Surface and wastewater quality monitoring: combination of liquid chromatography with (geno)toxicity detection,diode array detection and tandem mass spectrometry for identification of pollutants

Identification of unknown water pollutants with liquid chromatography and tandem mass spectrometry (LC–MS–MS) is often more complex and time consuming than identification with gas chromatography and mass spectrometry (GC–MS). In order to focus the identification effort on relevant compounds, unknown peaks need to be selected carefully. Based on its frequency of occurrence in the LC–Diode Array Detection (LC–DAD) chromatograms of surface and infiltrated waters, an unknown peak was selected for identification with LC–MS–MS. This compound was identified as hexamethoxymethylmelamine (HMMM), a chemical often used in the coating industry. This is the first time the presence of this chemical in surface waters has been reported. In addition to HMMM, two other structurally related compounds were found to be present in the investigated surface water. A standard mixture of HMMM and its by-products did not exhibit (geno)toxicity under the test conditions applied in this study. In another example, a genotoxic fraction of an industrial wastewater was isolated and examined by LC–MS–MS using a modern quadrupole–orthogonal acceleration-time-of-flight mass spectrometer (Q-TOF). Four compounds were detected. The structures of two compounds present are proposed to be 9-amino-2-hydroxy-acridine and 9-hydroxy-acridine-N-oxide or its structural isomer dihydroxy-acridine. Confirmation with standards could not be carried out, as pure compounds are not available. The other two compounds (structural isomers) could not be identified based on the data available within this study.     

Lower limits of detection in speciation analysis by coupling high-performance liquid chromatography and chemical-vapor generation

Speciation studies are much more important than total element determination because toxicity of many elements depends on their chemical forms. Nobody can claim that a foodstuff is very dangerous to eat by determining total arsenic due to the possibility that the arsenic could be present in non-toxic forms. Hence, speciation studies are crucial in any matrix relevant to human beings.Trace-element speciation requires sufficiently sensitive procedures to monitor each species at trace levels. One way to increase the sensitivity for elements forming volatile species is coupling high-performance liquid chromatography (HPLC) with chemical-vapor generation (CVG). This review aims to highlight not only development of HPLC-CVG techniques for ultratrace-elemental speciation in a variety of matrices but also their application. In addition, we discuss the advantages and the disadvantages of these techniques.     

Comparison between liquid chromatography-UV detection and liquid chromatography-mass spectrometry for the characterization of impurities and/or degradants present in trimethoprim tablets

The characterization of impurities and/or degradants present in pharmaceutical compounds is an important part of the drug development process. Although LC–UV is commonly employed for impurities and degradant compound determination, LC–MS techniques are proposed in this work to be a viable modern alternative for the characterization of these compounds. LC–UV and LC–MS were compared for the detection of impurities present in different brands of trimethoprim tablets by using an in-line LC–UV–MS system with atmospheric pressure chemical ionization source (APCI) coupled with a reversed-phase gradient HPLC system. It was shown that, although chemical noise was higher when using full-scan LC–MS compared to LC–UV, low level impurities were better detected by mass spectrometry (MS) when modern software algorithms are employed. These included the “Contour” chromatogram algorithm and/or the “component detection algorithm” (CODA). In addition, MS allowed for the simultaneous determination of the molecular masses and some structural information of the impurities and/or degradants. The results also showed a large difference in the purity of trimethoprim among different manufacturers. LC–MS and tandem MS techniques were employed to acquire fragmentation patterns for trimethoprim and its degradants to gain insight into their structures.     

Determination of theophylline and its metabolites in biological samples by liquid chromatography-mass spectrometry

Liquid chromatography–mass spectrometry (LC–MS) is a powerful tool for analysis of drugs and their metabolites. We used a column-switching system in combination with atmospheric pressure chemical ionization LC–MS (LC–APCI–MS) for the determination of theophylline and its metabolites in biological samples. The separation was carried out on a reversed-phase column using methanol–20 mM ammonium acetate as a mobile phase at a flow-rate of 1 ml/min in 30 min. In the mass spectrum, the molecular ions of these drugs and metabolites were clearly observed as base peaks. This method is sufficiently sensitive and accurate for the pharmacokinetic studies of these drugs.     

Separation and characterization of multicomponent peptide mixtures by liquid chromatography-electrospray ionization mass spectrometry. Application to crude products of the synthesis of leuprolide

Leuprolide is a synthetic structural analogue of luteinizing hormone-releasing hormone used for the treatment of a large number of diseases related with the regulation of sexual hormones. Solid-phase peptide synthesis is used to obtain leuprolide peptidic hormone, but this synthetic procedure results in complex mixtures that need separation and characterization. Here, liquid chromatography coupled with mass spectrometry using electrospray ionization, (LC–ES-MS), was used for the separation and characterization of multicomponent peptide mixtures of crudes of synthesis of leuprolide. To optimize the LC separation process, the method of linear solvation energy relationships was applied and the powerful coupling LC–ES-MS permitted rapid and reliable characterization of the reaction product.     

Mass spectrometric determination of ergosterol in a prairie natural wetland

Packed capillary liquid chromatography–electrospray mass spectrometry (LC–ESI-MS) was used for the analysis of a snow sample that was accidentally contaminated with an organophosphorus chemical warfare agent during the destruction of a chemical munition. Sarin, its hydrolysis products and a number of related compounds were identified on the basis of acquired LC–ESI-MS data. Full mass spectra were acquired for 14 compounds, with all exhibiting MH⁺, [MH+ACN]⁺ ions and/or protonated dimers that could be used to confirm molecular mass. Sampling cone voltages from 20 to 70 V were utilized with the higher sampling voltages enhancing formation of structurally important product ions in the ESI interface. All data were acquired with a time-of-flight mass spectrometer with a resolution of 5000 (50% valley definition), a resolution that aided in the assignment of elemental composition of the observed ions. The application of LC–ESI-MS to snow analysis appears to be an attractive alternative to the GC–MS methods, since both chemical warfare agents and their hydrolysis products may be analysed directly, eliminating the need for additional sample handling and derivatization steps.     

Applications of solid-phase microextraction in food analysis

Food analysis is important for the evaluation of the nutritional value and quality of fresh and processed products, and for monitoring food additives and other toxic contaminants. Sample preparation, such as extraction, concentration and isolation of analytes, greatly influences the reliable and accurate analysis of food. Solid-phase microextraction (SPME) is a new sample preparation technique using a fused-silica fiber that is coated on the outside with an appropriate stationary phase. Analyte in the sample is directly extracted to the fiber coating. The SPME technique can be used routinely in combination with gas chromatography (GC), GC–mass spectrometry (GC–MS), high-performance liquid chromatography (HPLC) or LC–MS. Furthermore, another SPME technique known as in-tube SPME has also been developed for combination with LC or LC–MS using an open tubular fused-silica capillary column as an SPME device instead of SPME fiber. These methods using SPME techniques save preparation time, solvent purchase and disposal costs, and can improve the detection limits. This review summarizes the SPME techniques for coupling with various analytical instruments and the applications of these techniques to food analysis.     

Energetic heterogeneity of the surface of a molecularly imprinted polymer studied by high-performance liquid chromatography

A sequential combination of reversed-phase liquid chromatography–mass spectrometry (LC–MS) and capillary electrophoresis (CE) has been explored in order to perform separation and characterization of a multicomponent peptide mixture from the synthesis of leuprolide. The mixture was first analyzed and fractionated by LC–MS, and the collected fractions were subsequently separated by CE. Unambiguous identification of the electrophoretic peaks was achieved by injecting the collected fractions separately and spiking the leuprolide crude mixture. Furthermore, structural information about the components of the mixture provided by several semi-empirical migration models has been used to check the accuracy of the structures previously proposed by LC–MS. Combination of the two orthogonal techniques results in an enhancement of their individual selectivity characteristics.     

Progress in liquid chromatography-mass spectrometry instrumentation and its impact on high-throughput screening

In the past 10 years, liquid chromatography–mass spectrometry (LC–MS) has rapidly matured to become a very powerful and useful analytical tool that is widely applied in many areas of chemistry, pharmaceutical sciences and biochemistry. In this paper, recent instrumental developments in LC–MS-related interfacing, ionization and mass analysis are reviewed from the perspective of the application of LC–MS in high-throughput screening of combinatorial libraries and the related high-throughput quantitative bioanalysis in early drug-discovery studies, such as early adsorption, distribution, metabolism and excretion studies.     

Speciation of nickel,copper, zinc,and manganese in different edible nuts: a comparative study of molecular size distribution by SEC–UV–ICP–MS

Molecular size distribution patterns of Cu, Mn, Ni, and Zn were determined in several nut species by size-exclusion liquid chromatography (SEC) coupled on-line to UV and inductively coupled plasma mass spectrometry (ICP–MS) for detection. The molecular weight (MW) fractionation of the different metals was performed with a Superdex Peptide column, injecting 100 L of the extracted solutions. The association of the elements with different MW fractions was observed with sequential detection by UV and ICP–MS. Various separation conditions were evaluated to obtain proper resolution and reproducible results with the size-exclusion column. Complete MW information of the elemental fractions in the nut samples was obtained within a retention time of 30 min. Fractionation of the above mentioned elements was done in nine different nut species commonly found in commercial markets. Variability of the fractionation patterns for two different extraction media, 0.05 mol L^–1 NaOH and 0.05 mol L^–1 HCl, was evaluated for every nut sample. Differences in the elemental fractionation patterns were found depending on the extraction procedure, nut species, and the type of element studied. It was also observed that the elements studied showed predominant association with high MW fractions when extracted with basic solution whereas with acidic extraction media only low MW fractions were obtained.     

Speciation of trace elements in biological materials: trends and problems.

In the determination of trace elements in biological materials, speciation is of particular importance as the essential effects or toxicity of an element and its metabolic behaviour depend to a large extent on the chemical forms in which it is present in the organism. Speciation is relatively easy if a property of a particular compound can be measured directly in the sample without interference from the other components of the material, e.g., the enzymic activities of the metallo-enzymes. Another possibility for speciation is immunoassay, which likewise allows direct determination of a particular trace element. At present, however, with most trace elements both fractionation methods and analytical procedures have to be combined and speciation has to be carried out by determining the elemental content in the separated fractions. The methods and apparatus used in taking, storing and preparing the samples can, therefore, not be selected solely according to the requirements of trace element determination, but it is also essential to ensure that the biological structures of the components to be separated remain intact. In this work the need for speciation in the investigation of the toxic and essential effects of trace elements is shown with the help of some examples, and the problems that can occur in the various steps of sampling, storage and sample preparation are discussed.     

Quantitative analysis of cytokinins in plants by liquid chromatography–single-quadrupole mass spectrometry

A sensitive method for the quantitative analysis of all natural isoprenoid cytokinins in plant material by electrospray single-quadrupole mass spectrometry is presented. A baseline chromatographic separation of 20 non-derivatised naturally occurring cytokinins has been developed. Precise analyses of O-glucoside and ribonucleotide fractions were also performed by the high-performance liquid chromatography–mass spectrometry (HPLC–MS) but run separately from the basic cytokinin metabolites. Using post-column splitting, the flux from narrow-bore (2.1 mm i.d.) reversed-phase liquid chromatography column was simultaneously introduced into the diode array and mass detector. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. When low cone voltage (20 V) was applied, all studied cytokinins were determined in aqueous methanol as dominant quasi-molecular ions of [M+H]⁺ with limits of detection ranging between 10 and 50 fmol. For routine analysis a linearity range between 25 (75) fmol and 100 pmol was obtained. Developed liquid chromatography–mass spectrometry (LC–MS) method in selective ion monitoring mode was employed to quantify cytokinin species in tobacco BY-2 suspension culture and poplar leaves (Populus×canadensis Moench, cv Robusta).

Purified plant cell (BY-2) and plant tissue (poplar leaves) extracts were obtained by using two different ion-exchange chromatography steps, in combination with immunoaffinity purification using a broad-spectrum monoclonal anti-cytokinin antibody. The antibody strongly recognises the presence of N⁶-substituent on purine skeleton and thus does not bind adenine and related compounds. The presence of authentic cytokinins in the extracts quantified by LC–MS was further verified by enzyme-linked immunosorbent assays (ELISAs) with prior LC preparation. The combination of liquid chromatography–single-quadrupole mass spectrometry with immunoaffinity chromatography offers an efficient and elegant method for detection and quantification of cytokinin metabolites.     

Simple ion chromatographic method for the determination of chlormequat residues in pears

Current methods for quantitative determination of chlormequat residues in food crops are characterized by rather low recoveries and the need for derivatization (in case of gas chromatography, GC), or by high capital investment (in case of liquid chromatography–mass spectrometry, LC–MS). We propose a cation-exchange chromatography method for the analysis of chlormequat in pears. The method is based on extraction of the target compound with 40 mM HCl, followed by centrifugation and filtration. The filtrate is directly injected into an ion chromatograph equipped with a commercially available cation-exchange column and a suppressed conductivity detection system. While the limit of detection (LOD) (0.5 mg/kg) may not be small enough to allow dietary analysis, the method meets all validation requirements and is an alternative for the existing GC and LC–MS methods in quality control.     

Chemical separation and inductively coupled plasma–atomic emission spectrometric determination of seventeen trace metals in thorium oxide matrix using a novel extractant – Cyanex-923

Comprehensive studies have been carried out on the extraction behaviour of thorium matrix vis-a-vis 17 trace metallic elements using a novel extractant viz. Cyanex-923. The near total extraction of thorium and quantitative separation of these metals has been established using inductively coupled argon plasma–atomic emission spectrometry (ICP–AES). The recovery of few representative elements has been confirmed by radio-active tracer studies. The studies carried out here have enabled determination of μg/l amounts of all analyte elements with a precision of better than 1% RSD with prior chemical separation from as low as 1 g thorium sample in just five chemical extractions.     

Improvements of Speciation Analyses in Environmental Matrices

The release of some organometallic compounds and other chemical forms of elements in the environment has led to great international concern because of their high toxicity. The validation of the analytical techniques became of paramount importance which led the Community Bureau of Reference (BCR) to decide on the organisation of a series of projects for the improvement of the quality of speciation analyses. In addition, it was found useful to discuss thoroughly the different sources of error likely occurring in speciation analyses and a workshop was organized for this purpose; the aim of this workshop was to discuss the state of the art of speciation determinations, to define use, applicability and necessity of determinations of element species, to investigate where limitations exist and discuss the work necessary to overcome these and to detect where techniques have sufficiently been developed to produce reliable and valuable results. This paper presents the organization of the workshop, its main issue and describe the state of the current BCR projects on speciation.Abbreviations AAS Atomic absorption spectrometry - CVAAS Cold vapour AAS - DPP Differential pulse polarography - ECD Electron capture detector - ETAAS Electrothermal AAS - FIA Flow injection analysis - FID Flame ionisation detector - FLUOR Spectrofluorimetry - FPD Flame photometric detector - GC Gas chromatography - HG Hydride generation - HPLC High performance liquid chromatography - HPLC-AES HPLC atomic emission spectrometry - INAA Instrumental neutron activation analysis - ICPAES Inductively coupled plasma AES - MS Mass spectrometry - QFAAS Quartz furnace AAS - RNAA Neutron activation analysis with radiochemical separation     

On-line coupled liquid chromatography-gas chromatography

On-line coupled liquid chromatography–gas chromatography (LC–GC) is a powerful technique that combines the best features of LC and GC and is ideal for the analysis of complex samples. This review describes the unique features of on-line coupled LC–GC. The different interfaces and evaporation techniques are presented, along with their advantages and disadvantages. Guidelines are given for selecting a suitable LC–GC technique and representative applications are noted.     

Analysis of selenium species by capillary electrophoresis

The presence of selenium in the form of different species in environmental and biological samples receives an increasing attention due to better understanding of its bioavailability, toxicity and transport mechanism. For many years, gas and liquid chromatography have been extensively explored in speciation analysis of this element. Recently, capillary electrophoresis (CE) has made much progress in this field. This review presents the developments in the application of CE for simultaneous separation and determination of different selenium compounds. Various separation approaches and detection methods as well as pre-concentration techniques are discussed. The speciation performance of CE is illustrated by a number of practically relevant applications.     

Speciation of essential and toxic elements in edible mushrooms: size‐exclusion chromatography separation with on‐line UV–inductively coupled plasma mass spectrometry detection

Size‐exclusion liquid chromatography was coupled to UV and inductively coupled plasma mass spectrometry (ICP‐MS) for detection to perform elemental speciation studies on different edible mushrooms. Molecular weight (MW) distribution patterns of several elements among different fractions present in various edible mushrooms are presented. The association of the elements with the high and low MW fractions was observed using sequential detection by UV and ICP‐MS. Separation was performed using a Superdex 75 column. Variability of the fractionation patterns with three different extraction media (0.05 mol l^?1 NaOH; 0.05 mol l^?1 HCl; hot water at 60°C) was evaluated for mushroom species. A comparative elemental speciation study was performed in order to determine the differences in the fractionation patterns of silver, arsenic, cadmium, mercury, lead, and tin in Boletus edulis, Agaricus bisporus, and Lentinus edodes. Differences in the fractionation patterns of the elements were found to depend on the mushroom species and the extraction medium. Most of the elements were associated with high mw fractions. It was not possible to assess the trace metal contributions from the mushroom growth media. Copyright © 2004 John Wiley & Sons, Ltd.     

高效液相色谱-电感耦合等离子体质谱联用技术用于环境中元素形态分析的最新进展

综述了高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)联用技术在环境科学中的应用情况.参考过去十年内的国内外文献,从方法学的角度论述了HPLC联用ICP-MS技术的发展,重点探讨了该技术在环境领域,尤其是在元素形态分析(包括生物学,生物无机化学和生物医学)中的应用.     

Dimensional and elemental characterization of suspended particulate matter in natural waters: quantitative aspects in the integrated ultrafiltration, splitt-flow thin cell and inductively coupled plasma–atomic emission spectrometry approach

The present study investigates the quantitative aspects of an analytical procedure for the trace element characterization of suspended particulate matter (SPM) in natural waters. The procedure consists of the following steps: (1) ultrafiltration (UF) concentration; (2) splitt-flow thin (SPLITT) cell fractionation (SF) into different micronic–submicronic dimensional ranges; and (3) inductively coupled plasma–atomic emission spectrometry (ICP–AES) elemental determination on both the separated fractions and the bulk phase. One specific feature of the UF/SF steps is that they are gentle and thus preserve the complexity of the colloidal features of SPM samples as far as possible. The investigation was performed on a real SPM sample (Po River, Italy). Two SF modes were considered: the so called conventional SPLITT fractionation (CSF) mode and the full feed depletion SPLITT fractionation (FFDSF) mode. These differ in terms of resolution, time (both better in CSF as compared to FFDSF) and operating mode (FFDSF does not require a diluting carrier). Quantitative aspects of the UF step recovery and of the CSF and FFDSF modes were investigated in terms of total mass balance proving that only the FFDSF mode is currently satisfactory for quantitative purposes. Mass balance versus the following elements: Cd, Cr, Cu, Mn, Ni and Pb, was performed using ICP–AES over the 0.2–1.5 and 1.5–20 μm FFDSF SPM fractions, proving that the analytical procedure based on UF/FFDSF/ICP–AES is consistent and useful in the investigation of trace element distribution in different SPM dimensional ranges versus that of the bulk phase. The relevance of aggregation–solubility equilibria concerning colloids of SPM phase is emphasized and further improvement of the procedure is discussed.