Health Risks of Chronic Exposure to Small Doses of Microcystins: An Integrative Metabolomic and Biochemical Study of Human Serum.

Health risks of chronic exposure to microcystins (MCs), a family of aquatic contaminants produced mainly by cyanobacteria, are critical yet unsolved problems. Despite a few epidemiological studies, the metabolic profiles of humans exposed to MCs remain unknown, hindering the deep understanding of the molecular toxicity mechanisms. Here, sensitive nuclear magnetic resonance (NMR)- and liquid chromatography-mass spectrometry (LC-MS)-based metabolomics were applied to investigate the serum metabolic profiles of humans living near Lake Chao, where toxic cyanobacterial blooms occur annually. MCs were positively detected in 92 of 144 sera by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with a median concentration of 0.016 µg/L. The estimated daily intake (0.15-0.27 µg MC-LReq/day) was less than the tolerable daily intake (TDI, 2.4 µg MC-LR for 60 kg adults) recommended by the World Health Organization (WHO). Obvious disruptions of the amino acid metabolism were confirmed and played important roles in renal impairments associated with serum MC burdens. Chronic oral exposure of mice to 30 µg MC-LR/kg body mass, which is less than the no observed adverse effect level, also led to obvious renal lesions and metabolic dysfunction. These observations provide the first evidence of metabolic disturbance of humans exposed to MCs and indicate that the WHO's TDI value determined traditionally should be lessened to protect human health effectively.

Association of fine particulate matter with glucose and lipid metabolism: a longitudinal study in young adults.

OBJECTIVES: This study aimed to evaluate whether PM2.5 exposure in a highly polluted area (>100 µg/m3) affects glucose and lipid metabolism in healthy adults. METHODS: We recruited 110 healthy adults in Baoding city, Hebei, China, and followed them up between 2017 and 2018. Personal air samplers were used to monitor personal PM2.5 levels. Eight glucose and lipid metabolism parameters were quantified. We performed the linear mixed-effect models to investigate the relationships between PM2.5 and glucose and lipid metabolism parameters. Stratified analyses were further performed according to sex and body mass index (BMI). RESULTS: The concentration of PM2.5 was the highest in spring, with a median of 232 µg/m3 and the lowest in autumn (139 µg/m3). After adjusting for potential confounders, we found that for each twofold increase in PM2.5, the median of insulin concentration decreased by 5.89% (95% CI -10.91% to -0.58%; p<0.05), and ox-LDL increased by 6.43% (95% CI 2.21% to 10.82%; p<0.05). Stratified analyses indicated that the associations were more pronounced in females, overweight and obese participants. CONCLUSIONS: Exposure to high PM2.5 may have deleterious effects on glucose and lipid metabolism. Females, overweight and obese participants are more vulnerable.

[Determination of indigo and brilliant blue in different types of food products by high performance liquid chromatography with solid phase extraction].

OBJECTIVE: To develop and validate a solid phase extraction-high performance liquid chromatographic( SPE-HPLC) method for the simultaneous determination of indigo and brilliant blue in different types of food products. METHODS: The artificial colors in food products were extracted by acetonitrile / water and purified by WAX SPE cartridges, The separation was achieved using a Waters Symmetry C_(18)( 5 µm, 4. 6 mm × 250 mm) column and a binary gradient mobile phase of methanol and 0. 02 mol/L ammonium acetate solution, detected by HPLC-PDA. RESULTS: The validated analytical method showed that there was a good linearity in the range of 0. 05- 20. 00 µg/mL for both indigo and brilliant blue( r > 0. 999). The lowest detection limits of indigo and brilliant blue were 0. 04 and 0. 02 mg/kg, respectively. The average recoveries were among 81. 8%- 101. 1%, with relative standard deviation( RSD) of 2. 1%- 4. 9%( n =6) for both artificial colors. CONCLUSION: The method has high selectivity, high sensitivity, good recovery and reproducibility. It is suitable to simultaneously monitor indigo and brilliant blue in several types of food products based on the food classification system of GB 2760-2014.

Why mammals more susceptible to the hepatotoxic microcystins than fish: evidences from plasma and albumin protein binding through equilibrium dialysis.

To elucidate the interspecies variation of susceptibility to microcystins (MCs), fresh plasma and purified albumin from six kinds of mammals and fish were used in toxins-substances binding test. Protein contents in the test plasma were analyzed and the binding characteristics to MCs were compared. Two kinds of widely observed MCs, microcystin-LR (MC-LR) and microcystin-RR (MC-RR) were tested and data were collected through the method of equilibrium dialysis. It was found that total plasma protein and albumin content in mammals were nearly two times and four times higher than that in fish, respectively. In the test range of 0-100 µg/mL, binding rates of fish plasma to MCs were considered significant lower (p < 0.01) than that of mammals. And human plasma demonstrated the highest binding rate in mammals. In all the test species, plasma protein binding rates of MC-RR were significantly higher than MC-LR (p < 0.01). Besides, binding profiles of albumin were acquired under the protein content of 0.67 mg/mL. Human serum albumin demonstrated the highest affinity to MCs throughout the six species and differences among the other five species were considered not significant (p > 0.05). From the view of protein binding, it is concluded that both the variation of plasma protein composition and albumin binding characteristic could influence the existing form of MCs in circulation, change MCs utilization, alter MCs half-life and further contribute to the difference of susceptibility between mammals and fish.

Comparative studies on the pH dependence of DOW of microcystin-RR and -LR using LC-MS.

Microcystins (MCs) are well known worldwide as hepatotoxins produced by cyanobacteria, but little is known about the physicochemical properties of these compounds. The dependence of the n-octanol/water distribution ratio (DOW) of MC-RR and -LR to pH was measured by high-performance liquid chromatography combined with mass spectrometry (LC-MS). There was a remarkable difference in such relationships between MC-RR and -LR. The log DOW of MC-LR decreased from 1.63 at pH 1.0 to -1.26 at pH 6.5, and stabilized between -1.04 and -1.56 at a pH of 6.5~12.0; log DOW of MC-RR varied between -1.24 and -0.67 at a pH of 1.00~4.00, and stabilized between -1.20 and -1.54 at a pH of 4.00~12.00. The difference of hydrophobicity in acidic condition between MC-RR and -LR is important, not only for the analytical method of both toxins, but perhaps also for understanding the difference of toxicity to animals between the two toxins.

Fine particulate matter exposure and perturbation of serum metabolome: A longitudinal study in Baoding, China.

Metabolomics represents a powerful tool for measuring environmental exposures and biological responses to unveil potential mechanisms. Few studies have investigated the effects of exposure to fine particulate matter (PM2.5) longitudinally on serum metabolomics in regions with high-level PM2.5. Therefore, we examined the changes of serum metabolomics corresponding to individual PM2.5 exposure levels in spring and autumn among 63 healthy college students in Baoding city, Hebei, China. The metabolic profiling was determined by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. The average level of individual PM2.5 in the spring was 1.82-fold higher than in the autumn (240 µg/m3 vs 132 µg/m3). Males were exposed to a higher level of PM2.5 than females in the spring. Metabolic profiling was clearly separated by orthogonal partial least square-discriminant analysis in males but not in females. In the analysis of the associations between the metabolome and PM2.5 of the two seasons, the changes of 14 serum metabolites were significantly associated with PM2.5 in males. The metabolites related to heme metabolism (bilirubin, biliverdin), energy metabolism and oxidative stress (2-Octenoylcarnitine, N-Heptanoylglycine, and acetylcysteine), phospholipid metabolism (lysophosphatidic acid, phospholipid acid, and lysophosphatidylethanolamine), and tryptophan metabolism (N-Acetylserotonin, indolepyruvate, and melatonin) were decreased in the range of 2.16%-6.80% for each 10 µg/m3 increase of PM2.5, while thyrotropin-releasing hormone, glutathione, and phosphatidylethanolamine related to energy metabolism and oxidative stress, and phospholipid metabolism were increased in the range of 2.95%-4.90% for each 10 µg/m3 increase of PM2.5. This longitudinal study suggests that higher PM2.5 exposure may induce perturbations in serum metabolic signaling related to oxidative stress and inflammation, and males may be more prone to these metabolic perturbations.

A nationwide study of the occurrence and distribution of atrazine and its degradates in tap water and groundwater in China: Assessment of human exposure potential.

Despite frequent detection of atrazine (ATZ) and its degradates (including hydroxyatrazine, ATZ-OH; deethylatrazine, DEA; deisopropylatrazine, DIA; and deethyldeisopropylatrazine, DACT) in a variety of water bodies, documentation of their occurrence and distribution in tap water in China is still scarce. A nationwide survey about ATZ and its degradates (ATZs) in tap water from 31 provinces in 7 regions of mainland China and Hong Kong was conducted during June 2019. At least one of the analytes was found in all the water samples (n = 884). The median sum concentrations of ATZs (ΣATZs) was 21.0 ng/L (range: 0.02 ng/L-3.04 µg/L). The predominant compounds of ATZs in tap water were ATZ and DEA, with a detection frequency of 99.5% and 98.0%, respectively, followed by ATZ-OH (87.3%), DACT (84.0%), and DIA (78.1%). Significant regional variations (p < 0.05) were found in the concentrations of ATZs in tap water, and the highest concentration of ΣATZs (median: 254 ng/L, range: 0.44 ng/L-3.04 µg/L) was found in Northeastern China, followed by Eastern (37.2 ng/L, 0.02-706 ng/L), Northern (30.2 ng/L, 0.04-317 ng/L), Central (29.3 ng/L, 0.04-256 ng/L), Southern (25.0 ng/L, 0.04-297 ng/L), Southwestern (17.2 ng/L, 0.02-388 ng/L), and Northwestern China (3.22 ng/L, 0.06-214 ng/L). The level of ΣATZs in groundwater from rural area of China was about 1/3 of that found in tap water. ATZs cannot be removed by boiling tap water. The highest estimated daily intake of ΣATZs (248 ng/kg-body weight/day) was found in the infant population of Changchun, Jilin, Northeastern China.

Determinations of MC-LR and [Dha(7)] MC-LR concentrations and physicochemical properties by liquid chromatography-tandem mass spectrometry.

A liquid chromatography electrospray mass spectrometry (LC/ESI/MS) method working in multiple reactions monitoring mode for the determination of trace amounts of microcystin variants (MC-LR and [Dha(7)] MC-LR) in waters was developed. The limit of quantification was 0.05 microg/L and the limit of detection was 0.015 microg/L for MC-LR and [Dha(7)] MC-LR, respectively. Recoveries for MCs were in the range of 68%-81%. MC-LR and [Dha(7)] MC-LR were chemically stable with similar physiochemical behavior.

Effects of water impoundment and water-level manipulation on the bioaccumulation pattern, trophic transfer and health risk of heavy metals in the food web of Three Gorges Reservoir (China).

The Three Gorges Reservoir (TGR) of China, the largest hydropower project over the world, has attracted much attention to the water impoundment and water-level manipulation. In this study, we evaluated potential effects of water impoundment and seasonal water-level manipulation on the bioaccumulation, trophic transfer and health risk of HMs (Cu, Fe, Zn, Hg, Cd and Pb) in food web components (seston, aquatic invertebrate and fish) in TGR. Our results show that, after the impoundment for eight years (2003-2010), all of the six metal concentrations in aquatic biota fell within the criteria of safety quality guidelines. The concentrations of Cu, Fe, Zn and Hg in fish and aquatic invertebrates were higher than those before impoundment, whereas Cd and Pb were lower than those before impoundment. Nonetheless, Hg, Cd and Pb in aquatic consumers underwent an increasing trend during the entire impoundment, implying potential reservoir effect in the future. Only the concentrations of Hg, Cd and Pb in aquatic consumers exhibited a declining trend towards the dam, showing consistent with the background level at the three reaches. Seasonal variations in HM concentrations of fish and aquatic invertebrates were ascribed to the water-level manipulation associated with reservoir management. Our findings show that Hg or Cd biomagnified through aquatic food web during different hydrological periods, whereas Pb, Cu, Fe and Zn exhibited weak biomagnification power. Overall, Hg, Cd and Pb showed a higher risk than that of Cu, Fe and Zn.

Spatial and interspecies differences in concentrations of eight trace elements in wild freshwater fishes at different trophic levels from middle and eastern China.

There have been numerous studies on concentrations of trace elements in aquatic ecosystems, but few have been conducted at a large spatial scale. This study collected 410 samples of five wild freshwater fishes at different trophic levels from middle and eastern China. Concentrations of eight trace elements, chromium (Cr), iron (Fe), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), lead (Pb) and cadmium (Cd) and stable isotope ratios (δ13C and δ15N) were determined in dorsal muscle of fishes. Spatially, concentrations of trace elements were least in fishes from the Hai River Basin, while those in fishes from the Taihu Lake Basin were greatest. The carnivorous topmouth culter and omnivorous common carp and crucian carp accumulated greater amounts of trace elements than did the planktivorous silver carp and bighead carp. Trophic biomagnification was for Cu, Fe and Zn, but not for Cr, Ni, As, Pb and Cd. Concentrations of As in 15 muscle samples (3.7%) from Taihu Lake Basin exceeded the guidelines (1.0 mg/kg, wet mass) provided by FAO/WHO (2014), while the total target hazard quotient (TTHQ) values were <1.0, indicating no obvious non-carcinogenic risks to humans that consume those fishes. However, people who consume larger amounts of fish products, or people who are vulnerable, such as pregnant women, children and people with poor health, might be at greater risk. Also, exposure to trace metals through other routes cannot be ignored. Accumulations of trace elements in Chinese freshwater fishes were affected by both geographical conditions and human activities.

Distribution of microcystins in various organs (heart, liver, intestine, gonad, brain, kidney and lung) of Wistar rat via intravenous injection.

The distribution of microcystins (MCs) in various tissues of Wistar rats was studied under laboratory conditions. Rats were injected intravenously (i.v.) with extracted MCs at a dose of 80 microg MC-LR(equivalent)/kg body weight. MCs concentrations in various tissues were detected at 1, 2, 4, 6, 12 and 24h post-injection using liquid chromatography-mass spectrometry (LC-MS). The highest concentration of MCs was found in kidney (0.034-0.295 microg/g dry weight), followed by lung (0.007-0.067 microg/g dry weight), stomach (0.010-0.058 microg/g dry weight) and liver (0.003-0.052 microg/g dry weight). The maximum MCs content in the whole body of rat, 2.9% of the injected dose, was observed at 2h post-injection. MCs concentration was higher in kidney than in liver during the experiment, and two peaks of MCs concentration (at 2 and 24h, respectively) were observed in kidney, indicating that MCs can be excreted directly via kidney of rat. Though heart, intestine, spleen, brain, gonad and stomach contained less than 0.2% of injected MCs during the whole experiment stage, the presence of MCs in these tissues represents potential damage to them.

Simultaneous determination of microcystin-LR and its glutathione conjugate in fish tissues by liquid chromatography-tandem mass spectrometry.

A sensitive and selective liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantitative determination of microcystin-LR (MC-LR) and its glutathione conjugate (MC-LR-GSH) in fish tissues. The analytes were extracted from fish liver and kidney using 0.01M EDTA-Na(2)-5% acetic acid, followed by a solid-phase extraction (SPE) on Oasis HLB and silica cartridges. High-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry, operating in selected reaction monitoring (SRM) mode, was used to quantify MC-LR and its glutathione conjugate in fish liver and kidney. Recoveries of analytes were assessed at three concentrations (0.2, 1.0, and 5microg g(-1) dry weight [DW]) and ranged from 91 to 103% for MC-LR, and from 65.0 to 75.7% for MC-LR-GSH. The assay was linear within the range from 0.02 to 5.0microg g(-1) DW, with a limit of quantification (LOQ) of 0.02microg g(-1) DW. The limit of detection (LOD) of the method was 0.007microg g(-1) DW in both fish liver and kidney. The overall precision was determined on three different days. The values for within- and between-day precision in liver and kidney were within 15%. This method was applied to the identification and quantification of MC-LR and its glutathione conjugate in liver and kidney of fish with acute exposure of MC-LR.

Distribution of toxins in various tissues of crucian carp intraperitoneally injected with hepatotoxic microcystins.

An acute toxicity experiment was conducted to examine the distribution and depuration of microcystins (MCs) in crucian carp (Carassius aurutus) tissues. Fish were injected intraperitoneally with extracted MCs at a dose of 200 microg MC-LR (where L=leucine and R=arginine) equivalent/kg body weight. Microcystin concentrations in various tissues and aquaria water were analyzed at 1, 3, 12, 24, and 48 h postinjection using liquid chromatography coupled with mass spectrometry. Microcystins were detected mainly in blood (3.99% of injected dose at 1 h), liver (1.60% at 1 h), gonad (1.49% at 3 h), and kidney (0.14% at 48 h). Other tissues, such as the heart, gill, gallbladder, intestine, spleen, brain, and muscle, contained less than 0.1% of the injected MCs. The highest concentration of MCs was found in blood (526-3,753 ng/g dry wt), followed by liver (103-1,656 ng/g dry wt) and kidney (279-1,592 ng/g dry wt). No MC-LR was detectable in intestine, spleen, kidney, brain, and muscle, whereas MC-RR was found in all examined fish tissues, which might result from organ specificity of different MCs. Clearance of MC-RR in brain tissue was slow. In kidney, the MC-RR content was negatively correlated with that in blood, suggesting that blood was important in the transportation of MC-RR to kidney for excretion.

Tissue distribution and depuration of the extracted hepatotoxic cyanotoxin microcystins in crucian carp (Carassius carassius) intraperitoneally injected at a sublethal dose.

An acute toxicity experiment was conducted by intraperitoneal injection with a sublethal dose of extracted microcystins (MCs), 50 microg MC-LR (where L = leucine and R = arginine) equivalent/kg body weight (BW), to examine tissue distribution and depuration of MCs in crucian carp (Carassius carassius). Liver to body weight ratio increased at 3, 12, 24, and 48 h postinjection compared with that at 0 h (p < 0.05). MC concentrations in various tissues and aquaria water were analyzed at 1, 3, 12, 24, 48, and 168 h postinjection using liquid chromatography coupled with mass spectrometry (LC-MS). The highest concentration of MCs (MC-RR + MC-LR) was found in blood, 2-270 ng/g dry weight (DW), followed by heart (3-100 ng/g DW) and kidney (13-88 ng/g DW). MC levels were relatively low in liver, gonad, intestine, spleen, and brain. MC contents in gills, gallbladder, and muscle were below the limit of detection. Significant negative correlation was present between MC-RR concentration in blood and that in kidney, confirming that blood was important in the transportation of MC-RR to kidney for excretion. Rapid accumulation and slow degradation of MCs were observed in gonad, liver, intestine, spleen, and brain. Only 0.07% of injected MCs were detected in liver. The recovery of MCs in liver of crucian carp seemed to be dose dependent.

[Methylamidation and normal phase chromatography with silica-hydride-based stationary phase for N-glycosylation characterization of monoclonal antibody drugs].

Monoclonal antibodies (mAbs) are efficacious therapeutic agents against various complex diseases. N-glycosylation characterization of mAbs is based primarily on fluorescence derivatization coupled with hydrophilic interaction liquid chromatography (HILIC). However, this method has some drawbacks. Presently, the N-glycosylation of mAbs was analyzed by methylamidation and silica-hydride-based normal phase chromatography (SiH-NPC). Samples were analyzed by liquid chromatography-mass spectrometry after enzymatic cleavage, methylamidation, and purification. SiH-NPC had some advantages compared to HILIC. It offered a novel separation mechanism that allowed for high resolution using salt-free mobile phases, avoiding contamination of the mass spectrometer. SiH-NPC may be applicable for rapid analysis. Its structure was more stable than that of HILIC, and it provided a long service life. In combination with the sialic acid derivatization, SiH-NPC presented a significant advantage in the analysis of sialylated glycans as well as isomeric oligosaccharides by liquid chromatography-mass spectrometry. The approach has potential applications in the biopharmaceutical industry.

Bioaccumulation of the hepatotoxic microcystins in various organs of a freshwater snail from a subtropical Chinese lake, Taihu Lake, with dense toxic Microcystis blooms.

In this paper, we describe the seasonal dynamics of three common microcystins (MCs; MC-RR, MC-YR, and MC-LR) in the whole body, hepatopancreas, intestine, gonad, foot, remaining tissue, and offspring of a freshwater snail, Bellamya aeruginosa, from Gonghu Bay of Lake Taihu, China, where dense toxic Microcystis blooms occur in the warm seasons. Microcystins were determined by liquid chromatography electrospray ionization mass spectrum. Microcystin (MC-RR + MC-YR + MC-LR) content of the offspring and gonad showed high positive correlation, indicating that microcystins could transfer from adult females to their young with physiological connection. This study is the first to report the presence of microcystins in the offspring of the adult snail. The majority of the toxins were present in the intestine (53.6%) and hepatopancreas (29.9%), whereas other tissues contained only 16.5%. If intestines are excluded, up to 64.3% of the toxin burden was allocated in the hepatopancreas. The microcystin content in the intestine, hepatopancreas, and gonad were correlated with the biomass of Microcystis and intracellular and extracellular toxins. Of the analyzed foot samples, 18.2% were above the tolerable daily microcystin intake recommended by the World Health Organization (WHO) for human consumption. This result indicates that public health warnings regarding human ingestion of snails from Taihu Lake are warranted. In addition, further studies are needed to evaluate the occurrence by Microcystis in relation to spatial and temporal changes in water quality.

Microwave-assisted purge-and-trap extraction device coupled with gas chromatography and mass spectrometry for the determination of five predominant odors in sediment, fish tissues, and algal cells.

Off-flavors are among the most troublesome compounds in the environment worldwide. The lack of a viable theory for studying the sources, distribution, and effect of odors has necessitated the accurate measurement of odors from environmental compartments. A rapid and flexible microwave-assisted purge-and-trap extraction device for simultaneously determining five predominant odors, namely, dimethyltrisulfide, 2-methylisoborneol, geosmin, ß-cyclocitral and ß-ionone, from the primary sources and sinks is demonstrated. This instrument facilitates the extraction and concentration of odors from quite different matrices simultaneously. This device is a solvent-free automated system that does not require cleaning and is timesaving. The calibration curves of the five odor compounds showed good linearity in the range of 1-500 ng/L, with correlation coefficients above 0.999 (levels=7) and with residuals ranging from approximately 77% to 104%. The limits of detection (S/N=3) were below 0.15 ng/L in algae sample and 0.07 ng/g in sediment and fish tissue samples. The relative standard deviations were between 2.65% and 7.29% (n=6). Thus the proposed design is ready for rapid translation into a standard analytical tool and is useful for multiple applications in the analysis of off-flavors.

Simultaneous determination of eight common odors in natural water body using automatic purge and trap coupled to gas chromatography with mass spectrometry.

Production and fate of taste and odor (T&O) compounds in natural waters are a pressing environmental issue. Simultaneous determination of these complex compounds (covering a wide range of boiling points) has been difficult. A simple and sensitive method for the determination of eight malodors products of cyanobacterial blooms was developed using automatic purge and trap (P&T) coupled with gas chromatography-mass spectrometry (GC-MS). This extraction and concentration technique is solvent-free. Dimethylsulfide (DMS), dimethyltrisulfide (DMTS), 2-isopropyl-3-methoxypyrazine (IPMP), 2-isobutyl-3-methoxypyrazine (IBMP), 2-methylisoborneol (MIB), ß-cyclocitral, geosmin (GSM) and ß-ionone were separated within 15.3 min. P&T uses trap #07 and high-purity nitrogen purge gas. The calibration curves of the eight odors show good linearity in the range of 1-500 ng/L with a correlation coefficient above 0.999 (levels=8) and with residuals ranging from approximately 83% to 124%. The limits of detection (LOD) (S/N=3) are all below 1.5 ng/L that of GSM is even lower at 0.08 ng/L. The relative standard deviations (RSD) are between 3.38% and 8.59% (n=5) and recoveries of the analytes from water samples of a eutrophic lake are between 80.54% and 114.91%. This method could be widely employed for monitoring these eight odors in natural waters.

Development and validation of a liquid chromatography-tandem mass spectrometry assay for the simultaneous quantitation of microcystin-RR and its metabolites in fish liver.

A novel method for identification and quantification of microcystin-RR (MC-RR) and its metabolites (MC-RR-GSH and MC-RR-Cys) in the fish liver was developed and validated. These analytes were simultaneously extracted from fish liver using water containing EDTA with 5% acetic acid, followed by a mixed-mode cation-exchange SPE (Oasis MCX) and subsequently determined by liquid chromatography-electrospray ionization ion trap mass spectrometry (LC-ESI-ITMS). Extraction parameters including volume and pH of eluting solvents, were optimized. Best recoveries were obtained by using 10 mL of 15% ammonia solution in methanol. The mean recoveries at three concentrations (0.2, 1.0, and 5.0 microg g(-1) dry weight [DW]) for MC-RR, MC-RR-GSH and MC-RR-Cys were 93.6-99%, 68.1-73.6% and 90.0-95.2%, respectively. Method detection limit (MDL) were 4, 7 and 5 ng g(-1) DW for MC-RR, MC-RR-GSH and MC-RR-Cys, respectively. Limits of quantification (LOQs) for MC-RR, MC-RR-GSH and MC-RR-Cys were calculated to be 10, 18 and 13 ng g(-1) DW, respectively. Finally, this method was successfully applied to the identification and quantification of MC-RR, MC-RR-GSH and MC-RR-Cys in the liver of bighead carp with acute exposure of MCs.

Determination of microcystin-LR and its metabolites in snail (Bellamya aeruginosa), shrimp (Macrobrachium nipponensis) and silver carp (Hypophthalmichthys molitrix) from Lake Taihu, China.

This paper describes seasonal changes of microcystin-LR (MC-LR) and its glutathione (MC-LR-GSH) and cysteine conjugates (MC-LR-Cys) in three aquatic animals--snail (Bellamya aeruginosa), shrimp (Macrobrachium nipponensis) and silver carp (Hypophthalmichthys molitrix) collected from Lake Taihu, China. MC-LR, MC-LR-GSH, and MC-LR-Cys were determined by liquid chromatography electrospray ionization mass spectrum (LC-ESI-MS). The mean MC-LR concentrations in the hepatopancreas of snail and shrimp and liver of silver carp were 6.61, 0.24, and 0.027 microg g(-1) dry weight (DW), respectively; while the average MC-LR-Cys concentrations were 0.50, 0.97, and 5.72 microg g(-1) DW, respectively. MC-LR-GSH was usually not detectable in these samples. The above results suggest that: (1) in aquatic animals, especially fish, the main excretion form of MC-LR could be MC-LR-Cys, but not MC-LR-GSH, whereas MC-LR-Cys might play an important role in detoxication of MC-LR and (2) that efficiency of MC-LR-Cys formation differs among species. The main detoxication pathway of MC-LR in aquatic animals is suggested as follows: when MC-LR enters into liver/hepatopancreas, it firstly conjugates with polypeptide or protein (including GSH, PP-1 and 2A) containing Cys residues, perhaps also some free cysteine; subsequently, MC-LR-Cys is degraded from these polypeptide or protein; and finally is excreted from animals by the compound of MC-LR-Cys.