Evaluation of a Fast and Simple Sample Preparation Method for

Feb 3, 2015 - E-mail: [email protected]. This article is part of the IUPAC - Analysis of Residues in Food special issue. Abstract. Abst...
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Evaluation of a Fast and Simple Sample Preparation Method for Polybrominated Diphenyl Ether (PBDE) Flame Retardants and Dichlorodiphenyltrichloroethane (DDT) Pesticides in Fish for Analysis by ELISA Compared with GC-MS/MS Yelena Sapozhnikova,* Tawana Simons, and Steven J. Lehotay Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, United States ABSTRACT: A simple, fast, and cost-effective sample preparation method, previously developed and validated for the analysis of organic contaminants in fish using low-pressure gas chromatography−tandem mass spectrometry (LPGC-MS/MS), was evaluated for the analysis of polybrominated diphenyl ethers (PBDEs) and dichlorodiphenyltrichloroethane (DDT) pesticides using enzyme-linked immunosorbent assay (ELISA). The sample preparation technique was based on the quick, easy, cheap, rugged, effective, and safe (QuEChERS) approach with filter-vial dispersive solid phase extraction (d-SPE). Incurred PBDEs and DDTs were analyzed in three types of fish with 3−10% lipid content: Pacific croaker, salmon, and National Institute of Standards and Technology (NIST) Standard Reference Material 1947 (Lake Michigan fish tissue). LPGC-MS/MS and ELISA results were in agreement: 108−111 and 65−82% accuracy ELISA versus LPGC-MS/MS results for PBDEs and DDTs, respectively. Similar detection limits were achieved for ELISA and LPGC-MS/MS. Matrix effects (MEs) were significant (e.g., −60%) for PBDE measurement in ELISA, but not a factor in the case of DDT pesticides. This study demonstrated that the sample preparation method can be adopted for semiquantitative screening analysis of fish samples by commercial kits for PBDEs and DDTs. KEYWORDS: gas chromatography−tandem mass spectrometry, PBDEs, DDT pesticides, ELISA, fish

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INTRODUCTION

DDD and DDE, which are also persistent in the environment and animal tissues. The most common tool for the analysis of both PBDEs and DDTs is gas chromatography with mass spectrometry (GCMS).6,7 GC-MS with electron ionization (EI) analysis offers high accuracy, sensitivity, and selectivity, but requires relatively expensive instrumentation and trained personnel for its operation and maintenance in a laboratory environment. Biochemical assays, such as enzyme-linked immunosorbent assay (ELISA), on the other hand, are simple and relatively inexpensive. ELISAs are often very sensitive and selective for the intended analyte. For example, because PBDE antibody was specifically developed for PBDE 47,8 the ELISA PBDE kit is very sensitive and selective to this congener, with some crossreactivity toward other congeners (28, 99, and 100) similar in structure to PBDE 47. In addition, another ELISA test kit specifically designed for a different type of contaminant (DDTs, for instance) is needed to analyze the same sample for additional analytes. ELISAs are often used as a screening tool for chemical contaminants, and positive findings subsequently are analyzed by a traditional method, such as GC-MS. Traditional methods for fish sample preparation for GC-MS analysis oftentimes include pressurized liquid extraction (PLE),

Simple, fast, and cost-effective sample preparation techniques are critical for the analysis of chemical contaminants in food and environmental samples to ensure food safety and ecosystem health. At the same time, the methods have to be reliable, accurate, and reproducible and provide low detection limits to meet regulatory needs. Persistent organic pollutants (POPs) are anthropogenic contaminants that are long-lived, oftentimes bioaccumulative, and toxic. Polybrominated diphenyl ethers (PBDEs) are brominated chemicals added as flame retardants to a variety of consumer products, including electronics, carpets, upholstery, textile, etc. Because they are not covalently bound to materials to which they are added, PBDEs freely liberate and make their way into the environment and foods. Multiple studies have reported PBDEs in air, dust, wastewater, fish, and other sample types. Recently, on the basis of their persistence and hazardous effects,1 some classes of PBDEs have been recognized as POPs by the Stockholm convention2 and therefore are being phased out and banned from consumer products.3 However, because of their persistence, they are still present in the environment and present a potential risk. Dichlorodiphenyltrichloroethane (DDT) pesticides (o,p′DDE, p,p′-DDE, o,p′-DDD, p,p′-DDD, o,p′-DDT, and p,p′DDT) are stable organochlorinated compounds, which were banned in the 1970s.4 DDTs are included in the “dirty dozen” chemical list,2 and similar to PBDEs, they are persistent (with half-lives >30 years), accumulate in fatty tissues, and cause toxic effects.5 DDT undergoes degradation, forming its metabolites This article not subject to U.S. Copyright. Published XXXX by the American Chemical Society

Special Issue: IUPAC - Analysis of Residues in Food Received: November 21, 2014 Revised: February 2, 2015 Accepted: February 3, 2015

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were homogenized with dry ice as described above and stored in glass containers in a −18 °C freezer. Standards of PBDE congeners (28, 47, 99, 100, 153, 154, and 183) and DDT pesticides (o,p′-DDE, p,p′-DDE, o,p′-DDD, p,p′-DDD, o,p′DDT, and p,p′-DDT) were purchased from AccuStandard (New Haven, CT, USA). All standards were ≥98% purity, and all solvents were of HPLC grade. Acetonitrile (MeCN), ethyl acetate, and hexane were from Fisher Scientific (Pittsburgh, PA, USA) and methanol (MeOH) was from VWR International (Radnor, PA, USA). Deionized water of 18.2 Ω-cm was prepared with an E-Pure model D4641 from Barnstead/Thermolyne (Dubuque, IA, USA). Primary−secondary amine (PSA) and C18 sorbents were purchased from UCT (Bristol, PA, USA). Z-Sep sorbent, which incorporated zirconium dioxide, was from Supelco (Bellefonte, PA, USA). Ammonium formate was from Sigma-Aldrich (St. Louis, MO, USA). PVDF 0.2 μm filter vials were from Thomson Instrument Co. (Oceanside, CA, USA). ELISA kits, 96-well microliter plates for PBDE and DDE/DDT, were from Abraxis (Warminster, PA, USA). Sample Preparation. The sample preparation procedure was based on the quick, easy, cheap, rugged, effective, and safe (QuEChERS) approach including the recently reported filter-vial dSPE cleanup technique.15 Homogenized fish samples (4 g) were placed into 50 mL polypropylene tubes, and after the addition of 4 mL of MeCN, the tubes were shaken for 10 min on a pulsed-vortexer (Glas-Col, Terre Haute, IN, USA) at 80% setting with maximum pulsing. Water (4 mL) was used as a reagent blank. After extraction, 2 g of ammonium formate was added to induce the phase separation,16 and the samples were shaken for 1 min on the pulsed-vortexer and centrifuged for 2 min at 3700 rcf. Extracts (0.5 mL) were transferred into the shell (bottom) portions of 0.2 μm PVDF filter-vials containing 75 mg of anhydrous MgSO4, 25 mg of PSA, 25 mg of C18, and 25 mg of Z-Sep sorbents. The filter-vial plungers (top pieces) were partially depressed, and the extracts were shaken by hand for 30 s in an autosampler vial tray. Then the plungers were fully depressed into the filter-vial shells, forcing the cleaned-up extract through the filter membrane. Filtered extracts (200 μL) were transferred to glass autosampler vials containing inserts for LPGC-MS/MS analysis. Another set of filtered extracts (200 μL) was transferred to glass tubes for ELISA analysis. These extracts were evaporated to dryness with nitrogen and reconstituted in 200 μL of 1:1 (v/v) MeOH/water for ELISA analysis. Different fish extracts were prepared in four replicates (except for SRM 1947) on different days for separate ELISA and LPGC-MS/MS analysis. The equivalent fish tissue concentration was 1 g/mL in all final extracts. Determination of Coextractives. Four types of fish tissues (croaker and salmon each from the specimen bank and from grocery stores) were extracted with MeCN, hexane, and ethyl acetate. Each solvent (15 mL) was added to 15.0 g each of the four fish tissues in 50 mL polypropylene tubes, and the tubes were shaken on the pulsedvortex shaker for 10 min as described above. In the case of MeCN, two replicate extracts were prepared (one was used to determine coextractives, and the other was to estimate d-SPE cleanup efficiency). Additional MeCN extracts were prepared in the same manner for store-purchased salmon and croaker to generate matrix-matched (MM) calibration standards. For MeCN extracts, 7.5 g of ammonium formate was added to induce phase separation. The tubes were then centrifuged for 2 min at 3700 rcf, and 10 mL of each corresponding solvent extract was transferred into a preweighed glass tube, evaporated to dryness, and dried in an oven at 120 °C, after which the glass tubes with residues were reweighed. d-SPE Removal Efficiency. Aliquots (10 mL) of MeCN extract for each fish type were placed into 50 mL tubes, to which 1500 mg of anhydrous MgSO4 and 500 mg each of PSA, C18, and Z-sep were added for d-SPE cleanup. The tubes were shaken for 30 s and centrifuged for 2 min at 3700 rcf. The treated extracts (5 mL) were placed into preweighed glass tubes and evaporated to dryness. After drying in the oven at 120 °C, the glass tubes with residues were weighed again. To prepare MM calibration curves for the ELISA procedure, another set of MeCN extracts after d-SPE of store-bought salmon and

followed by gel permeation chromatography (GPC) or column chromatography with sorbents to remove lipids and other interfering components from fish extracts.9 Nonpolar solvents, such as hexane, acetone, dichloromethane, and ethyl acetate, are commonly used for extraction. Similar techniques may be used to prepare fish samples for ELISA. For example, PLE with hexane and dichloromethane was used for extraction of fish tissues, followed either by cleaning with sulfuric acid or an acidified silica gel column to prepare fish samples for ELISA.10,11 PBDEs and DDTs are lipophilic and therefore accumulate in fatty tissues, such as in fish. In most method development studies, laboratory-spiked samples are used to assess method performance, but analytes are not internally integrated into the fat; thus, spiking is not necessarily acceptable to determine true extractability of the analytes from within fatty tissues. In incurred samples, contaminants are bound into cells and absorbed by fat and may not be readily available to the extraction solvent compared to spiked samples. The extractability of incurred contaminants is an important topic, but it is not given enough attention in method validation guidelines or in the scientific literature. A previous report on sample preparation for many POPs and pesticides in incurred fish with lipid content 3−10% showed acceptable total extractability and method performance using analysis by low-pressure gas chromatography−tandem mass spectrometry (LPGC-MS/ MS).12 This method should also be applicable to ELISA and provide superior ease, lower cost, and other benefits compared to PLE and GPC as described in the literature.9,11,13 The goal of this study was to evaluate a simple, rapid, and cost-effective sample preparation method, previously developed and validated for the analysis of diverse POPs in various fish tissues using LPGC-MS/MS, for the analysis of PBDEs and DDTs by commercial ELISA kits using incurred fish samples with various lipid content. Additionally, we sought to estimate matrix effects affecting the accuracy of measurements in the ELISAs for both contaminant groups. Comparison of ELISA and LPGC-MS/MS was also possible for the same samples, taking cross-reactivities of the ELISAs for individual analytes into account.

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MATERIALS AND METHODS

Samples of Pacific white croaker (Genyonemus lineatus) and salmon (unknown species) were received from the National Institute of Standard and Technology (NIST) Marine Environmental Specimen Bank in Charleston, SC, USA. The croaker was collected in 2002 from Palos Verdes Shelf Los Angeles Sanitation District Zone 1 EPA Superfund site in Palos Verdes, CA, USA. Croaker samples were received as a frozen, homogenized powder. The salmon was contributed to the specimen bank by Axys Analytical, Sidney, BC, Canada (collection location was unknown). The salmon was received frozen as a whole-body fish and was thawed, filleted into skinless and boneless chunks, refrozen, and then homogenized with dry ice using a Robot Coupe (Ridgeland, MS, USA) RSI 2Y1 chopper. It was stored in glass containers at −18 °C until portions were thawed for analysis. The measured moisture content was 63% for salmon and 79% for croaker, respectively. Reported average lipid levels were 3−4 and 5− 10% for croaker and salmon, respectively.14 Standard Reference Material (SRM) 1947 (Lake Michigan Fish Tissue) was purchased from NIST (Gaithersburg, MD, USA), and contained 73% moisture and 10% fat content. SRM 1947 contains PBDE congeners and DDT pesticides with certified/reference concentrations. In addition, samples of Pacific white croaker and salmon were purchased from local grocery stores as “clean” matrices for use in matrix-matched calibration. They B

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croaker (5 mL of each fish extract) were transferred into glass tubes, evaporated to dryness, and reconstituted in 5 mL of 1:1 (v/v) MeOH/ water. Aliquots of 0.45 mL of the corresponding fish extracts were placed into autosampler vials, and PBDE 47 standard solution was added to yield concentrations of 0, 0.1, 0.25, 0.5, 1, 2, and 4 ng/g, as was p,p′-DDE standard solution to yield concentrations of 0, 0.625, 1.25, 2.5, 5, 10, and 25 ng/g in a final volume of 0.5 mL. LPGC-MS/MS. Low-pressure vacuum outlet GC was carried out using an Agilent 7890 GC system with a multimode inlet (MMI) interfaced to a 7000B triple-quadrupole MS/MS. The GC instrument was upgraded with a 220 V rapid heating oven device. A capillary column (15 m × 0.53 mm i.d. × 1 μm film thickness) Rti-5 ms (Restek, Bellefonte, PA, USA) was coupled to a restriction capillary (5 m × 0.18 mm i.d.) (Restek HydroGuard). High-purity He was the carrier gas at a constant flow rate of 2 mL/min. The MMI was initially set at 80 °C for 0.31 min with vent at 50 mL/min, then ramped at 420 °C/min to 320 °C, and held until 9.5 min. Injection volume was 5 μL. The oven temperature started at 70 °C for a 1.5 min hold, followed by a ramp at 80 °C/min to 180 °C, then at 40 °C/min to 250 °C, and then at 70 °C/min to 290 °C, at which it was held until 9.5 min. The transfer line and ion source were set at 280 and 320 °C, respectively, and the quadrupole was at 150 °C. Two MRMs per each analyte were used with a dwell time of 2.5 ms. Calibration curves for PBDE congeners 28, 47, 99, 100, 153, 154, and 183 and p,p′-DDD, o,p′-DDT, o,p′-DDD, and p,p′-DDT were constructed at 0, 0.1, 1, 5, 10, 50, 100, 250, and 500 ng/g in MeCN. To quantify the high concentrations of DDE in the croaker, an additional calibration point of 1000 ng/g was added for o,p′-DDE. For p,p-DDE, the least intense MRM transitions were selected to avoid detector saturation and still achieve linear calibration with additional calibration points at 500, 1000, 2500, 5000, 7500, 10000, and 15000 ng/g of p,p′-DDE.12 ELISA Procedure. Because the analyte concentrations in the incurred samples were known, croaker and SRM 1947 extracts were diluted 1:50, 1:100, and 1:1000 times with MeOH/water (1:1 v/v) to fall within the calibration curve range. The ELISA procedure described in the commercial kit instructions was followed.17 For PBDE analysis, 50 μL of the final extracts and MM and solvent calibration standards (in MeOH/water (1:1 v/v) were placed into the wells, and 50 μL of PBDE antibody solution was added. After careful mixing and 30 min of incubation time at room temperature, 50 μL of PBDE enzyme conjugate solution was added. After further mixing, the solutions in the wells were incubated for another 30 min at room temperature. Then, the solutions were removed from the wells, which were rinsed with the washing buffer (provided with the commercial kit) four times. Afterward, 100 μL of color solution (hydrogen peroxide and 3,3′,5,5′-tetramethylbenzidine) was added, mixed, and allowed to incubate again for 30 min. Finally, 100 μL of stopping solution (0.5% of sulfuric acid) was added, and the optical density (OD) was read at 450 nm using the 96-well plate reader Synergy HT from Bio-Tek Instruments (Winooski, VT, USA). Quantification was based on MM calibration curves for croaker and salmon. For the DDE/DDT pesticide assay,18 25 μL of final extracts and MM and solvent calibration standards were incubated in wells with 50 μL of DDE/DDT antibody solution at room temperature for 30 min, after which 50 μL of enzyme conjugate solution was added, mixed, and incubated again for 30 min. After the incubation, all wells were emptied, rinsed four times with washing buffer, and 150 μL of color solution was added. Following another 30 min of incubation, 100 μL of stopping solution was added, and the OD was measured as before. Measured OD was converted to ng/g concentrations using semilog calibration curves after normalization to the OD control values (%B/ B0). In initial experiments, measured ODs from replicated wells were within 1% RSD; thus, only one well per sample was used for subsequent analyses.

Article

RESULTS AND DISCUSSION Sample Preparation. In our sample preparation method, MeCN was used as an extraction solvent, providing acceptable recoveries for relatively nonpolar and polar organic contaminants from fish tissues without extracting significant amounts of lipids and other possible interferences. For instance, hexane and ethyl acetate are commonly used for extraction of lipophilic POPs from fish and other fatty tissues.19,20 On the basis of studies using spiked samples, both solvents were shown to provide excellent recoveries. However, because hexane and ethyl acetate are rather nonpolar, they tend to extract significant amounts of lipids and similar coextractive components, creating a need for extensive cleanup to remove interfering coextractive components prior to analysis. Figure 1A demonstrates the

Figure 1. Comparison of matrix coextractive amounts (mg/g): (A) initial extraction; (B) coextractive removal efficiency (%) of MeCN extracts by d-SPE in the method.

difference in amounts of coextracted materials in four fish types in this study using hexane, ethyl acetate, and MeCN as extraction solvents. Hexane and ethyl acetate extracts contained up to 11 and 18 times, respectively, more coextractives compared to MeCN extracts. Furthermore, the filter-vial dSPE approach with the selected combination of sorbents (C18, PSA, and Z-Sep) removed 83−95% of coextracted matrix from the salmon and croaker extracts (Figure 1B), providing relatively clean extracts for analysis. This new QuEChERSbased sample preparation method is simple and rapid, enabling preparation of a batch of 10 prehomogenized fish in 1 h by one analyst, and it can be implemented in any laboratory or even in the field if shaking is done vigorously by hand and time is given for the salted-out MeCN layer to separate from the water. The cost per sample is approximately $3 for materials. The final MeCN extracts could be directly injected in GCMS. However, MeCN is not typically suitable for ELISA, and our experiments using 10, 25, and 50% MeCN in aqueous solutions for final extracts in analysis of PBDEs by ELISA failed C

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Table 1. Average Measured Concentrations of Incurred PBDEs and DDT Pesticides in Croaker and Salmon by LPGC-MS/MS, Measured Concentrations of Contaminants in SRM 1947, and Accuracy of Measurements versus Certified/Reference Concentrations av ± SD (ng/g) (n = 4) analyte

croaker

p,p-DDD + o,p′-DDT o,p′-DDD o,p′-DDE p,p′-DDE p,p′-DDT PBDE 28 PBDE 47 PBDE 99 PBDE 100 PBDE 153 PBDE 154

457 26 1210 10200 23 1.9 173 7.8 34.7 4.0 6.5

± ± ± ± ± ± ± ± ± ± ±

63 2 110 670 2 0.2 8 0.9 1.7 0.6 0.2

SRM 1947 salmon

ng/g

accuracy (%)

1.64 ± 0.15

57.7 3.9 2.9 753 57.5 1.95 68.3 14.5 11.9 3.8 8.3

94 119 87 105 97 86 93 76 70 100 122

9.8 ± 1.1

1.9 ± 0.1

Table 2. Comparison PBDEs and DDTs Concentrations between LPGC-MS/MS and ELISA Methods PBDE 47 equivalents, av ± SD (ng/g) croaker salmon SRM 1947

p,p′-DDE equivalents, av ± SD (ng/g)

calcd from LPGC-MS/MS data

measured by ELISA, (n = 4)

calcd from LPGC-MS/MS data

measured by ELISA, (n = 4)

181 ± 8 1.9 ± 0.1 82

195 ± 28 2.1 ± 1.0 89

10450 ± 670 11 ± 1 786

6800 ± 2400 9±2 560

to produce a dose−response curve, which is consistent with findings from a previous study.21 Therefore, the fish extracts had to be evaporated to dryness and reconstituted in 50% aqueous MeOH solution prior to ELISA. When aqueous solutions containing 140 pesticides in fish. J. Agric. Food Chem. 2014, 62, 3684−3689. (8) Shelver, W. L.; Keum, Y. S.; Kim, H. J.; Rutherford, D.; Hakk, H. H.; Bergman, A.; Li, Q. X. Hapten syntheses and antibody generation for the development of a polybrominated flame retardant ELISA. J. Agric. Food Chem. 2005, 53, 3840−3847. (9) Stapleton, H. M.; Keller, J. M.; Schantz, M. M.; Kucklick, J. R.; Leigh, S. D.; Wise, S. A. Determination of polybrominated diphenyl ethers in environmental standard reference materials. Anal. Bioanal. Chem. 2007, 387, 2365−2379. (10) Xu, T.; Cho, I. K.; Wang, D.; Rubio, F. M.; Shelver, W. L.; Gasc, A. M.; Li, J.; Li, Q. X. Suitability of a magnetic particle immunoassay for the analysis of PBDEs in Hawaiian euryhaline fish and crabs in comparison with gas chromatography/electron capture detection-ion trap mass spectrometry. Environ. Pollut. 2009, 157, 417−22. (11) Shelver, W. L.; Parrotta, C. D.; Slawecki, R.; Li, Q. X.; Ikonomou, M. G.; Barcelo, D.; Lacorte, S.; Rubio, F. M. Development

Figure 3. ELISA calibration curves for p,p′-DDE in solvent only (50% aqueous MeOH) and matrix-matched (MM) fish extracts. E

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of a magnetic particle immunoassay for polybrominated diphenyl ethers and application to environmental and food matrices. Chemosphere 2008, 73, S18−S23. (12) Sapozhnikova, Y.; Lehotay, S. J. Evaluation of different parameters in the extraction of incurred pesticides and environmental contaminants in fish. J. Agric. Food Chem. 2015, DOI: 10.1021/ jf506256q. (13) Sapozhnikova, Y.; Liebert, D.; Wirth, E.; Fulton, M. Polycyclic musk fragrances in sediments and shrimp tissues. Polycyclic Aromat. Compd. 2010, 30, 298−308. (14) USDA National Nutrient Database for Standard Reference; http://ndb.nal.usda.gov. (15) Han, L.; Sapozhnikova, Y.; Lehotay, S. J. Streamlined sample cleanup using combined dispersive solid-phase extraction and in-vial filtration for analysis of pesticides and environmental pollutants in shrimp. Anal. Chim. Acta 2014, 827, 40−46. (16) Gonzalez-Curbelo, M. A.; Lehotay, S. J.; Hernandez-Borges, J.; Rodriguez-Delgado, M. A. Use of ammonium formate in QuEChERS for high-throughput analysis of pesticides in food by fast, low-pressure gas chromatography and liquid chromatography tandem mass spectrometry. J. Chromatogr., A 2014, 1358, 75−84. (17) http://www.abraxiskits.com/wp-content/uploads/2014/03/ PBDE-PL-Users-Guide.pdf (accessed Jan 22, 2015). (18) http://www.abraxiskits.com/moreinfo/PN540041info.pdf (accessed Jan 22, 2015). (19) Kalachova, K.; Pulkrabova, J.; Drabova, L.; Cajka, T.; Kocourek, V.; Hajslova, J. Simplified and rapid determination of polychlorinated biphenyls, polybrominated diphenyl ethers, and polycyclic aromatic hydrocarbons in fish and shrimps integrated into a single method. Anal. Chim. Acta 2011, 707, 84−91. (20) Sapozhnikova, Y.; Zubcov, N.; Hungerford, S.; Roy, L. A.; Boicenco, N.; Zubcov, E.; Schlenk, D. Evaluation of pesticides and metals in fish of the Dniester River, Moldova. Chemosphere 2005, 60, 196−205. (21) Geis-Asteggiante, L.; Lehotay, S. J.; Fortis, L. L.; Paoli, G.; Wijey, C.; Heinzen, H. Development and validation of a rapid method for microcystins in fish and comparing LC-MS/MS results with ELISA. Anal. Bioanal. Chem. 2011, 401, 2617−2630. (22) Sapozhnikova, Y. Rapid sample preparation and fast GC-MS/ MS for the analysis of pesticides and environmental contaminants in fish. LCGC North Am. 2014, 32, 878−886.

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DOI: 10.1021/jf505651g J. Agric. Food Chem. XXXX, XXX, XXX−XXX