Determination of Pentachlorophenol in Seafood Samples from Zhejiang Province Using Pass-Through SPE-UPLC-MS/MS: Occurrence and Human Dietary Exposure Risk.

Pentachlorophenol (PCP) has attracted wide attention due to its high toxicity, persistence, and bioaccumulation. In this study, a sensitive UPLC-MS/MS method for the determination of PCP in seafood samples was developed and validated. The samples were ultrasonic extracted with acetonitrile containing 1% acetic acid-acetonitrile and followed by using a pass-through solid-phase extraction (SPE) cleanup on Captiva EMR-Lipid cartridges. The linearity of this method ranged from 1 to 1000 µg/L, with regression coefficients of >0.99. The detection limit and quantitation limit were 0.5 µg/kg and 1.0 µg/kg, respectively. The recoveries in different types of seafood samples ranged from 86.4% to 102.5%, and the intra-day and inter-day relative standard deviations (RSDs) were 3.7% to 11.2% and 2.9% to 12.1%, respectively (n = 6). Finally, the method has been successfully utilized for the screening of PCP in 760 seafood samples from Zhejiang Province. PCP was detected in 5.8% of all seafood samples, with the largest portion of detections found in shellfish, accounting for approximately 60% of the total. The average concentrations detected ranged from 1.08 to 21.49 µg/kg. The non-carcinogenic risk indices for adults and children who consume PCP ranged from 10-4 to 10-3 magnitudes. All of these indices stayed significantly below 1, implying that the health risk from PCP in marine organisms to humans is minimal.

Structure and Anticoagulant Activity of a Galactofuranose-Containing Sulfated Polysaccharide from the Green Seaweed, Codium isthmocladum.

A water-soluble sulfated polysaccharide, F2-1, was obtained from the marine green alga, Codium isthmocladum, using ion-exchange and size-exclusion chromatography. Structure analysis showed that the F2-1 was a sulfated arabinan comprising Ara, Rha, Man, Gal, and Xyl with an 18% sulfate content and a molecular weight of 100 kDa. Methylation analysis combined with desulfation, GC-MS, IR, and NMR spectroscopy showed that the backbone of F2-1 was →4)-ß-L-Arap(1→ residue. Its 2-O and/or 3-O positions showed sulfate modification; additionally, the 2-O or 3-O position showed branch points. The side chains were composed of →5)-ß-D-Galf, (1→2,6)-ß-D-Galf(1→, (1→2)-ß-L-Rhap4S, →4)-α-D-Glcp(1→, and terminal α-D-Galp(1→ and ß-D-Xylp(1→. Polysaccharides containing ß-D-galactofuranose are rarely found in seaweed. F2-1 exhibited significant anticoagulant activity in vitro. Our findings suggested that the green-tide alga, Codium isthmocladum, can be considered as a useful resource for bioactive polysaccharides.

Analysis of monosaccharide composition of water-soluble polysaccharides from Codium fragile by ultra-performance liquid chromatography-tandem mass spectrometry.

Codium fragile is a green alga belonging to Codiales family. The sulfated polysaccharides of this alga have anti-coagulation, antiviral, anti-angiogenesis, antioxidant, and immunoregulatory properties. In this study, we developed a reliable and rapid method for the analysis of 10 monosaccharides using ultra-performance liquid chromatography-tandem mass spectrometry in the negative electrospray ionization and multiple reaction monitoring mode. Monosaccharides, including two pentoses (xylose, arabinose); two deoxyhexoses (rhamnose, fucose); three hexoses (mannose, glucose, galactose); two hexuronic acids (glucuronic acid, galacturonic acid), and an N-acetyl-hexosamine (glucosamine), were derivatized using 1-phenyl-3-methyl-5-pyrazolone and simultaneously analyzed within 9 min. Optimization of the derivatization process, especially by using various 1-phenyl-3-methyl-5-pyrazolone concentrations, was studied. The calibration curves showed good linearity with a squared correlation coefficient > 0.995. The spiked recovery was determined to be 91.1-105.7% with the relative intra-day and inter-day standard deviations ranging from 2.58-6.71% and 3.15-7.67%, respectively. The limit of detection and limit of quantification for all 10 monosaccharides ranged from 0.02 to 0.10 µg/mL and 0.05 to 0.25 µg/mL, respectively. Using this method, the monosaccharides comprising the polysaccharides of Codium fragile were determined to be arabinose, galactose, and glucose.

Identification and quantification of nitrofurazone metabolites by ultraperformance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry with precolumn derivatization.

An ultraperformance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry method was developed and validated for the determination of nitrofurazone metabolites. Precolumn derivatization with 2,4-dinitrophenylhydrazine and p-dimethylaminobenzaldehyde as an internal standard was used successfully to determine the biomarker 5-nitro-2-furaldehyde. In negative electrospray ionization mode, the precise molecular weights of the derivatives were 320.0372 for the biomarker and 328.1060 for the internal standard (relative error 1.08 ppm). The matrix effect was evaluated and the analytical characteristics of the method and derivatization reaction conditions were validated. For comparison purposes, spiked samples were tested by both internal and external standard methods. The results show high precision can be obtained with p-dimethylaminobenzaldehyde as an internal standard for the identification and quantification of nitrofurazone metabolites in complex biological samples. Graphical Abstract A simplified preparation strategy for biological samples.

A selective biomarker for confirming nitrofurazone residues in crab and shrimp using ultra-performance liquid chromatography-tandem mass spectrometry.

Reliably detecting nitrofurazone (NFZ) residues in farmed crab and shrimp was previously hindered by lack of appropriately specific analytical methodology. Parent NFZ rapidly breaks down in meat, and the commonly used side-chain metabolite, semicarbazide (SEM), is non-specific as it occurs naturally in crustacean shell often leading to 'false positive' detections in meat. Using 5-nitro-2-furaldehyde (NF) as marker metabolite, following pre-column derivatization with 2,4-dinitrophenylhydrazine (DNPH), ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis in negative electrospray ionization mode enabled confirmation of NFZ residues in deliberately treated whole crab, crab meat and shrimp meat, with a limit of detection (LOD) and limit of quantification (LOQ) below 1 ng g(-1). Meanwhile, the derivatives of DNPH-NF were synthesized for the first time, purified by preparative liquid chromatography and structure characterized with nuclear magnetic resonance spectroscopy ((1)H-NMR). The purity of derivative was checked by ultra-performance liquid chromatography-tunable ultraviolet (UPLC-TUV), and the contents were beyond 99.9%. For comparison purposes, crustacean samples were analysed using both NF and SEM marker metabolites. NFZ treatment was revealed by both NF and SEM marker metabolites, but untreated crab also showed measurable levels of SEM which could potentially be misinterpreted as evidence of illegal NFZ use.

Hexabromocyclododecane-induced reproductive toxicity in Brachionus plicatilis: Impacts and assessment.

Hexabromocyclododecane (HBCD), third-generation brominated flame retardants (BRFs), has aroused worldwide concern because of its wide application and potentially negative impacts on marine ecosystems, but an information gap still exists regarding marine low-trophic organisms. Brachionus plicatilis, the model marine zooplankton, was used in the present study, and its reproductive responses were used as the endpoint to indicate HBCD-induced toxicity. HBCD was suggested to be extremely highly toxic compounds regarding the 96 h-LC50 of 0.58 mg L-1. The sublethal exposure of HBCD injured the reproduction of B. plicatilis: The total number of offspring per female and the key population index calculated from the life table, including the intrinsic rate of population increase (rm) and net reproductive rate (R0), were significantly influenced in a concentration-dependent manner. The reproductive process was also altered, as indicated by the first spawning time, first hatching time and oocyst development time. At the same time, individual survival and growth (body length) were also negatively affected by HBCD. Reactive oxygen species (ROS) were suggested to be responsible for reproductive toxicity mainly because the total ROS contents as well as the main components of •OH and H2O2 greatly increased and resulted in the oxidative imbalance that presented as malondialdehyde (MDA) elevation. Simultaneous activation of the glutathione antioxidant system was accompanied by the apoptosis marker enzymes Caspase-3 and 9, as well as the correlation between ROS content, physiological alteration and cell apoptosis, providing further evidence for this. The integrated biomarker response (IBR) and adverse outcome pathway (AOP) showed that HBCD had a significant toxic effect on B. plicatilis near the concentration range of 96 h-LC50. The establishment of this concentration range will provide a reliable reference for future environmental concentration warning of HBCD in marine.

Development and Application of Immunoaffinity Column Purification and Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry for Determination of Domoic Acid in Shellfish.

Domoic acid (DA) is a neurotoxin associated with amnesic shellfish poisoning (ASP). Though LC coupled to tandem mass spectrometry (LC-MS/MS) has become the preferred method for DA determination, traditional sample pretreatment is still labor-intensive. In this study, a simple, efficient and selective method for LC-MS/MS analysis of DA in shellfish was established by optimizing clean-up procedures on a self-assembly immunoaffinity column (IAC). Shellfish was extracted with 75% methanol twice and diluted with phosphate buffered saline (PBS, 1:2). The mixture was purified on IAC as follows: preconditioned with PBS, loaded with sample, washed by 50% MeOH, and eluted with MeOH containing 2% ammonium hydroxide. Concentrated analyte was monitored by multiple reaction monitoring (MRM) using electrospray (ESI) positive ion mode throughout the LC gradient elution. Based on the post-extraction addition method, matrix effects for various shellfish matrices were found to be less than 8%. The developed method was fully validated by choosing mussel as the representative matrix. The method had a limit of detection (LOD) of 0.02 µg·g-1, showed excellent linear correlation in the range of 0.05⁻40 µg·g-1, and obtained ideal recoveries (91⁻94%), intra-day RSDs (6⁻8%) and inter-day RSDs (3⁻6%). The method was successfully applied to DA determination in 59 shellfish samples, with a detection rate of 10% and contaminated content of 0.1⁻14.9 µg·g-1.

Physicochemical, antioxidant properties of giant croaker (Nibea japonica) swim bladders collagen and wound healing evaluation.

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were obtained from Nibea japonica swim bladders. The denaturation temperature (Td) of ASC and PSC was approximately 33.8 °C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy (FTIR) analyses indicated that ASC and PSC contained triple-helical type I collagen when compared to rat tail collagen type I. Moreover, the microstructure of collagen sponges was uniform and porous. In addition, ASC and PSC exhibited antioxidant properties and in vitro scratch assays showed that PSC at various concentrations (0, 12.5, 25, and 50 µg/mL) had significant effects on the scratch closure rate. Furthermore, collagen sponge from Nibea japonica swim bladders exhibited an increased efficacy of wound healing when compared to the control mice. The levels of interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α in the collagen sponge treated mice were significantly decreased when compared to the control group. Thus, our results suggested that collagen sponge from Nibea japonica swim bladders has potential wound healing applications.

A sensitive and selective immunoaffinity column clean up coupled to UPLC-MS/MS for determination of trace methyl-3-quinoxaline-2-carboxylic acid in animal tissues.

This paper described a reliable and simple method for the selective determination of MQCA in animal tissues using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A highly targeted immunoaffinity column was used for sample purification after enzymatic hydrolysis. The purified extracts were analyzed by reversed-phase HPLC-MS/MS in positive ESI and multiple reaction monitoring mode. The calibration curves showed good linearity with correlation coefficient (r2) larger than 0.995. The average recoveries at the spiked levels of 0.5, 2.0 and 20µgkg-1 were 90.2% to 103.5% with intra-day and inter-day relatives standard deviations (RSD, n=6) ranging from 1.8% to 6.7% and 3.5% to 7.6% respectively. The limit of quantification (LOQ) was 0.5µgkg-1, which can fulfil the maximum residue level (MRL) of 4.0µgkg-1 stipulated by the Agricultural Minister of China and the requirement of the confirmatory criteria according to the European Commission Decision 2002/657/EC. The method is sensitive, accurate, convenient and rapid, and has been successfully applied in real samples.

Identification and occurrence of endogenous semicarbazide in prawns and crabs from Zhejiang Province, China.

Semicarbazide (SEM) is a side-chain metabolite of the antibiotic drug nitrofurazone (NFZ) and is employed as a conclusive marker for the use of banned NFZ. Recent studies have shown that SEM in aquatic crustaceans can be derived natively or from other sources. The presence and distribution of endogenous SEM within aquatic crustaceans is examined in this paper, which finds that the SEM content varies amongst the muscle, shell, and viscera of various prawn and crab species within the range of 0.35-26.62 ng g(-1). The effects of heating and hypochlorite treatment on SEM levels were examined. The results indicate that thermal processing introduced a more significant impact, resulting in a maximum SEM value of 15.48 ng g(-1) in a sample of shell of Portunus trituberculatus crab, while SEM levels in muscle samples were not affected by the duration of heating. Though 6% active chlorine treatment led to SEM production ranging between 39.9 and 196.4 ng g(-1) in muscle samples from various crustaceans, SEM is unlikely to originate from hypochlorite or chlorine in practice where there are limits to actual chlorine in sanitation water and facilities. 5-Nitro-2-furaldehyde (NF) was proposed as a selective marker to differentiate between endogenous SEM and NFZ-derived SEM in seafood.

Immunoaffinity chromatography purification and ultrahigh performance liquid chromatography tandem mass spectrometry determination of tetrodotoxin in marine organisms.

A highly selective and sensitive method was developed for the determination of tetrodotoxin (TTX) in marine organisms by immunoaffinity chromatography (IAC) purification coupled with ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). An IAC column was prepared and used to cleanup the extracted samples. The operating conditions of the IAC column were optimized, and the capacity of new IAC column was found to be 1106 ng mL(-1), which was sufficient for TTX determination. The MS/MS conditions and UPLC mobile phase were also studied to optimize the operation conditions. Fortified marine organism samples at levels of 0.3-5.0 ng g(-1) were utilized, and the average recoveries were 86.5-103.6% with intra- and inter-day relative standard deviations less than 7.22 and 9.88%, respectively. The limits of detection and quantification were 0.1 and 0.3 ng g(-1), respectively. The method was later successfully applied for the determination of TTX in 100 marine organism samples collected from local markets.