Distribution and Probabilistic Risk Assessment of Antibiotics, Illegal Drugs, and Toxic Elements in Gastropods from Southeast China.

We investigated fourteen antibiotics, three illegal drugs, and two toxic elements in commercially available gastropods from southeast China. The data revealed high detection frequencies (DFs) for florfenicol (61.32%), florfenicol amine (47.33%), and thiamphenicol (39.88%), with maximum concentrations of 1110, 2222, and 136 µg/kg wet weight (ww), respectively. The DFs of illegal drugs were 3.54% for leucomalachite green and 0.3% for chloramphenicol. The average levels of Cd and As were 1.17 and 6.12 mg/kg ww, respectively. All chemicals presented diverse DFs in different sampling months. The highest DFs of florfenicol, florfenicol amine, and thiamphenicol were in July. The health risk assessment showed that targeted hazard quotients (THQs) of antibiotics, Cd, and As for children, teens, and adults were all less than one. Notably, the toxic elements (Cd and As) were identified as the primary health risk in gastropods, contributing to over 90% of the total THQs.

Antibiotic residues in commercial freshwater fish from southeast China: distribution and human health risk assessment.

We investigated 14 antibiotic residues in 8 marketed freshwater fish species from southeast China and estimated the associated health risks to local consumers. The antibiotic residues were determined by UPLC-MS/MS. Our findings revealed widespread distribution of quinolones (QNs), tetracyclines (TCs), and chloramphenicols (CAPs) in the freshwater fish. Notably, the average concentrations of enrofloxacin and ciprofloxacin reached levels as high as 62.5 µg/kg wet weight (ww) and 11.7 µg/kg ww, respectively, and detection frequencies were 68.7% for enrofloxacin and 31.6% for ciprofloxacin. Additionally, we detected chloramphenicol, a prohibited antibiotic, in samples with a detection frequency of 0.76%. Among the fish species, the mean concentration of total antibiotic residues was highest in bluntnose black bream (263.3 µg/kg), followed by English perch (52.4 µg/kg), crucian carp (46.3 µg/kg), black carp (28.6 µg/kg), yellowcheek carp (21.0 µg/kg), grass carp (15.3 µg/kg), bighead carp (3.78 µg/kg), and mandarin fish (3.69 µg/kg). We estimated the daily intake values of these antibiotic residues which were lower than the acceptable daily intake values and hazard indexes were much less than 1. It indicates that there is very low direct health risk to consumers. Despite that, investigation on the chronic impact, such as antibiotic-resistant bacteria, gut microbiota disruption, and allergic reactions, is urgently needed.

Toxic Elements in Beans from Zhejiang, Southeast China: Distribution and Probabilistic Health Risk Assessment.

This study described the distribution of As, Cd, Cr, Hg, and Pb in 692 bean samples from Zhejiang province, southeast China, and estimated the health risk using Monte Carlo simulation. The average levels of As, Cd, Cr, Hg, and Pb were 0.0349, 0.0379, 0.246, 0.0019, and 0.0246 mg kg-1. Correlation analyses showed very strong positive correlations for Cd-Pb in kidney beans and mung beans, Cd-As in black beans, and Pb-As in red beans. The target hazard quotients (THQs) were adopted for non-carcinogenic risk assessment, and THQs at the 50th percentile were all less than 1, indicating that there are no deleterious effects from rice exposure to these elements. When evaluating THQ for multiple elements, the certainty with a hazard index (HI) greater than 1 for children was 12.64%, for teens 11.54%, and for adults 1.01%. The sensitivity analysis reveals that the concentration of Cd in beans and ED (exposure duration) are the main principal factors that contributed to the total risk. The mean carcinogenic risks for children, teens, and adults were all less than 1 × 10-4, indicating no potential carcinogenic risk. Despite that, the routine monitoring of these elements, especially for Cd should be continued.

Analytical Method Optimization of Tetrodotoxin and Its Contamination in Gastropods.

Tetrodotoxin (TTX) is an extremely potent marine biotoxin. An analytical method was developed for both trace contamination and extremely high levels of TTX in gastropods by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with clean-up of cation exchange solid phase extraction (SPE) in this study. The limit of detection (LOD) in the sample matrix was 0.5 µg/kg. With the calibration of a screened internal standard (validamycin, IS), the linear range was 0.1-100 ng/mL (1.5-1500 µg/kg in sample matrix) with a correlation coefficient of r2 > 0.999. The average recoveries at three spiking levels (1.5 µg/kg, 44 µg/kg, and 1500 µg/kg) were 82.6-94.4% with relative standard deviations (RSDs) less than 8.4%. TTX levels in seven gastropods (741 samples) were studied. The contamination and analogues in Neverita didyma (N. didyma, 565 samples collected in Zhejiang province, China, from 2016 to 2022) were first reported. The detection rate of TTX in N. didyma was 34.2%. The average concentration was 23.1 µg/kg, and the maximum value was 2327 µg/kg. The time distribution study indicated that high contaminations of TTX occurred from May to August for N. didyma.

Quantification of 37 glucocorticoids in chicken muscle by UHPLC-Q-Orbitrap-MS with parallel reaction monitoring.

The quantification capability of high-resolution mass spectrometry (HRMS) has received increasing interest from analysts. In this study, we present a method for analyzing 37 glucocorticoids in chicken muscle using UHPLC-Q-Orbitrap MS with parallel reaction monitoring (PRM). The analytes were extracted using acetonitrile (ACN) containing 0.1% formic acid and subjected to commercial PRiME HLB solid-phase extraction (SPE) cartridge clean-up. Under optimized conditions, the analytes were separated on an analytical column and subsequently detected using a high-resolution hybrid quadrupole/Orbitrap mass spectrometer coupled with PRM scan mode. The Q-Orbitrap with PRM exhibited remarkable sensitivity, with limits of quantification (LOQs) ranging from 0.08 µg kg-1 to 7.59 µg kg-1. To validate the method, we conducted intra- and inter-day tests using a blank matrix sample at different spiking levels. The achieved results demonstrated satisfactory recovery values (74.1-97.5%) and precise results (RSDs < 15%) for all the studied analytes. In application, we found dexamethasone with 6.5 µg kg-1 and fluorometholone with 3.9 µg kg-1 in two chicken samples. These findings suggest that the UHPLC-Q-Orbitrap system, in conjunction with the SPE sample preparation method, has great potential as a routine quantification approach for multiple glucocorticoid residues in chicken samples.

Heavy metals accumulation in bivalve mollusks collected from coastal areas of southeast China.

The distribution of six heavy metal and metalloids (As, Cd, Cr, Hg, Ni and Pb) was analyzed in 597 bivalve mollusks (8 species) collected from coastal areas of southeast China. Target hazard quotient, total hazard index, and target cancer risk were calculated to evaluate potential human health risks from bivalve consumption. The mean concentrations of As, Cd, Cr, Hg, Ni and Pb were 1.83, 0.581, 0.111, 0.0117, 0.268 and 0.137 mg kg-1 wet weight in bivalves. The average estimated daily intakes for As, Cd, Cr, Hg, Ni and Pb were 1.156, 0.367, 0.07, 0.007, 0.167 and 0.087 µg kg-1 body weight/day. Health risk assessment showed that there was no non-carcinogenic health risk to general residents to these metals from consumption of bivalves. Cd intake through mollusks posed a potential cancer risk. Accordingly, regular monitoring for heavy metals, especially Cd is recommended with respect to potential contaminant on marine ecosystems.

Distribution of Cadmium in Fresh Vegetables Marketed in Southeast China and Its Dietary Exposure Assessment.

This study investigated concentrations of cadmium (Cd) in 2465 vegetable samples (52 species) from 2018 to 2022 and estimated the associated health risk for local consumers. The average concentration of Cd was 0.035 mg kg-1, and the percentage of samples exceeding the Chinese maximum allowed concentration was 3.89% (96/2465). The top five species with highest Cd levels were Lilium brownii F (0.182 mg kg-1), Allium chinense G (0.117 mg kg-1), Allium macrostemon Bunge (0.105 mg kg-1), Colocasia esculenta (0.064 mg kg-1), and Amaranthus tricolor L (0.054 mg kg-1). Bulb vegetables had a higher relative accumulation of Cd compared to other vegetables. The levels of Cd in vegetables varied significantly across sampling areas and years. The mean estimated daily intake (EDI) of cadmium through consumption of vegetables was 0.519 µg kg-1 bw per day for adults and 0.217 µg kg-1 bw per day for children. The target hazard quotients (THQs) were all less than the threshold of 1 for both adults and children. This indicates that there is low health risk for Cd through vegetable consumption. However, routine monitoring of Cd levels in food is still crucial to ensure food safety and protect public health.

Determination of steroid hormone residues in farmed fish using high-resolution orbital ion trap mass spectrometry.

A method for the determination of 15 steroid hormones in farmed fish by liquid chromatography-orbital ion trap mass spectrometry has been developed and validated. The method involved sample preparation with acetonitrile extraction and clean-up, and separation in the LC using a C18 column. The orbital ion trap MS was operated at a resolution of 35 000 FWHM in selected ion monitoring mode. An ion source with heated electrospray ionization was used in positive ionization mode. The samples were prepared by solid-phase extraction. The limit of quantification of steroid hormones in fish samples was 2 µg kg-1. Good linearity was observed since correlation coefficients were more than 0.99 for all compounds. Recoveries of spiked fish samples (2 µg kg-1 and 20 µg kg-1) ranged from 80.8% to 112.6% with relative deviations less than 15%. The method was successfully applied to detect steroid hormones in real farmed fish samples at the µg kg-1 level.

Distribution and safety assessment of heavy metals in fresh meat from Zhejiang, China.

There are increasing concerns on heavy metals in animal derived foods. We analyzed the levels of As, Cd, Cr, Cu, Hg, Ni, and Pb in 1066 fresh meat samples including pork, beef, mutton, chicken and duck from Zhejiang province, southeast China. The average levels of As, Cd, Cr, Cu, Hg, Ni, and Pb were 0.018, 0.002, 0.061, 0.801, 0.0038, 0.055, and 0.029 mg/kg wet weight respectively. There are significant positive correlations among Cd, Hg and Pb (P < 0.05) and negative correlations for Cu-Pb or Cu-Cd (P < 0.05). The exposure assessment showed that the health risk to humans by consuming these meat products was relatively low. However, regular monitoring of heavy metals in meat products is still recommended considering their intensive industrial activities.

Health risk assessment of heavy metals in marine fish to the population in Zhejiang, China.

Environmental pollution with toxic metals can lead to the possible contamination of the marine fish. We investigated the levels of As, Cd, Cr, Hg and Pb in 652 marine fish samples (15 species) collected from coastal areas of Zhejiang, China and estimated their health risk. Mean concentrations of As, Cd, Cr, Hg and Pb were 0.783, 0.009, 0.114, 0.031, 0.043 mg/kg wet weight. The average estimated daily intakes (EDIs) for As, Cd, Cr, Hg and Pb were 1.214, 0.014, 0.177, 0.048 and 0.067 µg/kg bw/day. The risk assessment at mean exposure level showed that there was no health risk associated with these elements through consumption of marine fish. However, potential health risk may exist for high exposure consumers considering the possible contamination of As and Hg. Given that the different levels of certain elements in marine fish in China, this study provides a scientific basis for food safety assessment and suggestions for risk management.

Analytical method development for α-amanitin and ß-amanitin in plasma at ultra-trace level by online solid phase extraction-high performance liquid chromatography-triple quadrupole mass spectrometry and its application in poisoning events.

α-Amanitin and ß-amanitin are the main fatal mushroom toxins. The toxins metabolize rapidly in blood and are reported hard to be detected 24 h after poisoning. The main challenge is that of developing a highly sensitive method at sub-pg mL-1 level in blood to diagnose intoxication cause and to study the poisoning mechanism and blood toxicity kinetics. An analytical method for α-amanitin and ß-amanitin at ultra-trace level was developed in this study by online solid phase extraction-high performance liquid chromatography-triple quadrupole mass spectrometry (online SPE-LC-MS/MS). Simple protein precipitation and liquid-liquid extraction were introduced to resolve the sample preparation problem of the online SPE-LC-MS/MS system with large-volume injection. A quick valve-switching technique with a quantitative loop as interface was used in the online system. This design can ensure the independence of flow path and pressure between the SPE and LC-MS/MS modules and can obtain the precise cleanup of the toxins. The limits of detection for α-amanitin and ß-amanitin in plasma were both 0.02 ng mL-1. The linear ranges were 0.05-20 ng mL-1 with a correlation coefficient r >0.99. The average recoveries at three spiking levels were 82.9 %-92.2 % with the relative standard deviations (RSD) of 5.4 %-8.0 % for α-amanitin and 84.5 %-93.9 % with RSDs of 4.5 %-7.8 % for ß-amanitin. The composition and concentration of the toxins in plasma from 18 patients in 5 mushroom poisoning events caused by aminitins were studied. The developed method has high positive confirmation ability and can identify toxins in plasma 40 h after poisoning.

Determination of ibotenic acid and muscimol in plasma by liquid chromatography-triple quadrupole mass spectrometry with bimolecular dansylation.

Ibotenic acid (IBA) is an amino acid and muscimol (MUS) is the decarboxyl derivative of IBA. They are mushroom neurotoxins with high polarity and low molecular weight. Only one transition (159->113 for IBA and 115->98 for MUS) can be found when directly measured by high performance liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). Therefore, the identification and quantification of trace amount of the toxins in biomaterial are difficult. A highly sensitive and accurate analytical method for IBA and MUS in plasma was developed by LC-MS/MS with the application of bimolecular dansylation and internal standard calibration. Acetonitrile was used for protein precipitation and for toxin extraction from plasma. The toxins and internal standards (L-tyrosine-13C9,15N for IBA and tyramine-d4 for MUS) were derivatized with dansyl chloride (DNSCl). The reaction conditions of the bimolecular dansylation were optimized and the fragmentation pathways of the derivatives in MS/MS were studied. Method validation was carried out according to the Bioanalytical Method Validation Guidance for Industry (FDA, USA, 2018). The limits of detection for IBA and MUS in plasma were 0.3 ng mL-1 and 0.1 ng mL-1, respectively. The linear ranges in plasma were 1-500 ng mL-1 and 1-200 ng mL-1 with the correlation coefficients of 0.998 and 0.999 for IBA and MUS, respectively. The recoveries at three spiked levels were 90.7-111.4% with relative standard deviations (RSDs) of 6.4-10.3% for IBA and the results were 85.1-94.2% with RSDs of 5.0-8.9% for MUS. The toxin levels in patients' plasma samples under different poisoning degree were presented.

Screening of multi-class antibiotics in pork meat by LC-Orbitrap-MS with modified QuEChERS extraction.

The quantification capability of high resolution mass spectrometry is of great interest to analysts. We described a method for analysis of multi-class antibiotics in pork meat by UPLC-quadrupole (Q)-Orbitrap-MS. The QuEChERS approach with a clean-up step using a sorbent of primary-secondary amine (PSA) and C18 was adopted for sample preparation, and 37 antibiotics including beta-lactams, tetracyclines, sulfonamides, fluoroquinolones and macrolides were analyzed. The Q-Orbitrap method showed high sensitivity with limits of detection (LODs) ranging from 0.8 µg kg-1 to 2.9 µg kg-1. The method was further validated by intra and inter-day tests with fortified samples. Recovery (85-105.6%) and precision values (RSDs < 15%) for all analytes were obtained. The result indicates that UPLC-Q-Orbitrap-MS coupled with QuEChERS preparation can serve as a routine method for multi-class antibiotic analysis in pork meat.

Distribution of metals and metalloids in dried seaweeds and health risk to population in southeastern China.

Concern about metals and metalloids, especially heavy metals in seaweeds has risen due to potential health risk. This study investigated the distribution of 10 metals and metalloids in 295 dried seaweeds (brown and red) and estimated the possible health risk via hazard index (HI). Elements in seaweeds can be sequenced in descending order by mean values: Al > Mn > As > Cu > Cr > Ni > Cd > Se > Pb > Hg. The levels of Cd, Cu, Mn and Ni in red seaweeds were significantly higher than those in brown seaweeds (P < 0.01). Correlation analysis showed contents of Ni-Cr (r = 0.59, P < 0.01) in seaweeds had moderate positive correlations. Seaweeds from different geographical origins had diverse element distribution. Risk assessment showed that HI at mean level was less than the threshold of 1. It indicates that for the general people there is low health risk to these elements by the intake of seaweeds. Furthermore, in terms of the confirmative toxicity of some metals, such as Cd, Pb and Hg, surveillance of metals in seaweeds should be performed continuously.

Fast and quantitative determination of saxitoxin and neosaxitoxin in urine by ultra performance liquid chromatography-triple quadrupole mass spectrometry based on the cleanup of solid phase extraction with hydrophilic interaction mechanism.

Saxitoxin (STX) and neosaxitoxin (NEO) are water-soluble toxins and their cleanup in bio-matrix is a hot topic but difficult problem. A fast and quantitative determination method for STX and NEO in urine was developed using ultra performance liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) based on the cleanup of solid phase extraction (SPE) with hydrophilic interaction (HILIC) mechanism. Acetonitrile/methanol/water mixture was used to extract the toxins in urine. Polyamide (PA) was used as HILIC SPE material to clean the toxins in sample matrix. The limits of detection were 0.2ngmL-1 for STX and 1ngmL-1 for NEO in urine. The linear ranges were 0.5ngmL-1-99.2ngmL-1 with the correlation coefficient of r=0.9992 for STX and 2.1ngmL-1-207ngmL-1 with r=0.997 for NEO in urine matrix. The recoveries at three spiking levels were 81.5%-117% with the relative standard deviations (RSDs) of 5.4%-8.5% for STX and 89.0%-118% with the RSDs of 6.7%-9.1% for NEO. STX was found in all the 6 patients' urines while NEO was only found in one sample from an intoxication case.

Authentication of pork in meat mixtures using PRM mass spectrometry of myosin peptides.

Adulteration of meat products is a major concern not only for economic fraud, but also for ethical reasons. In this study, we presented a parallel reaction monitoring (PRM) mass spectrometry approach for detection of trace pork in meat mixtures (chicken, sheep, and beef). Specific peptides were identified and screened by a shotgun proteomic approach based on tryptic digests of certain protein. Five surrogate peptides from myosin were screened and then used for pork detection by PRM of Orbitrap MS. When the most sensitive peptide was selected, the LOD in mixed meat can be up to 0.5%. The RSD values between detected and designated pork levels (1%, 5% and 50%) were 4-15%. The targeted method developed can be applied to identify and quantify the pork in meat mixture.

Quantification of 16 ß-lactams in chicken muscle by QuEChERS extraction and UPLC-Q-Orbitrap-MS with parallel reaction monitoring.

A method is described for the analysis of 16 ß-lactams in chicken muscle by UPLC-quadrupole(Q)-Orbitrap-MS with parallel reaction monitoring (PRM). QuEChERS approach includes clean-up step by sorbent of primary-secondary amine (PSA) and C18 was adopted for sample preparation. Q-Orbitrap with PRM showed high sensitivity with limits of detection (LODs) ranged from 0.01µgkg-1 to 0.35µgkg-1. The method was further validated by intra- and inter-day test with spiking levels less than MRLs (maximum residue limits, the European Union). Recovery (83-112%) and precision values (RSDs <15%) for all studied analytes were obtained. The result indicates that UPLC-Q-Orbitrap coupled with QuEChERS preparation can serve as a routine quantification method for ß-lactam residues in chicken muscles.

Analysis of monofluoroacetic acid in urine by liquid chromatography-triple quadrupole mass spectrometry and preparation of the positive sample by the bioconversion from monofluoroacetamide to monofluoroacetic acid in vitro.

Whether as a rodenticide or as a natural product, monofluoroacetic acid (FAcOH) may cause poisoning to humans or animals for its high acute toxicity. Urine is one of the most typical specimens for forensic diagnosis when poisoning case about FAcOH happens. The positive sample containing FAcOH plays a key role for the development of an accurate and reliable analytical method. The bioconversion from monofluoroacetamide (FAcNH2) to FAcOH in urine in vitro was studied for the preparation of positive urine sample containing FAcOH without standard spiking or animal experiment. The average bioconversion rates were 0%, 18.6% and 41.3% when incubated the FAcNH2 spiked urine in vitro for 21days at -20°C, room temperature (RT) and 37°C, respectively. Afterwards, a fast and sensitive analytical method was developed for determination of FAcOH in urine. Samples were diluted with water containing formic acid and cleaned with polymeric anion exchange (PAX) cartridge. The acid eluate was neutralized with ammonium hydroxide and directly measured by hydrophilic interaction liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) using basic mobile phase condition. The limit of detection and limit of quantification of FAcOH in urine were 2 and 5ngmL(-1), respectively. The linear range was 5-1000ngmL(-1) with a correlation coefficient of r=0.9993 in urine calibrated with internal standard. The recoveries at four spiking levels (5, 10, 50 and 500ngmL(-1) in urine) were 87.2%-107% with relative standard deviations ranged between 4.3%-8.8%.

Health risk assessment of heavy metals in rice to the population in Zhejiang, China.

Environmental pollution with toxic heavy metals can lead to the possible contamination of the rice. Selected metals (As, Cd, Hg and Pb) and their accumulation in rice collected from Zhejiang, China were analyzed to evaluate the potential health risk to the local adults and children. The mean levels found in rice were as follows: As, 0.080 mg/kg; Cd, 0.037 mg/kg; Hg, 0.005 mg/kg; Pb, 0.060 mg/kg. The estimated daily intakes (EDIs) were calculated in combination of the rice consumption data. The mean intakes of As, Cd, Hg and Pb through rice were estimated to be 0.49, 0.23, 0.03 and 0.37 µg/kg bw/day for adults, and 0.34, 0.29, 0.04 and 0.47 µg/kg bw/day for children. The 97.5th percentile (P97.5) daily intakes of As, Cd, Hg and Pb were 1.02, 0.64, 0.37 and 1.26 µg/kg bw/day for adults, and 0.63, 0.83, 0.47 and 1.63 µg/kg bw/day for children. The risk assessment in mean levels showed that health risk associated with these elements through consumption of rice was absent. However, estimates in P97.5 level of Cd and Pb for children, and Hg for adults have exceeded the respective safe limits.

Validation (in-house and collaboratory) of the quantification method for ethyl carbamate in alcoholic beverages and soy sauce by GC-MS.

A method for ethyl carbamate (EC) determination in alcoholic beverages and soy sauce was developed by GC-MS. We adopted the diatomaceous earth solid-phase extraction (SPE) column and elution solvent of ethyl acetate/diethyl ether (5:95 v/v) for sample cleaning. The in-house validation showed the limit of quantification (LOQ) was 5.0 µg/kg. In the accuracy assay, the total average recovery for was 96.7%. The relative standard deviations (RSDs) were <5%. Subsequently, a collaborative trial was organized for the further validation. The RSDs for repeatability and reproducibility were 1.2-7.8% and 2.3-9.6% respectively. It indicated that the present method performed well in different laboratories.