Development and validation of rapid screening of 192 veterinary drug residues in aquatic products using HPLC-HRMS coupled with QuEChERS.

The presence of veterinary drug residues in aquatic products represents a significant challenge to food safety. The current detection methods, limited in both scope and sensitivity, underscore the urgent need for more advanced techniques. This research introduces a swift and potent screening technique using high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) and a refined QuEChERS protocol, allowing simultaneous qualitative and semi-quantitative analysis of 192 residues. A comprehensive database, employing full scan mode and data-dependent secondary mass spectroscopy, enhances screening accuracy. The method involves efficient extraction using 90% acetonitrile, dehydration with Na2SO4, and acetic acid, followed by cleanup using dispersive solid-phase extract sorbent primary secondary amine. It is suitable for samples with varying fat content, offering detection limits ranging from 0.5 to 10 µg/kg, high recovery rates (60-120%), and low relative standard deviations (<20%). Practical application has validated its effectiveness for multi-residue screening, marking a significant advancement in food safety evaluation.

[Exposure Level and Risk Impact Assessment of Pesticides and Veterinary Drugs in Aquaculture Environment].

To explore the exposure level of pesticides and veterinary drugs in an aquaculture environment and its impact on the ecological environment, this study took the aquaculture environment in Shanghai as an example, and samples of water, sediment, and inputs from 40 major aquaculture farms were collected from July to September 2022. The types and contents of pesticides and veterinary drugs were screened using high-performance liquid chromatography-electrostatic field orbital ion trap mass spectrometry, and the risk quotient (RQ) method was used to assess the ecological risk of pesticide contamination in water and sediment. The results showed that 13 drugs were screened out from 204 samples (72 samples of water, 72 samples of mud, and 60 samples of input), namely, chlorpromazine, carbendazim, thiophanate, diazepam, florfenicol, simazine, amantidine, diazepam, trimethoprim, ciprofloxacin, ofloxacin, mebendazole, and enrofloxacin. Among them, 12 species were found in water samples with concentrations ranging from 0.016 µg·L-1 to 2.084 µg·L-1. The concentrations of seven species in the mud samples ranged from 0.018 µg·kg-1 to 23.101 µg·kg-1. The results showed that there were four types of inputs, ranging from 1.979 µg·kg-1 to 101.940 µg·kg-1. Seven drugs were found in both water and sediment. The risk quotient (RQ) results showed that there were some high and middle risks in both water and sediment samples of aquaculture farms, and the ecological risks of carbendazim were the highest in both water and sediment samples of aquaculture farms; the RQ values were 3.848 and 1.580, respectively, indicating high risk. It is suggested to strengthen the control and management of exogenous pesticides and veterinary drugs in aquaculture environments to protect the ecosystem health of the aquaculture environment.

Rapid Sample Enrichment, Novel Derivatization, and High Sensitivity for Determination of 3-Chloropropane-1,2-diol in Soy Sauce via High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

A novel, simplified derivatization method and a rapid sample preparation process using carbon yarn as a sorbent for the determination of 3-chloropropane-1,2-diol (3-MCPD) in soy sauce via high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. 3-MCPD was first enriched and purified with carbon yarn and then eluted with a methanol-water solution. Subsequently, the analyte underwent derivatization with p-(dimethylamino)-phenol for sensitive detection via HPLC-MS/MS. The limit of detection and the limit of quantitation for 3-MCPD were validated to be 0.5 and 1.0 µg/kg, respectively. Spiking experiments showed recoveries between 83 and 94%, with a relative standard deviation of ≤10%. The method was further validated with a certified reference material. Furthermore, 11 real soy sauce samples from local markets were tested by using this method. These results reveal the widespread 3-MCPD contamination. Consequently, this study offers a preferable alternative for the sensitive, accurate, and precise determination of 3-MCPD in soy sauce.

Multi-Residue Screening of Pesticides in Aquatic Products Using High-Performance Liquid Chromatography-Tandem High-Resolution Mass Spectrometry.

Pesticide residues in aquatic products are of great concern due to the risk of environmental transmission and their extensive use in aquaculture. In our work, a quick screening approach was developed for the qualitative and semi-quantitative screening of 87 pesticide residues in aquatic products. The sample preparation was investigated, including extract solvent, extract methods, buffer salts, lipid removal, cleanup materials and filter membranes for aquatic products. Samples were extracted using a modified QuEChERS procedure, and two clean-up procedures were developed for UHPLC-Q/Orbitrap MS analysis based on the fat content of the aquatic products. The screening detection limits for all studied pesticides were distributed between 1 and 500 µg/kg in the three representative matrices. Seventy-one pesticides could be analyzed with a screening limit between 1 and 25 µg/kg in grass carp and crayfish, sixty-one pesticides could be screened for limits between 1 and 50 µg/kg in crab. The accuracy results showed that recoveries ranged from 50 to 120% for 60, 56 and 52 pesticides at medium-level for grass carp, crayfish and crab, respectively. At high spiking levels, 74, 65 and 59 pesticides were recovered within the range of 50-120% for the three matrices, respectively. The relative standard deviations of most compounds in different matrices were less than 20%. With this method, the local farmed aquatic products were tested for pesticide residues. In these samples, ethoxyquinoline, prometryn and phoxim were frequently detected. The majority of these confirmed compounds did not exceed 2.00 µg/kg. A grass carp with trichlorfon at 4.87 µg/kg and two carps with ethoxyquinoline at 200 µg/kg were detected, indicating the potential dietary risk.

Simultaneous Determination of Tetrodotoxin in the Fresh and Heat-Processed Aquatic Products by High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Tetrodotoxin (TTX) was simultaneously detected in the fresh and heat-processed aquatic products by high-performance liquid chromatography-tandem mass spectrometry method. The detection conditions were investigated, including the chromatography column and mobile phase. Based on the optimized parameters, a sensitive determination method of TTX was established. The proposed method featured the merits of a good linear relationship between signal and TTX concentration (R2 = 0.9998), a wide detection matrix-based range of 0.2-100 ng/g, and a low detection limit of 0.2 ng/g, etc. The spiked assays evidenced its accuracy and reliability with recoveries of 90.5-107.2%. Finally, the developed method was simultaneously successfully applied in the determination of TTX in various fresh and heat-processed aquatic products.

Ratiometric Fluorescence Immunoassay Based on Carbon Quantum Dots for Sensitive Detection of Malachite Green in Fish.

Malachite green (MG) is a synthetic poisonous organic compound that has been banned in many countries as a veterinary drug for aquaculture. An efficient, fast and sensitive method is urgently needed for monitoring the illegal use of malachite green (MG) in aquaculture. In this study, a novel ratiometric fluorescence immunoassay was established. Nitrogen-doped carbon quantum dots were used as ratiometric fluorescent probes with a fluorescence peak at 450 nm. Horseradish peroxidase was employed to convert o-phenylenediamine to 2,3-diaminophenazine, with a new fluorescence peak at 580 nm and a strong absorption at 420 nm. The inner filter effect between N-CQD fluorescence and DAP absorption was identified. It allows for the ratiometric detection of MG using a fluorescent immunoassay. The results demonstrated a linear ratiometric fluorescence response for MG between 0.1 and 12.8 ng·mL-1. The limit of detection of this method was verified to be 0.097 µg·kg-1 with recoveries ranging from 81.88 to 108%, and the relative standard deviations were below 3%. Furthermore, this method exhibited acceptable consistency with the LC-MS/MS results when applied for MG screening in real samples. These results demonstrated a promising application of this novel ratiometric fluorescence immunoassay for MG screening with the merits of rapid detection, simple sample preparation, and stable signal readout. It can be an alternative to other traditional methods if there are difficulties in the availability of expensive instruments, and achieve comparable results or even more sensitivity than other reported methods.

Combined Effects of Temperature and Toxic Algal Abundance on Paralytic Shellfish Toxic Accumulation, Tissue Distribution and Elimination Dynamics in Mussels Mytilus coruscus.

This study assessed the impact of increasing seawater surface temperature (SST) and toxic algal abundance (TAA) on the accumulation, tissue distribution and elimination dynamics of paralytic shellfish toxins (PSTs) in mussels. Mytilus coruscus were fed with the PSTs-producing dinoflagellate A. catenella under four simulated environment conditions. The maximum PSTs concentration was determined to be 3548 µg STX eq.kg-1, which was four times higher than the EU regulatory limit. The increasing SST caused a significant decline in PSTs levels in mussels with rapid elimination rates, whereas high TAA increased the PSTs concentration. As a result, the PSTs toxicity levels decreased under the combined condition. Additionally, toxin burdens were assessed within shellfish tissues, with the highest levels quantified in the hepatopancreas. It is noteworthy that the toxin burden shifted towards the mantle from gill, muscle and gonad at the 17th day. Moreover, variability of PSTs was measured, and was associated with changes in each environmental factor. Hence, this study primarily illustrates the combined effects of SST and TAA on PSTs toxicity, showing that increasing environmental temperature is of benefit to lower PSTs toxicity with rapid elimination rates.

Characterization of the complete mitochondrial DNA sequence of the Lagocephalus gloveri (Tetraodontidae, Tetraodontiformes).

The complete mitochondrial genome of Lagocephalus gloveri is reported in the present study, which is 16,446 bp in length. It consists of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and a non-coding control region. The overall base composition of the genome is 27.58% for A, 25.07% for T, 30.83% for C and 16.52% for G. The phylogenetic tree, which is based on 12 protein coding gene sequences, suggested that L. gloveri was closest to L. lagocephalus. This study could give impetus to studies focused on population structure and molecular evolution of L. gloveri.

Antioxidant responses of triangle sail mussel Hyriopsis cumingii exposed to harmful algae Microcystis aeruginosa and high pH.

In lakes and reservoirs, harmful algal blooms and high pH have been deemed to be two important stressors related to eutrophication, especially in the case of CO2 depletion caused by dense blooms. However, the effects of these stressors on the economically important shellfish that inhabit these waters are still not well-understood. This study evaluated the combined effects of the harmful algae Microcystis aeruginosa (0%, 50%, and 100% of total dietary dry weight) and high pH (8.0, 8.5 and 9.0) on the antioxidant responses of the triangle sail mussel H. cumingii. The mussels were exposed to algae and high pH for 14 d, followed by a 7-day depuration period. Reactive oxygen species (ROS) in the mussel hemolymph, antioxidant and detoxifying enzymes, such as glutathione-S-transferase (GST), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) in the digestive glands were analyzed during the experimental period. GST, SOD and GPx activity levels and the content of GSH increased following exposure to toxic M. aeruginosa, whereas CAT activity was inhibited. pH showed no significant effects on the immune defense mechanisms and detoxification processes. However, a high pH could cause increased ROS and MDA levels, resulting in oxidative injury. After a 7-day depuration period, exposure to toxic M. aeruginosa or high pH resulted in latent effects for most of the examined parameters. The treatment group exposed to the highest pH (9.0) displayed an increased oxidation state compared with the other pH treatments (8.0 and 8.5) for the same concentrations of toxic M. aeruginosa. The trends observed for ROS, MDA, GPx, GST, SOD and GSH levels indicated that a high density of toxic algae could result in severe and continuous effects on mussel health.

Characterization of the complete mitochondrial DNA sequence of the Lagocephalus guentheri (Tetraodontidae, Tetraodontiformes).

The complete mitochondrial genome of Lagocephalus guentheri was reported in the present study, which was 16,461 bp in length. It consists of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and a non-coding control region. The overall base composition of the genome is 27.54% for A, 24.80% for T, 31.23% for C and 16.43% for G. The phylogenetic tree, which is based on 12 protein-coding gene sequences, suggested that L. guentheri was closest to L. spadiceus. This study could give impetus to studies focused on population structure and molecular evolution of L. guentheri.

Detection of Total and Pathogenic Vibrio parahaemolyticus in Shellfish Growing along the South Yellow Sea and the East China Sea.

This study was conducted to monitor the densities of total and pathogenic Vibrio parahaemolyticus in 300 samples of nine shellfish species harvested from the coasts of the South Yellow Sea and the East China Sea (N 23° to 34°, E 116° to 124°), People's Republic of China, between May and October 2015. Total V. parahaemolyticus densities were measured, and V. parahaemolyticus isolates were biochemically identified with probes for the thermostable direct hemolysin gene (tdh) and the thermostable direct hemolysin-related hemolysin gene (trh). We found that 202 of the 300 samples were positive for V. parahaemolyticus from all the sites: 58 of the 100 samples from the Fujian province, 71 of the 100 samples from the Zhejiang province, and 73 of the 100 samples from the Jiangsu province. In most (170) of the 300 samples, V. parahaemolyticus densities were 0.3 to 10 most probable number (MPN)/g; five lots exceeded 110 MPN/g, and two lots were estimated at 110 MPN/g. Among the 202 V. parahaemolyticus strains, only one was trh positive. Densities of V. parahaemolyticus in these shellfish were temperature dependent, with highest densities in June and July. Among the nine mollusk species, V. parahaemolyticus was most abundant in the agemaki clam (Sinonovacula constricta). The highest and lowest V. parahaemolyticus prevalences were found in oriental cyclina (Cyclina sinensis, 93.8%) and mussels (Mytilus edulis, 28.1%), respectively. Overall, although V. parahaemolyticus is widely distributed in marine environments, the density of V. parahaemolyticus was low and the prevalence of the main virulence factor was very low in shellfish along the coasts of the South Yellow Sea and East China Sea, which is important from a public health perspective. Data presented here will be useful for correlational research and can be utilized for developing risk management plans that establish food safety guidelines for V. parahaemolyticus in Chinese shellfish.

Loop-mediated isothermal amplification for visual detection of Vibrio parahaemolyticus using gold nanoparticles.

Loop-mediated isothermal amplification (LAMP) eradicates the need of thermocycler in DNA amplification. Signals are usually obtained via fluorometry or turbidimetry, but such methods need improvement in order to become more effortless and reliable. The authors describe a set of six specific primers targeting the species-specific tlh gene of Vibrio parahaemolyticus which were used in accelerated LAMP reaction. Gold nanoparticles (AuNPs) were functionalized with streptavidin (Avidin-AuNPs), and engineered to signal the LAMP reaction. Two of the loop primers for LAMP were biotinylated and then can produce a DNA that can cause clusterization of Avidin-AuNPs based on the formation of avidin-biotin complex. This leads to a color change of the solution from red to blue. Amplification is completed within 30 min and can be visually detected within 5 min. The detection limit of the method is found to be 8.6 cfu per reaction. This visual detection scheme does not require any fluorescent reagents and detection instruments. Conceivably, the method has a wide scope because such Avidin-AuNPs can be used as nanoprobes for a variety of other LAMP products. This rapid and universal strategy holds promise in point of care testing and food testing, particularly in resource-limited regions. Graphical abstract Six specific primers (two of them are biotinylated) were used to realize the accelerated Loop-Mediated Isothermal Amplification. Streptavidin modified gold nanoparticles (Avidin-AuNPs) cluster on the DNA products, leading to the apparent change of color from red to blue, which is readily identified even by unaided eye.

Correction to: Loop-mediated isothermal amplification for visual detection of Vibrio parahaemolyticus using gold nanoparticles.

The published version of this article, unfortunately, contained error. Modifications have been made to the Abstract, Introduction, Results and discussion, and Acknowledgements section. The original article has been corrected.

Chitosan-based adsorption and freeze deproteinization: Improved extraction and purification of synthetic colorants from protein-rich food samples.

A freeze method for deproteinization coupling with the chitosan purification process was developed for the determination of 8 synthetic food colorants in protein-rich samples. The solvents for extraction and different methods for deproteinization were examined and selected. Chitosan was employed for the purification after deproteinization, and further compared with the traditional polyamine purification method. Determination of the purified extract was conducted through the separation using high performance liquid chromatography and detection by multi-wavelength mode. Under the optimum conditions, the method showed good linearity between 0.6 and 10mg/kg, for the 8 synthetic colorants, and the limit of detection was between 0.1 and 0.4 mg/kg as was defined when the ratio of signal to noise was three. The recoveries of the spiked samples were found to be between 83% and 91%. The intra-day precision and inter-day precision was estimated to be 3-10% and 6-12%, respectively. The developed method could be applied to deproteinization and clean-up for pretreatment of protein-rich samples.

Effect of temperature on uptake and survival of Vibrio parahaemolyticus in oysters (Crassostrea plicatula).

This study investigated accumulation of Vibrio parahaemolyticus in Zhe oyster (Crassostrea plicatula) from culture water and effectiveness of frozen and chilled storage on reducing V. parahaemolyticus in oysters. Freshly harvested oysters were placed in artificial seawater containing V. parahaemolyticus (10(4)CFU/mL) at 16, 20, 26, and 32 degrees C for 96 h. Contaminated oysters were stored at chilled temperatures (0, 5, and 15 degrees C) and frozen at -18 and -30 degrees C and changes of V. parahaemolyticus populations in oysters were determined using the most probable number (MPN) method. Accumulations of V. parahaemolyticus in C. plicatula reached the peaks at 6.66 (32 degrees C), 5.72 (26 degrees C), 5.04 (20 degrees C), 4.72 (16 degrees C) log MPN/g after 32 h in contaminated artificial seawater. Holding contaminated Zhe oysters at 5 and 0 degrees C reduced V. parahaemolyticus populations in both shell stock and shucked oysters. Populations of V. parahaemolyticus in shell stock and shucked oysters declined by 1.42 and 2.0 log MPN/g, respectively, after 96 h of storage at 5 degrees C and by 2.11 and 2.38 log MPN/g, respectively, after 96 h of storage at 0 degrees C. However, populations of V. parahaemolyticus increased by 2.44 log MPN/g in shell stock oysters and by 1.64 og MPN/g in shucked oysters when stored at 15 degrees C for 60 h. Frozen storage was effective in inactivating V. parahaemolyticus. Populations of V. parahaemolyticus in shell stock and shucked oysters decreased from 5.46log MPN/g to 1.66 and 0.38 log MPN/g, respectively, after 75 days of storage at -30 degrees C. No V. parahaemolyticus cells were detected (<3 log MPN/g) in the shucked oysters after 60 days of storage at -18 degrees C. These results demonstrated that accumulation of V. parahaemolyticus in cultured C. plicatula increases as water temperature increases. Harvested C. plicatula should be stored at 5 degrees C or lower to control the hazard of V. parahaemolyticus.

[Determination of praziquantel residue in aquatic products using high performance liquid chromatography].

A sensitive and simple method for the determination of praziquantel residue in aquatic products has been established. The sample was extracted with ethyl acetate for three times (20 mL, 20 mL, 10 mL) and cleaned up with an LC-Si column. The extract was separated on a ZORBAX SB-C18 column (250 mm x 4.6 mm, 5 microm). Ultraviolet detection was performed at 214 nm. Acetonitrile-water (50: 50, v/v) was used as the mobile phase at a flow rate of 0.9 mL/min. The calibration curve of praziquantel in aquatic products at the concentration range of 0.02 - 20 mg/L was linear ( r = 0.999 98). The recovery of praziquantel was higher than 85%. The limit of detection was 10 microg/kg (S/N > 3).

[Identification of Vibrio campbellii isolated from cultured pacific oyster].

Four bacterial strains were isolated from cultured pacific oyster (Crassostrea gigia) in September 2003 at coast of Fujian province. Their morphological, physiological and biochemical characteristics and 16S rRNA sequence were analyzed. And the relationship between reproduction of the bacterium and NaCl concentrations, pH and temperature were also determined. The results showed that four strains were gram-negative rods with a single polar flagellum, glucose fermented without gas production, oxidase positive, required sodium ions for growth, no pigment, non-luminescence; They grew well on TCBS-plate as green colonies and were sensitive to the vibriostratic agent O/129. Therefore, it was confirmed that the strains belonged to the genus of Vibrio. The sequence analysis of 16S rRNA gene of SXS1 isolation and comparison with that of other related vibrios- showed that SXS1 was very close to Vibrio campbellii. The similarities was 99%. The distribution of V. campbellii in environment and its relationship to the diseases of aquaculture animals were discussed.