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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, K D  ~ 4.4 nM, was consistent with literature values., (© 2024. Crown.)

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 (ID 1 MS), double (ID 2 MS), and quintuple (ID 5 MS) 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 ID 1 MS, ID 2 MS, and ID 5 MS. 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 ID 1 MS compared to those by ID 2 MS and ID 5 MS. Upon scrutiny, these differences were attributed to an isotopic enrichment bias in the isotopically labelled internal standard [ 13 C 6 ]-OTA that was used for ID 1 MS, the OTAL-1 CRM. The advantages and limitations of each isotopic method are illustrated., (© 2023. Crown.)

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 ( 1 H-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., (© 2023. Crown.)

Cannabis vaping involves the vaporization of a cannabis vaping liquid or solid via a vaping accessory such as a vape pen constructed of various metals or other parts. An increasing number of reports advocate for expansion of the testing and regulation of metal contaminants in cannabis vape liquids beyond the metals typically tested such as arsenic, cadmium, mercury, and lead to reflect the possibility of consumers' exposure to other metal contaminants. Metal contaminants may originate not only from the cannabis itself but also from the vape devices in which the cannabis vape liquid is packaged. However, metal analyses of cannabis vape liquids sampled from cannabis vaping devices are challenged by poor precision and reproducibility. Herein, we present data on the metal content of 12 metals in 20 legal and 21 illegal cannabis vape liquids. The lead mass fraction in several illegal samples reached up to 50 μg g -1 . High levels of nickel (max 677 μg g -1 ) and zinc (max 426 μg g -1 ) were found in illegal samples, whereas the highest copper content (485 μg g -1 ) was measured in legal samples. Significant differences in metal mass fractions were observed in the legal cannabis vape liquid taken from two identical devices, even though the liquid was from the same lot of the same cannabis product. Metal particles in the vape liquids were observed by scanning electron microscopy, and laser ablation inductively coupled plasma mass spectrometry confirmed the presence of copper-, zinc-, lead-, and manganese-bearing particles, metals that are in common alloys that may be used to make vape devices. Colocalized particles containing aluminum, silica, and sodium were also detected. These results suggest that metal particles could be a contributing factor to poor measurement precision and for the first time, to the best of our knowledge, provide evidence of metal particles in cannabis vape liquids contained in unused cannabis vape pens., Competing Interests: The authors declare no competing financial interest., (Crown © 2022. Published by American Chemical Society.)

Rapid antigen tests have become a widely used COVID-19 diagnostic tool with demand accelerating in response to the highly contagious SARS-CoV-2 Omicron variant. Hundreds of such test kits are approved for use worldwide, predominantly reporting on the presence of the viral nucleocapsid (N) protein, yet the comparability among manufacturers remains unclear and the need for reference standards is recognized. To address this lack of standardization, the National Research Council Canada has developed a SARS-CoV-2 nucleocapsid protein reference material solution, NCAP-1. Reference value determination for N protein content was realized by amino acid analysis (AAA) via double isotope dilution liquid chromatography-tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in conjunction with UV spectrophotometry based on tryptophan and tyrosine absorbance at 280 nm. The homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. The molar concentration of the N protein in NCAP-1 was 10.0 ± 1.9 μmol L -1 ( k = 2, 95% confidence interval). Reference mass concentration and mass fraction values were subsequently calculated using the protein molecular weight and density of the NCAP-1 solution. Changes to protein higher-order structure, probed by size-exclusion liquid chromatography (LC-SEC) with UV detection, were used to evaluate transportation and storage stabilities. LC-SEC revealed nearly 90% of the N protein in the material is present as a mixture of hexamers and tetramers. The remaining low molecular weight species (<30 kDa) were interrogated by top-down mass spectrometry and determined to be autolysis products homologous to those previously documented for N protein of the original SARS-CoV [Biochem. Biophys. Res. Commun.2008t, 377, 429-433]., Competing Interests: The authors declare no competing financial interest., (Crown © 2022. Published by American Chemical Society.)

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., (© 2022. Crown.)

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., (© 2021. Crown.)

The number of approved peptide therapeutics, as well as those in development, has been increasing in recent years. Frequently, the biological activity of such peptides is elicited through the adoption of secondary structural elements upon interaction with their cellular target. However, many therapeutic peptides are unstructured in solution and accordingly exhibit a poor bioavailability due to rapid proteolysis in vivo . To combat this degradation, numerous naturally occurring peptides with therapeutic properties contain stabilizing features, such as N-to-C cyclization or disulfide bonds. Recently, hydrocarbon stapling via non-native amino acid substitution followed by ring-closing metathesis has been shown to induce a dramatic stabilization of α-helical peptides. Identifying the ideal staple location along the peptide backbone is a critical developmental step, and methods to streamline this optimization are needed. Mass spectrometry-based methods such as ion mobility (IM) and hydrogen-deuterium exchange (HDX) can detect multiple discrete peptide conformations, a significant advantage over bulk spectroscopic techniques. In this study we use IM-MS and HDX-MS to demonstrate that the native 36-residue enfuvirtide peptide is highly dynamic in solution and the conformational ensemble populated by stabilized constructs depends heavily on the staple location. Further, our measurements yielded results that correlate well with the average α-helical content measured by circular dichroism. The MS-based approaches described herein represent sensitive and potentially high-throughput methods for characterizing and identifying optimally stapled peptides.

Quantitative 1 H 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 13 C-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 13 C-satellites. Two decoupling schemes were explored to illustrate the benefits of 13 C-decoupling for qHNMR or qH{ 13 C}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{ 13 C}NMR through the quantitation of a diverse set of compounds, namely, small molecules (dimethyl terephthalate and zearalenone), a 13 C-labeled compound ( 13 C 6 -ochratoxin A), and an octapeptide (angiotensin II). Statistical comparisons confirmed that qH{ 13 C}NMR produced comparable data to qHNMR. However, with qH{ 13 C}NMR data providing added clarity about the presence of overlapping 13 C-satellites, impurities, and tautomers, it has an edge over qHNMR for accurate measurements.

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., (© 2020 National Research Council Canada. Rapid Communications in Mass Spectrometry © 2020 John Wiley & Sons, Ltd.)

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 r 2  ≥ 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 (Δ 9 -THC), delta-9-tetrahydrocannabinolic acid (Δ 9 -THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), and cannabinol (CBN) as well as more advanced interrogation of samples for both major and minor cannabinoids.

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.

Many methods, often depending on tandem mass spectrometry, have been developed for analysis of complex mixtures of triacylglycerols (TAGs), especially in clinical diagnostics and food authentication. Understanding the fragmentation mechanisms of cationized TAGs has proved problematic. To obtain a better understanding of viable mechanisms, detailed studies including double- and triple-stage tandem mass spectrometry were made using electrospray ionization on lithiated and sodiated tripropanoyl- and trihexanoylglycerols. Density functional theory computations, including a functional parameterized for van der Waals interactions, were used to correlate computed energies with mass spectra. Losses of both a neutral salt and a neutral acid corresponding to a glycerol side chain were observed as major product ions in MS 2 experiments. Signal intensities at low collision energies correlated well with computed energies. However, an important difference between the lithiated and sodiated ions was the appearance of the sodium cation as a major fragmentation product. Computations on the product ions resulting from the loss of a neutral acid indicated multiple structures for the lithiated ions but mainly a single structure for the sodiated ions. The lithiated product ions could be fragmented further (pseudo-MS 3 ) to give additional structural information, whereas the sodiated ions gave only m/z 23. The longer chain TAG, while giving a much less intense mass spectrum than the shorter chain TAG, gave comparable MS 2 and MS 3 product ion spectra. Taken together, the spectral and computational work described herein offer a new and detailed pathway for collision-induced fragmentation of lithiated and sodiated saturated TAGs.

Background: Among the regulated mycotoxins that contaminate global food supplies, ochratoxin A is particularly harmful as a nephrotoxin and suspected carcinogen. Objective: To support global measurement comparability, certified calibration solutions for ochratoxin A and [ 13 C 6 ]-ochratoxin A (OTAN-1 and OTAL-1, respectively) as well as a mycotoxin-contaminated rye flour certified reference material (CRM) known as MYCO-1 were developed. Methods: Quantitative proton NMR was used along with maleic acid as an external standard traceable to the Système international (SI) to measure the concentration of ochratoxin A and [ 13 C 6 ]-ochratoxin A for the calibration solutions. OTAN-1 and OTAL-1 were then used as a pair in double isotope dilution MS to certify the mass fraction of ochratoxin A in MYCO-1. The natural ochratoxin A CRM served as the primary standard for traceable quantitation, while the synthetic [ 13 C 6 ]-ochratoxin A CRM served as the internal standard. Results: The certified mass fraction of ochratoxin A or [ 13 C 6 ]-ochratoxin A in the two mycotoxin calibration solution standards was established to be 11.03 ± 0.32 µg/g ( k = 2) for OTAN-1 and 4.89 ± 0.18 µg/g ( k = 2) for OTAL-1. The mass fraction of ochratoxin A in the rye flour standard MYCO-1 was certified at 4.05 ± 0.88 µg/kg ( k = 2). Conclusions: These CRMs will support regulatory testing as they can be used in the method development, validation, calibration, and QC analysis of ochratoxin A. Highlights: This report highlights the methods used to certify OTAN-1, OTAL-1, and MYCO-1 as well as the challenges associated with producing such materials, which can be applied to a wide variety of other CRMs.

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.

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 1 H- 15 N- 13 C 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.

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 1 H-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 19 F-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).

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.

In this study, we propose a novel approach for the determination of total dissolved nitrogen (TDN) in seawater combining high-precision isotope dilution GC-MS with persulfate digestion. A 2 mL sample aliquot was digested with an alkaline solution of persulfate to convert nitrogen containing compounds to nitrate. Digested samples were spiked with 15 NO 3 - internal standard and treated with aqueous triethyloxonium to convert the analyte into volatile EtONO 2 . This derivative was readily separated from the matrix under gaseous form and could be sampled from the headspace before GC-MS analysis. The resulting chromatograms showed a stable flat baseline with EtONO 2 as the only eluting peak (retention time 2.75 min on a DB 5.625 column). Such an approach provides specificity and obviates the shortcomings of current detection methods employed to analyze seawater samples after digestion with persulfate. In negative chemical ionization mode, the method reached a detection limit of 0.5 μmol/kg TDN (7 ng/g N) and could be applied to quantify seawater samples with 1-25 μmol/kg TDN. On the upper end of the range, quantitation could be repeated within 1%, whereas on a 6 μmol/kg TDN sample repeatability was 2.3% on eight measurements. The method was employed in two proficiency testing exercises providing results in agreement with consensus values. We investigated the impact of reagent blank and we implemented a blank-matching optimal design to account for such contribution. Finally, we performed a study on the yield of persulfate oxidation for organic and inorganic nitrogen compounds typically present in seawater. Whilst nitrite and ammonium are fully converted to nitrate, more complex organic molecules showed recoveries varying from 70% to 100%., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

Many peptides with antimicrobial activity and/or therapeutic potential contain disulfide bonds as a means to enhance stability, and their quantitation is often performed using electrospray ionization mass spectrometry (ESI-MS). Disulfides can be reduced during ESI under commonly used instrument conditions, which has the potential to hinder accurate peptide quantitation. We demonstrate that this in-source reduction (ISR) is predominantly observed for peptides infused from acidic solutions and subjected to elevated ESI voltages (3-4 kV). ISR is readily apparent in the mass spectrum of oxytocin-a small, single disulfide-containing peptide. However, subtle m/z shifts due to partial ISR of highly charged (z ≥ 3) peptides with multiple disulfide linkages may proceed unnoticed. Ion mobility (IM)-MS separates ions on the basis of charge and shape in the gas phase, and using insulin as a model system, we show that IM-MS arrival time distributions (ATDs) are particularly sensitive to partial ISR of large peptides. Isotope modeling allows for the relative quantitation of disulfide-intact and partially reduced states of the mobility-separated peptide conformers. Interestingly, hepcidin peptides ionized from acidic solutions at elevated ESI voltages undergo gas-phase compaction, ostensibly due to partial disulfide ISR. Our IM-MS results lead us to propose that residual acid is the likely cause of disparate ATDs recently measured for hepcidin from different suppliers [Anal. Bioanal. Chem. 409, 2559-2567 (2017)]. Overall, our results demonstrate the utility of IM-MS to detect partial ISR of disulfide-bonded peptides and reinforce the notion that peptide/protein measurements should be carried out using minimally activating instrument conditions. Graphical Abstract ᅟ.

Many toxic secondary metabolites used for defense are also toxic to the producing organism. One important way to circumvent toxicity is to store the toxin as an inactive precursor. Several sulfated diesters of the diarrhetic shellfish poisoning (DSP) toxin okadaic acid have been reported from cultures of various dinoflagellate species belonging to the genus Prorocentrum. It has been proposed that these sulfated diesters are a means of toxin storage within the dinoflagellate cell, and that a putative enzyme mediated two-step hydrolysis of sulfated diesters such as DTX-4 and DTX-5 initially leads to the formation of diol esters and ultimately to the release of free okadaic acid. However, only one diol ester and no sulfated diesters of DTX-1, a closely related DSP toxin, have been isolated leading some to speculate that this toxin is not stored as a sulfated diester and is processed by some other means. DSP components in organic extracts of two large scale Prorocentrum lima laboratory cultures have been investigated. In addition to the usual suite of okadaic acid esters, as well as the free acids okadaic acid and DTX-1, a group of corresponding diol- and sulfated diesters of both okadaic acid and DTX-1 have now been isolated and structurally characterized, confirming that both okadaic acid and DTX-1 are initially formed in the dinoflagellate cell as the non-toxic sulfated diesters., (Copyright © 2017. Published by Elsevier B.V.)

Okadaic acid (OA) and its analogs dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic polyethers produced by marine dinoflagellates. These toxins accumulate in shellfish and cause diarrhetic shellfish poisoning (DSP) in humans. Regulatory testing of shellfish is essential to safeguard public health and for international trade. Certified reference materials (CRMs) play a key role in analytical monitoring programs. This paper presents an overview of the interdisciplinary work that went into the planning, production, and certification of calibration-solution CRMs for OA, DTX1, and DTX2. OA and DTX1 were isolated from large-scale algal cultures and DTX2 from naturally contaminated mussels. Toxins were isolated by a combination of extraction and chromatographic steps with processes adapted to suit the source and concentration of each toxin. New 19-epi-DSP toxin analogs were identified as minor impurities. Once OA, DTX1, and DTX2 were established to be of suitable purity, solutions were prepared and dispensed into flame-sealed glass ampoules. Certification measurements were carried out using quantitative NMR spectroscopy and LC-tandem MS. Traceability of measurements was established through certified external standards of established purity. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization, with components from the measurement, stability, and homogeneity studies being propagated into final combined uncertainties.

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.

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.

Phlorotannins are a class of polyphenols found in brown seaweeds that have significant potential for use as therapeutics, owing to their wide range of bioactivities. Molecular characterization of phlorotannin-enriched extracts is challenging due to the extreme sample complexity and the wide range of molecular weights observed. Herein, we describe a method for characterizing phlorotannins employing ultrahigh-pressure liquid chromatography (UHPLC) operating in hydrophilic interaction liquid chromatography (HILIC) mode combined with high-resolution mass spectrometry (HRMS).

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.

Analysis of triacylglycerols (TAGs), found as complex mixtures in living organisms, is typically accomplished using liquid chromatography, often coupled to mass spectrometry. TAGs, weak bases not protonated using electrospray ionization, are usually ionized by adduct formation with a cation, including those present in the solvent (e.g., Na(+)). There are relatively few reports on the binding of TAGs with cations or on the mechanisms by which cationized TAGs fragment. This work examines binding efficiencies, determined by mass spectrometry and computations, for the complexation of TAGs to a range of cations (Na(+), Li(+), K(+), Ag(+), NH4(+)). While most cations bind to oxygen, Ag(+) binding to unsaturation in the acid side chains is significant. The importance of dimer formation, [2TAG + M](+) was demonstrated using several different types of mass spectrometers. From breakdown curves, it became apparent that two or three acid side chains must be attached to glycerol for strong cationization. Possible mechanisms for fragmentation of lithiated TAGs were modeled by computations on tripropionylglycerol. Viable pathways were found for losses of neutral acids and lithium salts of acids from different positions on the glycerol moiety. Novel lactone structures were proposed for the loss of a neutral acid from one position of the glycerol moiety. These were studied further using triple-stage mass spectrometry (MS(3)). These lactones can account for all the major product ions in the MS(3) spectra in both this work and the literature, which should allow for new insights into the challenging analytical methods needed for naturally occurring TAGs.

Reversed-phase high performance liquid chromatography (RP-HPLC), followed by post-column addition of lithium salts and electrospray ionisation triple-stage mass spectrometry (ESI-MS(3)) of lithiated TAG adducts, is shown to provide a useful method for the positional analysis of triacylglycerols (TAGs) in fish oils containing eicosapentaenoic (EPA, 20:5) and docosahexaenoic acids (DHA, 22:6). One prominent fragmentation pathway in the ESI-MS(3) of these adduct ions involves the loss of a fatty acid from the sn-1/3 position in the first step followed by the loss of an α,β-unsaturated fatty acid from the sn-2 position in the second. Regioisomeric TAGs of the type ABA and AAB produced abundant product ions - [ABA+Li-RACOOH-R'BCHCHCOOH](+) and [AAB+Li-RACOOH-R'ACHCHCOOH](+) - the relative intensities of which were dependent on the position of acyl substituents. Standard solutions of TAGs containing different ratios of the regioisomeric pairs MME/MEM, PPE/PEP, PPD/PDP, EEP/EPE and DDP/DPD (M=14:0, P=16:0, E=20:5, D=22:6) were analysed by ESI-MS(3) with a quadrupole linear ion trap instrument. Methodology developed on the standards was applied to quantifying the relative isomeric abundances of EPA and DHA in several fish oil samples. DHA was preferentially located at the sn-2 position in both DHA-containing TAGs studied, while EPA was either observed at near equal levels in all positions, or predominantly at the sn-1 and -3 positions in some cases. The analysis protocol allows for quantification of the designated regioisomers in one simple, rapid chromatographic procedure using a single column and has the advantage of specificity over other methods for the positional analysis of TAGs, since it eliminates interferences associated with co-eluting TAGs of the same molecular weight that yield isobaric diacylglycerol-like product ions., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2013 John Wiley & Sons, Ltd.)

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., (Copyright © 2012 John Wiley & Sons, Ltd.)

A switchable hydrophilicity solvent (SHS) was studied for its effectiveness at extracting lipids from freeze-dried samples of Botryococcus braunii microalgae. The SHS N,N-dimethylcyclohexylamine extracted up to 22 wt.% crude lipid relative to the freeze-dried cell weight. The solvent was removed from the extract with water saturated with carbon dioxide at atmospheric pressure and recovered from the water upon de-carbonation of the mixture. Liquid chromatography-mass spectrometry (LC-MS) showed that the extracted lipids contained high concentrations of long chain tri-, di- and mono-acylglycerols, no phospholipids, and only 4-8% of residual solvent. Unlike extractions with conventional organic solvents, this new method requires neither distillation nor the use of volatile, flammable or chlorinated organic solvents., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 John Wiley & Sons, Ltd.)

To assess Soxhlet extraction as a method for quantifying fatty acids (FA) of microalgae, crude lipid, FA content from Soxhlet extracts and FA content from in situ transesterification (ISTE) were compared. In most cases, gravimetric lipid content was considerably greater (up to sevenfold) than the FA content of the crude lipid extract. FA content from Soxhlet lipid extraction and ISTE were similar in 12/18 samples, whereas in 6/18 samples, total FA content from Soxhlet extraction was less than the ISTE procedure. Re-extraction of residual biomass from Soxhlet extraction with ISTE liberated a quantity of FA equivalent to this discrepancy. Employing acid hydrolysis before Soxhlet extraction yielded FA content roughly equivalent to ISTE, indicating that acidic conditions of ISTE are responsible for this observed greater recovery of FA. While crude lipid derived from Soxhlet extraction was not a useful proxy for FA content for the species tested, it is effective in most strains at extracting total saponifiable lipid. Lipid class analysis showed the source of FA was primarily polar lipids in most samples (12/18 lipid extracts contained <5% TAG), even in cases where total FA content was high (>15%). This investigation confirms the usefulness of ISTE, reveals limitations of gravimetric methods for projecting biodiesel potential of microalgae, and reinforces the need for intelligent screening using both FA and lipid class analysis.

Biofuels from photosynthetic microalgae are quickly gaining interest as a viable carbon-neutral energy source. Typically, characterization of algal feedstock involves breaking down triacylglycerols (TAG) and other intact lipids, followed by derivatization of the fatty acids to fatty acid methyl esters prior to analysis by gas chromatography (GC). However, knowledge of the intact lipid profile could offer significant advantages for discovery stage biofuel research such as the selection of an algal strain or the optimization of growth and extraction conditions. Herein, lipid extracts from microalgae were directly analyzed by ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) using a benchtop Orbitrap mass spectrometer. Phospholipids, glycolipids, and TAGs were analyzed in the same chromatographic run, using a combination of accurate mass and diagnostic fragment ions for identification. Using this approach, greater than 100 unique TAGs were identified over the six algal strains studied and TAG profiles were obtained to assess their potential for biofuel applications. Under the growth conditions employed, Botryococcus braunii and Scenedesmus obliquus yielded the most comprehensive TAG profile with a high abundance of TAGs containing oleic acid.

Marine biotoxins pose a significant food safety risk when bioaccumulated in shellfish, and adequate testing for biotoxins in shellfish is required to ensure public safety and long-term viability of commercial shellfish markets. This report describes the use of a benchtop Orbitrap system for liquid chromatography-mass spectrometry (LC-MS) screening of multiple classes of biotoxins commonly found in shellfish. Lipophilic toxins such as dinophysistoxins, pectenotoxins, and azaspiracids were separated by reversed phase LC in less than 7 min prior to MS data acquisition at 2 Hz with alternating positive and negative scans. This approach resulted in mass accuracy for analytes detected in positive mode (gymnodimine, 13-desmethyl spirolide C, pectenotoxin-2, and azaspiracid-1, -2, and -3) of less than 1 ppm, while those analytes detected in negative mode (yessotoxin, okadaic acid, and dinophysistoxin-1 and -2) exhibited mass errors between 2 and 4 ppm. Hydrophilic toxins such as domoic acid, saxitoxin, and gonyautoxins were separated by hydrophilic interaction LC (HILIC) in less than 4 min, and MS data was collected at 1 Hz in positive mode, yielding mass accuracy of less than 1 ppm error at a resolving power of 100,000 for the analytes studied (m/z 300-500). Data were processed by extracting 5 ppm mass windows centered around the calculated masses of the analytes. Limits of detection (LOD) for the lipophilic toxins ranged from 0.041 to 0.10 μg/L (parts per billion) for the positive ions, 1.6-5.1 μg/L for those detected in negative mode, while the domoic acid and paralytic shellfish toxins yielded LODs ranging from 3.4 to 14 μg/L. Toxins were detected in mussel tissue extracts free of interference in all cases.

The production and certification of a series of azaspiracid (AZA) calibration solution reference materials is described. Azaspiracids were isolated from contaminated mussels, purified by preparative liquid chromatography and dried under vacuum to the anhydrous form. The purity was assessed by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The final concentration of each AZA in a CD(3)OH stock solution was determined by quantitative NMR spectroscopy. This solution was then diluted very accurately in degassed, high purity methanol to a concentration of 1.47 ± 0.08 μmol/L for CRM-AZA1, 1.52 ± 0.05 μmol/L for CRM-AZA2, and 1.37 ± 0.13 μmol/L for CRM-AZA3. Aliquots were dispensed into argon-filled glass ampoules, which were immediately flame-sealed. The calibration solutions are suitable for method development, method validation, calibration of liquid chromatography or mass spectrometry instrumentation and quality control of shellfish monitoring programs.

Herein we report a reversed-phase high-performance liquid chromatography tandem mass spectrometry (RP-HPLC/MS/MS) method for the analysis of positional isomers of triacylglycerols (TAGs) in vegetable oils. The fragmentation behavior of [M + X](+) ions (X = NH(4), Li, Na or Ag) was studied on a quadrupole-time-of-flight (Q-TOF) mass spectrometer under low-energy collision-induced dissociation (CID) conditions. Mass spectra that were dependent on the X(+) ion and the nature and position of the acyl substituents were observed for four pairs of 'AAB/ABA'-type TAGs, namely PPO/POP, OOP/OPO, LLO/LOL and OOL/OLO (where P is 16:0, palmitic acid; O is 18:1, oleic acid; and L is 18:2, linoleic acid). For the majority of [M + X](+) adducts, the loss of the fatty acid in the outer positions (sn-1 or sn-3) was favored over the loss in the central position (sn-2), which enabled the determination of the fractional abundance of the isomers. Ratios of the intensity of fragment ions at various AAB/ABA compositions produced linear calibration curves with positive slopes, comparable to those obtained traditionally by ESI-MS/MS of [M + NH(4)](+) adducts. The only exceptions were the [M + Ag](+) adducts of the PPO/POP system, which produced calibration curves with negative slopes. Sodium adducts provided the most consistent level of isomeric discrimination for the TAGs studied and also offered the most convenience in that they required no additive to the mobile phase. Therefore, calibration curve data derived from [M + Na](+) adducts were applied to the quantification of TAG regioisomers in sunflower and olive oils. The regiospecific analysis showed that palmitic acid was typically located at positions sn-1 or sn-3, whereas unsaturated fatty acids, oleic and linoleic acids were mostly found at the sn-2 position., (2010 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.)

It is well known that the flagellin of Campylobacter jejuni is extensively glycosylated by pseudaminic acid and the related acetamindino derivative, in addition to flagellin glycosylation being essential for motility and colonization of host cells. Recently, the use of metabolomics permitted the unequivocal characterization of unique flagellin modifications in Campylobacter, including novel legionaminic acid sugars in Campylobacter coli, which had been impossible to ascertain in earlier studies using proteomics-based approaches. To date, the precise identities of the flagellin glycosylation modifications have only been elucidated for C. jejuni 81-176 and C. coli VC167 and those present in the first genome-sequenced strain C. jejuni 11168 remain elusive due to lability and respective levels of individual glycan modifications. We report the characterization of the carbohydrate modifications on C. jejuni 11168 flagellin using metabolomics-based approaches. Detected as their corresponding CMP-linked precursors, structural information on the flagellin modifications was obtained using a combination of MS and NMR spectroscopy. In addition to the pseudaminic acid and legionaminic acid sugars known to be present on Campylobacter flagellin, two unusual 2,3-di-O-methylglyceric acid modifications of a nonulosonate sugar were identified. By performing a metabolomic analysis of selected isogenic mutants of genes from the flagellin glycosylation locus of this pathogen, these novel CMP-linked precursors were confirmed to be di-O-methylglyceric acid derivatives of pseudaminic acid and the related acetamidino sugar. This is the first comprehensive analysis of the flagellar modifications in C. jejuni 11168 and structural elucidation of di-O-methylglyceric acid derivatives of pseudaminic acid on Campylobacter flagellin.

Peptide dimethylation with isotopically coded formaldehydes was evaluated as a potential alternative to techniques such as the iTRAQ method for comparative proteomics. The isotopic labeling strategy and custom-designed protein quantitation software were tested using protein standards and then applied to measure proteins levels associated with Alzheimer's disease (AD). The method provided high accuracy (10% error), precision (14% RSD) and coverage (70%) when applied to the analysis of a standard solution of BSA by LC-MS/MS. The technique was then applied to measure protein abundance levels in brain tissue afflicted with AD relative to normal brain tissue. 2-D LC-MS analysis identified 548 unique proteins (p<0.05). Of these, 349 were quantified with two or more peptides that met the statistical criteria used in this study. Several classes of proteins exhibited significant changes in abundance. For example, elevated levels of antioxidant proteins and decreased levels of mitochondrial electron transport proteins were observed. The results demonstrate the utility of the labeling method for high-throughput quantitative analysis.

Here we report the first application of a matrix-assisted laser desorption/ionization (MALDI) triple-quadrupole mass spectrometer for targeted proteomics. Employing an amine-specific isotopic labelling approach, the technique was validated using five randomly selected bovine serum albumin peptides differentially labelled at known ratios. An indirect benefit of the isotopic labelling technique is a significant enhancement of the a1 ion in tandem mass (MS/MS) spectra of all peptides studied. Therefore, the a1 ion was selected as the fragment ion for multiple reaction monitoring (MRM) in all cases, eliminating tedious method development and optimization. Accurate quantification was achieved with an average relative standard deviation (RSD) of 5% (n = 5) and a detection limit of 14 amol. The technique was then applied to validate an important virulence biomarker of the fungal pathogen Candida albicans, which was not accurately quantified using global proteomics experiment employing two-dimensional liquid chromatography/electrospray ionization tandem mass spectrometry (2D-LC/ESI)-MS/MS. Using LC/MALDI-MRM analysis of five tryptic peptides, the protein PHR1 was found to be upregulated in the hyphal (pathogenic) form of C. albicans by a factor of 7.7 +/- 0.8., (Copyright 2006 John Wiley & Sons, Ltd.)

The study of peptide fragmentation is important to the understanding of chemical processes occurring in the gas phase and the more practical concern of peptide identification for proteomic analysis. Using the mobile proton model as a framework, we explore the effect of amino-group modifications on peptide fragmentation. Three aldehydes are used to transform the peptides' primary amino groups into either a dimethylamino or a heterocyclic structure (five- or six-membered). The observed fragmentation patterns deviate strongly from those observed for the analogous underivatised peptides. In particular, the a1 ion is the base peak in most tandem mass spectra of the derivatised peptides. The a1 ion intensity depends strongly on the N-terminal amino acid, with tyrosine and phenylalanine having the strongest enhancement. Despite the change in fragmentation patterns of the derivatised peptides, they still provide high-quality tandem mass spectra that, in many cases, are more amenable to database searching than the spectra of underivatised peptides. In addition, the reliable presence of the a1 ion facilitates rapid quantitative measurements using the multiple reaction monitoring approach., (Copyright 2006 John Wiley & Sons, Ltd.)

Traditional protein labeling reactions for capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection suffer from a variety of disadvantages. The reactions can be nonquantitative on a reasonable time scale, require relatively high concentrations of protein and fluorophore, and can give multiple reaction products that can not be separated. Herein, we describe a new noncovalent labeling technique that is rapid, selective for myoglobin, and gives a simple reaction product. Myoglobin is denatured with either 5.4 M urea or low pH (2.0). The denatured myoglobin releases its nonfluorescent heme group. A fluorescent porphyrin (protoporphyrin IX (PPIX) or its zinc (II) complex, Zn-PPIX), is added to the mixture and the solution conditions are altered (dilute to 0.54 M urea or adjust pH to 7.0) to allow myoglobin refolding. Upon refolding, the protein incorporates PPIX from solution, thus making the reaction product fluorescent. The experimental conditions have been optimized for both urea and low-pH denaturation of myoglobin. The latter procedure produces a detection limit of 50 nM. Alternatively, the reaction can be performed without denaturation by a simple exchange of the porphyrins. The use of Zn-PPIX yields the most efficient reaction. The low-pH reaction is unaffected by a 2000-fold excess of bovine serum albumin., (Copyright 2004 WILEY-VCH Verlag GmbH & Co.)

A highly sensitive technique for the analysis of urinary porphyrins using capillary electrophoresis (CE) coupled with laser-induced fluorescence (LIF) detection is reported. Separation of mesoporphyrin IX, coproporphyrin, uroporphyrin and the penta-, hexa- and heptacarboxylic acid porphyrins was achieved in 11 min using a 10 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES, pH 10) -75 mM sodium dodecyl sulfate (SDS) buffer. Migration time and peak area repeatability were less than 1 and 5% relative standard deviation (RSD), respectively. Limits of detection of 20 pM (2 x 10(-11) M) were achieved employing the recently introduced Nichia violet diode laser for excitation at 400 nm. This represents an enhancement in sensitivity of over two orders of magnitude compared to previous reports. This high sensitivity for urinary porphyrins was demonstrated through the quantification of coproporphyrin and uroporphyrin in urine samples after up to a 100-fold dilution.

This paper describes the adsorption mechanisms and aggregation properties of cetyltrimethylammonium bromide (CTAB) and didodecyldimethylammonium bromide (DDAB) surfactants that are used for dynamic coatings in capillary electrophoresis (CE). Atomic force microscopy is used to directly visualize surfactant adsorption on fused silica. It was found that the single-chained surfactant CTAB forms spherical aggregates on silica while the double-chained surfactant DDAB forms a bilayer. Aggregation at the surface occurs at approximately the same surfactant concentration in which EOF reversal is observed in CE. The nearest-neighbor distance between CTAB aggregates varies inversely with buffer pH and becomes constant at the point when the silanol groups are fully ionized. DDAB forms a flat, uniform coating independent of pH. Increasing the buffer ionic strength changes the morphology of the CTAB aggregates from spherical to cylindrical. The change in morphology can alter the surface coverage, which is related to the "normalized" EOF measured in identical buffers. The morphology of a surfactant coating is also shown to affect its ability to inhibit protein adsorption to the capillary wall. Specifically, the full surface coverage provided by DDAB proved superior in a head-to-head comparison with CTAB.

Capillary electrophoresis coupled with indirect UV absorbance detection was employed for the determination of the chemical warfare agent degradation products: methylphosphonic acid, ethyl methylphosphonate, isopropyl methylphosphonate, and pinacolyl methylphosphonate. Glutamic acid was used as a buffering agent at its isoelectric point (pH 3.22). In its zwitterionic form, glutamic acid does not act as a competing co-anion in the system, thus providing buffering capacity while maintaining high sensitivity. The indirect probe (phenylphosphonic acid) concentration was lowered to 1 mM from the 10 mM in previous literature studies, further enhancing sensitivity. Detection limits of 2 microM were achieved with hydrodynamic injection and up to 100-fold lower using electrokinetic injection. The increased buffering capacity of this system over previous methods led to migration time reproducibility RSD values of 0.18 to 0.22%. This represents a 10-fold improvement in reproducibility over previous studies with comparable or improved sensitivity.

The violet (415 nm) diode laser is used for indirect laser-induced fluorescence detection in capillary electrophoretic separations of inorganic anions and chemical warfare agent degradation products. Inorganic anions were detected using 8-hydroxypyrene-1,3,6-trisulfonic acid as the indirect probe and achieved submicromolar (40-80 ppb) detection limits in a 2-min separation. The chemical warfare agent degradation products methylphosphonic acid, ethyl methylphosphonate, isopropyl methylphosphonate, and pinacolyl methylphosphonate were detected using the porphyrin tetrakis(4-sulfophenyl)porphine as the indirect probe and achieved detection limits of 0.1 microM (9 ppb), which are 1 order of magnitude better than that achieved using indirect UV detection. Baseline stability achieved with the violet diode laser was excellent, with dynamic reserve (DR) values of > 1000, which are 15 times better than that achieved using an unstabilized HeCd laser.