RESEARCH PAPER
New environmentally friendly MSPD solid support basedon golden mussel shell: characterization and applicationfor extraction of organic contaminants from mussel tissue
Caroline Rombaldi1 & Jean Lucas de Oliveira Arias1 & Gabriel Ianzer Hertzog1 &
Sergiane Souza Caldas1 & João P. Vieira2 & Ednei Gilberto Primel1
Received: 6 February 2015 /Revised: 26 March 2015 /Accepted: 8 April 2015# Springer-Verlag Berlin Heidelberg 2015
Abstract The use of golden mussel shells as a solid sup-port in vortex-assisted matrix solid-phase dispersion(MSPD) was evaluated for the first time for extraction ofresidues of 11 pesticides and nine pharmaceutical and per-sonal care products from mussel tissue samples. After theyhad been washed, dried, and milled, the mussel shells werecharacterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy, infrared spectroscopy, andBrunauer—Emmett–Teller analysis. The MSPD procedurewith analysis by liquid chromatography–tandem massspectrometry allowed the determination of target analytesat trace concentrations (nanograms per gram), with meanrecoveries ranging from 61 to 107 % and relative standarddeviations lower than 18 %. The optimized methodconsisted of dispersion of 0.5 g of mussel tissue, 0.5 g ofNaSO4, and 0.5 g of golden mussel shell for 5 min, andsubsequent extraction with 5 mL of ethyl acetate. The ma-trix effect was evaluated, and a low effect was found for allcompounds. The results showed that mussel shell is aneffective material and a less expensive material than mate-rials that have traditionally been used, i.e., it may be usedin the MSPD dispersion step during the extraction of pes-ticides and pharmaceutical and personal care products fromgolden mussel tissues.
Keywords Vortex-assistedmatrix solid-phase dispersion .
Mussel shell . Solid support . Pesticides . Pharmaceutical andpersonal care products
Introduction
Matrix solid-phase dispersion (MSPD) is a fast and robusttechnique which is based on the dispersal of the sample withan appropriate solid and abrasive support with consecutiveelution of the analytes [1]. The technique has been used forthe extraction of organic contaminants in solid matrices suchas sediment, food, and biological samples [2–5].
More specifically with regard to biological samples,solid supports such as C18 [6], Florisil [7], and alumina[8] have been used. Although these materials have showngood results, they are usually expensive and syntheticallyproduced.
Alternative materials which are cheaper and environmen-tally friendlier than traditional materials have been little ex-plored in MSPD [9]. Some studies used sand and diatoma-ceous earth as dispersers, and these were shown to be inter-esting alternatives with effectiveness similar to that of thecommercial materials [3, 10, 11].
In this context, mussel shells seem to be an interestingoption, since they generally contain 95 % calcium carbonateand 5 % organic materials such as glycoproteins, polysaccha-rides, glycosaminoglycan, chitin, and other proteins [12].These components give the shell its abrasive and solid supportproperties [13]. Besides, the use of low-cost solid supportssuch as shell wastes that can be easily obtained in large quan-tities seems to be a good alternative [14]. Furthermore, musselshells are recognized as a worldwide environmental problemsince they reduce the liming value and render waste difficult torecycle [13, 14].
* Ednei Gilberto [email protected]
1 Escola de Química e Alimentos, Laboratório de Análise deCompostos Orgânicos e Metais (LACOM), Universidade Federal doRio Grande, Av Itália, km 8, Rio Grande, RS 96203-900, Brazil
2 Instituto de Oceanografia, Universidade Federal do Rio Grande, AvItália, km 8, Rio Grande, RS 96203-900, Brazil
Anal Bioanal ChemDOI 10.1007/s00216-015-8686-2
In the literature, mussel shells have been proposed asbiofillers for reinforcing polymers [15], as biosorbents toremove textile dyes from aqueous solutions [14], to obtaincalcium carbonate to be incorporated into polypropylene[16], and for retention of arsenic, chromium, and mercury[17]. From this information, it was supposed that thismaterial would be efficient in the dispersion step inMSPD.
The golden mussel (Limnoperna fortunei) is a specieswith great invasive potential. Originally from freshwaterhabitats of southeast Asia, this species invaded SouthAmerica (La Plata River, Argentina) through ballast wa-ters, and can now be found in parts of Bolivia, Paraguay,Uruguay, and Brazil [18–20]. Some bivalves are studiedas biological monitors of environmental changes sincethey are organisms that bioaccumulate because of theirfilter-feeding habits [21].
Pesticides and pharmaceutical and personal care products(PPCPs) are being widely investigated in environmental sam-ples because of their increasing misuse and disposal withoutprevious treatment, which is increasing the amount of envi-ronmental pollution. Pesticides and PPCPs have been found inmany biological matrices, such as mussels [22–24], fish [6,25], and seaweeds [26, 27].
The complex metabolism of the golden mussel which com-bines early sexual maturity, a high fecundity index, and wide
environmental tolerance enables its to adapt to different envi-ronmental conditions [28]. Thereby, the golden mussel is aninteresting matrix to investigate the presence of organiccontaminants.
In this context, the aim of this work was to characterize andevaluate the use of golden mussel shell in vortex-assistedMSPD as a new, environmentally friendly, efficient, andlow-cost solid support. We also evaluated its applicationthrough the optimization of a method based on vortex-assisted MSPD for the extraction of 11 pesticides and ninePPCPs from mussel tissue.
Experimental
Chemicals
High-purity (more than 99 %) analytical standards of atrazine,atrazine-d5, azoxystrobin, carbofuran, cyproconazole,clomazone, difenoconazole, diuron, epoxiconazol,fenoxaprop-P-ethyl, fipronil, and propiconazole were sup-plied by Sigma-Aldrich (Brazil), and the pharmaceuticalswere provided by Fiocruz (Fundação Oswaldo Cruz, Brazil).Triclocarban was provided by Dr. Ehrenstofer (Germany).Ketoconazole was bought from the US Pharmacopeial
Table 1 Chemical class and physicochemical properties of the compounds studied [29, 30]
Compound Chemical class Log Kow Water solubility (mg L-1)
Albendazole Benzimidazoles 2.7 Practically insoluble
Amitriptyline Dibenzocycloheptenes 4.92 9.71
Clarithromycin Macrolide 3.16 0.33
Diltiazem hydrochloride Phenylpropylamines, lactams, benzothiazepines 2.8 465
Flurazepam hydrochloride Benzodiazepines, lactams 3.8 500
Chlorpropamide Sulfonylureas 2.27 258
Haloperidol Piperidines 4.3 14
Nimesulide Sulfonanilides 2.6 18.2
Triclocarban Phenilurea 3.5 0.045
Atrazine Triazine 2.5 33
Azoxystrobin Methoxyacrylate 2.5 6
Carbofuran Carbamate 1.52 351
Cyproconazole Triazole 3.1 93
Clomazone Isoxazolidinone 2.5 1100
Difenoconazole Triazole 4.4 15
Diuron Urea 2.85 36.4
Epoxiconazole Triazole 3.44 0.663
Fenoxaprop-P-ethyl Aryloxyphenoxypropionate 4.58 0.7
Fipronil Fiprole 4.0 1.9 (pH 5), 2.4 (pH 9)
Propiconazole Triazole 3.72 100
C. Rombaldi et al.
Convention. The compounds investigated and their character-istics are listed in Table 1 [29, 30].
Anhydrous magnesium sulfate, sodium acetate, liquidchromatography (LC)-grade methanol, and acetonitrile werepurchased from J.T.Baker (USA). Ethyl acetate was boughtfrom Sigma-Aldrich (Brazil). Water was purified by a Direct-Q UV3® (resistivity 18.2 MΩ cm) water purification system(Millipore, USA). Acetic acid (98–100 %) was purchasedfrom Merck (Germany).
Preparation of standard solutions
Individual standard solutions with the target compounds at 1,000 mg L-1 were prepared in methanol or acetonitrile accord-ing to their solubility. These solutions were used to prepare theworking solutions, which were used for sample spiking andfor preparing the calibration curves. Working standard solu-tions were prepared monthly, whereas the dilutions were pre-pared daily.
Mussel sampling and preparation of the solid supportbased on mussel shells
Golden mussels were kindly donated by the Instituto deOceanologia, which collected them at CORSAN (SãoGonçalo Channel) and at Sangradouro (Mirim Lagoon), RioGrande, Brazil.
Adult golden mussels (shell total length more than 25 mm)were washed exhaustively with distilled water. Then, the tis-sue was separated from the shell, and the shells were dried at100 °C for 6 h. Then they were milled with a pestle in a mortarand sieved (2-mm mesh). The material obtained was stored ina glass bottle protected from humidity, and the mussel tissuewas frozen for further method development.
LC–tandem mass spectrometry
LC was performed with a Waters Alliance 2695Separations Module fitted to an autosampler, a membranedegasser, and a quaternary pump. Mass spectrometry wasperformed with a Micromass Quattro Micro API instru-ment with an electrospray ionization interface. The LCseparation was done by a Kinetex analytical column(C18, 2.6 μm, 50 mm×3-mm inner diameter; Waters,USA). Analytical instrument control, data acquisition,and treatment were performed by MassLynx version 4.1(Waters, USA). The mobile phase components were ultra-pure water with 0.1 % acetic acid (solvent A) and puremethanol (solvent B), with elution in the gradient mode.The initial composition was 20 % solvent B, which in-creased linearly to 90 % in 10 min. This composition wasmaintained for 13 min, and then the composition wasreturned to the initial composition in 0.5 min. This
composition was held for 6.5 min, giving a total analysistime of 30 min. The flow rate was 0.2 mL min-1, and theinjection volume was 10 μL.
The compounds were directly infused into the mass spec-trometer to optimize the mass spectrometer parameters—thatis, the ionization mode, the identification of the parent andproduct ions, and the selection of the cone energy and colli-sion energy.
Mussel shell characterization
The mussel shell solid support was characterized by scanningelectron microscopy (SEM), energy-dispersive X-ray (EDX)spectroscopy, Brunauer–Emmett–Teller (BET) analysis, andinfrared (IR) spectroscopy [13, 14, 31].
The morphology of the materials was analyzed bySEM using a JEOL model JSM 6610 LV instrument,and EDX spectroscopy was performed using a ThermoScientific UltraDry detector with a Noran System 7 X-ray microanalysis system. SEM was performed to providethe micromorphology and the surface fractures at a volt-age of 15 kV. EDX spectroscopy was performed togetherwith SEM. Whereas SEM gives clear images of the sur-face morphology, EDX spectroscopy provides immediatechemical identification. The experiments were done bycovering the samples with carbon, a sputtering step, inorder to ensure conductivity and the interaction of theelectron beam with the sample, as well as to improvethe intensity of the electron emission.
IR spectroscopy was used to identify and investigatesample constituents. The main goal of the IR spectroscop-ic analysis was to determine the chemical functionalgroups in the sample. IR spectroscopic analyses weredone with a Prestige-21 IR spectrometer (Shimadzu,Kyoto, Japan). The resolution of the spectrometer was2 cm-1. Finally, the BET technique provides informationon the specific surface area, so we could compare thespecific surface area of samples with that of the materialsusually used in MSPD. BET analyses were performedwith a TriStarII 3020 instrument from Micrometrics.
Vortex-assisted MSPD procedure
Amounts of 0.5 g of sample (mussel tissue) were transferred toa mortar (30-mL capacity). Then, the sample was homoge-nized for 5 min with 0.5 g of NaSO4 and 0.5 g of mussel shell.The mixture was then transferred to a polypropylene tube(15-mL capacity), to which 5 mL of ethyl acetate was added.The resulting mixture was vortexed for 1 min and centrifugedfor 5 min at 7,000 rpm. Finally, 10 μL was injected into theLC system.
New environmentally friendly MSPD solid support
Validation experiments and quality control criteria
Validation of the analytical method was performed by usingspiked control samples, and was assessed in accordance withSANCO guidelines [32]. The linearity of the method wasevaluated through matrix-matched calibration in the concen-tration range of the limit of quantification (LOQ) of each com-pound to a concentration of 10,000 ng g-1. Three replicates ofmixtures of the calibration standards at five different concen-trations were injected. Dilutions of the standard solution ofpesticides with the blank extract from the matrix extractedby MSPD were performed. An external calibration, in thesame concentrations, was also performed by the dilution ofthe standard solution of pesticides in acetonitrile.
The limit of detection (LOD) and LOQ were calculated onthe basis of the signal-to-noise ratio of individual peaks, as-suming a ratio of 3:1 and 10:1 for the LOD and LOQ, respec-tively. The LOQ was the lowest validated spiking level meet-ing the method performance acceptability criteria (mean re-coveries from 70 to 120 %, with relative standard deviation of20 % or less).
Recovery was evaluated for at least three levels, andthe experiments were performed in triplicate, with injec-tion three times (n=9). To calculate the recoveries, thefollowing equation was used: recovery (%)=[(C1- C2)/C3]×100, where C1 is the concentration of the analyte inthe final extract, C2 is concentration of the analyte in theblank sample, and C3 is the concentration of the analyteadded to the sample. All experiments were accompaniedby a blank in order to evaluate matrix interferences.Precision was calculated as the relative standard deviationfor each concentration. Repeatability was studied at threedifferent fortification levels, in triplicate, and intermediateprecision was estimated as the repeatability, but on differ-ent days and by different analysts.
The matrix effect (ME) was evaluated through a compari-son of the slope of the curve for the solvent and the slope ofthe curve prepared with the mussel tissue extracts:
ME %ð Þ ¼ 100� 1−SmSs
� �; ð1Þ
where Sm represents the slope of the matrix curve and Ss rep-resents the slope of the solvent curve.
Values above 100 % indicate ionization enhancement, andvalues below 100 % indicate ionization suppression. The MEis deemed to be low for a range of signal suppression/enhancement of ±20 %, medium for ranges of ±20 % and±50 %, and high for values higher than 50 % or lower than -50 % [33].
Internal quality controls were used during the analysis.To assess previous contamination from the mussel shellsas well as to eliminate false positives due to possible
contamination during the extraction procedure, in the in-strument, or in the chemicals, a blank matrix extract wasalso prepared and analyzed. A calibration curve to evalu-ate sensitivity as well as linearity in the working range ofconcentrations was constructed during all the experi-ments. In addition, atrazine-d5 was used as a surrogateduring the sample analysis.
Statistical analysis
For the statistical analysis performed with the fractional fac-torial design at the 95 % confidence level, Statistica 8.0(StatSoft, Tulsa, OX, USA) was used.
Results and discussion
LC–tandem mass spectrometry optimization
Nimesulide, triclocarban, and fipronil showed better responsein negative ionization mode, and the analysis of the rest of thecompounds was done in positive ionization mode. The mostintense ion transition was selected for quantification, and thesecond transition was used to confirm the analyte (Table 2).
Mussel shell characterization
The SEM image of mussel shell was compared with that of theC18 solid support, since it is one of the most used solid sup-ports [6]. The micrographs of the mussel shell and C18 areshown in Fig. 1. It is observed that the fractures in the shellare less polished and the particles are bigger than those of C18.However, it is noteworthy that these results were expected,because the shell powder was homemade, whereas C18 is anindustrial product.
The EDX spectrum (Fig. 2) shows that mussel shell isconstituted basically of calcium and oxygen, from the calciumcarbonate, with minor amounts of sodium, aluminum, andsilicon. The carbon peak is very intense because of the coatingpreviously done for SEM analysis.
As expected, the IR spectrum of golden mussel shellshowed a characteristic Fourier transform IR band at 1,483 cm-1, which was attributed to the C–O bond of the car-bonate groups present in abundance in the shell (data notshown).
Calcium carbonate can be found in three forms: calcite,aragonite, and vaterite [13, 31]. More specifically, theFourier transform IR band of golden mussel shell is character-istic of aragonite. Such a result is plausible given the trend forshells to be constituted of this crystal because of its greatersolidity, which derives from aragonite acicular crystals [34].As observed by Islam et al. [31], cockle shells also have ara-gonite as the major calcium carbonate form [35]. The
C. Rombaldi et al.
carbonate group band obtained is consisted with the bandsreported in the literature [14].
From the BET analyses, a surface area of 5.01 m2 g-1, anaverage pore diameter of 4.58 nm, and a pore volume of0.006298 cm3 g-1 were obtained. The surface area is aboutfour times greater than that of sand [36]. On the other hand,it is about 100 times smaller than the surface area of commer-cial C18 [37]. It is expected that the higher the surface area, thebetter the material, because it increases the interaction on dis-persion. Despite this, the shell performance was similar to thatof C18, and mussel shell has the advantages of being a material
that is widely available in nature, a material that is easilyprepared, and a material that costs less.
MSPD optimization
During the MSPD optimization, for the evaluation of the ex-traction efficiency, the samples were fortified using a solutioncontaining the analytes investigated. For this, 50 μL of a so-lution containing each compound at 10 mg L-1 was added tothe sample. The samples were extracted after solvent evapo-ration (approximately 1 h).
Table 2 Liquid chromatography–tandem mass spectrometry parameters for the compounds
ESI Transition (m/z) Cone voltage (V) Collision energy (eV) RT (min)
PPCPs
Albendazole + 266→234266→191a
3033
2032
13.8
Amitriptyline + 278.3→233.3a
278.3→116.93535
1515
14.2
Clarithromycin + 748.75→82.8748.75→158.3a
3535
5929
15.9
Diltiazem hydrochloride + 415→310415→178a
3535
2020
12.5
Flurazepam hydrochloride + 388.4→315.2a
388.4→288.23030
2525
11.2
Chlorpropamide + 277→175a
277→110.92727
2229
11.4
Haloperidol + 376→165a
376.4→1233035
2125
12.7
Nimesulide − 307→229307.2→198.1
3330
2025
13.3
Triclocarban − 313→160.1a
315→125.73030
2015
18.3
Pesticides
Atrazine + 216→174216→146
3335
2022
12.3
Azoxystrobin + 404→372a
404→3292015
2030
14.6
Carbofuran + 222→165222→123a
2020
2525
10.3
Cyproconazole + 292→125292→70a
3535
2020
15.4
Clomazone + 240→125a
240→2193026
2020
13.6
Difenoconazole + 406→251a
406→3373132
3220
17.9
Diuron + 233→72a
233→1602828
2025
12.9
Epoxiconazole + 330→123330→121a
2727
3030
16.1
Fenoxaprop-P-ethyl + 362.1→288.1a
362.1→1212222
2337
18.4
Fipronil − 435→330a
435→2503025
1526
16.7
Propiconazole + 342→159a
342>693230
2220
16.9
ESI electrospray ionization, PPCPs pharmaceutical and personal care products, RT retention timea quantification transition
New environmentally friendly MSPD solid support
Mussel shell as a solid support
To verify the possibility of using the shell as a support solidinstead of the traditional C18 [6], vortex-assisted MSPD wasperformed following a previously developed procedure.Samples were fortified, and after solvent evaporation (approx-imately 1 h) sample preparation continued by dispersing 0.5 gof spiked sample, 0.5 g of NaSO4, and 1.0 g of solid supportfor 5 min. This was followed by elution with 5 mL of aceto-nitrile, shaking for 1 min on a vortex mixer, and centrifugationat 2,545g for 10 min. From this test, recoveries from 43 to122 % were obtained for mussel shell and from 12 to 115 %for C18 (Fig. 3). These results suggest that the efficiency ofmussel shell as a solid support is similar to or even better thanthat of C18 for the extraction of these compounds from musseltissue. Thus, some extraction parameters were optimized.
The exact mechanism of extraction is not known, but somepoints can be discussed. The mussel shells used in this studywere composed mainly of CaCO3. Since CaCO3 is known forits abrasive property, the disruption process occurs during theblending process. Probably, when the mussel shell solid sup-port is blended with the sample, the shearing forces generatedby the blending process disrupt the sample architecture andprovide a more finely divided material for extraction. An ad-sorption process could also occur, since mussel shells havebeen proposed to act as biosorbents to remove textile dyes
from aqueous solution [38]. Mussel shell powder combinedwith natural clay was also used as a sorbent for removal ofcadmium from synthetic wastewater samples [39].
Type of solvent
Acetonitrile, ethyl acetate, andmethanol were tested as elutionsolvents because of their different polarities and properties.The recoveries were acceptable with the three solvents; how-ever, lower deviations and a lower ME were achieved withethyl acetate (Table 3). A Tukey test (p>0.05) was applied,and it was verified that ethyl acetate resulted in recoveriesbetter than or similar to those obtained with methanol andacetonitrile for most analytes, as shown in Table 3. The betterresults obtained with ethyl acetate can be explained by itspolarity, which is more similar to the polarities of the analytes,for which log Kow in most cases is higher than or equal to 2.5(Table 1). Also, according to the US Environmental ProtectionAgency [40], the overall acute toxicity of ethyl acetate is low,and it does not appear to be mutagenic. Ethyl acetate is readilybiodegradable and not potentially bioaccumulable (EURegulation 1272/2008), being considered as not dangerousto the environment. Furthermore, ethyl acetate is currentlyused in MSPD for food and biological samples [41].
Method validation
The validation procedure was carried out in accordance withEuropean Commission Directorate General for Health andFood Safety guidelines for food and feed.
Linearity was studied by injecting 10 μL of spiked blankextracts, from the LOQ to 10,000 ng g-1. The curve was plot-ted for each compound, and the correlation coefficients (r2)were higher than 0.9915 for all compounds except fornimesulide and triclocarban.
Fig. 1 Scanning electron microscopy images of a particle from golden mussel shell powder (a) and from C18 powder (b)
Fig. 2 Energy-dispersive X-ray spectrum of golden mussel shell
C. Rombaldi et al.
The procedure provided LODs that ranged from 3 to 30 ng g-1, and LOQs that ranged from 10 to 100 ng g-1 (Table 4). TheLOQs obtained are in accordance with and even lower thanthose reported in the literature. LOQs for atrazine, clomazone,and fipronil of 250, 50, and 100 ng g-1, respectively, were ob-tained for fish and crab tissue samples by using MSPD and gas
chromatography–mass spectrometry [6]. With use of a quick,easy, cheap, effective, rugged, and safe (QuEChERS) methodand fast low-pressure gas chromatography–triple-quadrupoletandem mass spectrometry, an LOQ of 5 ng g-1 for atrazine ina catfish muscle sample was obtained [42]. The LOQs obtainedare also in agreement with the concentrations that have been
Table 3 Recoveries (R) andrelative standard deviations(RSD) obtained using methanol,ethyl acetate, and acetonitrile asthe elution solvent with theanalytes at the 100 ng g-1 level
Methanol Ethyl acetate Acetonitrile
R (%) RSD (%) R (%) RSD (%) R (%) RSD (%)
PPCPs
Albendazole 32b 15 77a 6 79a 22
Amitriptyline 54a 25 82a 16 52a 23
Clarithromycin 22a 37 55a 16 43a 45
Diltiazem hydrochloride 71a 4 78a 10 71a 28
Flurazepam hydrochloride 94a 6 77a 9 77a 20
Chlorpropamide 96b 14 27a 16 46a 39
Haloperidol 108a 11 83a 5 78a 21
Nimesulide 93a 8 75a 6 78a 23
Triclocarban 58a 12 86b 10 83b 19
Pesticides
Atrazine 108a 15 89a 12 87a 16
Azoxystrobin 43a 49 73a 16 – –
Carbofuran 107a,b 5 118b 9 88a 15
Cyproconazole 45a 21 85b 5 81b 20
Clomazone 80a 9 100b 5 96b 9
Difenoconazole 87a 6 94a 6 82a 18
Diuron 199b 22 97a 14 82a 21
Epoxiconazole – – 86a 5 96a 29
Fenoxaprop-P-ethyl 106b 17 78a,b 13 74a 18
Fipronil 31a 57 81b 5 96b 23
Propiconazole 127a 17 92a 8 95a 24
Numbers followed by same letter indicate no significance difference, p>0.05.
Fig. 3 Recoveries obtained using C18 and golden mussel shell in vortex-assisted matrix solid-phase dispersion for the extraction of pesticides andpharmaceutical and personal care products frommussel tissue. Error barsindicate the relative standard deviation (n=9). The extraction conditions
were as follows: 0.5 g of sample, 0.5 g of NaSO4, 1.0 g of solid support,5-min dispersion, 5 mL of acetonitrile, 1 min on a vortex mixer, andcentrifugation at 4,000 rpm for 10 min
New environmentally friendly MSPD solid support
detected in environmental samples. Clomazone was detected inliver fish samples from 60 to 90 ng g-1 in southern Brazil [6]. InSan Francisco Bay, USA, triclocarban was detected in sedimentin amaximum concentration of 32.7 ng g-1 and inmussel tissuesin a concentration of 1.5 ng g-1 [43]. Amitriptyline was detectedin 13 % of mussel tissues samples from the California coast in amaximum concentration of 6.2 ng g-1 [44].
Recoveries were calculated using matrix-matched calibrationstandards. They ranged from 61 to 107%, with relative standarddeviations from 3 to 18% (Table 4). The accuracy and precisionobtained were adequate considering the standard criteria, sincebiological samples are matrices of high complexity. In other
studies dealing with extraction of organic compounds from bi-ological tissues, similar recoveries were obtained [6, 43].
Finally, suppression or enhancement of analytes was evalu-ated. For this, a solvent curve was prepared with the appropriatestandard solutions prepared in ethyl acetate, as was a curve forthe mussel blank extracts after MSPD. Comparison of theslopes showedME values lower than ±18% for all compounds.Values below 20% are considered low [33]. The lowME foundis an advantage of the proposedmethod since higherME valuesare normally found in the extraction of organic compoundsfrom biological matrices [42, 45]. However, although thesevalues are considered low, we performed a matrix-matched
Table 4 Analytical parameters
r2 Linear range (ng g-1) LOD (ng g-1) LOQ (ng g-1) R (%)±RSD (%)
10 ng g-1 50 ng g-1 100 ng g-1 500 ng g-1 1,000 ng g-1
PPCPs
Albendazole 0.9971 50–10,000 16 50 – 61±12 62±8 68±3 64±7
Amitriptyline 0.9975 100–10,000 30 100 – – 66±9 66±9 64±7
Clarithromycin 0.9986 50–10,000 16 50 – 62±10 65±15 63±8 61±15
Diltiazem hydrochloride 0.9983 10–10,000 3 10 69±11 79±8 76±10 80±7 76±8
Flurazepam hydrochloride 0.9991 50–10,000 16 50 – 83±15 74±7 76±9 73±6
Chlorpropamide 0.9988 100–10,000 30 100 – – 85±16 76±8 72±13
Haloperidol 09956 50–10,000 16 50 – 65±13 67±12 71±7 65±9
Nimesulide 0.9630 50–10,000 16 50 – 74±10 69±7 71±6 73±12
Triclocarban 0.9715 50–10,000 16 50 – 60±15 61±14 60±8 58±13
Pesticides
Atrazine 0.9987 50–10,000 16 50 – 90±10 91±7 96±5 93±7
Azoxystrobin 0.9982 50–10,000 16 50 – 91±9 85±8 91±5 86±12
Carbofuran 0.9970 100–10,000 30 100 – – 102±18 78±12 67±15
Cyproconazole 0.9994 50–10,000 16 50 – 92±6 86±10 88±6 82±8
Clomazone 09985 50–10,000 16 50 – 92±15 83±10 89±4 89±8
Difenoconazole 0.9983 50–10,000 16 50 – 85±9 90±7 89±4 91±8
Diuron 0.9984 100–10,000 30 100 – – 107±13 95±7 92±10
Epoxiconazole 0.9989 50–10,000 16 50 – 80±7 91±9 93±6 91±8
Fenoxaprop-P-ethyl 0.9985 100–10,000 30 100 – – 70±18 78±6 77±9
Fipronil 0.9938 50–10,000 16 50 – 71±8 62±8 62±5 64±7
Propiconazole 0.9915 100–10,000 30 100 – – 85±9 95±4 95±7
LOD limit of detection, LOQ limit of quantification
Table 5 Comparison ofcommercial C18 and mussel shell C18 (octadecyl-functionalized silica gel) [39] Mussel shell material
Price ($US for 50 g) 175.00 –
Particle size (mesh) 200–400 35
Surface area (m2 g-1) ~550 ~5
Recovery (%) 12–115 43–122
Relative standard deviation (%) 5–26 6–30
Availability Synthetic Renewable source
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calibration for quantification of compounds to compensate forthe ME effect, ensuring the reliability of the quantification.
Applicability
The optimized method was applied to the multiresidue analy-sis of 20 compounds in golden mussel collected in RioGrande, Brazil. Two samples were analyzed. Haloperidoland diltiazem hydrochloride were detected in one of the sam-ples in a concentration lower than the LOQ. Although fewcompounds were detected, the recording of those organic con-taminants in the biota is important. In this sense, the methoddeveloped herein constitutes a new tool for the survey of pes-ticides and PPCPs in mussel samples.
Comparison with commercial C18
A comparison between commercial C18 and the mussel shellmaterial is shown in Table 5. An experiment was performedusing the same conditions but changing only the solid supportas described in BMussel shell as a solid support.^ Then, theprice, particle size, surface area, recovery, precision, and avail-ability were compared. From the data it is clear that the use ofthe shell is cheaper and environmentally friendlier than the useof C18. Besides, mussel shells have similar or better recoveryand precision for the compounds studied. In this context, gold-en mussel shell, which is obtained from a renewable source,should receive special attention.
Conclusions
Golden mussel shells were successfully used by the first timeas an alternative material for use as a solid support in MSPD.This material is cheaper and environmentally friendlier thanthe materials traditionally used. The overall method proposedis fast, robust, and efficient, and it allows the cost of the meth-od to be reduced in comparison with the use of C18.
Further, the method developed constitutes an importanttool for the monitoring of pesticides and PPCPs in musselsamples.
Acknowledgments The authors acknowledge the financial support andfellowships granted by the Brazilian agencies CAPES, FINEP, CNPq,and FURG. Part of this study was supported by a grant from the Brazilianagencies CNPq/CAPES (process number 552318/2011-6), FAPERGS(process numbers 810-25.51/13-3 and 831-25.51/13-0), and FCT/CAPES (process number 336/13). E.G.P. received a productivity researchfellowship from CNPq (DT 310517/2012-5) and J.P.V. received a fellow-ship from CNPq (309575/2013-3). The authors thank the Electron Mi-croscopy Center (CEME-Sul) of the Federal University of Rio Grande.
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