Daniel G. Beach

Laser Ablation Electrospray Ionization-High Resolution Mass Spectrometry for Regulatory Screening of Domoic Acid in Shellfish.

Rationale Domoic acid (DA) is a potent neurotoxin that accumulates in shellfish. Routine testing involves homogenization, extraction and chromatographic analysis, with a run time of up to 30 min. Improving throughput using ambient ionization for direct analysis of DA in tissue would result in significant time savings for regulatory testing labs. Methods We assess the suitability of laser ablation electrospray ionization high‐resolution mass spectrometry (LAESI‐HRMS) for high‐throughput screening or quantitation of DA in a variety of shellfish matrices. The method was first optimized for use with HRMS detection. Challenges such as tissue sub‐sampling, isobaric interferences and method calibration were considered and practical solutions developed. Samples included 189 real shellfish samples previously analyzed by regulatory labs as well as mussel matrix certified reference materials. Results Domoic acid was selectively analyzed directly from shellfish tissue homogenates with a run time of 12 s. The limits of detection were between 0.24 and 1.6 mg DA kg−1 tissue, similar to those of LC/UV methods. The precision was between 27 and 44% relative standard deviation (RSD), making the technique more suited to screening than direct quantitation. LAESI‐MS showed good agreement with LC/UV and LC/MS and was capable of identifying samples above and below 5 mg DA kg−1 wet shellfish tissue, one quarter of the regulatory limit. Conclusions These findings demonstrate the suitability of LAESI‐MS for routine, high‐throughput screening of DA. This approach could result in significant time savings for regulatory labs carrying out shellfish safety testing on thousands of samples annually.

Sensitive determination of domoic acid in mussel tissue using dansyl chloride derivatization and liquid chromatography-mass spectrometry

This paper describes a new method for sensitive determination of domoic acid (DA), the causative toxin of amnesic shellfish poisoning (ASP), in shellfish. The method involves extraction of tissue homogenates with 50% methanol followed by a highly selective strong anion exchange solid phase extraction and a derivatization with dansyl chloride (DNS-Cl) to form the dansyl derivative of domoic acid (DNS-DA). Reaction times were very rapid and proceeded under ambient conditions to yield stable derivatives. A study of the collision-induced dissociation of ESI-produced protonated DNS-DA was carried out to identify the most sensitive transitions to use in development of a selected reaction monitoring detection method. Compared with un-derivatized DA, DNS-DA showed a 5-fold increase in sensitivity of MS/MS detection and improved retention on a reversed phase LC stationary phase. Resolution of DNS-DA and its isomers was achieved using isocratic elution in 15 min. A quantitative verification of the new method was carried out by analyzing a mussel tissue certified reference material (CRM) containing 49 mg kg−1 DA, as well as a toxin-free mussel tissue CRM spiked at levels ranging from 0.003 to 10 mg kg−1. Results showed good recovery (83–107%) with a between-sample variability of ≤5% RSD. The LC-MS/MS method presented is suitable for DA analysis over a broad range of concentrations spanning from above the regulatory limit of 20 mg DA per kg tissue down to near the method detection limit of 1.1 μg DA per kg mussel tissue. The resulting method serves as a confirmatory method with alternative selectivity to existing methods. It is also suitable for quantification of low levels of DA in shellfish as an early warning sign for toxic events or in forensic applications after intoxication has occurred.

Differential Mobility Spectrometry for Improved Selectivity in Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry Analysis of Paralytic Shellfish Toxins

AbstractParalytic shellfish toxins (PSTs) are neurotoxins produced by dinoflagellates and cyanobacteria that cause paralytic shellfish poisoning in humans. PST quantitation by LC-MS is challenging because of their high polarity, lability as gas-phase ions, and large number of potentially interfering analogues. Differential mobility spectrometry (DMS) has the potential to improve the performance of LC-MS methods for PSTs in terms of selectivity and limits of detection. This work describes a comprehensive investigation of the separation of 16 regulated PSTs by DMS and the development of highly selective LC-DMS-MS methods for PST quantitation. The effects of all DMS parameters on the separation of PSTs from one another were first investigated in detail. The labile nature of 11α-gonyautoxin epimers gave unique insight into fragmentation of labile analytes before, during, and after the DMS analyzer. Two sets of DMS parameters were identified that either optimized the resolution of PSTs from one another or transmitted them at a limited number of compensation voltage (CV) values corresponding to structural subclasses. These were used to develop multidimensional LC-DMS-MS/MS methods using existing HILIC-MS/MS parameters. In both cases, improved selectivity was observed when using DMS, and the quantitative capabilities of a rapid UPLC-DMS-MS/MS method were evaluated. Limits of detection of the developed method were similar to those without DMS, and differences were highly analyte-dependant. Analysis of shellfish matrix reference materials showed good agreement with established methods. The developed methods will be useful in cases where specific matrix interferences are encountered in the LC-MS/MS analysis of PSTs in complex biological samples. Graphical Abstractᅟ

Differential Mobility-Mass Spectrometry Double Spike Isotope Dilution Study of Release of β-Methylaminoalanine and Proteinogenic Amino Acids during Biological Sample Hydrolysis

The non-protein amino acid β-methylamino-L-alanine (BMAA) has been linked to neurodegenerative disease and reported throughout the environment. Proposed mechanisms of bioaccumulation, trophic transfer and chronic toxicity of BMAA rely on the hypothesis of protein misincorporation. Poorly selective methods for BMAA analysis have led to controversy. Here, a recently reported highly selective method for BMAA quantitation using hydrophilic interaction liquid chromatography-differential mobility spectrometry-tandem mass spectrometry (HILIC-DMS-MS/MS) is expanded to include proteinogenic amino acids from hydrolyzed biological samples. For BMAA quantitation, we present a double spiking isotope dilution approach using D3-BMAA and 13C15N2-BMAA. These methods were applied to study release of BMAA during acid hydrolysis under a variety of conditions, revealing that the majority of BMAA can be extracted along with only a small proportion of protein. A time course hydrolysis of BMAA from mussel tissue was carried out to assess the recovery of BMAA during sample preparation. The majority of BMAA measured by typical methods was released before a significant proportion of protein was hydrolyzed. Little change was observed in protein hydrolysis beyond typical hydrolysis times but the concentration of BMAA increased linearly. These findings demonstrate protein misincorporation is not the predominant form of BMAA in cycad and shellfish.

Isotope-labelling derivatisation: a broadly applicable approach to quantitation of algal toxins by isotope dilution LC-MS/MS

Two methods were developed for the analysis of algal biotoxins in complex biological and environmental samples to demonstrate the concept of isotope-labelling derivatisation for quantitation. These methods are based on dansyl chloride derivatisation of samples and dansyl-d6 chloride derivatisation of toxin standards. Derivatised sample and standard are then mixed to achieve isotope dilution calibration in liquid chromatography–tandem mass spectrometry analyses. Quantitation of the marine toxin domoic acid (DA) in mussel tissues and the freshwater toxins anatoxin-a (ATX) and homoanatoxin-a (hATX) in cyanobacteria is demonstrated. For DA, isotope-labelling was incorporated into existing dansylation methodology using inexpensive and commercially available reagents. For ATXs, a novel sample preparation procedure is presented that involves solid phase extraction on a mixed reverse phase/weak anion exchange column that facilitates simultaneous clean-up of the derivatised toxins and removal of excess dansylation reagent through covalent bonding. The challenge of achieving co-elution in LC between deuterated and non-deuterated dansylated toxins was addressed by modifying separation conditions from the usual reverse phase (RP) separation to hydrophilic interaction liquid chromatography in the case of DA and a shortened RP separation with high organic modifier content in the case of the ATXs. The new methods gave limits of detection between 10 and 60 μg kg−1 and allowed for precise, accurate and fast determination of toxins in spiked control samples and matrix reference materials. This work demonstrates that isotope-labelling derivatisation is broadly applicable to the field of algal toxin analysis where derivatisation is well established but isotopically-labelled standards are not available.

Screening of cyclic imine and paralytic shellfish toxins in isolates of the genus Alexandrium (Dinophyceae) from Atlantic Canada

The dinoflagellate genus Alexandrium Halim has frequently been associated with harmful algal blooms. Although a number of species from this genus are known to produce paralytic shellfish toxins (PST) and/or cyclic imines (CI), studies on comprehensive toxin profiling using techniques capable of detecting the full range of PST and CI analogues are limited. Isolates of Alexandrium spp. from Atlantic Canada were analyzed by targeted and untargeted liquid chromatography-tandem mass spectrometry (LC-MS). Results showed a number of distinct profiles and wide ranging cell quotas of PST and spirolides (SPX) in both A. catenella (Whedon & Kofoid) Balech and A. ostenfedii (Paulsen) Balech & Tangen. The concentration of PST in A. catenella ranged from 0.0029 to 54 fmol cell-1 with the major components being C2 and GTX4. In addition, putative PST metabolites were confirmed for the first time in A. catenella by high resolution MS/MS. By comparison, A. ostenfeldii isolates showed much lower concentrations of PST (<LOD to 2 fmol cell-1) and high total levels of SPX (14 to 43 fmol cell-1). The SPX profile of the A. ostenfeldii strains mainly included 13-desmethyl SPX-C, SPX-C and 20-methyl SPX-G, with low levels of other SPX and gymnodimine-like analogues detected by untargeted -high-resolution LC-MS. This work demonstrates the importance of using screening methods capable of detecting the full suite of PST and CI compounds when analyzing Alexandrium isolates for toxin production and adds further complexity to the known toxin profiles of this genus.

Collision induced dissociation mass spectrometry challenge

We would like to invite you to participate in the Analytical Challenge, a series of puzzles to entertain and challenge our readers. This special feature of “Analytical and Bioanalytical Chemistry” has established itself as a truly unique quiz series, with a new scientific puzzle published every other month. Readers can access the complete collection of published problems with their solutions on the ABC homepage at http://www. springer.com/abc . Test your knowledge and tease your wits in diverse areas of analytical and bioanalytical chemistry by viewing this collection. In the present challenge, mass spectrometry is the topic. And please note that there is a prize to be won (a Springer book of your choice up to a value of €100). Please read on...

Dynamics of paralytic shellfish toxins and their metabolites during timecourse exposure of scallops Chlamys farreri and mussels Mytilus galloprovincialis to Alexandrium pacificum

New C-11 hydroxyl metabolites of paralytic shellfish toxins (PSTs) have been reported in shellfish. To gain further information on these metabolites, as well as the potential for formation of phase-II metabolites and acyl esters of PSTs, bivalves were fed with the PSTs-producing dinoflagellate Alexandrium pacificum (strain ATHK). Through independent experiments, scallops (Chlamys farreri) were fed for 9 days and mussels (Mytilus galloprovincialis) for 5 days plus an additional 5 days of depuration, with representative samples taken throughout. Several common PSTs (C1-4, GTX1-6 and NEO) and metabolites including M1, M3, M5, M7, M9, M2 and M8 were detected in the hepatopancreas of scallops during toxin accumulation and in the hepatopancreas of mussels during both toxin accumulation and elimination periods. The relative molar ratio of metabolites to precursor molecules was used to estimate relative metabolic conversion rates. Conversion rates of C1/2 and GTX2/3 were higher than those of C3/4 and GTX1/4, in scallops and mussels. The first metabolites observed in both bivalve species investigated were M1/3, which are formed from C1/2. However, the conversion of GTX2/3 to M2 was more complete than other biotransformation reactions in both mussels and scallops. In general, metabolic conversion of PSTs was observed after a shorter time and to a greater extent in mussels than in scallops in the exposure period. No acyl esters or conjugation products of PSTs with glucuronic acid, glutathione, cysteine and taurine were detected by liquid chromatography with high resolution tandem mass spectrometry in the samples investigated. Additionally, only GTX1/4 and GTX2/3 were detected in the kidney of scallops, which demonstrates that PSTs are mainly metabolized through the hepatic metabolism pathway in bivalves. This work improves the understanding of PST metabolism during toxin accumulation and depuration in commercially harvested shellfish.

Solution to collision induced dissociation mass spectrometry challenge

All of the product ion peaks with m/z higher than the selected precursorm/z presented in the “Collision induced dissociation mass spectrometry challenge” [1] originate from ion–neutral reactions between analyte product ions and residual protic solvent molecules present in the mass spectrometer. In general, this is a well-documented phenomenon observed primarily, but not exclusively [2], in ion trap mass spectrometers operating at higher pressure and in which collision-induced dissociation (CID) experiments occur over a longer period of collisional activation compared with quadrupole CID [3, 4]. Depending on the instrument and the analyte, the origin of the solvent can be either residual neutral solvent molecules from electrospray ionization (ESI) [5, 6] or the collision gas [4, 7]. Unlike non-covalent solvent adducts or “clusters” formed during ESI that can be readily desolvated in the mass spectrometer inlet at lower energies than are typically required to break covalent bonds in CID, the ions under discussion represent the products of covalent bond forming ion–molecule reactions. In all the examples presented, the ions first undergo a loss of a neutral molecule (NH3, H2O, H2 or glutamic acid) to produce a reactive product ion that then reacts with water or methanol to give a stable product ion higher in mass than the selected precursor, as detailed in Table 1. The common structural feature of the reactive product ions that leads to solvent adduction in the examples presented is the ketene functional group (Structure 1). Ketenes are strong electrophiles with partial positive polarization at the α position, and are susceptible to nucleophilic attack with electrophilic participation. In tandem mass spectrometry (MS/MS), reactive ions with ketene functionality can be formed during collisional activation through ring opening and/or neutral loss reactions that result in a Cβ=Cα double bond being inserted adjacent to a carbonyl Cα=O. This is followed by nucleophilic attack by a neutral solvent molecule at the α position and electrophilic participation by the βcarbon or the oxygen [8, 9]. Guanine analogues are perhaps the best studied examples of ketene formation leading to ion–neutral reactions with water in mass spectrometry both as protonated ions and as deprotonated ions before and after dissociation [10–12]. During collisional activation, guanine undergoes tautomerization, ring opening and proton transfer reactions resulting in formation of ketene functionality. The most prominent of these occurs after a ring opening rearrangement accompanied by elimination of ammonia, as shown in Scheme 1. Reaction of this product ion with water leads to the significant peak 1 Da higher than the peak of the selected protonated guanine precursor [10, 11], as shown in Fig. 1 of the challenge [1]. Water adduction is also prominent in the CID of deprotonated guanine in negative ESI-MS/MS, which also results from ketene formation after ring opening [12]. The process by which water is eliminated from dicarboxylic acids was extensively studied by Grossert et al. [5], and involves intramolecular interactions between the ionized and non-ionized carboxyl groups. In the case of deprotonated dodecadedioic acid presented in Fig. 2 of the challenge [1], a +14-dalton peak was observed during This article is the solution to the Analytical Challenge to be found at https://doi.org/10.1007/s00216-017-0684-0

Capillary electrophoresis–tandem mass spectrometry for multiclass analysis of polar marine toxins

AbstractPolar marine toxins are more challenging to analyze by mass spectrometry-based methods than lipophilic marine toxins, which are now routinely measured in shellfish by multiclass reversed-phase liquid chromatography–tandem mass spectrometry (MS/MS) methods. Capillary electrophoresis (CE)–MS/MS is a technique that is well suited for the analysis of polar marine toxins, and has the potential of providing very high resolution separation. Here, we present a CE–MS/MS method developed, with use of a custom-built interface, for the sensitive multiclass analysis of paralytic shellfish toxins, tetrodotoxins, and domoic acid in seafood. A novel, highly acidic background electrolyte (5 M formic acid) was designed to maximize protonation of analytes and to allow a high degree of sample stacking to improve the limits of detection. The method was applied to a wide range of regulated and less common toxin analogues, and exhibited a high degree of selectivity between toxin isomers and matrix interference. The limits of detection in mussel tissue were 0.0052 mg/kg for tetrodotoxins, 0.160 mg/kg for domoic acid, and between 0.0018 and 0.120 mg/kg for paralytic shellfish toxins, all of which showed good linearity. Minimal ionization suppression was observed when the response from neat and mussel-matrix-matched standards was corrected with multiple internal standards. Analysis of shellfish matrix reference materials and spiked samples demonstrated good accuracy and precision. Finally, the method was transferred to a commercial CE–MS/MS system to demonstrate its widespread applicability for use in both R & D and routine regulatory settings. The approach of using a highly acidic background electrolyte is of broad interest, and can be considered generally applicable to simultaneous analysis of other classes of small, polar molecules with differing pKa values. Graphical abstractᅟ