Accelerated Solvent Extraction Followed by On-Line Solid-Phase

Feb 16, 2002 - Benzalkonium chlorides (BACs) were successfully extracted from sediment samples using a new methodology based on accelerated solvent ...
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Anal. Chem. 2002, 74, 1275-1280

Accelerated Solvent Extraction Followed by On-Line Solid-Phase Extraction Coupled to Ion Trap LC/MS/MS for Analysis of Benzalkonium Chlorides in Sediment Samples Imma Ferrer and Edward T. Furlong*

U.S. Geological Survey, P.O. Box 25046, MS 407, Denver Federal Center, Denver, Colorado 80225-0046

Benzalkonium chlorides (BACs) were successfully extracted from sediment samples using a new methodology based on accelerated solvent extraction (ASE) followed by an on-line cleanup step. The BACs were detected by liquid chromatography/ion trap mass spectrometry (LC/MS) or tandem mass spectrometry (MS/MS) using an electrospray interface operated in the positive ion mode. This methodology combines the high efficiency of extraction provided by a pressurized fluid and the high sensitivity offered by the ion trap MS/MS. The effects of solvent type and ASE operational variables, such as temperature and pressure, were evaluated. After optimization, a mixture of acetonitrile/water (6:4 or 7:3) was found to be most efficient for extracting BACs from the sediment samples. Extraction recoveries ranged from 95 to 105% for C12 and C14 homologues, respectively. Total method recoveries from fortified sediment samples, using a cleanup step followed by ASE, were 85% for C12BAC and 79% for C14BAC. The methodology developed in this work provides detection limits in the subnanogram per gram range. Concentrations of BAC homologues ranged from 22 to 206 µg/kg in sediment samples from different river sites downstream from wastewater treatment plants. The high affinity of BACs for soil suggests that BACs preferentially concentrate in sediment rather than in water. Benzalkonium chlorides (BACs) are quaternary ammonium surfactants with detergent and antimicrobial properties. BACs are produced as an industrial cleanser and primarily consist of C12, C14, C16, and C18 alkyl chain homologues.1 Commercially produced BAC mixtures are used as an active ingredient in many household products. In a recent study by the present authors, microgram per liter concentrations of BACs were found in wastewater samples and samples downstream of wastewater treatment plants.2 Other workers report levels of BACs in similar matrixes.3,4 We postulated that BACs would be predominantly associated with solids rather * Corresponding author. E-mail: [email protected]. (1) Suzuki, S.; Nakamura, Y.; Kaneko, M.; Mori, K.; Watanabe, Y. J. Chromatogr. 1989, 463, 188-191. (2) Ferrer, I.; Furlong, E. T. Environ. Sci. Technol. 2001, 35, 2583-2588. (3) Kummerer, K.; Eitel, A.; Braun, U.; Hubner, P.; Daschner, F.; Mascart, G.; Milandri, M.; Reinthaler, F.; Verhoef, J. J. Chromatogr., A 1997, 774, 281286. (4) Kawakami, S.; Callicott, R. H.; Zhang, N. Analyst 1998, 3, 489-492. 10.1021/ac010969l Not subject to U.S. Copyright. Publ. 2002 Am. Chem. Soc.

Published on Web 02/16/2002

than with water because of the high hydrophobicity of this compound. Due to the ionic character of BACs, these compounds also might be strongly adsorbed to mineral surfaces through an ion-exchange mechanism. Thus, BACs should be present at higher concentrations in sediment samples as compared to water samples. A methodology for the extraction of BACs from bed sediment was developed to test this hypothesis. Extraction is often the most time-consuming and error-prone step in identifying hydrophobic contaminants in complex environmental matrixes, such as soil or sediment. The most commonly used method for extracting compounds from soil or sediment matrixes is Soxhlet extraction (method 3540, U.S. Environmental Protection Agency5). Normally, several hundred milliliters of organic solvent and 10-12 h are required to extract semi- and nonvolatile organic analytes from sediment matrixes. In the past few years, various attempts have been made to replace this classical extraction technique. Ultrasonic extraction (method 3550) is a USEPA approved alternative,5 which is faster than Soxhlet extraction and uses less solvent, but requires substantial sample handling, with concomitant potential for sample loss and exposure to solvents. Recently, microwave extraction and supercritical fluid extraction have been successfully applied to sediment extraction.6,7 Although these techniques reduce the volume of extraction solvent required and shorten the sample preparation time, compared to Soxhlet extraction, all of these proceduressas well as Soxhlet extractionsinvolve multiple sample manipulations, thus making them time consuming and labor intensive. The recently developed accelerated solvent extraction (ASE; also referred to as pressurized fluid extraction) technique offers an order of magnitude additional reduction in solvent use with faster sample processing times, and with the potential of automated, unattended extraction of multiple samples. Briefly, using ASE, a solid sample is enclosed in a sample cell that is filled with an extraction solvent; after the cell is sealed, the sample is permeated by the extracting solvent under elevated temperature and pressure for short periods (5-10 min). Typically, the sample is extracted under static conditions, where the fluid is held in the cell for controlled time periods to allow sufficient contact between (5) Test Methods for Evaluating Solid Waste, 3rd ed.; USEPA SW-846; U.S. GPO: Washington, DC, July 1995; Update III. (6) Fernandez, P.; Alder, A. C.; Suter, M. J-F.; Giger, W. Anal. Chem. 1996, 68, 921-929. (7) David, M. D.; Campbell, S.; Li, Q. X. Anal. Chem. 2000, 72, 3665-3670.

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the solvent and the solid for efficient extraction. Alternatively, dynamic or flow-through techniques can be used. Compressed gas is used to purge the sample extract from the cell into a collection vessel. ASE achieves rapid extraction with small volumes of conventional organic solvents by using high temperatures (up to 200 °C) and high pressures (up to 20 000 kPa) to maintain the solvent in a liquid state. The use of liquid solvents at elevated temperatures and pressures enhances efficiency compared to extractions at or near room temperature and atmospheric pressure because of enhanced solubility and masstransfer effects and the disruption of surface equilibrium.8 ASE has been used to extract various hydrophobic organic compounds from different environmental samples.8-11 Some studies have carried out comparisons between ASE and conventional techniques, such as supercritical fluid extraction (SFE) and Soxhlet extraction.12,13 In the studies where method comparisons were made, the performance of ASE was consistently equivalent to or better than conventional methods, such as Soxhlet and sonication extraction. In this study, we evaluated the effect of ASE conditions in the recovery of BAC homologues, a class of commonly used cationic surfactants. No previous studies describing the extraction of quaternary ammonium compounds using ASE techniques have been reported. An additional problem with the analysis of sediment samples is the necessary cleanup of the extracts prior to chromatographic analysis. The cleanup of complex sample matrixes is usually carried out with alumina or silica columns (e.g., polyaromatic hydrocarbons14) or C18 solid-phase extraction (SPE) cartridges. In this work, we developed a methodology that uses ASE in combination with on-line SPE for extract cleanup. This step represents an important advance because the sample is processed through the cartridge and analyzed by liquid chromatography/ mass spectrometry (LC/MS), which is a sensitive technique for these compounds,2 thus achieving low nanogram per gram method detection levels on a small (∼10 g wet weight) sample. The methodology developed in this work was applied to the extraction of environmental sediment samples from different sites. This work is the first reported identification of BACs in sediment samples at ambient environmental conditions. It also represents a step forward in coupling automated extraction of solid samples to automated compound isolation, fractionation, and analysis. EXPERIMENTAL SECTION Chemicals. A technical grade mixture of BACs was obtained from Acros/Fisher Scientific (Pittsburgh, PA). Although the exact composition of the standard mixture for each one of the alkyl homologues was not provided, we assumed a technical composi(8) Richter, B. E.; Jones, B. A.; Ezzel, J. L.; Porter, N. L.; Avdalovic, N.; Pohl, C. Anal. Chem. 1996, 68, 1033-1039. (9) Fisher, J. A.; Scarlett, M. J.; Stott, A. D. Environ. Sci. Technol. 1997, 31, 1120-1127. (10) Gan, J.; Papiernik, S. K.; Koskinen, W. C.; Yates, S. R. Environ. Sci. Technol. 1999, 33, 3249-3253. (11) Hubert, A.; Wenzel, K. D.; Manz, M.; Weissflog, L.; Engewald, W.; Schuurman, G. Anal. Chem. 2000, 72, 1294-1300. (12) Heemken, O. P.; Theobald, N.; Wenclawiak, B. W. Anal. Chem. 1997, 69, 2171-2180. (13) Bandh, C.; Bjorklund, E.; Mathiasson, L.; Naf, C.; Zebuhr, Y. Environ. Sci. Technol. 2000, 34, 4995-5000. (14) Perez, S.; Ferrer, I.; Hennion, M.-C.; Barcelo, D. Anal. Chem. 1997, 70, 4996-5001.

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tion reported for the industrial mixtures used in the manufacturing of the products containing BACs, which is as follows: 50% C12, 30% C14, 17% C16, and 3% C18 homologues. These percentages were confirmed by flow injection analysis of the mixture, assuming the same mass spectrometry response for all homologues. Highperformance liquid chromatography (HPLC) grade acetonitrile, methanol, and water were purchased from Burdick and Jackson (Muskegon, MI). Ammonium formate and formic acid were obtained from Aldrich (Milwaukee, WI). The SPE cartridges used for on-line preconcentration of the aqueous sediment extracts were polymeric reversed-phase (PLRP-s; 10 mm by 2 mm inside diameter disposable precolumns that contained 20 mg of sorbent) obtained from Jones Chromatography (Lakewood, CO). Sampling. Sediment samples were collected downstream from different wastewater treatment plants and river sites near the plants. Grab sediment samples were collected from the South Platte River, Denver, CO, and from Indian Creek, Kansas City, MO. These streams receive discharge from wastewater treatment plants. The sediment samples were collected from depositional zones in wadeable sections of the stream. Sediment samples were collected into glass jars (baked at 300 °C) with Teflon-lined lids, chilled, and either directly transported to the laboratory or shipped chilled to the laboratory by overnight express. Samples were held at 4 °C until extraction. Sediment Fortification Experiments. The recoveries (percentage of standard added to sample that is recovered during extraction and cleanup) and reproducibility (relative standard deviation for triplicate analysis) of the method were determined by sediment fortification. Previously freeze-dried sediments from Evergreen Lake, Colorado, were fortified with BACs at 32.5 µg/ kg and atrazine (used as a surrogate standard) at 500 µg/kg and then incubated for at least 24 h to allow time for the analyte to interact with the matrix of the sediment and thus approximate real conditions. These sediment samples were previously used for validating a gas chromatography/mass spectrometry (GC/ MS) method for semivolatile compounds, such as polyaromatic hydrocarbons,15 and the ambient concentrations of other contaminants did not interfere in the method developed in this work. The BAC fortification solution (3.25 µg/mL) was prepared in methanol. Unfortified sediment samples also were analyzed for interferences and for determining whether ambient concentrations of BACs were present. No traces of BACs were found in the unfortified sediment samples. ASE Extraction. An automated Dionex-ASE 200 system (Dionex Co., Sunnyville, CA) was used for the sediment extractions. Ten grams of wet sediment was packed into a 33-mL stainless steel ASE vessel. The packed vessels were sealed at both ends with circular cellulose filters of 2.1-cm diameter (Whatman, Springfield Mill, Maidstone, Kent, U.K.). At the end of each extraction, nitrogen gas was used to expel the extract into glass collection vials (60-s purge). The final volume of extract was ∼45 mL. (15) Furlong, E. T.; Vaught, D. G.; Merten, L. M.; Foreman, W. T.; Gates, P. M. Methods of analysis by the U.S. Geological Survey National Water Quality Laboratory. Determination of semivolatile organic compounds in bottom sediment by solvent extraction, gel permeation chromatographic fractionation, and capillary-column gas chromatography/mass spectrometry. OpenFile Rep.-U.S. Geol. Surv. 1996, 95-719.

To determine the effect of solvent type on extraction performance, fortified sediments were extracted with miscible mixtures from among the solvents dichloromethane, hexane, acetone, methanol, acetonitrile, and water under identical conditions (120 °C, 10 340 kPa, and 10 min static time). Once the optimal solvent mixture was chosen, the optimal ASE temperature and pressure were determined. During optimization, temperature was varied from 75 to 200 °C, and the pressure was varied between 6900 and 13 800 kPa. For all optimizations, the extracts were analyzed directly by LC/MS because the sensitivity of this system allowed the detection of such low levels of concentration. Once temperature and pressure conditions were optimized, the total method recoveries for BAC homologues were determined for the entire methodology developed in this work (ASE followed by SPE/HPLC/ESI-MS). For this purpose, the extracts were brought up to 115 mL with distilled water and were treated as aqueous samples for the following cleanup and preconcentration step. Aqueous extracts were processed through automated SPE following the procedure of Ferrer and Furlong.2 Five replicates (n ) 5) was used to calculate the reproducibility of the method. Extract Cleanup and Concentration. The aqueous extracts were preconcentrated with an automated SPE system (Prospekt; Spark Holland, Emmen, The Netherlands) coupled on-line to a series 1100 HPLC (Hewlett-Packard, Palo Alto, CA). The SPE system consists of a cartridge-exchange module, a solvent delivery unit, and a low-pressure six-port valve that is connected directly to the gradient pumps of the HPLC system. Aqueous dilutions of ASE extracts were preconcentrated on PLRP-s cartridges.2 Prior to SPE, the aqueous sediment extracts were diluted to match the water/organic solvent composition of the previous optimized method.2 First, the cartridges were conditioned sequentially with 6 mL of acetonitrile and 4 mL of HPLC grade water. Then, a 50mL aliquot of sediment extract was preconcentrated through the cartridge at a flow rate of 2 mL/min. Finally, the compounds trapped on the sorbent were eluted onto the LC column with the chromatographic mobile phase by switching the valve into the elute position. LC/ESI-MS. Liquid chromatography/electrospray-ion trap mass spectrometry (LC/ESI-MS), in positive ionization, full-scan operation, was used to separate and identify the BAC homologues. The homologues were separated using an HPLC (series 1100, Agilent Technologies, Palo Alto, CA) equipped with a reversedphase C18 analytical column (Phenomenex RP18, Torrance, CA) of 250 mm by 3 mm and 5-µm particle diameter. Column temperature was maintained at 25 °C. The mobile phase used for eluting the analytes from the SPE and HPLC columns consisted of acetonitrile and 10 mM ammonium formate buffer, at a flow rate of 0.6 mL/min. A gradient elution was performed as follows: from 50% A (acetonitrile) and 50% B (10 mM ammonium formate) to 100% A and 0% B in 15 min and then held isocratically at 100% A for 10 min. This HPLC system was connected to an ion trap mass spectrometer LC/MS/MS (Esquire LC, Bruker Daltonics, Bellerica, MA) system equipped with an electrospray ionization probe. The operating conditions of the MS system were optimized in full-scan mode (m/z scan range, 50-400) by flow injection analysis of each compound at 10 µg/mL concentration.2 The maximum ion accumulation time was set at 200 ms.

Figure 1. Mass spectra for benzalkonium chlorides collected from a technical mixture using flow injection analysis. (a) C12 benzalkonium chloride homologue; (b) C14 benzalkonium chloride homologue.

MS and MS/MS detection. The operational conditions of the ESI interface and MS/MS conditions were optimized for the BAC homologues under study in a previous paper,2 and they are not described here. As expected, BAC homologues showed a high response under positive ESI conditions as a result of the fixed positive charge on the quaternary nitrogen. The mass spectrum for C12BAC and C14BAC is shown in Figure 1. The ion trap instrument also is used to perform multiple mass spectrometry (MS/MS) experiments that isolate and fragment specific ions. Use of MS/MS conditions is especially valuable in the analysis of a complex sediment extract where other compounds with the same molecular ion present in the mixture can coelute with the compound of interest. Isolating and fragmenting this molecular ion under MS/MS conditions provides specific fragmentation information that confirms analyte identification. The conditions for MS/MS confirmation also are described in the previous work.2 RESULTS AND DISCUSSION ASE Extraction. (a) Solvent Effect. Different extraction solvents and mixtures were evaluated for the extraction of BACs from fortified sediment samples. Preliminary experiments showed that dichloromethane, acetone, or hexane did not extract BACs from the samples efficiently, with best recoveries ranging from 5 to 10%. Surprisingly, organic solvents alone (i.e., methanol/ dichloromethane) did not remove BACs as efficiently from the sediment as might be expected. Instead, we observed that water is a necessary solvent component for the efficient extraction of BACs. This result might be explained because of the fixed ionic character of BACs, which could form ionic bonds with the humic and fulvic matter in the sediment or with mineral surfaces. Among all the solvents tested, a mixture of acetonitrile/water at a ratio of either 6:4 or 7:3 provided the highest recoveries (see Table 1). Analytical Chemistry, Vol. 74, No. 6, March 15, 2002

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Table 1. Mean Percent Recoveries of Benzalkonium Chloride Homologues Obtained after the Extraction from Sediment Samples Fortified at 3.25 µg/kg with Different Percentages of Solvents extraction recovery(%) solvent

conditionsa

MeOH/CH2Cl2 (7:3) MeOH/H2O (6:4) MeOH/H2O (5:5) MeOH/H2O (3:7) ACN/H2O (7:3) ACN/H2O (6:4) ACN/H2O (5:5) ACN/H2O (3:7) a

C12BAC

C14BAC

9 76 33 8 96 95 83 12

18 85 35 9 93 93 85 11

MeOH, methanol; ACN, acetonitrile.

Further variations in the percentage of both solvents had a negative effect in the recovery of BACs. Higher percentages of water than 60% (i.e., 70%) did not further improve extraction of BACs. The different extraction efficiencies by different solvents might be attributed to the different polarities of these solvents16 or the fact that the BAC mixture is a surfactant and has optimal solubility in a mixture of aqueous/organic solvents. Adjusting solvent pH using ammonium formate buffer solution did not improve recoveries, so solvents were not pH adjusted. A 6:4 acetonitrile/water mixture was chosen for subsequent extractions because it required less dilution for on-line SPE. (b) Optimization of Temperature and Pressure. For a given solvent mixture, temperature, pressure, and time are the three most important variables that can affect the extraction efficiency by ASE.10 We investigated the effects of changing the temperature and pressure on extraction efficiency. Temperaturesrather than pressure or timesis expected to have a greater effect on solubility and mass transfer for the performance of ASE. The increased solubility of water in organic solvents at elevated temperatures increases the contact of the solvent with the analytes in wet sediment, and they should be extracted more efficiently than at room temperature. Another effect of increasing extraction temperature is to overcome the energy barrier of desorption. Temperatures were varied between 75 and 200 °C at a constant pressure of 10 340 kPa, and the results are shown in Figure 2a. An increase in temperature (above 75 °C) leads to higher extraction recoveries. Above 120 °C, no substantial differences were observed for C12BAC, but a reverse effect was seen for C14BAC. The pressure applied to the cell is necessary to keep the solvents in a liquid state when temperatures greater than their boiling points at 1 atm are used. Secondarily, pressure enhances permeation of solvent into the sample matrix. The effect of pressure on analyte recoveries was investigated by extracting fortified sediment samples with BACs at pressures ranging from 6900 and 13 800 kPa at a constant temperature of 100 °C, as described in the Experimental Section, and at a constant extraction solvent composition of 6:4 acetonitrile/water. The purpose of this test was to determine whether elevated pressures improved (16) Hawthorne, S. B.; Yang, Y.; Miller, D. J. Anal. Chem. 1994, 66, 29122920.

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Figure 2. Optimization of (a) temperature and (b) pressure for the accelerated solvent extraction of benzalkonium chloride homologues from sediment samples.

solvent diffusivity into the matrix pores, where the analytes might be sequestered. In this case, no substantial differences were observed in the extraction recoveries for BACs within this pressure range (see Figure 2b). The number of cycles carried out for the static pressurization is also an important parameter in ASE extraction. A total of three cycles provided higher extraction recoveries and improved precision in the results (data not shown). Recoveries were not improved by increasing the number of extraction cycles beyond three; no homologues were detected in the fourth cycle of extraction. Finally, a total volume of 40% fresh solvent after each static cycle was found to be appropriate for the extraction of the analytes. The optimized conditions for the extraction of BACs from sediment samples and used in subsequent extractions are shown in Figure 3. (c) ASE Recoveries for the Fortified Sediment. The extraction recoveries for BACs from fortified sediment using only the ASE extraction followed by detection by LC/ESI-MS are listed in Table 2 (second column). These recoveries were obtained by direct injection of a 20-µL aliquot of the extracted volume of 45 mL obtained after the ASE. The purpose here was to determine the performance of the ASE extraction without potential variability introduced by on-line SPE. No additional cleanup step was necessary because the sensitivity of the LC/MS ion trap system was sufficient to detect low microgram per liter levels, and the matrix of the sediment was not complex. When wastewaterinfluenced sediments are analyzed, a cleanup step is necessary before injecting them into the mass spectrometer because the samples contain substantial coextracted interferences, which could decrease recoveries.

Table 3. Concentrations (µg/kg) of Benzalkonium Chloride Homologues in Sediment Samples, Analyzed by Accelerated Solvent Extraction (ASE) Followed by On-Line SPE/LC/ESI-MS under Positive Mode of Operationa

b

Figure 3. Diagram of the methodology developed in this work. Table 2. Mean Percent Recoveries, Total Method Recoveries, and Relative Standard Deviations (RSD, n ) 3) of Benzalkonium Chloride Homologues by Accelerated Solvent Extraction (ASE), Obtained after Extraction of 10 g of Sediment Fortified at 32.5 µg/kg with Benzalkonium Chloride compound C12BAC C14BAC

% extraction recovery using ASE (RSD)

% total method recoveries (RSD)

103 (9) 95 (12)

85 (12) 79 (15)

Solid-Phase Extraction Cleanup. The retention of BAC homologues on C18 and polymeric cartridges in the extraction of water samples was investigated and is reported in a previous paper.2 In that study, quantitative addition of acetonitrile to the water sample to make a 25% acetonitrile solution was the approach chosen because adsorption of BACs to the glass bottle surface was minimized while high recovery from the polymeric SPE cartridge was maintained. Total method recovery values for BACs from aqueous extracts were ∼90% when using PLRP-s cartridges. In this method, we are using a mixture of acetonitrile and water to extract BACs from the sediment samples; dilution of the sediment extract to 25% acetonitrile/75% water allowed preconcentration through a polymeric cartridge as if the extract were a water sample. This result is convenient because BACs are preconcentrated in the cartridge, and a cleanup step also is performed simultaneously. A flowchart of the procedure for the determination of BACs in sediment samples is shown in Figure 3. Analytical Performance. Samples require little manipulation using this method because the two main steps of the procedures sample extraction and extract isolation with cleanupsare fully automated, and as a consequence, better precision can be obtained. The precision of the method was evaluated by using three independent extractions of BACs from fortified sediment samples. The relative standard deviations were 12 and 15% for C12BAC and C14BAC, respectively, indicating good reproducibility of the method developed in this work. Mean total method recoveries of the fortified sediments were 85% for C12BAC and 79% for C14BAC (see Table 2), indicating quantitative recovery when fortified at 32.5 µg/kg BAC (technical

sediment location

C12BAC m/z 304

C14BAC m/z 332

C16BAC m/z 360

Indian Creek at 69 Hwy (KS) Indian Creek at Farley (KS) Indian Creek at 103rd St. (MO) South Platte River (CO)

24 23 105 35

55 54 206 22

67b 70b 260b 21b

a Relative standard deviation (n ) 3) vaired between 8 and 15%. Estimated from response of C14BAC.

composition). These recoveries are lower than those found in water and may be attributed to either inefficient recoveries or losses during the ASE extraction and transfer of the extract to different vessels for on-line SPE plus HPLC/ESI-MS. Calibration curves were constructed by passing 50 mL of reagent water, fortified with a solution containing BAC, through a PLRP-s cartridge. The curves obtained were linear across the concentration range studied, from 10 to 300 µg/kg (n ) 7), and the correlation coefficients (r2) were higher than 0.98 for both compounds. Method detection limits were calculated by using a signal-tonoise ratio of 3 (the ratio between the peak intensity under selected-ion monitoring conditions and the noise). These detection limits were calculated from the analysis of extracts derived from processing sediments fortified at low concentrations through the entire method. Method detection limits were 0.16 µg/kg for C12BAC and 0.2 µg/kg for C14BAC using this criterion. Thus, the method developed in this work has the capability of selectively detecting low concentrations of the BAC homologues in environmental sediment samples. No BACs were detected in blank samples routinely analyzed between environmental samples. Environmental Sediment Samples. The performance of the method was evaluated by analyzing sediment samples suspected to contain environmentally relevant concentrations of BACs. Sediment samples were collected from several locations in Indian Creek within metropolitan Kansas City, on the border between Missouri and Kansas, and one sediment sample collected in the South Platte River in Denver. Sample-specific sediment properties were not determined, but in general, the clay fraction (defined as that portion of the sediments