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...     

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.     

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     

芯片液相色谱-质谱法高灵敏度检测水产品中微囊藻毒素

建立了一种芯片液相色谱-高分辨质谱测定3种微囊藻毒素（MC-RR、MC-YR和MC-LR）的分析方法.在最优条件下,3种藻毒素在16 min内实现基线分离,并选择电喷雾源正离子模式进行质谱测定.方法在2.5~2 000 ng/mL范围内线性良好,线性相关系数大于0.996 5,检测限介于0.5~2.0 ng/mL之间.鱼肉和虾肉样品经固相萃取法净化处理后未测得上述毒素残留,加标回收率为82.8%~120.3%.方法具有样品用量少、定性快速准确、灵敏度高等优点,适合用于水产品等生物样品中藻毒素的痕量监测.     

固相萃取高效液相色谱法测定水中痕量-微囊藻毒素

微囊藻毒素是有害的蓝藻水华释放的有毒代谢物,对人类及环境具有很大危害性。建立了固相萃取－高效液相色谱测定水中痕量藻毒素的方法。该法对两种常见微囊藻毒素MC－LR、MC－RR的检测限为0．02－0．05μg/L,线性定量范围为0．1－50μg/L。应用该法分析了天然水样,表明方法具有实用性。     

Extraction and clean-up strategies for the analysis of poly- and perfluoroalkyl substances in environmental and human matrices

The rapidly expanding field of per- and polyfluorinated alkyl substances (PFASs) research has resulted in a wide range of analytical methodologies to determine the human and environmental exposure to PFASs. This paper reviews the currently applied techniques for sample pre-treatment, extraction and clean-up for the analysis of ionic and non-ionic PFASs in human and environmental matrices. Solid phase extraction (SPE) is the method of choice for liquid samples (e.g. water, blood, serum, plasma), and may be automated in an on-line set-up for (large volume) sample enrichment and sample clean-up. Prior to SPE, sample pre-treatment (filtration or centrifugation for water or protein precipitation for blood) may be required. Liquid-liquid extraction can also be used for liquid samples (and does not require above mentioned sample pretreatment). Solid-liquid extraction is the commonly applied method for solid matrices (biota, sludge, soil, sediment), but automation options are limited due to contamination from polytetrafluorethylene tubings and parts applied in extraction equipment. Air is generally preconcentrated on XAD-resins sandwiched between polyurethane foam plugs. Clean-up of crude extracts is essential for destruction and removal of lipids and other co-extractives that may interfere in the instrumental determination. SPE, (fluorous) silica column chromatography, dispersive graphitized carbon and destructive methods such as sulphuric acid or KOH treatment can be applied for clean-up of extracts. Care should be taken to avoid contamination (e.g. from sample bottles, filters, equipment) and losses of PFASs (e.g. adsorption, volatilization) during sampling, extraction and clean-up. Storage at -20 degrees C is generally appropriate for conservation of samples.     

Use of gel permeation chromatography for automatic and rapid extract clean-up for the determination of diarrhetic shellfish toxins (DSP) by liquid chromatography-mass spectrometry

Summary A new analytical technique was established to improve the performance of DSP toxin determination in different sample matrices. High performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algal cells and mussel tissue prior to the determination of DSP toxins by LC/MS. The proposed protocol can be performed totally automatically and enables fast and sensitive analysis of large sample numbers. The recovery of the entire method protocol (consisting of extraction, clean-up and LC/MS determination) was approximately 70% with good repeatability (standard deviation ranging from 1.9% to 5.0% in the concentration range analyzed).     

Clean-up techniques in the pressurized liquid extraction of abiotic environmental solid samples

Pressurized liquid extraction (PLE) uses high pressure and temperature to perform exhaustive extraction and is one of the extraction techniques widely used for determining contaminants in environmental solid samples. However, under these conditions, compounds from the matrix are also extracted. Therefore, clean-up strategies must be often applied to remove interferences and avoid matrix effect in the subsequent determination. There are different clean-up strategies that can be used in PLE, some of which are applied during the extraction procedure and some are applied to the PLE extract. The aim of this review is to critically discuss these clean-up techniques used in PLE of abiotic environmental solid samples in the last 10 years. We provide the readers with information about the weaknesses and strengths of each strategy so they can select the most suitable clean-up technique for a specific environmental analytical problem.     

全自动在线固相萃取-高效液相色谱法测定水体中痕量微囊藻毒素

建立了全自动化在线固相萃取-高效液相色谱法快速、准确测定水体中3种痕量微囊藻毒素的新方法.样品用自动进样器连续注入固相萃取小柱完成富集后,通过双梯度高效液相系统中的一个泵(上样泵)实现净化,再通过阀切换将固相萃取小柱切换至分析流路中,用另一个泵(分析泵)将待测物冲洗至分析柱进行测定.方法使用Acclaim PA柱芯(1...     

Simplified determination of polycyclic aromatic hydrocarbons

Accurate quantitative analysis for selected polycyclic aromatic hydrocarbons present on urban dust can be obtained by using a simple procedure consisting of sonic-probe extraction with cyclohexane; clean-up with Florisil((R))-XAD-4((R)), and measurement by high-resolution gas chromatography with flame-ionization detection (HRGC/FID). The analysis can be further simplified by eliminating the clean-up step if HRGC/electron-impact mass-spectrometry (MS) is available. Both the FID and MS methods give results consistent with those obtained by standard procedures. The direct HRGC/MS procedure, combined with chemical ionization, can also be applied to the determination of polycyclic organic materials present in solvent-refined coal, shale oil and crude oil.     

Bestimmung von isomeren 4-Nonylphenolen in B?den

Summary It was reported that 4-nonylphenol isomers had been found in high concentrations in sewage sludge. When this sludge is applied to fields there is a risk that soils may be contaminated with nonylphenols. Therefore, a method is described here for the determination of 4-nonylphenol isomers in soil. The steam distillation/solvent extraction technique is used. An essential part of the method is the clean-up step by column chromatography. The determination is performed on a fused silica capillary by a flame ionization or a mass selective detector. Average recoveries from soil samples spiked with 0.1 to 1 mg/kg ranged from 90 to 116%. The detection limit is 0.1–0.05 mg/kg soil (this limit corresponds to the technical mixture of the isomers).     

Determination of dexamethasone in feed by TLC and HPLC

Two detection methods are described for the determination of dexamethasone in feed, which may be used as screening and confirmatory methods. The procedures were developed after studying different extraction and clean-up procedures: feed extraction was optimized using methylene chloride and hexane followed by a tandem solid-phase clean-up step with silica and C18 cartridges. The confirmatory method presented is based on normal-phase high-performance liquid chromatography on a Diol 5 microns column, with hexane-propan-2-ol (90:10 v/v) as mobile phase, followed by diode array detection and confirmation. All parameters are discussed, especially the extraction and clean-up steps. Recovery studies after application of the proposed method to different kinds of feed samples spiked with dexamethasone at levels between 120 and 1600 ng g-1 yielded a mean value of 55.4% with an overall standard deviation of 15.6%. The proposed procedure allows the determination of dexamethasone in feed at levels lower than 50 ng g-1.     

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.     

Methodological Challenges of Protein Analysis in Blood Serum and Cerebrospinal Fluid by Capillary Electrophoresis

Summary High-performance capillary electrophoresis (HPCE or CE) is an ultrasensitive analytical technique with high resolving power and a wide area of applications including peptide/protein analysis. Its applicability is greatly enhanced by the short separation times, the ease of method development and the minimum sample and organic solvent requirements. Various HPCE modes have been developed for peptide/protein analysis, including capillary zone electrophoresis, micellar elektrokinetic capillary chromatography, capillary isoelectric focusing, isotachophoresis, capillary gel electrophoresis and microemulsion elektrokinetic chromatography. HPCE can easily be applied to quality control of manufacturing processes or to clinical routine for diagnostic purposes due to its potential to provide information on the identity, the purity of the samples and the quantities of the constituents. Furthermore, interactions of a peptide or a protein with other molecules can be studied by HPCE. The separation principles of the various operation modes applied to peptide/protein analysis are presented in this article. Furthermore, in order to exemplify the application of the separation principles in the area of serum protein analysis, which is of importance in clinical practice, the capillary electrophoretic methods developed for analysis of serum and cerebrospinal fluid proteins are also reviewed.Presented at: International Symposium on Separation and Characterization of Natural and Synthetic Macromolecules, Amsterdam, The Netherlands, February 5–7, 2003     

色谱／质谱定量分析技术在纺织品禁用偶氮染料测试中的应用

建立了偶氮染料印染的纺织品中有害芳胺的ＧＣ／ＭＳ定量分析方法。     

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 emerging organic contaminants in environmental solid samples

Spreading sewage sludge on agricultural lands has been actively promoted by national authorities as an economic way of recycling. However, as by-product of wastewater treatment, sewage sludge may contain toxic substances, which could be incorporated into agricultural products or be distributed in the environment. Moreover, sediments can be contaminated by the discharge of wastewater effluents into rivers. This article reviews the determination of emerging contaminants (surfactants, flame retardants, pharmaceuticals and personal care products) in environmental solid samples (sludge, soil and sediment). Sample preparation, including extraction and clean-up, as well as the subsequent instrumental determination of contaminants are discussed. Recent applications of extraction techniques, such as Soxhlet extraction, ultrasound assisted extraction, pressurised liquid extraction, microwave assisted extraction and matrix solid-phase dispersion to the analysis of emerging contaminants in environmental solid samples are reviewed. Determination of these contaminants, generally carried out by gas chromatography and liquid chromatography coupled with different detectors, especially mass spectrometry for the identification and quantification of residues, is also summarised and discussed.      

Use of solid phase extraction for the isolation and clean-up of a derivatised furazolidone metabolite from animal tissues

A method is presented for the determination of protein-bound residues of furazolidone in animal tissue. The use of furazolidone in food-producing animals has been banned in the EU. Illegal use of furazolidone can be monitored most effectively by testing for bound residues containing the 3-amino-2-oxazolidone (AOZ) moiety which, unlike the parent drug, is stable and can be detected for prolonged periods after cessation of treatment. This paper reports the development of an extraction and clean-up procedure for AOZ from liver using solid phase extraction. The method replaces solvent extraction and provides extensive sample clean-up with removal of approximately 99% of the derivatising agent, 2-nitrobenzaldehyde, which may interfere with the determination. It also offers the advantage of being suitable for automation, thereby increasing throughput of samples. The extraction procedure may be used for HPLC and ELISA screening techniques. The method has been validated in fortified and incurred pig liver samples, yielding mean recovery of AOZ in excess of 60%.     

High-performance capillary electrophoresis for characterization of hapten—protein conjugates used for production of antibodies against soyasaponin I

Micellar electrokinetic capillary chromatography using sodium cholate as the micellar phase has been investigated for characterization of hapten—protein conjugates. Special focus has been placed on the hapten soyasaponin I which is a quantitatively dominating glycoside in seeds of several legumes including pea (Pisum sativum L.) and soybean [Glycine max (L.) Merr.]. Soyasaponin I has been isolated from pea and used as hapten for production of anti-saponin specific polyclonal antibodies. Soyasaponin I was coupled to Kunitz soybean trypsin inhibitor (KSTI) and bovine serum albumin. The degree of coupling was determined by high-performance capillary electrophoresis (HPCE). Capillaries dynamically coated with zwitterions were found to be efficient for reduction of interaction between the silica capillary surface and the proteins. The applicability of HPCE for determination of coupling density was confirmed by investigation of a model hapten (p-nitrophenyl-- -galactoside; PNPG) coupled to KSTI. The PNPG—KSTI conjugates were examined by both HPCE and by spectrophotometric determination of the PNPG density on KSTI. The HPCE method was shown to be efficient in studies of the formation of hapten—protein conjugates and to be more specific than alternative techniques applied for determination of coupling densities.     

Validation and robustness testing of a HPLC method for the determination of avermectins and moxidectin in animal liver samples using an alumina column clean-up

A multi-residue method has been developed for the quantitative determination of moxidectin, abamectin, doramectin and ivermectin in liver samples, with capability for qualitative identification of the presence of eprinomectin. Liver samples are extracted with isooctane, followed by clean-up on alumina-N solid phase extraction (SPE) cartridges. Extracts are derivatised and determined by high-performance liquid chromatography (HPLC) with fluorescence detection. The method was validated using bovine liver fortified at levels of 4 and 20 micrograms kg-1 with the drugs. The mean recovery from bovine liver ranged between 90 and 96%. The intra and inter-assay variations showed RSD typically of < 5% and < 10%, respectively. The procedure was applied also to ovine and porcine liver, giving similar results. A robustness study, carried out on the alumina clean-up step, indicated that the step is relatively insensitive to method changes. However, significant differences overall were found for the type of alumina and/or commercial SPE cartridge used. The limit of quantitation of the method is 2 micrograms kg-1 (ppb).