ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2015, том 70, № 7, с. 700-709
ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
OPTIMIZATION OF SOXTEC EXTRACTION PROCEDURE FOR DETERMINATION OF POLYBROMINATED DIPHENYL ETHERS
IN BIVALVE MOLLUSC © 2015 N. Negreira, J. Cobas, N. Carro1
Institute Tecnoloxico para o Control do Medio Marino de Galicia, Conselleria do Medio Rural e do Mar. Xunta de Galicia Peirao de Vilaxoan s/n. 36611, Vilagarcia de Arousa, Pontevedra, Received Spain 1E-mail: ncarro@intecmar.org Received 25.04.2013; in final form 23.10.2014
A comprehensive and efficient method is presented for the determination of 9 polybrominated diphenyl ether congeners (PBDEs), 2,4,4'-tribromodiphenyl ether (PBDE-28), 2,3',4,4'-tetrabromodiphenyl ether (PBDE-47), 2,2',4,4',6-pentabromodiphenyl ether (PBDE-66), 2,2',4,4',5-pentabromodiphenyl ether (PBDE-100), 2,2',3,4,4'-pentabromodiphenyl ether (PBDE-99), 2,2',4,4',5,6'-hexabromodiphenyl ether (PBDE-85), 2,2',4,4',5,5'-hexabromodiphenyl ether (PBDE-154), 2,2',4,4',5,5'-hexabromodiphenyl ether (PBDE-153) and 2,2',3,4,4',5,6'-heptabromodiphenyl ether (PBDE-183), in mollusc bivalve samples. An experimental design was used to evaluate the influence of several operational parameters on the extraction performance and to obtain the best experimental conditions. Under the final working conditions, freeze-dried mollusc was placed in the Soxtec thimble in order to extract PBDEs with 100 mL of n-hexane—acetone (4 : 1) after 1.5 and 1 h of boiling and rinsing times, respectively. Soxtec extracts were additionally purified on a multilayered column and after injected in the gas chromatography combined with mass spectrometry (GC—MS) system. Quantitative recoveries of the analytes at three concentration levels ranged between 80 and 108%, and limits of quantification of the method (LOQs) between 0.5 and 20 ng/g. Analysis of mussel and clam samples showed that PBDE-47 and PBDE-99 were presented in almost all samples with concentrations between 0.6 and 2.6 ng/g dry weight. Moreover, a certified reference material (NIST 2974a) was used. The obtained concentrations were in agreement with the certified values.
Keywords: polybrominated diphenyl ethers, soxtec device, GC—MS, bivalve mollusc.
DOI: 10.7868/S0044450215050126
Polybrominated diphenyl ethers are a group of persistent organic pollutants (POPs) consists of 209 PBDE congeners, distinguished by the number and position of their bromine atoms in the molecule [1—3]. Many of these compounds are commonly used in building materials, furnishing textiles and electronic equipment as additive flame retardants [4—5]. Their octanol—water partitioning coefficients (lgKow) are high which implies poor water solubilities and a significant hydrophobic and lipophilic nature. PBDEs have been shown to accumulate in sediments [6—7], dust [8—9] and to bioaccumulate and biomagnify in wildlife [1, 3, 4, 6, 10—16]. Whereas the lower brominated congeners increase in concentration up the food chain [2], the higher brominated compounds appear to be more prevalent in aquatic environments and sediments [11]. S ev-eral of the PBDE congeners present in commercial mixtures degrade slowly in the environment. These compounds are absorbed by the organisms and are hardly excreted [17—18]. Although toxicological data are still limited, it is known that exposure to these compounds during pregnancy can cause neurological
problems in fetal development, changes in behaviour, learning ability and memory. Moreover, they behave as endocrine disruptors causing changes in the thyroid gland and reproduction system [19—22].
Despite the efforts made by relevant agencies and institutions to regulate the use and disposal of these compounds, PBDEs levels in the environment are increasing in contrast to the decreasing levels of most of POPs over the last 20 years [8, 23].
Bivalve molluscs, mainly mussel and oyster, are chosen as bio-indicator for the monitoring of environmental contaminants since they are characterized by sedentary behaviour, tolerance to polluted areas and inclusion in the human diet. The determination of contaminants in biota gives a representative description of pollution patterns surrounding the area where it was caught.
Determination of the lipophilic contaminants in biota is directly related with the extraction of the fat content. Although recent techniques, such as pressurized liquid extraction (PLE) and solid phase extraction (SPE) are often used [10, 15, 23, 24], Soxhlet de-
Table 1. Stock solution of native polybrominated diphenyl ethers (PBDE—MXF)
Isomer PBDE CAS number
28 2,4,4'-Tribromodiphenyl ether 41318-75-6
47 2,2',4,4'-Tetrabromodiphenyl ether 5436-43-1
66 2,3',4,4'-Tetrabromodiphenyl ether 189084-61-5
100 2,2',4,4',6-Pentabromodiphenyl ether 189084-64-8
99 2,2',4,4',5-Pentabromodiphenyl ether 60348-60-9
85 2,2',3,4,4'-Pentabromodiphenyl ether 182346-21-0
154 2,2',4,4',5,6'-Hexabromodiphenyl ether 207122-15-4
153 2,2',4,4',5,5'-Hexabromodiphenyl ether 68631-49-2
183 2,2',3,4,4',5,6'-Heptabromodiphenyl ether 207122-16-5
vice is still an attractive option because of its general robustness, relatively low cost and its simple sample preparation. Soxhlet extraction was used in some studies [1, 4, 12, 13] for determination of PBDEs in bivalve mollusc. However this technique requires large quantities of organic solvent and high extraction times, 24 [12] or 48 h [16]. In order to reduce these parameters, Foss commercialized the Soxtec in 1982 by introducing the Randall's patented two staged technique for boiling and rinsing [25]. The main difference with the conventional Soxhlet is the addition of a previous step in which the sample, introduced into a cellulose thimble, is completely submerged in the extraction solvent in order to facilitate the rapid solubilisation of analyte in boiling solvent. The boiling step allows a more efficient extraction and highly reduces the extraction time and solvent volume. Soxtec devices consist of other three steps. In the rinsing step, the sample thimble is hung above the surface of the solvent, the sample is rinsing by condensed solvent. In the recovery step, the distilled solvent is automatically collected. In the pre drying step, there is an elimination of boil dry maintaining the temperature of the hot plate. The commercial apparatus are equipped with the necessary safety features regarding the use of flammable solvents. In general, Soxtec system shows improved performance [26].
Due to the low levels of PBDEs in the samples and the large number of interfering substances, the use of selective techniques is required. GC—electron capture negative ionization—quadrupole MS (GC—NCI— qMS), GC-high-resolution MS (GC-HRMS) and GC-ion trap MS (GC-ITMS) are most commonly used MS techniques for the determination of PBDEs [15]. Selected ion monitoring mode (SIM) is often selected [6, 10, 12, 14] because it minimizes or even removes matrix interferences by selecting adequate ions.
The current study expands the knowledge regarding PBDEs concentrations in bivalve mollusc coming from Galicia coast (Norwest of Spain). Our objective was to optimize an efficient and fast method for routine determination of PBDEs by using a Soxtec de-
vice. Purification steps were also studied in order to eliminate and minimize possible matrix interferences before gas chromatography coupled to mass spectrometry (mode SIM) determination. Method performance was evaluated in terms of recovery, precision, accuracy, and LOQs. The applicability of the optimized method was explored by analyzing several real samples collected from Galician coast.
EXPERIMENTAL
Standards, sorbents and material. «-Hexane for pesticide residue analysis, acetone and isooctane for gas chromatography were supplied by Merck (Darmstadt, Germany). Dichloromethane was purchased from Fluka (Steinheim, Germany). Sulphuric acid (purity 95%) was acquired by Panreac (Barcelona, Spain). The stock solution of native PBDEs (certificated concentration 2000 ng/mL in toluene—nonane 67.6 : 32.4) was obtained from Wellington Laboratories (Canada) (Table 1).
PBDEs mixture was used to fortify the freeze-dried mussel samples used during the optimization of the extraction conditions. Further dilutions were prepared in isooctane to optimize GC—MS determination conditions (calibration standards). The 2,2',4,4',6,6'-hexachlorobiphenyl, PCB-155 (400 ng/mL), used as surrogate standard was purchased from Dr. Ehrenstor-fer (Augsburg, Germany). The Certified Reference Material used was freeze-dried mussel tissue, NIST 2974a (National Institute of Standards and Technology, Gaithersburg, USA). Anhydrous sodium sulphate for analysis, florisil PR (60—100 mesh), silica extra pure for column chromatography (70—230 mesh) and alumina (activity stage I) for column chromatography (70—230 mesh) were acquired from Merck. Normalphase materials were activated at 130°C, for 24 h. After activation, they were allowed to cool down in a desiccator before being used in the purification process. Acid silica gel was prepared from activated silica gel impregnated with 44% (w/w) concentrated sulphuric acid.
Table 2. Performance of the GC—MS system for PBDE standards in isooctane
Compound Retention time, min Selected ions*, m/z R2** RSD, %*** LOQ, ng/mL
PBDE-28 11.39 404 + 406 + 408 + 410 0.9993 2.5 0.4
PBDE-47 12.71 484 + 486 + 488 0.9997 3.7 0.3
PBDE-66 12.86 484 + 486 + 488 0.9997 4.3 0.6
PBDE-100 13.69 562 + 564 + 566 + 568 0.9995 4.0 0.6
PBDE-99 13.96 562 + 564 + 566 + 568 0.9992 2.6 1
PBDE-85 14.65 562 + 564 + 566 + 568 0.9997 1.1 2
PBDE-154 15.02 641 + 643 + 645 0.9992 2.5 5
PBDE-153 15.62 641 + 643 + 645 0.9963 2.1 5
PBDE-183 18.14 719 + 721 + 723 + 725 0.9994 2.6 20
* Underlined ions were used for quantification purposes; ** linearity range 1—1000 ng/mL; *** RSD — relative standard deviation, repeatability of injection 50 ng/mL.
Samples and sample preparation. Optimization of sample preparation conditions was performed with a f
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