Environ. Sci. Technol. 1999, 33, 1916-1925
Class-Selective Immunosorbent for Trace-Level Determination of Polycyclic Aromatic Hydrocarbons in Complex Sample Matrices, Used in Off-line Procedure or On-line Coupled with Liquid Chromatography/Fluorescence and Diode Array Detections in Series MARIANNE BOUZIGE, VALERIE PICHON, AND MARIE-CLAIRE HENNION* Ecole supe´rieure de Physique et de Chimie Industrielles de Paris, Laboratoire Environnement et Chimie Analytique (ERS CNRS 657), 10 rue Vauquelin, 75231 Paris Cedex 05, France
A new immunoaffinity solid-phase extraction (SPE) methodology based on antigen-antibody interactions was evaluated and optimized for the selective extraction of polycyclic aromatic hydrocarbons (PAHs) in various complex environmental matrices. This immunosorbent (IS) consists of anti-pyrene antibodies immobilized on a silica support and is used as a classical SPE sorbent. The cross-reactivity of the antibodies for analytes structurally related with pyrene allows the simultaneous extraction of the priority PAHs included in the European Union and/or U.S. EPA priority lists. In addition, extraction, trace enrichment, and cleanup are achieved in one step due to the selectivity of the antigen-antibody interaction. For aqueous samples, limitation of unwanted adsorption of the PAHs on vessels or tubing due to their high hydrophobicity is obtained by adding acetonitrile in samples before percolation. Losses due to the volatility of the two- or three-ring PAHs are avoided by coupling on-line the extraction using the antipyrene IS with LC. From a sample volume of 80 mL, the sensitivity of the fluorescence associated with the selectivity of the IS allows the quantification of individual PAHs in contaminated or surface water below the 0.02 µg/L level and therefore the fulfillment of the EU regulation for monitoring contaminated surface water used as a source for drinking water. The presence of several PAHs at 0.02 µg/L could be confirmed by spectral identification using the diode array detector. Off-line extraction procedures were also set up for the extraction of PAHs from complex solid environmental matrices such as sludge or mussel extracts. The whole offline procedure was validated using a sewage sludge reference material containing several PAHs at a concentration varying from 0.5 to 2.2 mg/kg of dry sludge. The high selectivity provided by the antibodies permitted extraction of the PAHs and elimination of the great number of interferents in only one step, so that identification of compounds could be achieved using UV diode array detection.
Introduction Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that mainly result from incomplete 1916
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combustion of organic materials, in particular petroleum fuels. Many of them have mutagenic and carcinogenic properties and consequently have been included in U.S. EPA (1) and European Union (EU) priority lists of pollutants (2). EU regulations are the tighter ones and require the control of six of them in surface water used for drinking purposes, the sum of their concentration not exceeding 0.2 µg/L with a concentration limit of 0.02 µg/L for benzo[a]pyrene (2). New EU regulations are being set up with maximum concentrations of three PAHs in the range 2-5 mg/kg in sewage sludge used for soil amendment. PAHs have been measured in a variety of environmental matrices and reference materials have been certified in solid matrices such as river and marine sediments, urban and diesel particulate matter, mussel tissues, shale oil, petroleum crude oils, and sewage sludge. Liquid chromatography (LC) using fluorescence detection and gas chromatography (GC) coupled to mass spectrometry (MS) are the most powerful techniques for their monitoring. Nevertheless, an advantage of LC/fluorescence is the ability to measure some PAH isomers that cannot be easily quantified by GC/MS. Because of this excellent separation and detection selectivity, LC has been specified as the method of choice by the U.S. EPA for the analyses of aqueous effluents for the determination of PAHs (3). To reach low concentrations, concentration and isolation from matrices are required, and this is all the more difficult because PAHs are very hydrophobic and only slightly soluble in water. Consequently, they tend to adsorb everywhere, leading to losses during the sampling and storage. Moreover, some are volatile and losses cannot be avoided when the sample handling includes evaporation steps. Common procedures are off-line extraction using classical sorbents such as n-alkyl silica or polymers followed by LC separation with fluorescence detection (3-11). More specific sorbents such as Chromspher p, Boos silica, or Blue Pearls have been tested, leading to lower recoveries of extraction (10). Some automated on-line systems in which the preconcentration is directly coupled to the LC separation have also been reported (10, 11). Whatever the system used, a modifier has to be added to the sample prior to the extraction to avoid the adsorption of PAHs on sample containers or connecting tubing. This modifier is usually an organic solvent (2propanol, methanol, acetonitrile) (4-11) or a surfactant, for example, Brij 35 (8, 10). Because of the selectivity of the fluorescence detection, some PAHs can be quantified in environmental extracts from simple C18 extractions. However, one main drawback of the use of LC/fluorescence detection is that analytes are identified only by their retention time. When samples are complex and many peaks are detected, identification has to be confirmed. This can be achieved either by using in series a diode array absorption detector (DAAD), which provides the match with UV spectra because many PAHs possess very specific UV spectra, or by using GC/ or LC/MS. However, LC/DAAD, LC/MS, and GC/MS can be used provided an additional cleanup of samples is performed, even with extracts from surface water samples. These cleanup steps using silica or Florisil are always laborious and time-consuming. Although a fluorescence detector able to provide fluorescence spectra has been recently commercially available, most environmental laboratories are equipped with DAAD * Corresponding author phone 33 1 40 79 46 51; fax: 33 1 40 79 47 76; e-mail:
[email protected] or marie-claire.hennion@ espci.fr. 10.1021/es981031l CCC: $18.00
1999 American Chemical Society Published on Web 04/21/1999
TABLE 1. Structures, Log Kow Coefficients, and Fluorescence Wavelength Programming Conditions of the Hapten and of the 16 PAHs Listed by the U.S. EPA
and a simple fluorescence detector. Therefore, there is great interest in providing selective extraction procedures that eliminate the coextraction of matrix interferences and can be simply coupled on-line with LC with fluorescence and DAAD detection. These highly selective extractions have been recently obtained using new types of sorbent involving analyte-antibody interactions. Antibodies produced against a target compound are immobilized on a silica-based support to form a so-called immunosorbent (IS) that is used just like a classical SPE sorbent. Because of the structural similarity of the target molecules, the antibodies are able to recognize not only the antigen that was used to initiate the immune response but also compounds from the same family. In contrast to previous development of IS for single analytes (12-14), our group took advantage of this cross-reactivity of the antibodies to develop class-selective ISs for triazine and phenylurea pesticides, including their degradation products in various matrices such as water, foodstuffs, soils and sediments (15-22). Recently, an immunosorbent based on anti-fluorene antibodies was developed (23). Fluorene is one of the most volatile PAHs and was chosen as a target
compound representative of the two or three aromatic ring PAHs. The IS was connected on-line with a LC/DAAD in series with a fluorescence detector. The sensitivity of the fluorescence detection associated with the selectivity of the extraction sorbent allows detection of PAHs between 2 and 10 ng/L from a sample volume as low as 20 mL. Moreover, the structural similarity within this group of pollutants showed the possibility of using this sorbent for the extraction of the 16 priority PAHs, but confirmation using the DAAD was obtained for only a few of them. To increase the potential of the method for the four- to six-ring PAHs, anti-pyrene antibodies were produced. Pyrene is one of the most hydrophobic PAHs and is well adapted to the trapping of the four- to six-ring aromatic ring PAHs. Unlike the two- or three-ring PAHs, which are volatile, the four- to six-rings PAHs can be analyzed either in an on-line system or off-line. The objective of this work was to evaluate the performance of the anti-pyrene IS (1) in on-line procedures for the extraction of the 16 PAHs in surface water by on-line coupling to LC/fluorescence and DAAD in series for identity confirmation and (2) in off-line procedures for the selective VOL. 33, NO. 11, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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cleanup of extracts from complex matrices such as sewage sludge or mussel extracts. This requires the study, for each type of system, of (i) the efficiency of the IS for trapping the various PAHs, (ii) the effect of the necessary addition of solvent in the sample on recoveries and breakthrough volumes, (iii) the possibility of the methods for the quantification at the parts per trillion level with confirmation using DAAD, (iv) the selectivity of the IS, compared to classical sorbents for the extraction of PAHs from real samples, and (v) the validation of the procedure using a certified reference matrix.
Experimental Section Chemicals. HPLC grade acetonitrile, tetrahydrofuran, and methanol were from Mallinckrodt Baker (Deventer, The Netherlands). LC quality water was prepared by purifying demineralized water in a Milli-Q filtration system (Millipore, Bedford, MA). Other chemicals came from Prolabo, Merck, SDS, and Fluka. PAHs, obtained from Mallinckrodt Baker, were dissolved in acetonitrile and stored at 4 °C. These stock solutions were further used for the preparation of the samples. The phosphate-buffered solution (PBS) consists of a 0.01 M sodium phosphate buffer containing 0.15 M NaCl (pH 7.4). Sewage sludge certified reference material (CRM No. 088) was prepared by the Community Bureau of Reference from Brussels and was purchased from Promochem (Molsheim, France). Materials. LC analyses were performed with a Varian LC System Workstation including a Varian Star 9010 solventdelivery system, a model 9065 Polychrom diode array detector, and a Varian 9075 fluorescence detector. Immunoprecolumn and analytical column switching was accomplished with two Rheodyne (Berkeley, CA) valves. The on-line preconcentration was performed with a Milton Roy pump. This system of preconcentration was totally constituted by stainless steel tubing to minimize adsorption processes. Stationary Phases and Columns. The analytical column was a 250 × 3 mm i.d. column prepacked with the PAH16Plus silica from Mallinckrodt Baker. The immunosorbent consisted of anti-pyrene antibodies bonded onto glutardialdehyde-activated silica particles of 30 nm pore size (Mallinckrodt Baker). Polyclonal antibodies were supplied by Prof. F. Le Goffic (ENSCP, Laboratory of Bioorganic and Biotechnologies, Paris). To induce an immunogen response, the pyrene has been derivatized and linked to a carrier protein, that is, bovine serum albumin. The immunizing reagent thus obtained has been injected into a rabbit, and the serum has been collected 6-9 months after the first immunization and purified; 40 mg of the purified IgG fraction was bound to 1 g of silica. The whole procedure is similar to that previously described (15). The immunosorbents have been used either in an on-line system and packed in a stainless steel precolumn (30 × 4.6 mm i.d.) or for an off-line preconcentration, cartridges being filled with 250 mg of sorbent. Off-line preconcentrations on nonselective sorbents were carried out on C18 disposable extraction columns containing 500 mg of sorbent (Mallinckrodt Baker), whereas for on-line preconcentration, a precolumn of 20 × 4.6 mm i.d. prepacked with Pelliguard C18 silica (Supelco, Bellefonte, PA) was used. LC Conditions. For the separation of the 16 PAHs using the on-line coupling of the IS precolumn with LC, the mobile phase was a mixture of acetonitrile and water according to the following elution gradient: 50% acetonitrile and 50% water during the first 5 min to 100% acetonitrile at 20 min and then isocratic for 15 min, with a flow rate of 0.5 mL min-1. When off-line preconcentration was performed, another gradient starting from 65% acetonitrile and 35% water and increasing to 100% acetonitrile in 15 min was applied. 1918
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TABLE 2. Variation of the Mean Extraction Recoveries (Percent) and Standard Deviations with the Percentage of Acetonitrile after On-line Preconcentration on the Anti-pyrene Immunoextraction Sorbent of 10 mL of LC Grade Water Spiked with 10 ng of Each PAH
naphthalene acenaphthene fluorene phenanthrene anthracene fluoranthene pyrene b[a]anthracene chrysene b[b]fluoranthene b[k]fluoranthene b[a]pyrene dib[ah]anthracene b[ghi]perylene i[123cd]pyrene a
10%, n)3
25%, n)3
40%, n)2
13 ( 2 23 ( 3 27 ( 3 52 ( 6 48 ( 7 62 ( 3 58 ( 10 41 ( 4 45 ( 4 32 ( 3 34 ( 3 31 ( 2 31 ( 6 31 ( 5 30 ( 6
6(1 7(1 6(1 12 ( 2 11 ( 2 15 ( 4 21 ( 5 27 ( 4 24 33 36 ( 3 36 42 45 ( 3 43 ( 5
4 4(1 4 5(1 4(1 5(1 7(2 5 nda nd 6(1 nd nd 9(1 nd
nd, not determined.
TABLE 3. Average Recoveries (Percent) Obtained with Increasing Volumes of the Percolated Sample (On-line Preconcentration of LC Grade Water Spiked with 10 ng of Each PAH and Containing 10% of Acetonitrile)
naphthalene acenaphthene fluorene phenanthrene anthracene fluoranthene pyrene b[a]anthracene chrysene b[b]fluoranthene b[k]fluoranthene b[a]pyrene dib[ah]anthracene b[ghi]perylene i[123cd]pyrene a
10 mL n)3
20 mL, n)3
50 mL, n)3
80 mL, n)6
100 mL, n)3
13 ( 2 23 ( 3 27 ( 3 52 ( 6 48 ( 7 62 ( 3 58 ( 10 41 ( 4 45 ( 4 32 ( 3 34 ( 3 31 ( 2 31 ( 5 30 ( 6 nda
7(1 12 14 ( 1 32 ( 4 33 ( 2 56 ( 2 62 ( 2 42 ( 3 43 ( 1 32 ( 3 43 ( 3 29 ( 3 27 25 ( 2 nd
3(2 6(4 6(3 14 ( 3 15 ( 3 29 ( 5 41 ( 5 47 ( 4 48 ( 3 37 ( 3 45 ( 5 32 ( 4 24 ( 5 24 ( 4 nd
3(1 4(1 5(1 13 ( 3 13 ( 1 31 ( 3 35 ( 2 60 ( 1 53 ( 2 47 ( 4 52 ( 1 46 ( 6 40 ( 8 37 ( 7 nd
1 2 3 8 9 18 ( 5 24 ( 4 41 ( 5 43 ( 4 41 ( 4 38 ( 5 37 ( 5 30 ( 6 30 ( 5 19 ( 4
nd, not determined.
The fluorescence detector was programmed to have the most appropriate excitation and emission wavelengths for each group of PAHs with close retention times. The detailed fluorescence program is given in Table 1. Immunoextraction Procedure. (a) On-line Procedure. The on-line device involves several steps. The IS precolumn is conditioned with 6 mL of PBS followed by 6 mL of LC grade water with the switching valve in load position. The sample is then percolated through the IS precolumn at a flow rate of 2 mL min-1. After the valve is switched to the inject position, the trapped analytes are desorbed and transferred to the analytical column with the acetonitrile/ water mobile phase gradient. After each experiment, the antibodies are washed with 20 mL of a 70:30 acetonitrile/ water mixture. They are then regenerated by 10 mL of water and 10 mL of a solution of PBS containing 0.02% of azide. When not in use, they are stored at 4 °C in this solution. (b) Off-line Procedure. For off-line preconcentration the IS is packed in an empty disposable cartridge. The immunosorbent is first conditioned with 6 mL of PBS and 6 mL of LC grade water. The sample is then flushed through the cartridge at a flow rate of ∼2 mL min-1. The elution is achieved
FIGURE 1. On-line preconcentration of a marina harbor water (Atlantic coast, France) containing 10% of acetonitrile and spiked at 0.1 µg L-1 with PAHs on a C18 silica (a) and on the anti-pyrene IS (b). UV detection was at 249 nm (a, b). Compounds: see Table 1. by the percolation of 5 mL of a mixture of acetonitrile and LC grade water (70:30). A pure organic solvent was not selected because the IS is still at the laboratory-stage study and the mixture of acetonitrile and LC grade water (70:30) is more appropriate for its regeneration after use. However, the evaporation of this solution would cause evaporation of the PAHs. Therefore, a second C18 cartridge was used and desorption was performed with the more volatile THF. This C18 cartridge was previously conditioned by 6 mL of THF, 6 mL of MeOH, and 6 mL of a 70:30 acetonitrile/LC grade water mixture. The PAHs are finally eluted from the C18 cartridge with 6 mL of THF. The THF is dried over anhydrous sodium sulfate and evaporated under a gentle stream of nitrogen. The evaporation is conducted to dryness, and the residue is brought up to 200 µL with acetonitrile/water 80:20. The sample containers are rinsed with 5 mL of the 70:30 acetonitrile/water mixture and with 6 mL of THF before their percolation on the sorbent for elution. After elution, the immunosorbent is washed with 20 mL of a 70:30 acetonitrile/water mixture. The antibodies are regenerated with 10 mL of LC grade water and 10 mL of a PBS solution containing 0.02% of azide. When not in use, they are stored at 4 °C in this solution. Sample Preparation. (a) Water. Surface water samples came from a marina harbor, in the west of France and from the River Seine in Paris. These samples were on-line percolated after addition of 10% acetonitrile. (b) Mussel Extract. Ninety grams of mussels (20 g dried weight) were crushed in 500 mL of acetonitrile/methanol/ water 40:40:20 (v/v). The mixture was centrifuged and the supernatant collected. Fifty milliliters was evaporated to dryness and the residue redissolved in 5 mL of acetonitrile. The aliquot was then spiked with the 16 PAHs to have a final
concentration of 55 ng of each PAH/g of dried mussel. One milliliter was evaporated to dryness and redissolved in 200 µL of an 80:20 acetonitrile/water mixture for direct injection in LC. One milliliter was used for the cleanup on the IS. Each injection is equivalent to the analysis of 90 mg of dried mussel. (c) Sludge Extracts. The reference material certified from the BCR (CRM No. 088) was used. The sludge was extracted by supercritical fluid extraction as described by Mie`ge et al. (24). One gram of dried sludge was extracted with supercritical carbon dioxide modified by 5% of toluene. The pressure was set at 500 atm and the temperature at 150 °C. The final extract consisted of pure acetonitrile, and 200 µL was diluted with 50 µL of water to give an extract ready for direct injection into LC. One hundred microliters was used for the cleanup on the anti-pyrene IS. Each injection corresponded to an equivalent of 5.4 mg of dried sludge.
Results and Discussion To obtain an immune response of the animal, pyrene has to be derivatized. A hapten was synthesized by introducing a carboxylic moiety to pyrene as shown in Table 1. The hapten carrier conjugate was obtained by a covalent bonding between the hapten and the lysine amino side chains of bovine serum albumin. It was injected into the rabbit to produce the so-called anti-pyrene antibodies. Table 1 presents the structures of the 16 PAHs listed by the EPA. The structural similarity within the PAH family allows one to predict that the antibodies will recognize not only pyrene but the other PAHs. For that reason the evaluation of the immunosorbent is made on the whole group of PAHs. Optimization of the On-line Procedure for Aqueous Samples. On-line coupling of the IS with LC is the most appropriate method for drinking and surface water monitorVOL. 33, NO. 11, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 4. Calibration Curves, Correlation Coefficients, and Limits of Detection of Each PAH for the On-line Preconcentration on Anti-pyrene IS of 80 mL of River Seine Water Containing 10% of Acetonitrile (Fluorescence Detection)
naphthalene acenaphthene fluorene phenanthrene anthracene fluoranthene pyrene b[a]anthracene chrysene b[b]fluoranthene b[k]fluoranthene b[a]pyrene dib[ah]anthracene b[ghi]perylene a
calibration curvea
R2
LDDb (pg/L)
extraction recoveries (%)
y ) 185335x + 5600 y ) 299712x + 3807 y ) 183263x + 8475 y ) 173195x + 11248 y ) 1305748x + 50932 y ) 1291157x + 6089 y ) 101294x + 7365 y ) 3221025x + 63436 y ) 569341x + 1838 y ) 2940065x - 9475 y ) 15235975x + 261346 y ) 4186898x + 32848 y ) 1791527x + 7474 y ) 822051x - 395
0.9941 0.9912 0.9965 0.9744 0.9838 0.9451 0.9781 0.9963 0.9998 0.9992 0.9955 0.9994 0.9972 0.9963
15 10 10 9 2 2 12 0.8 5 0.9 0.1 0.7 2 2
3 ( 0.5 4 ( 0.4 5(1 14 ( 3 14 ( 3 32 ( 8 34 ( 7 58 ( 5 53 ( 2 46 ( 3 52 ( 9 43 ( 6 38 ( 8 38 ( 6
Least-squares regression equation, y for peak area, x for concentration in µg/L.
ing, because the most water-soluble PAHs are also the most volatile ones. Therefore, avoiding evaporation steps and the resulting losses is very important. (a) Effect of the Addition of Organic Solvent on Solubilization and Breakthrough of Analytes. To make the PAHs soluble in the sample and to avoid their adsorption on the flasks or on the tubing of the on-line device, one must add to the sample an organic solvent or a surfactant. Because of the presence of such a modifier, the retention on classical supports such as C18 or PRP1 is lowered because breakthrough occurs (10, 11). Binding between antibodies and analytes occurs if the spatial complementarity between molecules is good and involves several types of interactions such as van der Waals, electronic, hydrogen, and hydrophobic interactions. In a previous study (23), we have shown that the interaction with the anti-fluorene antibodies was also affected by the solvent or surfactant added to the sample. 2-Propanol or acetonitrile was shown to be the more appropriate organic solvent to be added, which allowed a good regeneration of the ISs. However, their addition caused a breakthrough of the PAHs due to an insufficient retention and resulted in measurements of incomplete extraction. Therefore, a compromise had to be found between a good solubilization of the two- to three-ring volatile PAHs in the spiked sample and sufficient recoveries from the IS for quantification and identification. The problem here is more complicated with the most hydrophobic PAHs. As shown in Table 1, they have octanolwater coefficients, log Kow, close to 5 or 6 and are consequently more difficult to dissolve. A sample of 10 mL of LC grade water spiked with 1 µg/L of each of the 16 PAHs was preconcentrated onto the IS, on-line eluted, and analyzed. Various amounts of acetonitrile were added, and the effect on the extraction recoveries is shown in Table 2. Using 10% acetonitrile, the recoveries range from 20 to 60% depending on the compounds. The cross-reactivity of the anti-pyrene antibodies for the PAH family is demonstrated, and pyrene and fluroanthene show the highest recoveries. The recoveries of extraction are lower for the first eluted PAHs because they are more affected by the solvent and because their structures are slightly different from that of the pyrene (only two or three rings compared to four rings for the pyrene). When 10% acetonitrile is added to the sample, the recoveries of the more hydrophobic PAHs range from 45 to 30%. This can be explained by the fact that either they are not solubilized enough or because, once they are trapped on the antibodies, they are very difficult to desorb and the mobile phase used for the analytical separation is not strong enough to desorb the compounds. Increasing the amount of acetonitrile to 1920
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b
Corresponding to a signal-to-noise ratio of 3.
25% causes breakthrough for PAHs up to chrysene and recoveries obtained for 25% are lower than those obtained with 10%, whereas there is a slight increase for the four more hydrophobic PAHs, certainly due to a better solubilization. Increasing the amount of acetonitrile to 40% indicates that breakthrough occurs for all of the PAHs and is a too strong amount for allowing interactions, despite a complete solubilization. To trap the maximum of compounds, 10% of acetonitrile was chosen as a good compromise between solubilization and elution. (b) Effect of the Sample Volume on Recoveries. To reach the required limits of 0.02 µg/L for individual identification of analytes in surface water using the DAAD detector, it is important to concentrate the highest possible volume of sample on the IS. To evaluate the volume of sample that can be percolated on the IS, increasing volumes of LC grade water, containing 10 ng of each PAH and 10% of acetonitrile, were percolated on the IS. The corresponding extraction recoveries are reported in Table 3. Up to 20 mL the extraction recoveries remain constant for all of the compounds except for the three first PAHs, which are eluted very early. When the preconcentrated volume is increased to 50 or 80 mL, the recoveries for the first PAHs drop, but they remain the same for the most hydrophobic PAHs. With a 100 mL volume, only the last PAHs keep satisfactory extraction recoveries. To analyze the whole group of PAHs, 20 mL would be the most suitable volume. Nevertheless, the recoveries obtained for the two- to three-ring PAHs, from naphthalene to anthracene, on anti-fluorene antibodies were higher (23), showing thus that the anti-pyrene antibodies are more appropriate for the extraction of the more hydrophobic PAHs, which are structurally closer, whereas the two- to three-ring PAHs are better recognized by the anti-fluorene antibodies. If one wants to focus on the most hydrophobic PAHs using the anti-pyrene IS, volumes up to 80 mL can be preconcentrated without any major losses in extraction recoveries, with the advantage of providing a higher amount of analytes for detection. However, because some analytes are lost by incomplete solubilization, it is important to rinse the whole system after each run and to make a blank run with a nonspiked sample before a spiked or unknown sample is analyzed. (c) Selectivity of the IS in Real Samples. Nonselective interactions between the PAHs and the silica-based support or the nonspecific part of the antibodies may occur in addition to the selective interactions with the recognition sites of the antibodies (25). The contribution of the nonselective interactions to the retention on the IS has been evaluated in our previous study by comparing anti-fluorene antibodies with antibodies not designed to retain PAHs, that is, anti-atrazine
FIGURE 2. On-line preconcentration on anti-pyrene IS of 80 mL of River Seine water containing 10% of acetonitrile and spiked at 20 ng L-1 with PAHs: UV detection at 239 nm (a); fluorescence detection (b). The insets correspond to the match of the UV spectrum of some PAHs, with the spectra of the library plotted in dashed lines. Compounds: see Table 1. antibodies (23). Extraction recoveries were higher on the antifluorene IS, but recoveries on anti-atrazine IS were not negligible, showing nonspecific retention. Dealing with this type of hydrophobic compounds, there will be a part of nonspecific retention that cannot be avoided but, in addition, the anti-PAH antibodies bring their specificity.
Another means for demonstrating the selectivity of the antibodies and the contribution of the specially designed anti-pyrene antibodies is to apply the IS to the extraction of PAHs from real sample matrices. An 80 mL volume of harbor water (taken in a marina harbor, Atlantic coast, France) was preconcentrated either on the anti-pyrene IS or on a classical VOL. 33, NO. 11, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 5. Extraction Recoveries (Percent) Obtained for Off-line Preconcentration of Different Volumes of LC Grade Water Spiked with 20 ng of Each PAH and Containing 25% of Acetonitrile on the Anti-pyrene Immunosorbent
anthracene fluoranthene pyrene b[a]anthracene chrysene b[b]fluoranthene b[k]fluoranthene b[a]pyrene dib[ah]anthracene b[ghi]perylene i[123cd]pyrene
10 mL, n)3
20 mL, n)5
50 mL, n)3
68 ( 2 89 ( 3 60 54 ( 4 53 51 60 ( 3 49 47 47 ( 8 47
38 ( 8 53 ( 6 56 ( 10 49 ( 9 47 ( 9 55 ( 6 56 ( 5 44 ( 6 48 ( 7 50 ( 5 48 ( 4
13 ( 5 27 ( 6 28 ( 6 26 ( 3 30 ( 8 34 ( 5 28 ( 4 27 ( 5 30 ( 8 37 ( 6 35 ( 5
C18 extraction sorbent. The water contained 10% of acetonitrile and was spiked with the 16 PAHs at 0.1 µg/L. The corresponding UV chromatograms obtained at 249 nm are shown in parts b and a of Figure 1. The effect of the IS on the matrix obviously demonstrates the gain in selectivity provided by the antibodies. The huge hump at the beginning of the chromatogram and the baseline noise corresponding to coextracted compounds highly present in this contaminated water have been greatly reduced. Coeluting compounds hindering the identification of the first PAHs in the case of the C18 preconcentration (Figure 1a) have been eliminated after the preconcentration on the IS (Figure 1b). Even with fluorescence detection (not shown here), which is more selective than UV detection, a difference between the two sorbents exists and higher recoveries are obtained, especially for peaks 1-7 with the IS. (d) Quantification Limits and Detection Limits for Identity Confirmation in Real Samples. Quantification is reliable using on-line preconcentration and LC, provided that calibration curves are constructed using the on-line system and with the same experimental conditions as those used for analyzing the unknown samples (80 mL with 10% acetonitrile, same precolumn and analytical column, same composition of the mobile phase gradient for elution and separation). Calibration curves have been constructed with River Seine water spiked with each PAH in the range 0.020.5 µg/L. For each experiment, several replicates were performed. The resultant standard deviations range from 1 to 10%, showing a good reproducibility of the whole on-line system, as previously pointed out (23). Table 4 shows the calibration curves and correlation coefficients calculated. A good linearity with high correlation coefficients for all of the PAHs was obtained, and this is a probe of the reproducible extraction recoveries, although they are not equal to 100%. The linearity up to 0.5 µg/L for each analyte also shows that the capacity of the IS has not been overloaded when 16 analytes are present, each one at a concentration of 0.5 µg/L in the real sample. Quantification at a level 50) without losses in reproducibility of results. Optimization of the Off-line Procedure for Solid Matrices. For solid samples an extraction is usually first applied using a solvent or a surpercritical fluid. The extract can then be dissolved in water/acetonitrile, and the on-line procedure described previously can be applied. However, the size of on-line precolumns is limited, and there can be an interest in using an off-line procedure because then the amount of sorbent can be increased. Off-line procedures using immunosorbent are easy and rapid, requiring simple apparatus and allowing one to obtain in a few steps selectively cleanedup extracts that can be analyzed either by LC or by other systems such as GC/MS. (a) Losses during Evaporation. Numerous studies have shown that the lightest PAHs are quite sensitive to the evaporation step (6, 7). To evaluate the losses during this step, aliquots of tetrahydrofuran or acetonitrile spiked with a mixture of PAHs were evaporated under nitrogen. Evaporation leads to drastic losses of the first PAHs with very irreproducible results. Using that method, it will be very difficult to obtain reliable results for the first six PAHs, and the on-line system will be the only way to quantify them properly. For the more hydrophobic PAHs (starting from anthracene), no losses occur during the evaporation, even when conducted to dryness. The residue is brought up to 200 µL with acetonitrile/water 80:20. Special care must be taken to be sure to dissolve all of the PAHs adsorbed on the surface of the vial. (b) Optimization of the Experimental Conditions. The desorption conditions were first studied and 5 mL of a mixture containing 70% acetonitrile and 30% water was used, because the antibodies could be affected by the use of a pure organic solvent. This mixture allows complete desorption of each PAH. However, such a mixture is difficult to evaporate and leads to great losses even for some hydrophobic PAHs, so that a second C18 cartridge was used for reconcentration and desorption occurred with tetrahydrofuran, which is easier to evaporate (see experimental conditions). No losses due to an insufficient retention on the C18 silica are noticed, because only 5 mL of the acetonitrile/water mixture is percolated. Solubilization of the PAHs contained in extracts also required the addition of an organic solvent. With 25% of acetonitrile, the recoveries are ∼50% for all of the compounds using a cartridge packed with 250 mg of IS. The recoveries obtained are slightly higher than those obtained using the on-line system with the same amount of sorbent, and this is explained by the different procedure that has been set up to minimize the losses due to adsorption: the vial containing
FIGURE 3. Analysis of a certified reference sludge (CRM No. 088) without (a, c) or after cleanup on the anti-pyrene IS (b, d) by UV detection at 249 nm (a, b) and fluorescence detection (c, d). Compounds: see Table 1. the sample is rinsed with the desorption solvent (5 mL of the 70:30 acetonitrile/water mixture and then 6 mL of tetrahydrofuran). The PAHs adsorbed on the surface of the vial are dissolved and analyzed, whereas using the on-line system there is no way to analyze this adsorbed part, which must be quite important in the case of the very hydrophobic PAHs. Moreover, the elution solvent (70:30 acetonitrile/water) has a stronger eluting power than the initial mobile phase of the analytical system, which is used to elute the compounds from the IS in the on-line device. A percentage of acetonitrile of 40% in the sample was tested, but the recoveries were very low for all of the PAHs, as observed in on-line procedures. Twenty-five percent of acetonitrile is then the best choice for the solubilization of the most hydrophobic PAHs. The volume that can be percolated without exceeding too much the analyte breakthrough was also evaluated. Various volumes of LC grade water spiked with 20 ng of each PAH and containing 25% of acetonitrile were percolated on the anti-pyrene IS. Table 5 shows the effect of the volume on the extraction recoveries. Increasing the volume from 10 to 20 mL does not affect the recoveries except that for anthracene, but when 50 mL is preconcentrated, the recoveries decrease from 50 to 30% for all of the compounds. Because the off-line system is more devoted to solid samples for cleanup, there is no need to percolate high volumes of sample and 20 mL is enough. Five replicates have been performed, and the standard deviations calculated range from
5 to 10%, which is good if we take into account the number of steps in the method. (c) Application to Real Samples and Validation Using Certified Reference Materials. Sewage sludge were selected as reference material for validation of the whole sequence: extraction using supercritical fluid extraction, cleanup of the extract using the anti-pyrene IS, quantification and identity confirmation using LC/fluorescence and diode array detections in series. Because PAHs, especially the four- to sixrings ones, are very hydrophobic compounds, so they tend to adsorb on solid materials instead of remaining in the water, one can expect them to be accumulated in sewage sludge during the wastewater treatment. Because a way of using sludge is soil amendment, a new regulation is being set up for the monitoring of fluoranthene, benzo[b]fluoranthene, and benzo[a]pyrene at concentrations of 5, 2.5, and 2 mg/kg of dried sludge, respectively. Sludge is a very complex matrix, containing high amounts of lipids, and a selective cleanup step with the anti-pyrene IS could be of great interest. PAHs were extracted from a sludge certified reference material using supercritical fluid extraction (SFE) and was either directly analyzed using LC/fluorescence and diode array detections or cleaned up on the anti-pyrene IS. For the cleanup, the SFE extract in pure acetonitrile has to be diluted with water to obtain a sample of 20 mL containing only 25% of acetonitrile. The comparison of the two resulting chromatograms, corresponding to the analysis of 5.4 mg of sludge, is shown in VOL. 33, NO. 11, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 6. PAH Contents (Milligrams per Kilogram of Dry Sludge) in the Reference Certified Sludge from the BCR (CRM No. 088) Evaluated after Cleanup on the Anti-pyrene IS and Comparison with the Certified Valuesa
pyrene b[a]anthracene b[b]fluoranthene b[k]fluoranthene b[a]pyrene i[123cd]pyrene a
cleanup on anti-pyrene IS
certified value
1.84 (21) 0.74 (15) 1.10 (10) 0.57 (12) 0.79 (12) 0.96 (12)
2.16 (4) 0.93 (10) 1.17 (7) 0.57 (9) 0.91 (10) 0.81 (7)
Mean values of three replicates with relative standard deviations
(%).
Figure 3. A great part of the interfering compounds present on the UV chromatogram at 249 nm (Figure 3a,b) has been eliminated by the IS cleanup, especially the polar ones. The confirmation of the identification of five certified PAHs could be done using the match with their UV spectrum. Some interferents remain either because they have similar structures or because they are retained by nonspecific interactions, which are unavoidable when one is dealing with such hydrophobic compounds. The fluorescence chromatogram in Figure 3d is cleaner after cleanup as compared to that of Figure 3c, especially at the beginning of the chromatogram. The peaks corresponding to the PAHs could be shifted to the beginning of the chromatogram without hindering their detection using a gradient allowing a more rapid separation. Table 6 indicates the certified values and the mean concentration calculated using the recoveries of Table 5. There is a good agreement between the determined and the certified values. The recoveries of extraction measured by comparing the results of the direct injection and of the cleanup on the IS are similar to those obtained with LC grade water. No effect of matrix occurs. This allows us to quantify the PAHs in the sludge extract by using the results calculated for LC grade water. The values obtained after the cleanup on the IS and the certified reference values match completely, showing that the whole off-line procedure can be validated for the quantification of PAHs in sludge. Another type of real matrix in which the purification of the extracts is needed are biological matrices. They are very complex, and the procedures currently used for the cleanup of extracts involving alkaline digestion are laborious and timeconsuming. In this case again the selectivity given by the antibodies can be of great benefit. A mussel extract, spiked with the PAHs at 55 ng/g of dried mussel, which is a level close to the concentration of the certified reference material (3), was percolated on the anti-pyrene IS. The extract was diluted to 20 mL in a PBS solution containing 25% of acetonitrile. PBS was used instead of LC grade water to decrease ionic interactions. We believe that the PBS ions may occupy the ionic interaction sites and prevent the ionized interferents from being retained by ionic interactions with antibodies. Nonspecific ionized interactions are removed, and only PAHs are retained by specific interactions. The resulting chromatograms equivalent to the analysis of 90 mg of dried mussel are presented in Figure 4 and compared to the one corresponding to the direct injection without any cleanup of the extract. The UV detection at 215 nm (Figure 4a,b) allows us to evaluate the effect of the antibodies on the matrix. A lot of interfering compounds have been eliminated. The elimination of the coextracted compounds, the reduction of the hump in the beginning of the chromatogram, and the decrease of the overall absorbency can be of great interest if one wants to use a type of detector other than fluorescence as, for example, mass spectrometry. With only one cleanup step on an anti-pyrene cartridge, a great number of com1924
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FIGURE 4. Analysis of mussel tissue without (a) or after cleanup on the anti-pyrene IS (b) by UV detection at 215 nm. pounds that polluted the source or could modify the quantification are eliminated. Mass spectrometry can be used not only as a qualitative but also as a quantitative method, and the number of analyses without any cleanup of the source can be greatly enhanced. The off-line cleanup has been shown to be an easy way to clean up highly contaminated extracts such as sludge or mussel extracts. The whole procedure coupling the cleanup on the anti-pyrene and LC analysis with fluorescence and UV diode array detections in series has been validated on a certified reference sludge. The high selectivity of the antibodies offers the prospective of using this cleanup before injections on GC/MS systems. However, the experiments described above are still at the laboratory stage. The polyclonal antibodies used are of limited supply and vary from batch to batch. Reproducibility of immunosorbents can be obtained only with monoclonal antibodies. For that reason and to develop this method for routine usage, monoclonal antibodies are now in preparation.
Acknowledgments This work was supported by the Environment and Climate Program 1994-1998 from the Commission of European Communities (Contract ENV4-CT95-0016). We thank our partners of the program, especially N. Fisher-Durand and F. Le Goffic (ENSCP, Paris, France), for providing the anti-pyrene antibodies. We thank also Mallinckrodt Baker for the supply of columns and various chemical products.
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Received for review October 6, 1998. Revised manuscript received February 23, 1999. Accepted March 1, 1999. ES981031L
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