxins, maitotoxins, and gambierones, with varying compositions and toxicities across species and strains. Given this chemical diversity there is a need to develop selective and sensitive methods for secondary metabolite profiling. In this study, we used a cultured Caribbean strain of Gambierdiscus silvae to develop sample preparation and analysis strategies for characterizing vic-diol containing secondary metabolites. A pooled cellular extract was first screened by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for ciguatoxin-related compounds, which resulted in the confirmation of gambierone (1) and a novel isomer of 44-methylgambierone (3). Treatment of the extract with periodate confirmed that the gambierones each contained one reactive vic-diol, which was exploited for the development of a selective extraction procedure using m-aminophenylboronic acid gel and the non-aqueous binding solvent chloroform. Using this non-traditional boronate affinity procedure, LC-HRMS also revealed the presence of additional sulfated polycyclic ethers in the gambierone-containing vic-diol fraction, while pigments and other contaminants were removed. The developed tools could be applied to screen collections of Gambierdiscus and other benthic algae to provide additional chemical characterization of gambierone-related compounds. The selective extraction procedure may also prove useful as a step in the isolation of these sulfated polyethers for structural, toxicological and biotransformation studies.

Reductive Amination for LC-MS Signal Enhancement and Confirmation of the Presence of Caribbean Ciguatoxin-1 in Fish.

Ciguatera poisoning is a global health concern caused by the consumption of seafood containing ciguatoxins (CTXs). Detection of CTXs poses significant analytical challenges due to their low abundance even in highly toxic fish, the diverse and in-part unclarified structures of many CTX congeners, and the lack of reference standards. Selective detection of CTXs requires methods such as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) or high-resolution MS (LC-HRMS). While HRMS data can provide greatly improved resolution, it is typically less sensitive than targeted LC-MS/MS and does not reliably comply with the FDA guidance level of 0.1 µg/kg CTXs in fish tissue that was established for Caribbean CTX-1 (C-CTX-1). In this study, we provide a new chemical derivatization approach employing a fast and simple one-pot derivatization with Girard's reagent T (GRT) that tags the C-56-ketone intermediate of the two equilibrating C-56 epimers of C-CTX-1 with a quaternary ammonium moiety. This derivatization improved the LC-MS/MS and LC-HRMS responses to C-CTX-1 by approximately 40- and 17-fold on average, respectively. These improvements in sensitivity to the GRT-derivative of C-CTX-1 are attributable to: the improved ionization efficiency caused by insertion of a quaternary ammonium ion; the absence of adduct-ions and water-loss peaks for the GRT derivative in the mass spectrometer, and; the prevention of on-column epimerization (at C-56 of C-CTX-1) by GRT derivatization, leading to much better chromatographic peak shapes. This C-CTX-1-GRT derivatization strategy mitigates many of the shortcomings of current LC-MS analyses for C-CTX-1 by improving instrument sensitivity, while at the same time adding selectivity due to the reactivity of GRT with ketones and aldehydes.

Production and stability of Oxygen-18 labeled Caribbean ciguatoxins and gambierones.

Ciguatoxins (CTXs) and gambierones are ladder-shaped polyethers associated with ciguatera poisoning and Gambierdiscus spp. Several of these compounds contain carbonyl or hemiketal groups, which have the potential to exchange with 18O-labeled water under acidic conditions. The effects of solvent composition and acid on the rate of exchange and on the stability of the labels at various pH values were assessed to optimize the incorporation of 18O into Caribbean ciguatoxin-1 and -2 (C-CTX1/2), gambierone, and 44-methylgambierone. LC-HRMS results showed that 18O-labeling occurred at the hydroxy group of the hemiketal at C-56 in C-CTX1/2, and at the hydroxy group of the hemiketal at C-4 and the ketone at C-40 in gambierones. Labeling occurred very rapidly (complete in <30 min) for C-CTX1/2, and more slowly (complete in ca. 16 h) for both gambierones. Labeled C-CTX1/2 was reduced with sodium borohydride to produce 18O-labeled C-CTX3/4. The incorporated 18O labels in the gambierones and C-CTXs were retained in aqueous solvent mixtures under neutral conditions in a short-term stability study, demonstrating that these 18O-labeled toxins have the potential to be used in isotope dilution and metabolism studies.

In Vitro Metabolism of Azaspiracids 1-3 with a Hepatopancreatic Fraction from Blue Mussels (Mytilus edulis).

Azaspiracids (AZAs) are a group of biotoxins produced by the marine dinoflagellates Azadinium and Amphidoma spp. that can accumulate in shellfish and cause food poisoning in humans. Of the 60 AZAs identified, levels of AZA1, AZA2, and AZA3 are regulated in shellfish as a food safety measure based on occurrence and toxicity. Information about the metabolism of AZAs in shellfish is limited. Therefore, a fraction of blue mussel hepatopancreas was made to study the metabolism of AZA1-3 in vitro. A range of AZA metabolites were detected by liquid chromatography-high-resolution tandem mass spectrometry analysis, most notably the novel 22α-hydroxymethylAZAs AZA65 and AZA66, which were also detected in naturally contaminated mussels. These appear to be the first intermediates in the metabolic conversion of AZA1 and AZA2 to their corresponding 22α-carboxyAZAs (AZA17 and AZA19). α-Hydroxylation at C-23 was also a prominent metabolic pathway, producing AZA8, AZA12, and AZA5 as major metabolites of AZA1-3, respectively, and AZA67 and AZA68 as minor metabolites via double-hydroxylation of AZA1 and AZA2, but only low levels of 3ß-hydroxylation were observed in this study. In vitro generation of algal toxin metabolites, such as AZA3, AZA5, AZA6, AZA8, AZA12, AZA17, AZA19, AZA65, and AZA66 that would otherwise have to be laboriously purified from shellfish, has the potential to be used for the production of standards for analytical and toxicological studies.

Determination of Cannabinoids in Cannabis sativa Dried Flowers and Oils by LC-UV: Single-Laboratory Validation, First Action 2018.10.

BACKGROUND: Legalization of Cannabis across many U.S. states and in Canada had led to an urgent need for validated analytical methods for the quantitation of cannabinoids in Cannabis sativa L. flowers and finished products. The AOAC Stakeholder Panel on Strategic Food Analytical Methods Cannabis Expert Review Panel (ERP) approved an HPLC-diode-array detection (DAD) method for First Action Official MethodsSM status. OBJECTIVE: To present Official Methods of AnalysisSM (OMA) 2018.10 method details, validation results, and additional method extension data as approved by the ERP and further requirements for Final Action Official MethodsSM status. METHODS: This previously published method used 80% aqueous methanol via sonication for extracting eight cannabinoids-tetrahydrocannabidiolic acid, tetrahydrocannabinol, cannabidiolic acid, cannabidiol, tetrahydrocannabivarin, cannabigerol, cannabinol, and cannabichromene-in dried flowers followed by reversed-phase chromatographic separation and UV detection. RESULTS: The original method underwent extensive method optimization and a single-laboratory validation. Additional requirements requested by the Standard Method Performance Requirement (SMPR®) included a method extension, which was performed to collect repeatability data on two additional cannabinoids: cannabidivarinic acid and cannabigerolic acid. The methods performance was compared with the AOAC SMPR 2017.002 and 2017.001. RSDr ranged from 0.78 to 10.08% and recoveries from 90.7 to 99.2% in several different chemotypes. CONCLUSIONS: The ERP adopted the method and provided recommendations for achieving Final Action status. HIGHLIGHTS: After submission of additional validation data, an HPLC-DAD method for quantitation of cannabinoids in dried flowers and oils was accepted for First Action Official Method status (OMA 2018.10).

Determination of Curcuminoids in Turmeric Dietary Supplements by HPLC-DAD: Multi-laboratory Study Through the NIH-ODS/NIST Quality Assurance Program.

BACKGROUND: Turmeric is a medicinal herb containing curcuminoids, used as quality markers in dietary supplements. In 2016, an AOAC First Action Official MethodSM was adopted for quantitation of curcuminoids and requires multi-laboratory reproducibility data for Final Action status. OBJECTIVE: To collect reproducibility data for the quantitation of curcuminoids in dietary supplements through the National Institutes of Health Office of Dietary Supplements/National Institute of Standards and Technology Quality Assurance Program (QAP). METHOD: Laboratories that participated in the QAP by following the Official Methods of AnalysisSM Method 2016.16, submitted data for ten turmeric products. The data were analyzed for mean, repeatability, and reproducibility standard deviations, repeatability, and reproducibility. RESULTS: The initial data collection resulted in insufficient replicates (five) for each test sample to determine reproducibility, therefore laboratories were provided additional materials resulting in an incremental data approach. For homogenous products, reproducibility for curcumin ranged from 3.4 to 10.3%, bisdemethoxycurcumin with reproducibility ranging from 6.4 to 14.8%, and demethoxycurcumin ranging from 5.6 to 9.9%. The method was unsuitable for the quantitation of curcuminoids in complex smoothie products, products containing microbeads, or tinctures based on interlaboratory variances. Recommendations were provided for future multi-laboratory studies performed through QAPs and incremental approaches. CONCLUSIONS: Method 2016.16 is suitable for the quantitation of curcuminoids and should be adopted for Final Action status for single and multi-ingredient dietary supplements containing dried roots, dried powders/extracts in bulk material, capsules, and softgels. HIGHLIGHTS: Reproducibility for Method 2016.16 was collected through a non-traditional incremental data multi-laboratory study. The method is suitable for quantitation of curcuminoids in most common dietary supplements.

Fatty acid esters of azaspiracids identified in mussels (Mytilus edulis) using liquid chromatography-high resolution mass spectrometry.

Azaspiracids (AZAs) are lipophilic polyether toxins produced by Azadinium and Amphidoma species of marine microalgae. The main dinoflagellate precursors AZA1 and AZA2 are metabolized by shellfish to produce an array of AZA analogues. Many marine toxins undergo fatty acid esterification in shellfish, therefore mussel tissues contaminated with AZAs were screened for intact fatty acid esters of AZAs using liquid chromatography-high resolution mass spectrometry. Acyl esters were primarily observed for AZAs containing hydroxy groups at C-3 with 3-O-palmitoylAZA4 identified as the most abundant acyl ester, while other fatty acid esters including 18:1, 16:1, 17:0, 20:2 and 18:0 acyl esters were detected. The structures of these acyl derivatives were determined through LC-MS/MS experiments, and supported by periodate cleavage reactions and semi-synthesis of palmitate esters of the AZAs. Esters of the hydroxy groups at C-20 or C-21 were not observed in mussel tissue. The relative proportion of the most abundant AZA ester was less than 3% of the sum of the major free AZA analogues. These findings reveal an additional metabolic pathway for AZAs in shellfish.