Quantitative determination of paralytic shellfish poisoning toxins in shellfish using prechromatographic oxidation and liquid chromatography with fluorescence detection: interlaboratory study

An interlaboratory study was conducted for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish. The method used liquid chromatography with fluorescence detection after prechromatographic oxidation of the toxins with hydrogen peroxide and periodate. The PSP toxins studied were saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 and 3 (GTX2,3 together), gonyautoxins 1 and 4 (GTX1,4 together), decarbamoyl saxitoxin (dcSTX), B-1 (GTX5), C-1 and C-2 (C1,2 together), and C-3 and C-4 (C3,4 together). B-2 (GTX6) toxin was also included, but for qualitative identification only. Samples of mussels, both blank and naturally contaminated, were mixed and homogenized to provide a variety of PSP toxin mixtures and concentration levels. The same procedure was followed with samples of clams, oysters, and scallops. Twenty-one samples in total were sent to 21 collaborators who agreed to participate in the study. Results were obtained from 18 laboratories representing 14 different countries.     

Storage Stability Study of Salicylate-based Poly(anhydride-esters)

Storage stability was evaluated on a biodegradable salicylate-based poly(anhydride-ester) to elucidate the effects of storage conditions over time. The hydrolytically labile polymer samples were stored in powdered form at five relevant storage temperatures (−12 °C, 4 °C, 27 °C, 37 °C, 50 °C) and monitored over four weeks for changes in color, glass transition temperature, molecular weight, and extent of hydrolysis. Samples stored at lower temperatures remained relatively constant with respect to bond hydrolysis and molecular weight. Whereas, samples stored at higher temperatures displayed significant hydrolysis. For hydrolytically degradable polymers, such as these poly(anhydride-esters), samples are best stored at low temperatures under an inert atmosphere.     

Development and validation of a high‐throughput online solid‐phase extraction–liquid chromatography–tandem mass spectrometry method for the detection of gonyautoxins 1&4 and gonyautoxins 2&3 in human urine

Paralytic shellfish toxins (PSTs), including gonyautoxins and saxitoxins, are produced by multiple species of microalgae and dinoflagellates, and are bioaccumulated by shellfish and other animals. Human exposure to PSTs typically occurs through ingestion of recreationally harvested contaminated shellfish and results in nonspecific symptomology. Confirmation of exposure to PSTs has often relied on the measurement of saxitoxin, the most toxic congener; however, gonyautoxins (GTXs), the sulfated carbamate derivatives of saxitoxin, may be present in shellfish at higher concentrations. To improve identification of PST exposures, our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography method to identify GTX1–4 in human urine with tandem mass spectrometry. The reportable range varied for each analyte, with all falling within 0.899 and 250 ng/mL in urine with precision <15% and >85% accuracy as determined for all quality control samples. This new online method quantitates GTX1–4 following exposures to PSTs, supporting the work of public health authorities.     

Quantitative determination of paralytic shellfish poisoning toxins in shellfish using prechromatographic oxidation and liquid chromatography with fluorescence detection: collaborative study

A collaborative study was conducted for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish. The method used liquid chromatography with fluorescence detection after prechromatographic oxidation of the toxins with hydrogen peroxide and periodate. The PSP toxins studied were saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 and 3 (GTX2,3; together), gonyautoxins 1 and 4 (GTX1,4; together), decarbamoyl saxitoxin (dcSTX), B-1 (GTX5), C-1 and C-2 (C1,2; together), and C-3 and C-4 (C3,4; together). B-2 (GTX6) toxin was also included, but for qualitative identification only. Mussels, both blank and naturally contaminated, were mixed and homogenized to provide a variety of PSP toxin mixtures and concentration levels. The same procedure was followed with clams, oysters, and scallops. Twenty-one test samples in total were sent to 21 collaborators who agreed to participate in the study. Results were obtained from 18 laboratories representing 14 different countries. It is recommended that the method be adopted First Action by AOAC INTERNATIONAL.     

Freeze-drying for the stabilisation of shellfish toxins in mussel tissue (<Emphasis Type="Italic">Mytilus edulis</Emphasis>) reference materials

Two samples of mussels (Mytilus edulis) were collected from the southwest of Ireland. One sample contained domoic acid, the other sample contained okadaic acid, dinophysistoxin-2 and azaspiracid-1, -2 and -3. Wet and freeze-dried reference materials were prepared from each of the two samples to test for differences in homogeneity, stability and extractability of the analytes in either condition. Wet materials were homogenised, aliquoted and hermetically sealed under argon and subsequently frozen at −80 °C. Dry materials were similarly homogenised but frozen in flat cakes prior to freeze-drying. After grinding, sieving and further homogenisation, the resulting powder was aliquoted and hermetically sealed. Domoic acid materials were characterised using HPLC–UV, while LC–MS was used for the determination of lipophilic toxins. The extractabilities of all phycotoxins studied were comparable for wet and freeze-dried materials once a sonication step had been carried out for reconstitution of the freeze-dried materials prior to extraction. Homogeneity was assessed through replicate analysis of the phycotoxins (n = 10), and was found to be similar for wet and freeze-dried materials, for both hydrophilic and lipophilic toxins. Water contents were determined for both wet and freeze-dried materials, and particle size was determined for the freeze-dried materials. Stability was evaluated isochronously over eight months at four temperatures (−20, +4, +20 and +40 °C). The freeze-dried material containing domoic acid was stable over the whole duration at all temperatures, while in the wet material domoic acid degraded to some extent at all temperatures except −20 °C. In freeze-dried and wet materials containing lipophilic toxins, okadaic acid, dinophysistoxin-2, azaspiracid-1 and azaspiracid-2 were stable over the whole duration at all conditions, while concentrations of azaspiracid-3 changed significantly in both materials at some storage temperatures. Figure Aliquots of freeze-dried and wet mussel tissue reference materials containing the various shellfish toxins examined in the study     

Rapid liquid chromatography for paralytic shellfish toxin analysis using superficially porous chromatography with AOAC Official Method 2005.06

The bioaccumulation of paralytic shellfish toxins in mussels, oysters, cockles, hard clams, razors, and king scallops is monitored in England, Scotland, and Wales by AOAC Official Method 2005.06 LC-with fluorescence detection (FLD). One of the commonly perceived disadvantages of using this method is the long turnaround time and low throughput in a busy laboratory environment. The chromatographic analysis of each sample typically utilizes a 15 min cycle time to achieve toxin oxidation product separation and column equilibration prior to subsequent analysis. A standard RP C18 analytical column, used successfully in recent years, achieves good separation with a long column lifetime. The analysis of a 40 sample qualitative screening batch takes approximately 18 h, including blanks, standards, and other QC samples. The availability of superficially porous column technology has offered the potential to reduce analysis time while retaining column performance on existing hardware. In this study, AOAC Official Method 2005.06 with LC-FLD was transferred to two different commercially available superficially porous columns, and the method performance characteristics were evaluated. Both columns separated all toxins adequately with cycle times less than half that of the existing method. Linearity for each toxin was acceptable up to two times the European maximum permitted limit of 800 microg di-HCl saxitoxin equivalent/kg flesh. LOD and LOQ values were substantially improved for the majority of toxins, with gonyautoxin 1&4 and neosaxitoxin showing up to a two- and fourfold improvement, respectively, depending on the column used. Quantification results obtained from parallel analysis of contaminated samples were acceptable on both columns. Comparative screen results gave a slight increase in the occurrence of contaminated samples, which was attributed to the improved detection limit for most toxins. Issues with rapidly increasing back pressure, however, were identified with both columns, with a limit of around 500 injections. This compares to the >3000 cycles routinely obtained with the standard RP-C18 HPLC columns currently in use. Overall, the gain achieved with these columns through shorter analysis time and improved analytical sensitivity is potentially of benefit in a high-throughput environment. For the routine high-throughput screening of shellfish samples, however, an improved column lifetime is desirable.     

Thermal degradation behaviour of aromatic poly(ester-amide) with pendant phosphorus groups investigated by pyrolysis-GC/MS

The thermal stability of a novel phosphorus-containing aromatic poly(ester-amide) ODOP-PEA was investigated by thermogravimetric analysis (TGA). The weight of ODOP-PEA fell slightly at the temperature range of 300-400 °C in the TGA analysis, and the major weight loss occurred at 500 °C. The structural identification of the volatile products resulted from the ODOP-PEA pyrolysis at different temperatures was performed by pyrolysis-gas chromatography/mass spectrometry (pyrolysis-GC/MS). The P-C bond linked between the pendant DOPO group and the polymer chain disconnected first at approximately 275 °C, indicating that it is the weakest bond in the ODOP-PEA. The P-O bond in the pendant DOPO group was stable up to 300 °C. The cleavage of the ester linkage within the polymer main chain initiated at 400 °C, and the amide bond scission occurred at greater than 400 °C. The structures of the decomposition products were used to propose the degradation processes happening during the pyrolysis of the polymer.     

Loss of selenium in water samples at natural levels during storage

Selenium losses in river, ground, snow-melt and tap water samples, and the recovery of selenite, selenate and selenomethionine added to purified water have been studied. In 1-litre high-density polyethylene bottles, tap, river and snow-melt water samples (at Se concentrations of 44.5–138 ng/l) could be stored at 4 °C for up to 15 days without Se losses. In similar samples stored at room temperature Se losses of 13–25% after 15 days were found, except for groundwater, which showed no Se losses during storage for 13 months at room temperature or at 4 °C. Selenite and selenate added to purified water were recovered without losses after 15 days at 4 °C, while 7.5% of selenomethionine was lost. The stability of different chemical forms of Se during storage followed the order: selenate > selenomethionine > selenite. It is recommended that unacidified water samples should not be kept in polyethylene bottles at room temperature for more than 1 week, nor stored at 4 °C for more than 2 weeks, before analysis for Se.     

Determination of vanadium in mussels by electrothermal atomic absorption spectrometry without chemical modifiers

A method was developed for the quantitative determination of total vanadium concentration in mussels via electrothermal atomic absorption spectrometry (ETAAS). After the microwave digestion of the samples, a program using temperatures of 1600 °C and 2600 °C for ashing and atomization respectively, without any matrix modifiers, allowed us to obtain results that were satisfactory since they agreed closely with certified reference material values. The detection limit was 0.03 mg kg^–1 (dry weight), indicating that the method is suitable for the analysis of mussel samples. This determination was compared with matrix modifiers that have been reported previously. The method was applied to various cultivated and wild mussels from the Galician coast, yielding levels below 1 mg kg^–1 (wet weight).     

Thermal oxidation of poly(1-trimethylsilylprop-1-yne) studied by IR spectroscopy

Thermal oxidation of poly(1-trimethylsilylprop-1-yne) was studied by IR spectroscopy in the 20—245 °C temperature interval. In the 20—160 °C temperature range, the reaction proceeds predominantly at the C—Me group as revealed by the decrease in the intensity of the bands of the methyl group bound to the C atom and the appearance of the bands of the hydroperoxide and methylene groups. The decomposition of hydroperoxides produces aldehydes and ethers. At 160—200 °C, oxidation occurs via two routes: at the C—Me and C=C groups, while the Me₃Si group remains unchanged. At 230—240 °C, the rate of the reaction occurring at the C=C bond is higher than the rates of the processes involving the MeC and Me₃Si groups. The relative content of the structural units was calculated for the samples oxidized at different temperatures. Plausible mechanisms of thermal oxidation of poly(1-trimethylsilylprop-1-yne) were considered on the basis of the data obtained.     

Potential use of gamma irradiation in the production of mussel and oyster reference materials for paralytic shellfish poisoning toxins

Bivalve shellfish samples containing paralytic shellfish poisoning toxins were subjected to gamma irradiation dosage trials in order to assess the potential suitability of the technique in the production of toxin reference materials. Two candidate reference materials of tissue homogenates, mussels (Mytilus sp.) and native oysters (Ostrea edulis), were prepared in-house. Both were subjected to gamma irradiation at four different dose levels, 3.0, 6.0, 13.0 and 18.1 kGy. Bacterial levels were shown to be eliminated in the mussels and significantly reduced in the oysters following irradiation at all four dose levels. Paralytic shellfish poisoning (PSP) toxin concentrations were not significantly reduced in any of the samples indicating the treatment had no adverse affect on the initial stability of any of the PSP toxins monitored. Chromatographic results showed near-identical profiles for treated and non-treated samples inferring that no fluorescent toxin degradation products or matrix interferences were produced during the irradiation process. Results therefore proved that gamma irradiation treatment reduced bacterial levels within paralytic shellfish poisoning reference materials without compromising analyte content, with the subsequent potential to enhance the stability of future candidate reference materials treated in this manner.     

Thermal Denaturation of Bacterial Cells Examined by Differential Scanning Calorimetry

Thermal stability of vegetative cells of Listeria monocytogenes, Escherichia coli and Lactobacillus plantarum was studied by counting viable fractions and determining DSC curves of their suspensions. DSC curves in the 5–99°C range showed a series of endothermic transitions between 50 and 60°C, where the heat destruction of cells occurred. Heat denaturation of DNA required a higher temperature than cell killing. Thermal death was strongly influenced by the pH, composition and NaCl content of the suspending buffer. A mathematical model developed by us enabled comparison of DSC peak temperatures and temperatures required for loss of viability.This revised version was published online in November 2005 with corrections to the Cover Date.     

Spectral Characteristic,Storage Stability and Antioxidant Properties of Anthocyanin Extracts from Flowers of Butterfly Pea (Clitoria ternatea L.)

Anthocyanins from flowers of the butterfly pea (Clitoria ternatea L.) are promising edible blue food colorants. Food processing often faces extreme pHs and temperatures, which greatly affects the color and nutritional values of anthocyanins. This study explored the color, spectra, storage stability, and antioxidant properties of C. ternatea anthocyanin extract (CTAE) at different pHs. The color and absorption spectra of CTAEs at a pH of 0.5–13 were shown, with their underlying structures analyzed. Then, the storage stability of CTAEs were explored under a combination of pHs and temperatures. The stability of CTAE declines with the increase in temperature, and it can be stored stably for months at 4 °C. CTAEs also bear much resistance to acidic and alkaline conditions but exhibit higher thermal stability at pH 7 (blue) than at pH 0.5 (magenta) or pH 10 (blue-green), which is a great advantage in food making. Antioxidant abilities for flower extracts from the butterfly pea were high at pH 4–7, as assessed by DPPH free radical scavenging assays, and decreased sharply when the pH value exceeded 7. The above results provide a theoretical basis for the application of butterfly pea flowers and imply their great prospect in the food industry.     

Screening for multiple classes of marine biotoxins by liquid chromatography-high-resolution mass spectrometry

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.     

Influence of Citrates and EDTA on Oxidation and Decarboxylation of Betacyanins in Red Beet (Beta vulgaris L.) Betalain-Rich Extract

The influence of stabilizing activity of citric buffers on betacyanins, as well as their thermal dehydrogenation and decarboxylation in a beetroot betalain-rich extract (BRE), was studied at pH 3–8 and temperature 30, 50 and 85 °C with an additional effect of EDTA. In acetate/phosphate buffers, the highest stability is observed at pH 5 and it decreases toward pH 3 as well as pH 8, which is more remarkable at 85 °C. For the citrates, a contradictory effect was observed. Citric buffers tend to stabilize the substrate pigments and their intermediary products in acidic solutions, although increase their reactivity at pH 6–8. The highest impact of EDTA addition on pigment retention in acetate buffers is observed at 85 °C and pH 3–5 as well as 8, reflecting the preserving activity of EDTA at the most unfavorable conditions. At lower temperatures, pigment stability in more acidic conditions is still at higher levels even without addition of citrates or EDTA. The most striking effect on generation of betanin derivatives during heating is 2-decarboxylation which preferentially proceeds in the most acidic environment and this generation rate at 85 °C is much higher in the citrate buffers compared to acetates.     

Characterization of Alumina Gel Catalysts by Emanation Thermal Analysis (ETA)

Alumina gels made from the metal alkoxide is known to have high catalyst activity for the selective reduction of NO _x by hydrocarbons. It is also reported that the fine structure of the gels effects the activity. In this study, the effect of the preparation method on the fine structure and catalyst activity of the gels was investigated. Monolithic gels were obtained by hydrolysis of Al(sec-C₄H₉O)₃. The wet gels were dried at 90°C (xerogels), supercritically dried (aerogels), or dried after immersion in an ethanol solution of methyltrimethoxysilane (modified xerogels). The changes in the microstructure during heating were discussed using the results of TG-DTA, ETA and N₂ adsorption. The ETA curves show the ²²⁰Rn-release rate, E, of the samples, previously labelled with ²²⁸Th and ²²⁴Ra, during heating. The decrease in E of the xerogel at temperatures higher than 400°C indicates a gradual decrease in the surface area and porosity. A remarkable decrease in the BET surface area of the xerogel was found after heat-treating at 500°C. On the other hand, constant E of the aerogels and modified xerogels above 450°C suggests high thermal stability. The pore radii, estimated by BJH method, and the catalyst activities at 500°C of the aerogels and the modified xerogels were higher than those of the xerogels. The temperature range in which the alumina gels are applicable as catalysts was determined.     

Solid-state characterization of chloroprocaine hydrochloride

Summary Chloroprocaine hydrochloride (2-CPCHC) is a local anaesthetic agent of the ester type preferentially used for epidural anaesthesia. The compound, official in the USP, was found to exist in two polymorphic crystal forms which have been characterized by thermomicroscopy, differential scanning calorimetry (DSC), pycnometry, FTIR-, FT-Raman-spectroscopy as well as X-ray powder diffractometry. Based on these data the relative thermodynamic stability of the two forms was determined and is represented in a semi-schematic energy/temperature diagram. Mod. I° is the thermodynamically stable form at room temperature. This form is present in commercial products and can be crystallized from ethanol. Mod. II can be obtained by annealing the supercooled melt in a temperature range between 100 and 130°C. Upon heating mod. II exhibits an exothermic phase transition (Δ_trsH_II-I: -5.0±0.5 kJ mol^-1) at about 134°C to mod. I° (melting point 175°C, Δ_fusH_I: 46.6±0.6 kJ mol^-1). The exothermic transformation of mod. II to mod. I° confirms that mod. I° is thermodynamically stable in the entire temperature range (heat of transition rule) whereas mod. II is monotropically related to mod. I°, i.e. is metastable at all temperatures below its melting point. Mod. II is of low kinetic stability at room temperature and the transformation to mod. I° starts within a few minutes at room temperature. The N-H band in the infrared spectrum of mod. I° (3433 cm^-1) lies at significantly higher wavenumbers than that of mod. II (3413 cm^-1) indicating differences in the hydrogen bonding arrangement. Furthermore, the measured density of mod. I° is lower than the density of mod. II and thus both, the IR- and the density-rule are violated in this polymorphic system.     

Ionization of five aqueous fluorobenzoic acids: Conductance and thermodynamics

The three monofluorobenzoic acids together with 2,4-difluoro and 2,6-difluorobenzoic acids in aqueous solution are the subject of precision conductance measurements. The experimental data are analyzed to give ionization constants and limiting conductances at temperatures from 0 to 100°C. Walden products for the acid anions are derived from the limiting conductances while the ionization consatants are fitted by statistical methods to the function pK _a (m)=A+B/T+ C logT+DT. Only the 2,6- acid requires the fourth term of the function to fit the data to a precision of better than 0.03%. Mathematical analysis of the pK function gives the standard changes in enthalpy, entropy, and heat capacity. All the acids studied are more acidic than the parent, benzoic acid, as well as more acidic than the isoelectronic methylbenzoic acids. In general the increased acidity is tied to decreases in enthalpy while entropy changes on ionization differn little from those found for the parent acid.     

Degradation of benzoic acid and its derivatives in subcritical water

In this research, the stability of benzoic acid and three of its derivatives (anthranilic acid, salicylic acid, and syringic acid) under subcritical water conditions was investigated. The stability studies were carried out at temperatures ranging from 50 to 350 °C with heating times of 10–630 min. The degradation of the benzoic acid derivatives increased with rising temperature and the acids became less stable with longer heating time. The three benzoic acid derivatives showed very mild degradation at 150 °C. Severe degradation of benzoic acid derivatives was observed at 200 °C while their complete degradation occurred at 250 °C. However, benzoic acid remained stable at temperatures up to 300 °C. The degradation products of benzoic acid and the three derivatives were identified and quantified by HPLC and confirmed by GC/MS. Anthranilic acid, salicylic acid, syringic acid, and benzoic acid in high-temperature water underwent decarboxylation to form aniline, phenol, syringol, and benzene, respectively.     

Dual Effect of Organomercury Compounds on Peroxide Oxidation of Oleic Acid

Oxidation of oleic acid with atmospheric oxygen in the presence of HgCl₂ and various organo- mercury compounds (methylmercury iodide, isopropylmercury bromide, n-hexylmercury bromide, phenylmercury bromide, diphenylmercury, p-tolylmercury bromide, bis-p-tolylmercury) was studied. Mercury compounds exert a dual effect on accumulation of oleic acid hydroperoxide in the temperature range 20-90°C. Below 50°C, the concentration of the hydroperoxides formed in the presence of mercury compounds is lower, and at higher temperatures, higher than in the experiments performed without mercury compounds. Comparison of the concentrations of oleic acid hydroperoxides with those of their transformation products, carbonyl compounds, determined spectrophotometrically, shows that actually organomercury compounds and HgCl₂ accelerate peroxide oxidation at all the studied temperatures. Decreased accumulation of peroxides below 50°C is apparently due to the fact that the rate of their reaction with organomercury compounds is higher than the rate of their formation.