Bull Vet Inst Pulawy 54, 549-555, 2010
COMPARISON OF EXTRACTION AND CLEANUP PROCEDURES FOR THE DETERMINATION OF PCDD/Fs IN FEED SAMPLES WITH THE CALUX BIOASSAY SYLWIA STYPUŁA-TRĘBAS1,2 AND JADWIGA PISKORSKA-PLISZCZYŃSKA1 1
Department of Radiobiology, 2Department of Pharmacology and Toxicology, National Veterinary Research Institute, 24-100 Pulawy, Poland
[email protected] Received for publication April 6, 2010
Abstract The efficiency of four extraction techniques (shaking extraction, column extraction, accelerated solvent extraction (ASE), and ultrasonic assisted extraction) and two clean-up procedures was evaluated in order to find the best procedure. The ASE proved to be the best extraction method with the highest recovery rates (84%-91%) and imprecision less than 15%. Application of the threecolumn cleanup procedure delivered slightly better cleanup efficiency in comparison with two-step cleanup but the use of two columns is less time consuming, cost-effective, and enabled to process more samples with the bioassay.
Key words: feed, polychlorinated dibenzo-p-dioxins, dibenzofurans, bioassay, extraction, cleanup. Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) represent a diverse group of persistent halogenated aromatic hydrocarbons (HAHs). Among 210 possible congeners specifically the 17 PCDD/Fs with chlorine substitution in the 2,3,7, and 8 positions are considered to be the most toxic (30). Several dioxin contamination incidents have shown that these compounds pose an important threat to the quality of food of animal origin (7, 19, 22). To reduce the PCDD/Fs uptake by animals and to ensure consumer protection, the European Community (EC) set maximum limits for feed and implemented monitoring programmes, focusing on high numbers of randomly taken samples (8). Analysis of feed samples for the presence of toxic PCDD/Fs is a challenge for analysts not only because of the complexity of the matrix and the need for simultaneous determination of many analytes, but mainly because of their ultra trace concentrations in the sub pg/g range (13, 23). The need for determination of PCDD/Fs at these low levels makes the instrumental quantitative methods expensive and difficult to perform, which limits the scope of research, especially during emerging situations (23). The effectiveness of the instrumental analysis in revealing PCDD/Fs contamination can be improved significantly by using high throughput in vitro bioassays (6, 19). At present, the most commonly used for screening purposes is the chemically activated luciferase gene expression (CALUX) bioassay (6, 26, 31, 32). The XDS-CALUX® bioassay uses genetically engineered mouse hepatoma cells, expressing a luciferase reporter
gene in the presence of compounds that activate the aryl hydrocarbon receptor (AhR), a transcription factor responsible for mediating toxicity of PCDD/Fs (6, 5, 26, 32). Since sample extraction and clean-up are critical stages in the CALUX bioassay, the aim of our study was to evaluate the efficiency of different techniques for isolation of PCDD/Fs from feed matrices as well as to evaluate effectiveness of different clean-up procedures. The evaluation was made in accordance with the official EC requirements for screening methods for the monitoring of dioxins in feed (9).
Material and Methods Reagents and standards. All used chemicals were of pesticide or HPLC grade. Cell culture media were purchased from GIBCO (UK) and Hyclone (USA). PCDD/Fs standard solutions (50 μg/mL, purity >90%) were purchased from Wellington Laboratories Ltd (Canada) and AccuStandard (USA). The Luciferase Assay System was purchased from Promega Corp. (USA). A fortification solution was prepared as a mixture of 17 PCDD/Fs in DMSO (0.422 ng WHOTEQ/mL). Study design. The study was divided into two stages. In the first stage, the performance of four commonly applied extraction techniques (shaking, column extraction, ultrasonic assisted extraction (UAE), and accelerated solvent extraction (ASE)) was investigated. The fish meal and compound feed samples
550 with known background concentrations of PCDD/Fs (Table 1) were used. In the second stage, the effectiveness of twostep (acid silica/activated carbon) column clean-up was compared with three-column (acid silica/Florisil/activated carbon) cleanup. The fish meal and fish oil samples were selected on the basis of false positive CALUX bioassay results that were 5 to 8 times higher than results of the chemical analysis (high resolution gas chromatography/ high resolution mass spectrometry, HRGC/HRMS), (Table 1). Table 1 PCDD/Fs content (ng WHO-TEQ/kg dw) of experimental feed samples, determined using the CALUX bioassay and instrumental HRGC/HRMS analysis Matrix Extraction study Compound feed Fish meal Clean-up study Fish meal Fish oil
PCDD/Fs ng WHO-TEQ/kg dw CALUX HRGC/HRMS 0.03 0.21
0.05 0.11
1.74 11.02
0.21 2.08
Extraction. The samples were mixed thoroughly, weighed (10 g of compound feed, 5 g of fish meal) and fortified at concentrations of 0.42 and 0.68 ng WHO-TEQ/kg dry weight (dw) for compound feed and fish meal, respectively. The fortification level corresponded to the half of the official maximum limits set for the chosen matrices (9). An analytical run consisted of six fortified samples, six sample blanks, and two solvent blanks. Each sample was extracted three times with methanol:toluene (2:8) mixture, and all elutes were pooled together. The fat content was determined gravimetrically after complete solvent evaporation. The residues were then re-dissolved in n-hexane and twocolumn cleanup protocol was performed as it was described below for the cleanup study. The recovery was determined as the measured concentration in the fortified sample corrected by subtraction of the signal from the unfortified material, divided by the known concentration and expressed as a percentage: % recovery=100 x (measured concentration/fortification level). The shaking extraction was performed with the use of a mechanical shaker (Heidolph, Germany). The samples were shaken with extraction solution (20 ml) for 120 min and then centrifuged (5 min, 1,000 rpm) to allow for phase separation. The shaking time in the last two steps was 60 min. In the column extraction, the glass columns (14 mm of diameter) were filled from bottom to top with Celite 545 (2 g), anhydrous sodium sulfate (5 g), and feed samples. The columns were then eluted with the extraction solution (20 ml).
The UAE was carried out in an ultrasonic water bath (290 W, 55 Hz, Elmasonic S40 H, Elma, Germany). The glass tubes were filled with the samples and extraction solution (20 ml), shaken manually for 3 min and immersed into the ultrasonic water bath. The extraction was performed at 350C in the three consecutive steps of 60 min. The ASE was performed using an ASE 300 extractor (Dionex, USA). The feed samples were mixed with Hydromatrix (3 g, Varian) and placed in the 66 ml extraction cells (Fig. 1). The following ASE parameters were used: temperature 1200C, pressure 1.500 psi, 6 min heating time, 2 min static time, 75% flush volume, 200 s purge time, and three cycles. The extracts were then concentrated to 1 mL in a Turbovap (Zymark, USA).
Filter Hydromatrix (3 g)
Feed/Hydromatrix (3 g)
Hydromatrix (2 g) Filter
Fig. 1. ASE cell packing. Cleanup. The ASE extraction was performed before the cleanup of fish meal samples (5 g). Fish oil samples (3 g) were cleaned without the extraction step. Analytical series consisted of four highly contaminated samples, four sample blanks, four fortified samples, and two solvent blanks. For the recovery determination fish meal and fish oil with the low background PCDD/Fs levels of 0.21 and 0.54 ng WHO-TEQ/kg dw, respectively, were spiked with PCDD/Fs at 0.85 ng WHO-TEQ/kg dw (fish meal) and 3 ng WHO-TEQ/kg dw (fish oil). The recovery was calculated as described above for the extraction experiment. In the two-column cleanup procedure the samples were shaken for 2 min with acidified (sulphuric acid, 33% w/w) silica gel (Fluka, Germany) and nhexane (20 ml) in three consecutive steps. For 1 g of fat, 10 g of aciditified silica was used. The supernatants were then passed through columns packed with acidified silica gel (10 g) and anhydrous sodium sulfate (1.2 g). The packed columns were conditioned with 50 ml nhexane prior to adding the sample extracts. Elution was performed with 60 ml of n-hexane. The extracts were then purified on the patented XCARB columns (4). A Florisil column was used after silica gel column cleanup in the three-column cleanup procedure. The Florisil cleanup step was performed according to the EPA 1613B procedure (29). The extracts were then purified on the XCARB columns (4). Calux bioassay. The bioassay was carried out using mouse hepatoma H1L6.1c3 cells, which were stably transfected with AhR-regulated, dioxin-inducible
551 luciferase expression vector pGudLuc6.1 (17). The cells were obtained from Xenobiotic Detection Systems Inc. (XDS, USA). The conditions for cell culture and details of the CALUX bioassay procedure have been described elsewhere (26). The luminescence was measured using an Orion II luminometer (Berthold, USA). The luciferase activity of an extract was estimated from 2,3,7,8-TCDD standard curve with a four-parameter Hill equation using a least squares algorithm. Statistical evaluation. The four extraction protocols were examined by one-way ANOVA with individual means compared following a significant Fvalue by Tukey’s post hoc procedure. The means from the two cleanup protocols were compared using a paired Student’s t-test. Significance was inferred at P
