The role of analytical chemistry in exposure science: Focus on the aquatic environment.

Exposure science, in its broadest sense, studies the interactions between stressors (chemical, biological, and physical agents) and receptors (e.g. humans and other living organisms, and non-living items like buildings), together with the associated pathways and processes potentially leading to negative effects on human health and the environment. The aquatic environment may contain thousands of compounds, many of them still unknown, that can pose a risk to ecosystems and human health. Due to the unquestionable importance of the aquatic environment, one of the main challenges in the field of exposure science is the comprehensive characterization and evaluation of complex environmental mixtures beyond the classical/priority contaminants to new emerging contaminants. The role of advanced analytical chemistry to identify and quantify potential chemical risks, that might cause adverse effects to the aquatic environment, is essential. In this paper, we present the strategies and tools that analytical chemistry has nowadays, focused on chromatography hyphenated to (high-resolution) mass spectrometry because of its relevance in this field. Key issues, such as the application of effect direct analysis to reduce the complexity of the sample, the investigation of the huge number of transformation/degradation products that may be present in the aquatic environment, the analysis of urban wastewater as a source of valuable information on our lifestyle and substances we consumed and/or are exposed to, or the monitoring of drinking water, are discussed in this article. The trends and perspectives for the next few years are also highlighted, when it is expected that new developments and tools will allow a better knowledge of chemical composition in the aquatic environment. This will help regulatory authorities to protect water bodies and to advance towards improved regulations that enable practical and efficient abatements for environmental and public health protection.

Hyphenation of coupled-column liquid chromatography and thermospray tandem mass spectrometry for the rapid determination of beta 2-agonist residues in bovine urine using direct large-volume sample injection. Set-up of single-residue methods for clenbuterol and salbutamol.

In a previous work, coupled-column LC (LC/LC) with direct large-volume sample injection for the analysis of beta 2-agonists in bovine urine was investigated, in view of its combination with thermospray (TSP) tandem mass spectrometric (MS/MS) detection. In this work, the potential of both conventional LC and LC/LC coupled to TSP-MS/MS for the rapid determination of beta 2-agonists in bovine urine was evaluated. It was first established that, in order to avoid disturbances in TSP-MS/MS detection due to changes in pressure and flow rate of the mobile phase, LC/LC is preferable to conventional LC. Then, selecting clenbuterol and salbutamol as model compounds for the class of beta 2-agonists, two single-residue LC/LC/TSP-MS/MS methods were developed, permitting the determination of the analytes in bovine urine samples with a limit of quantification of at least 0.1 ng, ml-1, with a sample throughput of 4 h-1. The methods were validated by analysing both spiked urine samples and samples containing clenbuterol or salbutamol residues. Mean recoveries from urine samples spiked with clenbuterol at levels of 5 and 0.22 ng ml-1 were 107% (n = 9, RSD = 7.4%) and 102% (n = 6, RSD = 14%), respectively. A bovine urine sample containing 1.28 ng ml-1 clenbuterol residue (previously quantified by GC/MS) was re-analysed by LC/LC/TSP-MS/MS on four different days (n = 3, each day) and yielded a mean concentration of 1.31 ng ml-1, with a reproducibility of 8.4%. Bovine urine samples spiked with salbutamol at levels of 10.8 and 0.55 ng ml-1 yielded mean recoveries of 101% (n = 3, RSD = 1%) and 89% (n = 3, RSD = 13%), respectively. In bovine urine samples containing salbutamol residues, the concentrations of unconjugated salbutamol were found to be in the range 8-57 ng ml-1.

Rapid determination of glyphosate in cereal samples by means of pre-column derivatisation with 9-fluorenylmethyl chloroformate and coupled-column liquid chromatography with fluorescence detection.

A rapid procedure for the determination of glyphosate in cereals has been developed. Convenient sample pretreatment is carried out by (i) a overnight standing extraction of 1.0 g homogenized sample with 20 ml of water, (ii) centrifugation of the samples, (iii) a passing of 2.5 ml of the clear layer through a 100 mg C18 solid-phase extraction cartridge and (iv) collection of the last 1.5 ml of the eluent into a calibrated tube. For the instrumental analysis, the efficient approach developed earlier for environmental water samples [J.V. Sancho, F. Hernández, F.J. LUpez, E.A. Hogendoorn, E. Dijkman, P. van Zoonen, J. Chromatogr. A, 737 (1996) 75] was successfully adopted for the determination of glyphosate in the obtained cereal extracts. The procedure includes a 15 min derivatisation step of the analyte with 9-fluorenylmethyl chloroformate and a 16 times dilution step prior to instrumental analysis employing coupled-column LC with fluorescence detection. The developed procedure has a sample throughput of more than 25 samples per day and a limit of quantification of 0.5 mg/kg. The method was validated by analyzing freshly spiked cereal samples and samples with aged residues at levels between 1.0 and 10 mg/kg. The overall recovery of the freshly spiked samples was 86% (n = 10) with a repeatability of 6.5% and a reproducibility of 9.5%. For samples with aged residues recoveries performed at different time intervals (range 80-150 days) did not differ significantly; the overall recovery (n = 10) was 74% with a repeatability and reproducibility of 14 and 20%, respectively.

Comparison of various extraction techniques for the determination of polycyclic aromatic hydrocarbons in worms.

Two less laborious extraction methods, viz. (i) a simplified liquid extraction using light petroleum or (ii) microwave-assisted solvent extraction (MASE), for the analysis of polycyclic aromatic hydrocarbons (PAHs) in samples of the compost worm Eisenia andrei, were compared with a reference method. After extraction and concentration, analytical methodology consisted of a cleanup of (part) of the extract with high-performance gel permeation chromatography (HPGPC) and instrumental analysis of 15 PAHs with reversed-phase liquid chromatography with fluorescence detection (RPLC-FLD). Comparison of the methods was done by analysing samples with incurred residues (n=15, each method) originating from an experiment in which worms were exposed to a soil contaminated with PAHs. Simultaneously, the performance of the total lipid determination of each method was established. Evaluation of the data by means of principal component analysis (PCA) and analysis of variance (ANOVA) revealed that the performance of the light petroleum method for both the extraction of PAHs (concentration range 1-30 ng/g) and lipid content corresponds very well with the reference method. Compared to the reference method, the MASE method yielded somewhat lower concentrations for the less volatile PAHs, e.g., dibenzo[ah]anthracene and benzo[ghi]perylene and provided a significant higher amount of co-extracted material.

Comparison of various liquid chromatographic methods involving UV and atmospheric pressure chemical ionization mass spectrometric detection for the efficient trace analysis of phenylurea herbicides in various types of water samples.

The performance of mass spectrometric (MS) detection and UV detection in combination with reversed-phase liquid chromatography without and with the use of coupled column RPLC (LC-LC) has been compared for the trace analysis of phenylurea herbicides in environmental waters. The selected samples of this comparative study originated from an inter-laboratory study. For both detection modes, a 50 mm x 4.6 mm I.D. column and a 100 mm x 4.6 mm I.D. column packed with 3 microm C18 were used as the first (C-1) and second (C-2) column, respectively. Atmospheric pressure chemical ionization mass spectrometry was performed on a magnetic sector instrument. The LC-LC-MS analysis was carried out on-line by means of direct large volume (11.7 ml) injection (LVI). The performance of both on-line (LVI, 4 ml of sample) and off-line LC-LC-UV (244 nm) analysis was investigated. The latter procedure consisted of a solid-phase extraction (SPE) of 250 ml of water sample on a 500 mg C18 cartridge. The comparative study showed that LC-LC-MS is more selective then LC-LC-UV and, in most cases, more sensitive. The LVI-LC-LC-MS approach combines direct quantification and confirmation of most of the analytes down to a level of 0.01 microg/l in water samples in less then 30 min. As regards LC-LC-UV, the off-line method appeared to be a more viable approach in comparison with the on-line procedure. This method allows the screening of phenylurea's in various types of water samples down to a level of at least 0.05 microg/l. On-line analysis with LVI provided marginal sensitivity (limits of detection of about 0.1 microg/l) and selectivity was sometimes less in case of surface water samples. Both the on-line LVI-LC-LC-MS method and the LC-LC-UV method using off-line SPE were validated by analysing a series of real-life reference samples. These samples were part of an inter-laboratory test and contained residues of herbicides ranging from 0.02 to 0.8 microg/l. Beside good correlation between the methods the data agreed very well with the true values of the samples.

Determination of linuron and related compounds in soil by microwave-assisted solvent extraction and reversed-phase liquid chromatography with UV detection.

The combination of microwave-assisted solvent extraction (MASE) and reversed-phase liquid chromatography (RPLC) with UV detection has been investigated for the efficient determination of phenylurea herbicides in soils involving the single-residue method (SRM) approach (linuron) and the multi-residue method (MRM) approach (monuron, monolinuron, isoproturon, metobromuron, diuron and linuron). Critical parameters of MASE, viz, extraction temperature, water content and extraction solvent were varied in order to optimise recoveries of the analytes while simultaneously minimising co-extraction of soil interferences. The optimised extraction procedure was applied to different types of soil with an organic carbon content of 0.4-16.7%. Besides freshly spiked soil samples, method validation included the analysis of samples with aged residues. A comparative study between the applicability of RPLC-UV without and with the use of column switching for the processing of uncleaned extracts, was carried out. For some of the tested analyte/matrix combinations the one-column approach (LC mode) is feasible. In comparison to LC, coupled-column LC (LC-LC mode) provides high selectivity in single-residue analysis (linuron) and, although less pronounced in multi-residue analysis (all six phenylurea herbicides), the clean-up performance of LC-LC improves both time of analysis and sample throughput. In the MRM approach the developed procedure involving MASE and LC-LC-UV provided acceptable recoveries (range, 80-120%) and RSDs (<12%) at levels of 10 microg/kg (n=9) and 50 microg/kg (n=7), respectively, for most analyte/matrix combinations. Recoveries from aged residue samples spiked at a level of 100 microg/kg (n=7) ranged, depending of the analyte/soil type combination, from 41-113% with RSDs ranging from 1-35%. In the SRM approach the developed LC-LC procedure was applied for the determination of linuron in 28 sandy soil samples collected in a field study. Linuron could be determined in soil with a limit of quantitation of 10 microg/kg.

Screening and analysis of polar pesticides in environmental monitoring programmes by coupled-column liquid chromatography and gas chromatography-mass spectrometry.

Screening and analysis of polar pesticides based on coupled-column reversed-phase liquid chromatography (LC-LC) and GC- or LC-MS is a powerful tool in the execution of environmental monitoring programmes. This paper presents a unified approach utilising LC-LC screening followed by GC-MS confirmation. As polar pesticides are not generally amenable to GC a widely applicable derivation technique is used. The results demonstrate that the proposed LC and MS techniques are capable of analysing a wide range of polar pesticides down to levels of 0.1 microgram/l (EU limit for drinking water). LC switching techniques for group analysis or individual compounds rely on the reversed-phase retention and the UV detectability of the pesticides in combination with the choice of the LC columns. Fast miniaturised derivatization prior to GC-MS forms an integral part in the proposed strategy. In order to avoid extraction losses, derivation in the aqueous sample, preferably with electrophoric reagents with enhanced sensitivity in GC-NICI-MS are employed where possible. In this communication, method development and validation fitting in the strategy are evaluated and the results of the combined approach are discussed.

Semi-permeable surface analytical reversed-phase column for the improved trace analysis of acidic pesticides in water with coupled-column reversed-phase liquid chromatography with UV detection. Determination of bromoxynil and bentazone in surface water.

The coupled-column (LC-LC) configuration consisting of a 3 microm C18 column (50 x 4.6 mm I.D.) as the first column and a 5 microm C18 semi-permeable-surface (SPS) column (150 x 4.6 mm I.D.) as the second column appeared to be successful for the screening of acidic pesticides in surface water samples. In comparison to LC-LC employing two C18 columns, the combination of C18/SPS-C18 significantly decreased the baseline deviation caused by the hump of the co-extracted humic substances when using UV detection (217 nm). The developed LC-LC procedure allowed the simultaneous determination of the target analytes bentazone and bromoxynil in uncleaned extracts of surface water samples to a level of 0.05 microg/l in less than 15 min. In combination with a simple solid-phase extraction step (200 ml of water on a 500 mg C18-bonded silica) the analytical procedure provides a high sample throughput. During a period of about five months more than 200 ditch-water samples originating from agricultural locations were analyzed with the developed procedure. Validation of the method was performed by randomly analyzing recoveries of water samples spiked at levels of 0.1 microg/l (n=10), 0.5 microg/l (n=7) and 2.5 microg/l (n=4). Weighted regression of the recovery data showed that the method provides overall recoveries of 95 and 100% for bentazone and bromoxynil, respectively, with corresponding intra-laboratory reproducibilities of 10 and 11%, respectively. Confirmation of the analytes in part of the samples extracts was carried out with GC-negative ion chemical ionization MS involving a derivatization step with bis(trifluoromethyl)benzyl bromide. No false negatives or positives were observed.

Potential of a simple mechanical extraction system for the rapid extraction of pesticides from foodstuffs. Application to the determination of organochlorine pesticides in lettuce.

The efficiency of a simple mechanical extraction system as applied in veterinary drug analysis has been tested in the field of pesticide residue analysis. As a first application, the system was used for the extraction of organochlorine pesticides from vegetables. The convenience of this simple extraction system consists of performing mechanical extraction in disposable polyethylene-based extraction bags, reducing considerably manual operation and cross-contamination. For the tested compounds and matrix (lettuce), recoveries of the 10 organochlorine pesticides performed at two spiking levels (n = 4 each level and compound) ranging between 0.06 and 3.3 mg/kg were between 60 and 80%, with most standard deviations <5%. The extraction method appeared to be simple and fast with a great potential for the analysis of many pesticide-matrix combinations.

Liquid chromatographic determination of the fungicide iprodione in surface water, using on-line preconcentration.

A new method for on-line sample preparation of surface water extracts for the reversed phase HPLC analysis of the fungicide iprodione at the ppb level is presented. Water samples are extracted with dichloromethane and after concentration and evaporation to dryness, taken up in a mixture of acetonitrile/water. 2-Ml aliquots are injected onto a small precolumn, which is subsequently flushed with 20% acetonitrile in water as a clean-up step. The precolumn is switched on-line with the analytical column, and, using 47.5% acetonitrile in water as the eluent, the concentrated zone of iprodione is desorbed and transported to the analytical column. Detection takes place with UV at 229 nm. The resulting chromatograms are free of interferences. The detection limit of the method in 0.02 ppb. Good reproducibility and linear calibration curves are obtained. The results of the method are in agreement with those of capillary GC analysis with cold-on-column injection. The advantage of the method compared to GC is the lower susceptibility to errors due to the presence of interferences.

Liquid chromatographic determination of bromide in human milk and plasma.

A liquid chromatographic method for the determination of bromide present in human body fluids at the level of 0.5-5.0 ppm is presented. The method involves liquid--liquid extraction of lipids and other lipophilic compounds, destruction of the aqueous phase and analysis of the residue on an aminopropyl bonded silica column with UV detection at 214 nm. The method was applied to the analysis of 278 samples of Dutch human milk. Comparison of the results obtained with those from a routinely used colorimetric procedure for plasma indicated excellent agreement. The ease of automation of the described procedure and its excellent reproducibility make it a good alternative to existing methods for bromide analysis in body fluids.

Liquid chromatographic determination of the herbicide diuron and its metabolite 3,4-dichloroaniline in asparagus.

A liquid chromatographic method is presented for the determination of the phenylurea herbicide diuron and its major metabolite, 3,4-dichloroaniline in asparagus. The method involves a simple isolation step using dichloromethane extraction, followed by reversed phase chromatography on a C18 column, using a basic aqueous-organic eluent and UV detection at 254 nm. The limit of determination is 0.02 mg/kg for both analytes.

Improved coupled column liquid chromatographic method for high-speed direct analysis of urinary trans,trans-muconic acid, as a biomarker of exposure to benzene.

A coupled column liquid chromatographic (LC-LC) method for high-speed analysis of the urinary ring-opened benzene metabolite, trans,trans-muconic acid (t,t-MA) is described. Efficient on-line clean-up and concentration of t,t-MA from urine samples was obtained using a 3 microm C18 column (50x4.6 mm I.D.) as the first column (C-1) and a 5 microm C18 semi-permeable surface (SPS) column (150x4.6 mm I.D.) as the second column (C-2). The mobile phases applied consisted, respectively, of methanol-0.05% trifluoroacetic acid (TFA) in water (7:93, v/v) on C-1, and of methanol-0.05% TFA in water (8:92, v/v) on C-2. A rinsing mobile phase of methanol-0.05% TFA in water (25:75, v/v) was used for cleaning C-1 in between analysis. Under these conditions t,t-MA eluted 11 min after injection. Using relatively non-specific UV detection at 264 nm, the selectivity of the assay was enhanced remarkably by the use of LC-LC allowing detection of t,t-MA at urinary levels as low as 50 ng/ml (S/N>9). The study indicated that t,t-MA analysis can be performed by this procedure in less than 20 min requiring only pH adjustment and filtration of the sample as pretreatment. Calibration plots of standard additions of t,t-MA to blank urine over a wide concentration range (50-4000 ng/ml) showed excellent linearity (r>0.999). The method was validated using urine samples collected from rats exposed to low concentrations of benzene vapors (0.1 ppm for 6 h) and by repeating most of the analyses of real samples in the course of measurement sequences. Both the repeatability (n=6, levels 64 and 266 ng/ml) and intra-laboratory reproducibility (n=6, levels 679 and 1486 ng/ml) were below 5%.

The potential of restricted access media columns as applied in coupled-column LC/LC-TSP/MS/MS for the high-speed determination of target compounds in serum. Application to the direct trace analysis of salbutamol and clenbuterol.

This study investigated the potential of restricted access media (RAM) columns used as a first column in coupled-column LC hyphenated to thermospray tandem mass spectrometry (LC/LC-TSP/MS/MS) for the fast, selective, and sensitive determination of target drugs in serum samples. Because of their wide range in polarity, salbutamol and clenbuterol were chosen as model compounds and representatives of the class of beta 2-agonists. Three types of RAM columns were tested: (i) Pinkerton ISRP (internal surface reversed phase, 5 microns), (ii) SPS (semipermeable surface, 5 microns C18), and (iii) RP-18 ADS (alkyl-diol silica, 25 microns). A 3-micron C18 column (50 mm x 4.6 mm i.d.) was chosen as the second column. Tandem mass spectrometric detection was carried out in the selected reaction monitoring (one parent-->one daughter) mode. With regard to retention and, moreover, the peak elution volume of the analytes, the ISRP material was found to perform best: a 50-mm x 4.6-mm i.d. ISRP column in combination with a 100% aqueous buffer (pH of 7.0 +/- 0.2) allowed the injection of large volumes (up to 200 microL) of sample without additional band broadening of the analytes and provided sufficient preseparation between analytes and large-molecule serum constituents. Under the selected conditions, both analytes could be determined in serum samples up to a limit of quantification (LOQ) of 0.5 ng/mL, with a sample throughput of 7 and 5 h-1 for salbutamol and clenbuterol, respectively. Method validation was carried out by analyzing, in the course of several days, calf and human serum samples spiked with the analytes. In the case of salbutamol, the overall recovery from serum samples spiked at levels between 0.5 and 50 ppb (n = 33) was 103.4%, with a repeatability of 12.7% and reproducibility of 14.3%. The overall recovery for clenbuterol was 99.6% (n = 15, spiked level 0.5-5 ppb), with a repeatability of 15.2% and reproducibility of 16.4%. The adopted LC/LC-TSP/MS/ MS analyzer appeared to be very robust under the selected conditions, and, after the period of analysis involving the processing of more than 100 mL of serum, neither loss of chromatographic performance nor pressure increase of columns or of the interface was observed.

Improvement of selectivity and sensitivity by column switching in the determination of glycyrrhizin and glycyrrhetic acid in human plasma by high-performance liquid chromatography.

A sensitive method is described for the simultaneous determination of glycyrrhizin and glycyrrhetic acid in human plasma. Quantitation is by high-performance liquid chromatography using ion-pair chromatography on a reversed-phase column. Variable-wavelength ultraviolet detection is employed. For the extraction of both compounds from plasma, a new solid-phase ion-pair extraction procedure using octadecylsilane columns was developed. Because of the strong forces involved in the protein binding of glycyrrhizin, quantitative extraction of this compound from plasma was possible only after an alkaline pH shift. A considerable improvement in selectivity and sensitivity was obtained by automated column switching involving on-line preseparation of the solid-phase extract on a short precolumn and chromatographic analysis of a heart-cut from the precolumn eluate. The limit of detection of both glycyrrhizin and glycyrrhetic acid was 0.1 mg/l in 0.5 ml of plasma. From a preliminary study in human volunteers, it was concluded that glycyrrhetic acid rather than glycyrrhizin is preferred in a study in human volunteers to assess the zero effect level of glycyrrhizin.

Fast sample preparation involving MASE and coupled column normal phase liquid chromatography for the rapid trace analysis of dioxins in air-dust samples from fire catastrophe emissions.

A new approach has been developed and tested for the urgent analysis of dioxins in samples of air-dust filters originating from catastrophe emissions. The procedure consists of a fast extraction of the sample with microwave solvent extraction (MASE) and acetone as solvent followed by a fast cleanup of the extract with normal phase coupled column liquid chromatography (LC/LC). The multi-dimensional LC/LC system employs a 50 mm x4.6 mm i.d. column packed with 3mum silica and a 150 mm x4.6 mm i.d. column packed with 5mum PYE as the first and second analytical column, respectively. Iso-hexane is used on both columns to perform cleanup and dichloromethane to perform efficient back-flush elution of the compounds from the second column. The obtained polarity-based separation in the first dimension and molecular-structure based separation in the second dimension provides a fast and powerful cleanup. Validation was done by analysing samples of homemade RIVM air-dust with aged residues (n=8, spiking level about 15pgmg(-1) per compound) of dioxins/furans and samples of reference Urban Dust SRM 1649a (n=4) with both the new approach and the existing conventional procedure and were instrumentally analyzed with capillary gas chromatography and high resolution mass spectrometric detection (GC/HRMS). In comparison to the existing conventional procedure, the new approach reduces sample processing from several days to several hours per sample. As regards the aged-residue air-dust samples, the new method shows a good accuracy, precision and high selectivity providing a performance in good agreement with the existing procedure. In SRM air-dust, the concentration of a few compounds obtained by the new method was below (10-50%) the certified value.