Comparison of calibration strategies for accurate quantitation by isotope dilution mass spectrometry: a case study of ochratoxin A in flour.

Analysis of low-level organic contaminants in complex matrices is essential for monitoring global food safety. However, balancing sample throughput with complex experimental designs and/or sample clean-up to best reduce matrix effects is a constant challenge. Multiple strategies exist to mitigate these effects, with internal standard-based methods such as isotope dilution mass spectrometry (IDMS) being the most advantageous. Here, multiple internal calibration strategies were investigated for the quantification of ochratoxin A (OTA) in wheat samples by liquid chromatography-mass spectrometry (LC-MS). Internal standard-based quantitation methods such as single (ID1MS), double (ID2MS), and quintuple (ID5MS) isotope dilution mass spectrometry, as well as external standard calibration, were explored and compared. A certified reference material (CRM) of OTA in flour, MYCO-1, was used to evaluate the accuracy of each method. External calibration generated results 18-38% lower than the certified value for MYCO-1, largely due to matrix suppression effects. Concurrently, consistently lower OTA mass fractions were obtained for the wheat samples upon quantitation by external calibration as opposed to ID1MS, ID2MS, and ID5MS. All isotope dilution methods produced results that fell within the expected range for MYCO-1 (3.17-4.93 µg/kg), validating their accuracy. However, an average 6% decrease in the OTA mass fraction was observed from results obtained by ID1MS compared to those by ID2MS and ID5MS. Upon scrutiny, these differences were attributed to an isotopic enrichment bias in the isotopically labelled internal standard [13C6]-OTA that was used for ID1MS, the OTAL-1 CRM. The advantages and limitations of each isotopic method are illustrated.

Production and certification of BOTS-1: bovine muscle-certified reference material for incurred veterinary drug residues.

A freeze-dried bovine muscle-certified reference material (CRM), known as BOTS-1 (DOI: https://doi.org/10.4224/crm.2018.bots-1 ), containing incurred residues of commonly used veterinary drugs was produced and certified for the mass fraction of eight veterinary drug residues. Value assignment was carried out using liquid chromatography tandem mass spectrometry (LC-MS/MS) methods in conjunction with isotope dilution and standard addition approaches involving stable isotope internal standards. Data from the National Research Council of Canada (NRC), Canadian Food Inspection Agency (CFIA), United States Department of Agriculture (USDA), and the Federal Office of Consumer Protection and Food Safety in Germany (BVL) were used for value assignment. Results for two drug residues were also obtained through an international inter-laboratory comparison CCQM-K141/P178 organized under the auspices of the International Bureau of Weights and Measures (BIPM). Quantitative NMR (1H-qNMR) was used to characterize primary standards of all veterinary drugs certified. The certified mass fractions of the veterinary drug residues were 490 ± 100 µg/kg for chlorpromazine, 44 ± 4.4 µg/kg for ciprofloxacin, 3.3 ± 1.4 µg/kg for clenbuterol, 9.5 ± 0.8 µg/kg for dexamethasone, 57 ± 4.8 µg/kg for enrofloxacin, 3.0 ± 0.4 µg/kg for meloxicam, 12.4 ± 1.2 µg/kg for ractopamine, and 2290 ± 120 µg/kg for sulfadiazine with expanded uncertainties quoted (95% confidence) which include the effects due to between-bottle inhomogeneity, instability during long-term storage and transportation, and characterization.

Characterization of biotinylated human ACE2 and SARS-CoV-2 Omicron BA.4/5 spike protein reference materials.

Accurate diagnostic and serology assays are required for the continued management of the COVID-19 pandemic yet spike protein mutations and intellectual property concerns with antigens and antibodies used in various test kits render comparability assessments difficult. As the use of common, well-characterized reagents can help address this lack of standardization, the National Research Council Canada has produced two protein reference materials (RMs) for use in SARS-CoV-2 serology assays: biotinylated human angiotensin-converting enzyme 2 RM, ACE2-1, and SARS-CoV-2 Omicron BA.4/5 spike protein RM, OMIC-1. Reference values were assigned through a combination of amino acid analysis via isotope dilution liquid chromatography tandem mass spectrometry following acid hydrolysis, and ultraviolet-visible (UV-Vis) spectrophotometry at 280 nm. Vial-to-vial homogeneity was established using UV-Vis measurements, and protein oligomeric status, monitored by size exclusion liquid chromatography (LC-SEC), was used to evaluate transportation, storage, and freeze-thaw stabilities. The molar protein concentration in ACE2-1 was 25.3 ± 1.7 µmol L-1 (k = 2, 95% CI) and consisted almost exclusively (98%) of monomeric ACE2, while OMIC-1 contained 5.4 ± 0.5 µmol L-1 (k = 2) spike protein in a mostly (82%) trimeric form. Glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection facilitated calculation of corresponding mass concentrations. To confirm protein functionality, the binding of OMIC-1 to immobilized ACE2-1 was investigated with surface plasmon resonance and the resulting dissociation constant, KD ~ 4.4 nM, was consistent with literature values.

Characterization of a SARS-CoV-2 spike protein reference material.

Development of diagnostic testing capability has advanced with unprecedented pace in response to the COVID-19 pandemic. An undesirable effect of such speed is a lack of standardization, often leading to unreliable test results. To assist the research community surmount this challenge, the National Research Council Canada has prepared a SARS-CoV-2 spike protein reference material, SMT1-1, as a buffered solution. Value assignment was achieved by amino acid analysis (AAA) by double isotope dilution liquid chromatography-tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in combination with ultraviolet-visible spectrophotometry (UV-Vis) based on tryptophan and tyrosine absorbance at 280 nm. Homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. Transportation and long-term storage stabilities were assessed by monitoring relative changes in oligomeric state by size-exclusion liquid chromatography (LC-SEC) with UV detection. The molar concentration of the spike protein in SMT1-1 was 5.68 ± 0.22 µmol L-1 (k = 2, 95% CI), with the native trimeric form accounting for ~ 94% of the relative abundance. Reference mass concentration and mass fraction values were calculated using the protein molecular weight and density of the SMT1-1 solution. The spike protein is highly glycosylated which leads to analyte ambiguity when reporting the more commonly used mass concentration. After glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection, we thus reported mass concentration values for both the protein-only portion and intact glycoprotein as 0.813 ± 0.030 and 1.050 ± 0.068 mg mL-1 (k = 2), respectively.

Thermal stability of cannabinoids in dried cannabis: a kinetic study.

This study was undertaken to quantitatively explore the effect of temperature on the degradation of cannabinoids in dried cannabis flower. A total of 14 cannabinoids were monitored using liquid chromatography and tandem mass spectrometry in temperature environments from - 20 to + 40 ∘C lasting up to 1 year. We find that a network of first-order degradation reactions is well-suited to model the observed changes for all cannabinoids. While most studies focus on high-temperature effects on the cannabinoids, this study provides high-precision quantitative assessment of room temperature kinetics with applications to shelf-life predictions and age estimates of cannabis products.

13C-Satellite Decoupling Strategies for Improving Accuracy in Quantitative Nuclear Magnetic Resonance.

Quantitative 1H nuclear magnetic resonance (qHNMR) with an appropriate internal standard is a well-established quantitation method for assigning purity to organic molecules. For accurate measurements, the premise of qHNMR relies on the careful selection of integrals, for both the analyte and the standard, in such a way that the selected integrals are free from interferences. The 13C-satellite signals of adjacent integrals, low-level impurities, and tautomer signals are among the common integral interferences that are typically encountered. One of the simplest ways to identify and avoid these interferences is to decouple the 13C-satellites. Two decoupling schemes were explored to illustrate the benefits of 13C-decoupling for qHNMR or qH{13C}NMR: GARP and bilevel adiabatic broadband decoupling. Unwanted sample heating and nuclear Overhauser effect (NOE) enhancements are the two main drawbacks of decoupling schemes. We show that with careful optimization of acquisition parameters and decoupling power, no excessive sample heating occurred during acquisition at 400 MHz. At 900 MHz, only bilevel adiabatic decoupling could be safely implemented. Furthermore, any undesirable NOE enhancements were completely avoided if acquisition was executed with an inverse-gated pulse sequence. We explored and confirmed the benefits of qH{13C}NMR through the quantitation of a diverse set of compounds, namely, small molecules (dimethyl terephthalate and zearalenone), a 13C-labeled compound (13C6-ochratoxin A), and an octapeptide (angiotensin II). Statistical comparisons confirmed that qH{13C}NMR produced comparable data to qHNMR. However, with qH{13C}NMR data providing added clarity about the presence of overlapping 13C-satellites, impurities, and tautomers, it has an edge over qHNMR for accurate measurements.

Rapid quantitative screening of cyanobacteria for production of anatoxins using direct analysis in real time high-resolution mass spectrometry.

RATIONALE: Anatoxins (ATXs) are a potent class of cyanobacterial neurotoxins that are increasingly problematic in drinking water reservoirs and recreational water bodies worldwide. Because of their high polarity and low molecular weight, analysis of ATXs is challenging and they can be considered underreported compared with other classes of cyanobacterial toxins. Improved screening methods are therefore needed to effectively assess their occurrence and concentrations in the environment. METHODS: A rapid screening method was developed for ATXs in cyanobacteria using direct analysis in real time combined with high-resolution mass spectrometry (DART-HRMS), requiring less than 2 min per sample for triplicate analysis. The developed method was evaluated for its quantitative capabilities, applied to the screening of 30 cyanobacterial culture samples for the presence of anatoxin-a, homoanatoxin-a and dihydroanatoxin-a, and compared with a more typical liquid chromatography (LC)/HRMS method. RESULTS: Excellent linearity was observed in the analysis of a matrix-matched calibration curve using DART-HRMS, with ionization suppression of about 50% and relative standard deviations between replicate analyses of approximately 30%. Limits of detection for both anatoxin-a and homoanatoxin-a were estimated as 1 ng/mL. Excellent agreement was observed between DART-HRMS and LC/HRMS with all ATX-producing cultures correctly identified and only one false positive culture by DART-HRMS. CONCLUSIONS: DART-HRMS shows excellent promise for the rapid, quantitative screening of ATXs in cyanobacteria and could be expanded in the future to include the analysis of field samples and drinking water, as well as additional ATX analogues.

Quantitative determination and validation of 17 cannabinoids in cannabis and hemp using liquid chromatography-tandem mass spectrometry.

The increase in production of cannabis for medical and recreational purposes in recent years has led to a corresponding increase in laboratories performing cannabinoid analysis of cannabis and hemp. We have developed and validated a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that is simple, reliable, specific, and accurate for the analysis of 17 cannabinoids in cannabis and hemp. Liquid-solid sample extraction coupled with dilution into a calibration range from 10 to 10,000 ng/mL and LC-MS/MS analysis provides quantification of samples ranging from 0.002 to 200 mg/g (0.0002 to 20.0%) in matrix. Linearity of calibration curves in methanol was demonstrated with regression r2 ≥ 0.99. Within-batch precision (0.5 to 6.5%) and accuracy (91.4 to 108.0%) and between-batch precision (0.9 to 5.1%) and accuracy (91.5 to 107.5%) were demonstrated for quality control (QC) samples in methanol. Within-batch precision (0.2 to 3.6%) and accuracy (85.4 to 111.6%) and between-batch precision (1.4 to 6.1 %) and accuracy (90.2 to 110.3%) were also evaluated with a candidate cannabis certified reference material (CRM). Repeatability (1.5 to 12.4% RSD) and intermediate precision (2.2 to 12.8% RSD) were demonstrated via analysis of seven cannabis samples with HorRat values ranging from 0.3 to 3.1. The method provides enhanced detection limits coupled with a large quantitative range for 17 cannabinoids in plant material. It is suitable for a wide range of applications including routine analysis for delta-9-tetrahydrocannabinol (Δ9-THC), delta-9-tetrahydrocannabinolic acid (Δ9-THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), and cannabinol (CBN) as well as more advanced interrogation of samples for both major and minor cannabinoids.

Cannabis Inflorescence for Medical Purposes: USP Considerations for Quality Attributes.

There is an active and growing interest in cannabis female inflorescence (Cannabis sativa) for medical purposes. Therefore, a definition of its quality attributes can help mitigate public health risks associated with contaminated, substandard, or adulterated products and support sound and reproducible basic and clinical research. As cannabis is a heterogeneous matrix that can contain a complex secondary metabolome with an uneven distribution of constituents, ensuring its quality requires appropriate sampling procedures and a suite of tests, analytical procedures, and acceptance criteria to define the identity, content of constituents (e.g., cannabinoids), and limits on contaminants. As an independent science-based public health organization, United States Pharmacopeia (USP) has formed a Cannabis Expert Panel, which has evaluated specifications necessary to define key cannabis quality attributes. The consensus within the expert panel was that these specifications should differentiate between cannabis chemotypes. Based on the secondary metabolite profiles, the expert panel has suggested adoption of three broad categories of cannabis. These three main chemotypes have been identified as useful for labeling based on the following cannabinoid constituents: (1) tetrahydrocannabinol (THC)-dominant chemotype; (2) intermediate chemotype with both THC and cannabidiol (CBD); and (3) CBD-dominant chemotype. Cannabis plants in each of these chemotypes may be further subcategorized based on the content of other cannabinoids and/or mono- and sesquiterpene profiles. Morphological and chromatographic tests are presented for the identification and quantitative determination of critical constituents. Limits for contaminants including pesticide residues, microbial levels, mycotoxins, and elemental contaminants are presented based on toxicological considerations and aligned with the existing USP procedures for general tests and assays. The principles outlined in this review should be able to be used as the basis of public quality specifications for cannabis inflorescence, which are needed for public health protection and to facilitate scientific research on cannabis safety and therapeutic potential.

Corona discharge electrospray ionization of formate-containing solutions enables in-source reduction of disulfide bonds.

Disulfide bonds are critical linkages for maintaining protein structure and enzyme activity. These linkages, however, can limit peptide sequencing efforts by mass spectrometry (MS) and often require chemical reduction and alkylation. Under such conditions, information regarding cysteine connectivity is lost. Online partial disulfide reduction within the electrospray (ESI) source has recently been established as a means to identify complex cysteine linkage patterns in a liquid chromatography-MS experiment without the need for sample pre-treatment. Corona discharge (CD) is invoked as the causative factor of this in-source reduction (ISR); however, evidence remains largely circumstantial. In this study, we demonstrate that instrumental factors-nebulizing gas, ESI capillary material, organic solvent content, ESI spray needle-to-MS distance-all modulate the degree of reduction observed for the single disulfide in oxytocin, further implicating CD in ISR. Rigorous analysis of solution conditions, however, reveals that corona discharge alone can induce only minor disulfide reduction. We establish that CD-ESI of peptide solutions containing formic acid or its conjugate base results in a dramatic increase in disulfide reduction. It is also determined that ISR is exacerbated at low pH for complex peptides containing multiple disulfide bonds and possessing higher-order structure, as well as for a small protein. Overall, our results demonstrate that ESI of formate/formic acid-containing solutions under corona discharge conditions facilitates disulfide ISR, likely by a similar reduction pathway measured in γ-radiolysis studies nearly three decades ago.

Gramillin A and B: Cyclic Lipopeptides Identified as the Nonribosomal Biosynthetic Products of Fusarium graminearum.

The virulence and broad host range of Fusarium graminearum is associated with its ability to secrete an arsenal of phytotoxic secondary metabolites, including the regulated mycotoxins belonging to the deoxynivalenol family. The TRI genes responsible for the biosynthesis of deoxynivalenol and related compounds are usually expressed during fungal infection. However, the F. graminearum genome harbors an array of unexplored biosynthetic gene clusters that are also co-induced with the TRI genes, including the nonribosomal peptide synthetase 8 ( NRPS8) gene cluster. Here, we identify two bicyclic lipopeptides, gramillin A (1) and B (2), as the biosynthetic end products of NRPS8. Structural elucidation by high-resolution LC-MS and NMR, including 1H-15N-13C HNCO and HNCA on isotopically enriched compounds, revealed that the gramillins possess a fused bicyclic structure with ring closure of the main peptide macrocycle occurring via an anhydride bond. Through targeted gene disruption, we characterized the GRA1 biosynthetic gene and its transcription factor GRA2 in the NRPS8 gene cluster. Further, we show that the gramillins are produced in planta on maize silks, promoting fungal virulence on maize but have no discernible effect on wheat head infection. Leaf infiltration of the gramillins induces cell death in maize, but not in wheat. Our results show that F. graminearum deploys the gramillins as a virulence agent in maize, but not in wheat, thus displaying host-specific adaptation.

Assessing MS-based quantitation strategies for low-level impurities in peptide reference materials: application to angiotensin II.

Identification and quantitation of related impurities is vital in obtaining corrected purity values for peptide certified reference materials. The sensitivity and selectivity of high-resolution mass spectrometry (MS) renders it an indispensable technique in this arena. Typical quantitation efforts involve constructing external calibration curves, although analysis of dilute peptide solutions can be complicated by analyte adsorption to vial walls, instrument tubing, etc. The standard addition method alleviates many concerns associated with this sample loss as the calibrant solutions more closely match the matrix of the samples. Yet, both strategies require acquisition of synthetic impurity peptide standards. Label-free proteomics relies on electrospray ionization (ESI)-MS signals to quantify identical peptides across multiple samples; however, peptides of differing sequence can exhibit widely disparate ESI-MS responses. This study explores the use of peak area ratios to quantitate sequence-related peptide impurities in an angiotensin II candidate certified reference material. Using synthetic standards of five abundant substances, impurity mass fractions calculated via the relative response method are in reasonable agreement with those determined from standard addition experiments, whereas external calibration measurements frequently overestimate impurity amounts. For a synthetic peptide and its related sequence impurities, the relative response method can expedite analysis and lower expenditures, and in some cases improve data quality.

Purity assignment for peptide certified reference materials by combining qNMR and LC-MS/MS amino acid analysis results: application to angiotensin II.

The purity value assignment of metrologically traceable peptide reference standards requires specialized primary methods. Conventionally, amino acid analysis by isotope dilution tandem mass spectrometry (LC-MS/MS) following peptide hydrolysis is employed as a reference method. By contrast, quantitative nuclear magnetic resonance (qNMR) spectroscopy allows for quantitation of intact peptides, thus eliminating potential bias due to hydrolysis. Both methods are susceptible to interference from related peptide impurities, which need to be accurately measured and accounted for. The mass balance approach has also been employed for peptide purity measurements, whereby the purity is defined by the sum of the mass fraction of all impurities identified. Ideally, results from these three orthogonal methods can be combined for final purity assignment of peptide reference standards. Here we report a novel strategy for correcting both LC-MS/MS and 1H-qNMR results for related peptide impurities and combining results from both methods using a Bayesian statistical approach using mass balance results as prior knowledge. The mass balance method relied on a validated 19F-qNMR method to measure the trifluoroacetic acid (TFA) counter-ion, considered an impurity in this case at nearly 25% by mass. Using a candidate certified reference material (CRM) for angiotensin II, excellent agreement was achieved with the three methods. The final purity value assignment of the candidate CRM was 691 ± 9 mg/g (k = 2).

Hydroxylation of Longiborneol by a Clm2-Encoded CYP450 Monooxygenase to Produce Culmorin in Fusarium graminearum.

A second structural gene required for culmorin biosynthesis in the plant pathogen Fusarium graminearum is described. Clm2 encodes a regio- and stereoselective cytochrome P450 monooxygenase for C-11 of longiborneol (1). Clm2 gene disruptants were grown in liquid culture and assessed for culmorin production via HPLC-evaporative light scattering detection. The analysis indicated a complete loss of culmorin (2) from the liquid culture of the ΔClm2 mutants. Culmorin production resumed in a ΔClm2 complementation experiment. A detailed analysis of the secondary metabolites extracted from the large-scale liquid culture of disruptant ΔClm2D20 revealed five new natural products: 3-hydroxylongiborneol (3), 5-hydroxylongiborneol (4), 12-hydroxylongiborneol (5), 15-hydroxylongiborneol (6), and 11-epi-acetylculmorin (7). The structures of the new compounds were elucidated by a combination of HRMS, 1D and 2D NMR, and X-ray crystallography.

Analysis of paralytic shellfish toxins using high-field asymmetric waveform ion mobility spectrometry with liquid chromatography-mass spectrometry.

The analysis of paralytic shellfish toxins (PSTs) by liquid chromatography-mass spectrometry remains a challenge because of their high polarity, large number of analogues and the complex matrix in which they occur. Here we investigate the potential utility of high-field asymmetric waveform ion mobility spectrometry (FAIMS) as a gas-phase ion separation tool for analysis of PSTs by mass spectrometry. We investigate the separation of PSTs using FAIMS with two divergent goals: using FAIMS as a primary separation tool for rapid screening by electrospray ionization (ESI)-FAIMS-MS or combined with LC in a multidimensional LC-ESI-FAIMS-MS separation. First, a survey of the parameters that affect the sensitivity and selectivity of PST analysis by FAIMS was carried out using ESI-FAIMS-MS. In particular, the use of acetonitrile as a gas additive in the carrier gas flow offered good separation of all PST epimeric pairs. A second set of FAIMS conditions was also identified, which focussed PSTs to a relatively narrow CV range allowing development of an LC-ESI-FAIMS-MS method for analysis of PST toxins in complex mussel tissue extracts. The quantitative capabilities of this method were evaluated by analysing a PST containing mussel tissue matrix material. Results compared favourably with analysis by an established LC-post-column oxidation-fluorescence method with recoveries ranging from 70 to 106%, although sensitivity was somewhat reduced. The current work represents the first successful separation of PST isomers using ion mobility and shows the promise of FAIMS as a tool for analysis of algal biotoxins in complex samples and outlines some critical requirements for its future improvement.

Sulfide oxidations for LC-MS analysis of methionine-containing microcystins in Dolichospermum flos-aquae NIVA-CYA 656.

Microcystins are cyclic heptapeptides produced by a range of cyanobacteria. More than 150 microcystin analogues have been reported from cultures, algal blooms, or other contaminated samples. Relatively few analytical standards are available, making identification and quantitation of these toxins a challenge, even with LC-MS technology. We developed a two-step oxidative procedure that allows LC-MS identification of microcystins containing methionine and methionine sulfoxide, and reveals the oxidation state of the methionyl sulfur atom. The procedure was used in parallel with mercaptoethanol derivatization and LC-MS(2) analysis to demonstrate the presence of [Asp(3)]MC-MR (12) and MC-MR (17) in a culture of Dolichospermum flos-aquae, together with low levels of [Asp(3)]MC-M(O)R (5) and MC-M(O)R (7), as well as 20 other microcystins. Fresh culture contained only traces of sulfoxides 5 and 7, but these increased during storage or sample extraction and preparation. This suggests that microcystins containing methionine sulfoxide are primarily postextraction oxidation artifacts, rather than being produced by biosynthesis in cyanobacteria. A simple, rapid extraction under inert gas followed promptly by LC-MS analysis minimized oxidation artifacts for D. flos-aquae.

Applicability of non-linear versus linear fractional abundance calibration plots for the quantitative determination of triacylglycerol regioisomers by tandem mass spectrometry.

RATIONALE: Regioisomeric analysis of triacylglycerols is important in understanding lipid biochemistry and the involvement of lipids in disease and nutrition. The use of calibration plots employing fractional abundances provides a simple and rapid method for such analyses. These plots are believed to be linear, but evidence exists for non-linearity. The behavior of such plots needs to be understood to allow for proper interpretation of regioisomeric data. METHODS: Solutions of five regioisomer pairs were prepared from pure standards and used to construct calibration plots using triple-stage tandem mass spectrometry (MS(3) ) with electrospray ionization (ESIMS(3) ) and cationization by lithium ions. The data were taken by direct infusion with an AB SCIEX QTRAP 2000 QqLIT mass spectrometer. RESULTS: Non-linear calibration plots were observed for the four isomer pairs containing the polyunsaturated eicosapentaenoic (20:5) and docosahexaenoic (22:6) acids paired with palmitic acid (16:0) or myristic acid (14:0), while the pair including palmitic and stearic (18:0) acids provided a linear plot. A non-linear model was developed for these plots and then verified experimentally. CONCLUSIONS: The fractional abundance calibration plots used in regioisomeric analysis of triacylglycerols are intrinsically non-linear, but may appear linear if the scatter in data points obscures the curvature, if the curvature is slight, or if the response factors for the two isomers in the regioisomer pair are similar. Therefore, linearity should not be assumed for these types of measurements until confirmed experimentally.

Strategic identification of in vitro metabolites of 13-desmethyl spirolide C using liquid chromatography/high-resolution mass spectrometry.

A strategy to identify metabolites of a marine biotoxin, 13-desmethyl spirolide C, has been developed using liquid chromatography coupled to high-resolution mass spectrometry (LC/HRMS). Metabolites were generated in vitro through incubation with human liver microsomes. A list of metabolites was established by selecting precursor ions of a common fragment ion characteristic of the spirolide toxin which was known to contain a cyclic imine ring. Accurate mass measurements were subsequently used to confirm the molecular formula of each biotransformation product. Using this approach, a total of nine phase I metabolites was successfully identified with deviations of mass accuracy less than 2 ppm. The biotransformations observed included hydroxylation, dihydroxylation, oxidation of a quaternary methyl group to hydroxymethyl or carboxylic acid groups, dehydrogenation and hydroxylation, as well as demethylation and dihydroxylation reactions. In a second step, tandem mass spectrometry (MS/MS) was performed to elucidate structures of the metabolites. Using the unique fragment ions in the spectra, the structures of the three major metabolites, 13,19-didesmethyl-19-carboxy spirolide C, 13,19-didesmethyl-19-hydroxymethyl spirolide C and 13-desmethyl-17-hydroxy spirolide C, were assigned. Levels of 13-desmethyl spirolide C and its metabolites were monitored at selected time points over a 32-h incubation period with human liver microsomes. It was determined that 13,19-didesmethyl-19-carboxy spirolide C became the predominant metabolite after 2 h of incubation. The stability plot of 13-desmethyl spirolide C showed first-order kinetics for its metabolism and the intrinsic clearance was calculated to be 41 µL/min/mg, suggesting first-pass metabolism may contribute to limiting oral toxicity of 13-desmethyl spirolide C.

Profiling phlorotannins in brown macroalgae by liquid chromatography-high resolution mass spectrometry.

INTRODUCTION: Phlorotannins, phenolic compounds produced exclusively by Phaeophyceae (brown algae), have recently been associated with a wide variety of beneficial bioactivities. Several studies have measured the total phenolic content in extracts from various species, but little characterisation of individual phlorotannin components has been demonstrated. OBJECTIVE: The purpose of this study was to develop a liquid chromatography-mass spectrometry (LC-MS) based method for rapid profiling of phlorotannins in brown algae. METHODOLOGY: Phlorotannin-enriched extracts from five phaeophyceaen species were analysed by ultrahigh-pressure liquid chromatography (UHPLC) operating in hydrophilic interaction liquid chromatography (HILIC) mode combined with high resolution mass spectrometry (HRMS). The method was optimised using an extract of Fucus vesiculosus; separation was achieved in less than 15 min. The basic mobile phase enhanced negative-ion electrospray ionisation (ESI), and generated multiply charged ions that allowed detection of high molecular weight phlorotannins. RESULTS: The phlorotannin profiles of Pelvetia canaliculata, Fucus spiralis, F. vesiculosus, Ascophyllum nodosum and Saccharina longicruris differed significantly. Fucus vesiculosus yielded a high abundance of low molecular weight (< 1200 Da) phlorotannins, while P. canaliculata exhibited a more evenly distributed profile, with moderate degrees of polymerisation ranging from 3 to 49. HRMS enabled the identification of phlorotannins with masses up to 6000 Da using a combination of accurate mass and ¹³C isotopic patterns. CONCLUSION: The UHPLC-HRMS method described was successful in rapidly profiling phlorotannins in brown seaweeds based on their degree of polymerisation. HILIC was demonstrated to be an effective separation mode, particularly for low molecular weight phlorotannins.