Laser irradiation desorption of microcystins from protein complex in fish tissue and liquid chromatography‐tandem mass spectrometry analysis

Microcystins are a group of cyanotoxins which interact with the C‐terminal region of PP1 and PP2A proteins, so denaturation and inactivation are necessary for breaking covalent binding to release microcystins. In this study, a novel extraction method was developed by laser irradiation desorption of microcystins from fish protein. The sample was mixed with aqueous methanol and irradiated by a 450 nm laser, with an optimized value of laser power density at 8 W and exposure time at 5 min. ThenLC–MS/MS was applied for the determination of microcystins in fish extracts. The ionization behaviors of microcystins were investigated firstly, and doubly charged microcystins were selected as precursor ions in multiple reaction monitoring scan for quantification. This proposed quantitative method was well validated in terms of selectivity, linearity, sensitivity, accuracy, recovery, and stability. The successful application of this LC–MS/MS method showed its ability for the analysis of microcystins in low concentration, and it would be of significant interest for environmental and food safety applications to ensure the safety of fish and related products.     

Rapid clean-up and effective sample preparation procedure for unambiguous determination of the cyclic peptides microcystin and nodularin

Summary A new sample preparation strategy has been established to improve the identification and determination of nodularin and microcystins. The sample preparation consisted of enrichment of the analytes by solid phase extraction with C18 cartridges followed by clean-up of the enriched raw extracts by high performance size exclusion gel permeation chromatography. In contrast to established clean-up procedures based on polarity, related distribution of microcystins and nodularin in non-miscible phases (e. g. a C18 cartridge as stationary phase and a water-containing eluent as mobile phase) this strategy separates microcystins from interfering compounds by molecular size differences. The sample preparation procedure can be automated easily and was validated for both water samples as well as raw extracts of algal cells. The method was success-fully applied during an experiment with natural algae communities from the Baltic Sea to investigate the influence of different nutrient limitations on toxicity ofNodularia sp...     

Comparison of liquid chromatography/mass spectrometry, ELISA, and phosphatase assay for the determination of microcystins in blue-green algae products

More than 100 samples of blue-green algae products (consisting of Aphanizomenon, Spirulina, and unidentified blue-green algae) in the form of pills, capsules, and powders were collected from retail outlets from across Canada. The samples were extracted with 75% methanol in water and centrifuged to remove solids. Aliquots of the extracts along with spiked blank sample extracts were sent to each participating laboratory and independently analyzed for microcystins by enzyme-linked immunosorbent assay (ELISA), protein phosphatase inhibition assay, and by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after sample cleanup using C18 solid-phase extraction. The results obtained by ELISA and LC-MS/MS agreed very well over a concentration range of about 0.5-35 microg/g. The colorimetric phosphatase results generally agreed with the other 2 methods. While the 2 biochemical assays measured total microcystin content compared with a standard of microcystin LR, the LC-MS/MS method measured specific microcystins (LA, LR, RR, YR) using external standards of these for identification and quantitation. Microcystin LR was found in all positive samples by LC-MS/MS. Microcystin LA was the only other microcystin found in the samples analyzed. These 2 microcystins represent essentially all the microcystins that were present in the extracts. Otherwise, the LC-MS/MS results would have been significantly lower than the results of the biochemical assays had other unknown microcystins been present.     

超高效液相色谱–串联质谱法检测果蔬中的吡氟甲禾灵残留

建立了超高效液相色谱–串联质谱(UPLC–MS/MS)法测定果蔬中吡氟甲禾灵残留的方法。样品以乙腈匀浆提取,并采用Sep-Pak Vac型氨基固相萃取柱净化样品,采用超高效液相色谱柱WATERS ACQUITY C_(18)柱(50mm×2.1 mm,1.7μm)分离,以乙腈–0.1%甲酸水溶液作为淋洗液进行梯度洗脱。吡氟甲禾灵在1.0~50.0 ng/m L范围内线性关系良好,相关系数r~2=0.997 7,加标回收率为84.1%~88.6%,测定结果的相对标准偏差为1.18%~3.58%(n=6)。该方法操作简便,分析快速,提取效率高,重现性好,有实用价值。     

Optimization of Dispersive Liquid–Liquid Microextraction of Irganox 1010 and Irgafos 168 from Polyolefins Before Liquid Chromatographic Analysis

A new dispersive liquid–liquid microextraction method has been established for extraction of two antioxidants, Irganox 1010 and Irgafos 168, from polyolefins. The extracts were analyzed by liquid chromatography. Carbon tetrachloride at microliter levels and acetonitrile at milliliter levels were used as extraction and dispersive solvents, respectively. Central-composite design and response-surface methodology were used as experimental strategies for modeling and optimization. The effects of experimental conditions on extraction were investigated by modeling extraction recovery as the response. The experimental design was performed at five levels of the operating conditions. Nearly the same results for optimization were obtained by using the one-variable-at-a-time and central-composite-design methods: sample size 5–10 mg, dispersive solvent acetonitrile (2 mL), extraction solvent carbon tetrachloride (200 μL); extraction temperature 100 °C, and extraction time 3 h. Under the optimum conditions the calibration plots were linear over the range 50–2,000 μg L^?1 in solution. The relative standard deviation of the method for six replicate experiments was 7.0 and 4.9% for Irganox 1010 and Irgafos 168, respectively.     

Analysis of microcystins by capillary high performance liquid chromatography using a polymethacrylate-based monolithic column

A polymethacrylate-based monolithic column was prepared and its application to the separation of three kinds of similar microcystins (MCs) in capillary high performance liquid chromatography (capillary-HPLC) with ultraviolet detection was studied. The monolithic matrix contains both hydrophobic and cation-exchange interaction sites. Factors influencing the separation performance have been investigated. A baseline separation could be achieved by means of Tris(hydroxymethyl)aminomethane buffer of mildly alkaline pH and acetonitrile as the mobile phases in less than 5 min. The calibration curves were linear with a correlation coefficient r>0.9990 over a range of 0.25-18.00 mg/L. This method was successfully applied to the separation of microcystins from other compounds in spiked uncontaminated lake water after performing solid-phase extraction. The whole procedure provided low LODs, e. g. the LODs for MC-LR, MC-YR, and MC-RR were found to be 0.49, 0.67, 0.30 microg/L, respectively. The LODs, precision, efficiency, and the results obtained for the real samples demonstrate the potential of polymethacrylate-based monolithic columns as fast separation tools for routine use in the monitoring of microcystins in real water samples.     

Analysis of microcystins using high‐performance liquid chromatography and magnetic solid‐phase extraction with silica‐coated magnetite with cetylpyridinium chloride

Microcystins (MCs), produced by freshwater cyanobacteria, can be serious water pollutants, so it is important to monitor their concentration in drinking water. We have developed a method for rapid and accurate determination of microcystin levels in environmental water, using magnetic solid‐phase extraction and high‐performance liquid chromatography with UV detection. The magnetic composite material, which was combined with cetylpyridinium chloride, was prepared by hydrothermal synthesis. The optimal extraction of microcystins in water sample was achieved by optimizing the amount of adsorbent, time of adsorption, ratio of eluting solvent, and volume of eluent. Under the optimal conditions, the limit of detection of MC‐LR was 0.001 μg/L, and the limit of quantification was 0.0028 μg/L. The limit of detection of MC‐RR was 0.001 μg/L, and the limit of quantification was 0.003 μg/L. These values are far lower than those established by the International Health Organization for the maximum concentration of microcystins in drinking water. The magnetic solid‐phase extraction adsorbent used in this method has the advantages of simple preparation, low price, and easy solid–liquid separation, and it can be used for the rapid and sensitive monitoring of trace microcystins in environmental water samples.     

通过型固相萃取-液相色谱-串联质谱法同时快速测定鱼肉中7种微囊藻毒素

采用通过型固相萃取净化去除样品基质中脂类物质的干扰,建立了鱼肉中7种微囊藻毒素的液相色谱-串联质谱快速分析方法。样品经80℃水浴热处理后用体积分数为90%的甲醇水溶液进行提取,使用Oasis PRiME HLB通过型固相萃取柱净化。净化后的样品采用Waters XSelect HSS T3色谱柱分离,以0.1%(体积分数)甲酸乙腈溶液和0.1%(体积分数)甲酸水溶液为流动相,梯度洗脱,多反应监测正离子模式扫描,采用基质匹配溶液外标法定量。研究了7种微囊藻毒素的质谱离子化特征,结果表明,酸能显著增加双电荷离子的响应强度。7种目标物在相关范围内线性关系良好,相关系数不低于0.99,定量限为0.30~2.0μg/kg,基质加标回收率为70.6%~96.1%,相对标准偏差为3.4%~9.6%。该方法前处理操作简便,灵敏度和准确度高,可实现鱼肉中多种微囊藻毒素的同时快速测定。     

甲基丙烯酸酯毛细管整体柱分离微囊藻毒素的研究

以甲基丙烯酸丁酯为单体,乙二醇二甲基丙烯酸酯为交联剂,在致孔剂存在的条件下原位聚合制备了甲基丙烯酸丁酯毛细管整体柱(150 μm i.d.)。实验中优化了用此整体柱分离3种微囊藻毒素(MC-LR,-YR和-RR)的色谱条件(流动相种类、缓冲溶液浓度、pH、流动相流速),建立了微囊藻毒素的整体柱毛细管液相色谱分离方法,该法可以在9 min之内实现3种微囊藻毒素的基线分离。将该方法应用于实际水样中微囊藻毒素的分析,成功实现了培养水样和巢湖水样中微囊藻毒素的快速分离,两种样品中均检测到MC-LR。结果表明,所制备的甲基丙烯酸酯毛细管整体柱具有良好的重现性、渗透性,在微囊藻毒素的常规检测中具有很好的应用前景。     

Optimization of the extraction of polycyclic aromatic hydrocarbons from wood samples by the use of microwave energy

A simple and rapid microwave-assisted extraction (MAE) procedure was developed and optimized for benzo[a]anthracene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[a]pyrene in wood samples. The spiked wood used was prepared 3 months before analysis to simulate weathering processes and to allow the formation of analyte-matrix interaction. The samples, immersed in acetonitrile were irradiated with microwaves in a closed-vessel system. Optimization of the method was achieved by using a factorial design approach on parameters such as extraction time, temperature and sample amount. The analysis of extracts has been carried out by reversed-phase high-performance liquid chromatography with fluorescence detection for quantification and UV-diode-array detection for confirmation. The MAE procedure yielded extracts that could be analyzed directly without any preliminary clean-up or solvent exchange steps.     

New strategy for the determination of microcystins and diarrhetic shellfish poisoning (DSP) toxins, two potent phosphatases 1 and 2A inhibitors and tumor promoters

A new analytical strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal.     

Analysis of aged sulfadiazine residues in soils using microwave extraction and liquid chromatography tandem mass spectrometry

An efficient extraction of sulfadiazine residues from soils is difficult, as sulfadiazine is known to form quickly sequestering residues. The objective of this study was to optimize an exhaustive extraction for aged residues of sulfadiazine and its two major metabolites, N-acetylsulfadiazine and 4-hydroxysulfadiazine, from soil. For this purpose two representative used agricultural soils (Luvisol, Cambisol) were blended with manure derived from [¹⁴C]sulfadiazine-treated pigs and incubated at 10 °C in the laboratory. After different extraction tests with various solvent mixtures (two- to four-component mixtures with water, methanol, acetonitrile, acetone, and/or ethyl acetate), different pH values (pH 4 and 9), and extraction temperatures (up to 200 °C), soil extracts were measured by liquid scintillation counting and liquid chromatography coupled to tandem mass spectrometry. With respect to sulfadiazine yields, stability of soil extracts, and the amount of coextracted matrix, a microwave extraction of soil (15 min, 150 °C) using acetonitrile/water 1:4 (v/v) is the method of choice for the exhaustive extraction of aged sulfadiazine residues from soils.     

分散固相萃取/高效液相色谱-串联质谱法检测大葱中虫酰肼、涕灭威及其衍生物的农药残留

采用分散固相萃取法净化(DSPE),高效液相色谱-串联质谱法(HPLC-MS/MS)在分时段多反应监测模式下对大葱中的虫酰肼、涕灭威及其衍生物进行测定,外标法定量,并对比了乙酸乙酯和乙腈作为提取剂的提取效果.结果表明,采用乙腈为提取剂时,4种农药在10～120μg/L范围内线性关系良好,且方法的定量下限(LOQ)均低于...     

New strategy for the determination of microcystins and diarrhetic shellfish poisoning (DSP) toxins, two potent phosphatases 1 and 2A inhibitors and tumor promoters

A new analytical strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal. Received: 23 July 1999 / Revised: 9 September 1999 / Accepted: 16 September 1999     

Establishment of pressurized liquid extraction followed by HPLC–MS/MS method for the screening of adrenergic drugs,steroids, sedatives,colorants and antioxidants in swine feed

A facile and sensitive multi‐residue detection approach of pressurized liquid extraction following high‐performance liquid chromatography tandem mass spectrometry was established to detect the residues of adrenergic drugs, steroids, sedative, colorant and antioxidant in feed. The conditions employed for pressurized liquid extraction involved acetonitrile/ethyl acetate (1:1, v/v) as the extracting solvent, the temperature 80°C, two cycles and a static time of 10 min. The extraction was followed by a solid‐phase extraction clean‐up step. The separation of samples was done by C₁₈ column with the mobile phase of 5 mM ammonium acetate solution and acetonitrile with 0.1% formic acid. The limits of quantification ranged from 0.03 to 1 μg/kg, limits of detection were in a range of 0.01–0.5 μg/kg, and average recoveries were 70.4–98.6%. The pressurized liquid extraction procedure was optimized and overall method was validated in terms of sensitivity, linearity, selectivity, matrix effect, accuracy, recovery and stability of the target drugs in the pressurized liquid extraction extracts solution. The screening method was proved to be fast, selective, accurate and sensitive for screening drugs.     

Determination of Microcystins in Cyanobacteria by Supercritical Fluid Extraction and Liquid Chromatography‐Tandem Mass Spectrometry

Abstract

A method for the detection of microcystins (microcystin LR, RR, and YR) in cyanobacteria by supercritical fluid extraction (SFE) and liquid chromatography‐mass spectrometry (LC/MS) has been developed. Supercritical fluids for the analytical extraction of nonvolatile, higher molecular weight compound, and microcystins from cyanobacteria were investigated. The microcystins included in this study are sparsely soluble in neat supercritical fluid CO₂. However, the microcystins was successfully extracted with a ternary mixture (90% CO₂, 9.5% methanol, 0.5% water) at 40°C and 250 atm. The polar carbon dioxide‐aqueous methanol fluid system gave high extraction efficiency for the extraction of the polar microcystins from cyanobacteria. The microcystins were determined by liquid chromatography‐tandem mass spectrometry (LC/MS/MS).     

Comparison of supercritical fluid and Soxhlet extractions for the isolation of nitro compounds from soils

Supercritical fluid extraction (SFE) with CO(2), a clean and rapid alternative to conventional Soxhlet extraction, was investigated for the extraction of nitro compounds from soil samples. Quantitative extraction by SFE was accomplished at a pressure of 25 MPa and an extraction temperature of 60 degrees C, for 30 min in dynamic mode and using acetonitrile as modifier, and the results were comparable with those obtained by acetonitrile Soxhlet extraction (3 h) for all soil samples. Extracts from these two procedures were analyzed by gas chromatography coupled with mass spectrometry. Quantitative reproducibility for SFE extracts was acceptable (RSD 2-10%), and the quantity of solvent was reduced from 160 mL for Soxhlet extraction to 5 mL in the case of SFE.     

Isolation and preparative purification of microcystin variants

Preparative reversed-phase liquid chromatography was successfully used to purify two microcystins (microcystin LR and microcystin LA) from a cyanobacterial process waste. The separation protocol involved extraction of lyophilized cells by methanol, isolation and concentration by solid-phase extraction, and purification by reversed-phase HPLC. Milligram-level loading of microcystins was obtained on a solid-phase extraction cartridge packed with 0.5 g of C18 stationary phase. The separations were first carried out on an analytical column and then scaled-up to a preparative column. The microcystins were quantified by HPLC and enzyme-linked immunosorbent assay. A method to remove microcystins rapidly and economically from the cyanobacterial process waste is also described.     

Determination of chlorpyrifos in soils using multi-throughput dynamic microwave-assisted extraction coupled online with salting-out-assisted liquid–liquid extraction followed by LC–MS/MS

A simple and rapid method based on multi-throughput dynamic microwave-assisted extraction coupled online with salting-out-assisted liquid–liquid extraction was developed for the analysis of chlorpyrifos in soil. First, the chlorpyrifos was extracted with acetonitrile aqueous (50%, v/v) under the action of microwave energy. Then the obtained extract was separated clearly and easily into an acetonitrile phase and an aqueous phase with the assistance of ammonium acetate. The acetonitrile phase containing chlorpyrifos was concentrated and determined by liquid chromatography–tandem mass spectrometry. The effects of parameters on extraction efficiency including microwave power, extraction solvent, volumes and flow rate of extraction solvent, sample pH, types and amount of salt were studied and optimised. To eliminate the matrix effect, validation of the method was carried out using the matrix-based calibration curve. The limits of detection and quantification for chlorpyrifos were 0.17 and 0.5 ng g^?1, respectively. The proposed method was applied to analyse chlorpyrifos in five soil samples and verified by the recovery test. The recoveries of chlorpyrifos at three spiked levels (5, 50, 200 ng g^?1) were in the range of 90.0–100.5%, with relative standard deviations varying from 1.3% to 5.7%. Compared with the methods reported previously, the proposed method can simplify the operation procedure and reduce solvent consumption in sample pretreatment.     

Optimization of flavanones extraction by modulating differential solvent densities and centrifuge temperatures

Understanding the factors influencing flavonone extraction is critical for the knowledge in sample preparation. The present study was focused on the extraction parameters such as solvent, heat, centrifugal speed, centrifuge temperature, sample to solvent ratio, extraction cycles, sonication time, microwave time and their interactions on sample preparation. Flavanones were analyzed in a high performance liquid chromatography (HPLC) and later identified by liquid chromatography and mass spectrometry (LC-MS). The five flavanones were eluted by a binary mobile phase with 0.03% phosphoric acid and acetonitrile in 20 min and detected at 280 nm, and later identified by mass spectral analysis. Dimethylsulfoxide (DMSO) and dimethyl formamide (DMF) had optimum extraction levels of narirutin, naringin, neohesperidin, didymin and poncirin compared to methanol (MeOH), ethanol (EtOH) and acetonitrile (ACN). Centrifuge temperature had a significant effect on flavanone distribution in the extracts. The DMSO and DMF extracts had homogeneous distribution of flavanones compared to MeOH, EtOH and ACN after centrifugation. Furthermore, ACN showed clear phase separation due to differential densities in the extracts after centrifugation. The number of extraction cycles significantly increased the flavanone levels during extraction. Modulating the sample to solvent ratio increased naringin quantity in the extracts. Current research provides critical information on the role of centrifuge temperature, extraction solvent and their interactions on flavanone distribution in extracts.