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Headspace solid phase microextraction-gas chromatography for the determination of trihalomethanes in fish
Microchemical Journal 133 (2017) 539–544
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Headspace solid phase microextraction-gas chromatography for the determination of trihalomethanes in fish Nelson A. Delvaux Júnior a, Maria E.L.R. de Queiroz a, Antônio A. Neves a, André F. Oliveira a, Marcos R.F. da Silva a, Lêda R.A. Faroni b, Fernanda F. Heleno b,⁎ a b
Department of Chemistry, Universidade Federal de Viçosa, Viçosa, MG, Brazil Department of Agricultural Engineering, Universidade Federal de Viçosa, Viçosa, MG, Brazil
a r t i c l e
i n f o
Article history: Received 11 November 2016 Received in revised form 13 March 2017 Accepted 9 April 2017 Available online 13 April 2017 Keywords: Trihalomethanes Disinfection by products Sample preparation GC/ECD
a b s t r a c t The aim of this work is to develop a method for determining trihalomethanes (THMs) in fish samples. The proposed method uses solid phase microextraction and gas chromatography with an electron capture detector. Factors such as temperature, extraction time and type of fiber were assessed to maximize the performance of the extraction technique. The performance of the method was evaluated using selectivity, linearity, precision, accuracy and limits of detection (LOD) and quantification (LOQ). The new method allows analysis of THMs with appropriate selectivity and linearity, with coefficient of correlation N 0.98. The LOD and LOQ of the analytes of interest are from 0.11 to 0.35 μg kg−1 and 0.35 to 1.18 μg kg−1, respectively. In addition, the relative standard deviation (RSD) was between 1.6 and 8%, and the relative recovery was between 76 and 113%. The optimized and validated method was applied to fish samples purchased from the Viçosa (MG) local market. At least three of the THMs of interest were detected in most of the analyzed fish samples with maximum values for the concentration of chloroform, bromodichloromethane and bromoform at 8.33, 0.42 and 2.41 μg kg−1, respectively. © 2017 Elsevier B.V. All rights reserved.
1. Introduction In the fishing industry, the expression “quality of fish” is often related to species, marketable size and commercial value. Fish considered of low quality for a fish processor could be too small or in bad condition due to incorrect processing or the presence of wounds or spots on their back, resulting in low prices and thus low profits. Very often, however, quality means freshness and good appearance, and it is related to the degree of deterioration inherent to fish [1]. To insure the quality of fish, the slaughtering and cleaning processes are very important. The processing is designed to prevent the action of digestive enzymes before evisceration, which affect parameters such as pH, total volatile nitrogenous bases, shear force, color and the sensory attributes of the fish [2]. For initial processing, the fish is immediately washed to remove mucus, which is composed of glycoproteins released by the skin glands. This washing procedure associated with hyperchlorination (10 mg L−1), also reduces the microbiota on the fish surface [3]. After slaughtering, the fish scales and guts are removed. Then, fish are washed with chlorinated drinking water to remove adhered residues. At this point, the fish are clean and could be packed and cooled or frozen for commercialization or continue to be filleted. Since fish ⁎ Corresponding author at: Av. P. H. Rolfs s/n° - Campus UFV, 36570-900, Brazil E-mail address: [email protected] (F.F. Heleno).
http://dx.doi.org/10.1016/j.microc.2017.04.019 0026-265X/© 2017 Elsevier B.V. All rights reserved.
deteriorates easily, ice must be used for cooling. Ice cubes (or any other shape) with a maximum volume of 1 cm3 are used, in the ratio ice:fish of 1:1 [4]. After filleting, another step of washing with treated water is necessary. For commercialization, the fish can be kept fresh, cooled or frozen. “Fresh” fish are consumed without having undergone additional conservation process, except for being kept on ice action. “Cooled” fish are kept properly packaged in ice and at temperatures between −0.5 and − 2 °C, and “frozen” fish are kept frozen with temperatures not to exceed −25 °C [5]. For fish stored under refrigeration, microbial proliferation has been identified as the main cause of deterioration [6]. An alternative method that has been used in washing and storing the fish is the use of chlorinated water and ice. Scherer et al. [6] observed that the use of chlorinated water and ice effectively reduces the psychotropic, mesophilic and aerobic microorganisms in grass carp meat, increasing the shelf life of the whole fish stored under refrigeration by approximately three days. However, some by-products, as trihalomethanes (THMs), may be formed during chlorination process. The chloroform (CF), dichlorobromomethane (DCBM) chlorodibromomethane (CDBM) and bromoform (BF) are the most important THMs. THMs are mutagenic, cause various types of cancer, and may take more than thirty years to degrade in the environment [7]. The World Health Organization (WHO) recommends that the highest levels of chloroform, dichlorobromomethane,
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chlorodibromomethane and bromoform allowed in drinking water are 200, 60, 100 and 100 μg L−1, respectively [8]. The maximum contaminant level allowed by Brazilian legislation for the total concentration of THMs in water is 100 μg L− 1, which corresponds to the sum of the concentrations of the four major compounds: chloroform, dichlorobromomethane, chlorodibromomethane and bromoform. Some studies from England testing several kinds of foods have found the presence of chloroform at the following concentrations: 1.4 to 33 μg kg− 1 in dairy products; 1 to 4 mg kg− 1 in meat; 2 to 10 mg kg− 1 in vegetable oils; 0.4 to 18 mg L−1 in beverages; and 2 to 18 mg kg−1 in fruits and vegetables [9]. In fish meat, the concentration of CF was from 4 to 52 μg kg− 1; in chicken meat, between 2 and 76 μg kg−1; and in pork meat, between 2 and 68 μg kg−1 of CF and 11 μg kg−1 of DCBM [10–12]. Since fish is a product with rapid deterioration and quality loss, the use of preservatives and methods to increase its shelf life, especially compared to fresh fish, is essential for the success of aquaculture activity. However, monitoring the by-products from the employed methods is very important. Thus, a headspace solid phase microextraction (HS/ SPME) with gas chromatography (GC) method was optimized and validated to simultaneously determinate the four major THMs in fish samples.
Fig. 2. The influence of temperature on the chromatographic peak areas of THMs extracted from fish samples at 1 μg kg−1. Sample mass, 2 g; extraction time, 30 min.
which were bought at the local market in Viçosa/MG. Extractions and chromatographic analysis, were carried out for different storage periods.
2. Material and methods 2.3. Chromatographic conditions 2.1. Chemicals and standard solutions All reagents used were of analytical grade. The stock solutions of trihalomethanes in methanol (Merck, Darmstad, Germany), were prepared from standard mixture obtained from Supelco, Inc. (Bellefort, PA, USA), containing chloroform, bromodichloromethane, dibromochloromethane and bromoform at a concentration of 2000 mg mL− 1. The solutions were stored at − 18 °C. The working solutions were prepared daily from the stock solutions.
A gas chromatograph Shimadzu model 2014 with an electron capture detector was used to determine the trihalomethanes. Separations were performed on a Restek RTX-5MS capillary column, with the stationary phase composed of 5% diphenyl and 95% dimethylpolysiloxane (30 m × 0.25 mm i.d. and 0.10 μm thick film). The optimized chromatographic conditions were injector temperature, 200 °C; column oven temperature, 45 °C (2 min); heating ramp of 40 °C, min−1 to 100 °C; detector temperature, 300 °C; flow of carrier gas (N2) of 1.2 mL min−1; and flow division (split) 1:10.
2.2. Samples 2.4. HS/SPME procedure For procedure optimization and method validation, fish (Nile tilapia fillets) that were slaughtered and sanitized with water (free of chlorine) was purchased from subsistence farms in Viçosa/MG. The optimized and validated method was applied to 13 samples of fish fillets from different size fish farms, small, medium and large,
Fig. 1. The influence of fiber type on the chromatographic peak areas of THMs extracted from fish samples at 1 μg kg−1. Sample mass, 2 g; extraction time, 15 min. (CF = chloroform, DCBM = dichlorobromomethane, CDBM = chlorodibromomethane and BF = bromoform).
Method optimization tests to determine trihalomethanes in fish were made with a manual holder for SPME (Supelco, Bellefort, PA, USA) equipped with a fiber. The THM free fish samples (Nile tilapia fillets) were fortified with the 4 analytes of interest (chloroform, bromodichloromethane, dibromochloromethane and bromoform) at concentrations of 1 μg kg−1. The optimized method was performed on 2.0 g samples of spiked fish in 20 mL glass flasks equipped with PVC covers and PTFE skiving
Fig. 3. The influence of exposure time on the chromatographic peak areas of THMs extracted from fish samples at 1 μg kg−1. Sample mass, 2 g; extraction temperature, 30 °C.
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2.6. Evaluation of the method for incurred samples The method for the determination of trihalomethanes was employed in samples of fresh fish (A1 and A2) and frozen fillets (A3 to A13). The samples of Nile tilapia fillets were acquired at the local market of Viçosa and the origin of the fish and the storage time were considered. Some samples were kept frozen in a freezer and were periodically analyzed (A10 to A13).
3. Results and discussion 3.1. Optimization of headspace solid phase microextraction
Fig. 4. Chromatograms obtained from the HS/SPME fish samples: (Standard) spiked with THMs (1 μg L−1) and non-spiked (Blank). Chloroform, Rt = 2.1 min; bromodichloromethane, Rt = 2.8 min; dibromochloromethane, Rt = 3.5 min and bromoform, Rt = 4.2 min.
silicone septum. The flasks were transferred to a thermostatic bath (Tecnal TE-184) with a set temperature of 30 °C. The analytes were extracted with a fiber of divinylbenzenecarboxen-polydimethylsiloxane-50/30 μm (DVB-CAR-PDMS-50/30 μm). During the sorption process, the fiber was immersed in the gaseous part of the flask (headspace) for 15 min. After that, the fiber was immediately collected and introduced to the chromatograph injector where it remained exposed until the end of the run to avoid any carry-over effects. The analytes were thermally desorbed from the fiber under the flux of the carrier gas. All analyses were performed in triplicate. 2.4.1. Parameters for HS/SPME To establish the best conditions for the extraction of the analytes from the fish, three commercially available fibers were compared. The 100 μm thick polydimethylsiloxane (PDMS) fiber, the 65 μm thick polydimethylsiloxane and divinylbenzene (PDMS-DVB) fiber and the 50/30 μm thick divinyl benzene-carboxen-polydimethylsiloxane (DVB-CARPDMS) fiber from Supelco (Bellefort, PA, USA) were tested. The temperature of the thermostatic bath (10, 20, 30, 40, 60 and 80 °C) and the exposure time of the fiber (5, 10, 20, 30, 60 and 80 min) were optimized for the selected fiber. The best extraction conditions were determined by comparing the peak areas related to the analytes of interest. 2.5. Method validation The method (HS/SPME-GC/ECD) was validated based on the recommendations of the National Health Surveillance Agency (Agência Nacional de Vigilância Sanitária - ANVISA) [13], the US Food and Drug Administration (USFDA) [14] and the Ministry of Agriculture, Livestock and Supply (Ministério da Agricultura, Pecuária e Abastecimento MAPA) [15] which establish validation criteria for analytical methods. The performance of the method was assessed by studying the selectivity, linearity, precision, accuracy and limits of detection and quantification.
The temperature, extraction time and fiber type were optimized univariately. In each test, the chromatographic peak areas for each of the analytes were considered. The appropriate fiber choice is essential for the establishment of a sensitive method for solid phase microextraction and depends on the chemical nature of the compound of interest. The normalized peak areas obtained for each fiber are presented in Fig. 1. We chose to present the results of normalized peak areas for each compound to evaluate the most suitable fiber for each compound. The DVB-CAR-PDMS fiber had the highest extraction efficiency of the volatiles among when compared to the PDMS-DVB and PDMS fibers using an analysis of variance (ANOVA) with a Turkey post hoc test (Fig. 1). Guillén et al. [16] and Iglesias and Medina [17] also reported that DVB-CAR-PDMS fiber was the best for the extraction of volatile substances from fish samples. The extraction efficiency increased in the following sequence: PDMS-100 μm b PDMS-DVB-65 μm b DVB-CAR-PDMS-50/30 μm. This observation is mainly due to the carboxen porous phase, which captures small analytes between two to twelve carbon atoms. The pore size determines which analytes will be retained. The porous phase of carboxen contains micro (2–20 Å), meso (20–500 Å) and macropores (N500 Å). The DVB-CAR-PDMS fiber has two porous phases connected to the PDMS polymer [18]. These features make it more efficient to extract compared to the other fibers. The samples were maintained at 10, 20, 30, 40, 60 and 80 °C for 30 min to evaluate the effect of temperature on fiber extraction efficiency. It was observed that at the extraction temperature of 30 °C, the results were significantly equal to the results obtained at 10 to 20 °C and better than other temperatures for all compounds when evaluated using an analysis of variance (ANOVA) with a Turkey post hoc test (Fig. 2). We chose a temperature of 30 °C because it is easiest to maintain during extraction. The influence of temperature extraction on the chromatographic response of compounds was also observed in other studies that used the HS/SPME technique for the extraction of one or more classes of compounds in samples of drinking water and soft drinks [19–21]. The effect of the exposure time of the fiber in the headspace was evaluated at 5, 10, 20, 30, 60 and 80 min at an extraction temperature of 30 °C. There was an increase in the chromatographic responses of all analytes when the extraction time increased from 5 to 10 min. After 10 min, there was no significant difference for the extraction of
Table 1 The figures of merit for the method HS/SPME-GC/MS for analysis of THMs in fish samples. Analyte
Regression equation
ra
Linear range
LODb μg kg−1
LOQc μg kg−1
Chloroform Bromodichloromethane Dibromochloromethane Bromoform
80,905× + 37,796 483,556× + 312,338 582,368× + 93,028 222,972× + 38,774
0.9879 0.9918 0.9967 0.9913
1.18–8 0.43–8 0.35–8 0.78–8
0.35 0.14 0.11 0.26
1.18 0.43 0.35 0.78
a b c
Correlation coefficient. Limit of detection. Limit of quantification.
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THMs according to the variance analysis (ANOVA) with a Tukey post hoc test, indicating that equilibrium was reached (Fig. 3). The results for variable exposure times were lower than those reported in the literature for the extraction of THMs from drinking water. San Juan et al. [22] obtained an optimal extraction time of 40 min for CHCl3, CHCl2Br, CHClBr2 and higher than 40 min for CHBr3. The difference between the results may be related to the type of matrix being evaluated since at lower temperatures, THMs tend to be poorly absorbed by meat [23], making them more available to extraction by the fiber, which causes a shorter extraction time.
Table 2 The percentage of the relative recovery of the analytes at different concentrations. Analyte
Concentration μg kg−1
% Relative recovery
Chloroform
1.2 1.5 7.8 0.78 1.5 7.8 0.78 1.5 7.8 0.78 1.5 7.8
76.5 ± 7.7 89.9 ± 6.2 78.1 ± 1.3 120 ± 4.2 114 ± 1.6 92.6 ± 4.2 102 ± 3.7 103 ± 2.9 104 ± 3.7 98.7 ± 7.7 94.6 ± 1.3 95.9 ± 3.9
Bromodichloromethane
Dibromochloromethane
Bromoform
3.2. HS/SPME-GC/ECD method validation To evaluate the selectivity, the optimized method was applied to samples of Nile tilapia fillets free of THMs acquired from properties that slaughter fish without using treated water. Subsequently, those samples were spiked with THMs to concentrations of 1 μg kg−1 and again submitted to the method of extraction and analysis. The chromatograms of these samples were compared and no interference in the retention time of the analytes of interest was observed (Fig. 4). These results indicate the selectivity of the method and confirm the efficiency of the method for determining the analytes in the presence of other compounds in the matrix. Linearity was assessed from analytical curves for the four THMs. Correlation coefficients (r) N0.98 were obtained for all analytes (Table 1). The limits of detection (LOD) and quantification (LOQ) of the method were determined using advanced statistical resources in Excel (PROJ.LIN). Fish samples spiked with the analytes of interest (THMs) in different concentrations between 0.26 and 8.00 μg kg−1 were subjected to the extraction and quantification procedure. The LOD shown in Table 1 are lower than those obtained for the methods used for the analysis of these compounds in other matrices such as soil and water [19,24]. The optimized method was capable of detecting concentrations of chloroform, bromodichloromethane, dibromochloromethane and bromoform approximately 220, 520, 530 and 317 times lower, respectively, than the maximum concentration of THMs permissible in drinking water according to the USEPA [25]. To evaluate the accuracy of the method of determination of trihalomethanes in fish samples, we spiked fish samples free from the compounds of interest (blanks) with different levels of THMs to obtain concentrations of 1.2, 1.5 and 7.8 μg kg−1 of CF and 0.78, 1.5 and 7.8 μg kg−1 for the other THMs. The results, relative recovery percentages for each analyte concentration, are presented in Table 2. The accuracy was assessed through a recovery test. The percentage of the relative recovery was calculated by the ratio of between the average concentration determined experimentally and the corresponding theoretical concentration. The results for the relative recovery were within the acceptable range of 70 to 120% [15], indicating that the proposed method has adequate accuracy for the determination of these compounds in fish. The precision of the method was evaluated by carrying out repeatability and intermediate precision tests. The method was applied to fish samples spiked with concentrations of 1.2, 1.5 and 7.8 μg kg−1 of CF and 0.78, 1.5 and 7.8 μg kg−1 for the other THMs. For the repeatability analysis, we made nine consecutive determinations of three THM concentrations in nine samples of fillets on the same day with the same equipment and by the same analyst. The intermediate precision test was performed on three different days. The results are presented in Table 3. The method is considered precise if the coefficient of variation is b15% according to the criteria adopted by the USFDA [14]. The relative standard deviations (RSD) for repeatability and intermediate precision tests were below 6.3% and 12%, respectively, which demonstrates good precision of the established method.
3.3. Analysis of the fish samples acquired in the city of Viçosa, MG The optimized and validated method was used for the determination of the trihalomethanes chloroform, bromodichloromethane, dibromochloromethane and bromoform in samples of Nile tilapia fillets destined for human consumption in the city of Viçosa/MG. Samples A1 and A2 were purchased from aquacultures that slaughter the fish with untreated water. The other samples of fish fillets were purchased in the Viçosa local market. These samples were slaughtered and sanitized with properly chlorinated water and stored on chlorinated ice. Seventy percent of the fish samples were contaminated by the trihalomethanes chloroform, bromodichloromethane, dibromochloromethane and bromoform. The maximum values for the concentration of chloroform, bromodichloromethane, dibromochloromethane and bromoform found were 7.98, 0.55, 0.72 and 2.41 μg kg−1, respectively (Table 4). The sum of the concentrations of the trihalomethanes in a sample was b 9 μg kg−1. This value is lower than the maximum contaminant level (MCL) for these compounds in potable water established by Brazilian legislation, which is 100 μg L− 1 for total THMs and very close to the MCL of 10 μg L−1 allowed in Germany and France [26]. According to the Health Surveillance's Glossary, a “Cold Chain” consists of cooling the product during production and keeping it cool until its final consumption [27]. Several cold storage operations with the product under controlled relative temperature and humidity are involved in the cold chain. The sequence of operations can be simple or much more complex, depending on the characteristics of the processed product and its commercialization process [28]. The type of equipment used for keep the stability of the temperature is an important issue for the preservation and safety of products stored at low temperatures. Equipment for domestic use, due to its lower price, is often used by small businesses instead of commercial equipment that achieves adequate and stable temperatures [29]. According to Walker et
Table 3 Repeatability and intermediate precision tests of the method for the determination of the analytes in different concentrations. Analyte
Concentration μg kg−1
Repeatability %RSD
Intermediate precision %RSD
Chloroform
1.2 1.5 7.8 0.78 1.5 7.8 0.78 1.5 7.8 0.78 1.5 7.8
5.9 5.1 1.3 2.4 1.1 3.8 3.0 2.6 3.6 6.3 1.1 3.8
10 5.3 2.7 3.4 6.4 12 2.3 5.1 4.0 5.5 2.1 3.6
Bromodichloromethane
Dibromochloromethane
Bromoform
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Table 4 Application of HS/SPME-GC/ECD for the determination of THMs in fish samples. Aquaculture size
Sample identification
Days in storage
Chloroform
Bromodichloromethane
Dibromochloromethane
Bromoform
Total THMs
Small size
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A10 A10 A11 A11 A11 A12 A12 A12 A13 A13
7
– – 7.98 2.75 3.54 1.05 bLOD 3.55 bLOD 2.16 2.27 7.87 bLOD bLOD bLOD bLOD bLOD bLOD bLOD bLOD
– – bLODa bLOD bLOD bLOD bLOD 0.42 bLOD bLOD 0.52 0.55 bLOD bLOD bLOQ bLOD 0.55 0.56 bLOQ bLOQ
– – bLOD bLOD bLOD bLOD bLOD bLOD bLOD bLOD bLOD bLOD bLOD 0.55 0.57 bLOD bLOD bLOD bLOD bLOD
–
– – 7.98 5.16 3.54 1.05 bLOQb 3.97 bLOD 2.16 3.31 8.97 bLOD 1.42 1.47 bLOD 0.55 0.56 bLOQ 1.21
Medium size
Large size a b
15 15 20 25 39 41 45 39 41 45 100 110
bLOD 2.41 bLOD bLOD bLOD bLOD bLOD bLOD 0.52 0.55 bLOD 0.87 0.90 bLOD bLOD bLOD bLOQ 1.21
Limit of detection. Limit of quantification; A1 and A2 = fresh fish; A3 to A13 = frozen fillets.
al. [29], in the United Kingdom, 60% of companies use domestic refrigerators. Generally, cold storage, transport trucks and refrigerated display counters in supermarkets do not maintain the required temperatures [28]. Due to high costs and lack of information, the cold chain is frequently broken, which presents problems for the quality and the safety of the food. According to the Regulation of Industrial and Sanitary Inspection of Animal Products (RIISPOA) from 1952, under article 439 at the third paragraph: “It is understood that frozen fish is the fish treated with suitable freezing processes, at temperatures not higher than −25 °C”. Article 440 establishes that, after freezing, the fish must be kept in cold storage at −15 °C. The Codex Alimentarius establishes that, for several species of fish, quick freezing is complete when the product has reached the stable temperature of −18 °C at thermal center. The samples used for the analysis of the applicability of the method presented in Table 4 were acquired in the city of Viçosa/MG from aquacultures of different economic sizes that have cold storage facilities more or less adequate to meet the requirements of the food. Samples from A3 to A10 were processed in small size aquacultures; samples A11 and A12, medium size; and sample A13, large size. Since the freezing slows down the physicochemical and biochemical reactions that spoil the food [30], the differences in the storage time relative to the formation of THMs may be associated with the different freezing speeds of fish in the aquacultures. 4. Conclusions A method using HS/SPME and GC/ECD was optimized and validated for the determination of THMs in samples of fish. Through optimization, a DVB-CAR-PDMS fiber, an exposure time of 15 min and an extraction temperature of 30 °C were chosen as the best conditions for the determination of THMs. The validation parameters confirmed that the method has low LOD and LOQ levels, for the four THMs and adequate accuracy and precision, thus becoming an important tool for assessing the quality of fish. Chloroform, bromoform and bromodichloromethane residues were found in 70% of the samples analyzed. Since it is a method that employs no organic solvents and offers simple procedures, the proposed analysis protocol enables routine analysis of THMs in fish intended for human consumption.
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[24] S.H. Martín, C.G. Pinto, J.L.P. Pavón, B.M. Cordero, Determination of trihalomethanes in soil matrices by simplified quick, easy, cheap, effective, rugged and safe extraction and fast gas chromatography with electron capture detection, J. Chromatogr. A 1217 (2010) 4883–4889. [25] USEPA, National primary drinking water regulations: long term 1 enhanced surface water treatment rule, Final Rule, Federal Register 67 (2002) 1811. [26] A. Serrano, M. Gallego, Rapid determination of total trihalomethanes index in drinking water, J. Chromatogr. A 1154 (2007) 26–33. [27] Brasil, Secretaria de Estado de Saúde de Mato Grosso do Sul, Glossário da Vigilância Sanitária, http://www.saude.ms.gov.br 2011 accessed 20/09/2016. [28] J.F.D. Nantes, J.G.C.F. Machado, Aspectos Competitivos da Indústria de Alimentos no Brasil, Resultados/Workshop Identificação de Gargalos Tecnológicos na Agroindústria Paranaense, IPARDES, Curitiba, 2005 3–28. [29] E. Walker, C. Pritchard, S. Forsythe, Hazard analysis critical control point and prerequisite programme implementation in small and medium size food businesses, Food Control 14 (2003) 169–174. [30] M.S. Rahman, J.F.V. Ruiz, Food preservation by freezing, in: M.S. Rahman (Ed.), Handbook of Food Preservation, CRC press, Boca Raton 2007, pp. 635–657.
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Headspace solid phase microextraction-gas chromatography for the determination of trihalomethanes in fish Nelson A. Delvaux Júnior a, Maria E.L.R. de Queiroz a, Antônio A. Neves a, André F. Oliveira a, Marcos R.F. da Silva a, Lêda R.A. Faroni b, Fernanda F. Heleno b,⁎ a b
Department of Chemistry, Universidade Federal de Viçosa, Viçosa, MG, Brazil Department of Agricultural Engineering, Universidade Federal de Viçosa, Viçosa, MG, Brazil
a r t i c l e
i n f o
Article history: Received 11 November 2016 Received in revised form 13 March 2017 Accepted 9 April 2017 Available online 13 April 2017 Keywords: Trihalomethanes Disinfection by products Sample preparation GC/ECD
a b s t r a c t The aim of this work is to develop a method for determining trihalomethanes (THMs) in fish samples. The proposed method uses solid phase microextraction and gas chromatography with an electron capture detector. Factors such as temperature, extraction time and type of fiber were assessed to maximize the performance of the extraction technique. The performance of the method was evaluated using selectivity, linearity, precision, accuracy and limits of detection (LOD) and quantification (LOQ). The new method allows analysis of THMs with appropriate selectivity and linearity, with coefficient of correlation N 0.98. The LOD and LOQ of the analytes of interest are from 0.11 to 0.35 μg kg−1 and 0.35 to 1.18 μg kg−1, respectively. In addition, the relative standard deviation (RSD) was between 1.6 and 8%, and the relative recovery was between 76 and 113%. The optimized and validated method was applied to fish samples purchased from the Viçosa (MG) local market. At least three of the THMs of interest were detected in most of the analyzed fish samples with maximum values for the concentration of chloroform, bromodichloromethane and bromoform at 8.33, 0.42 and 2.41 μg kg−1, respectively. © 2017 Elsevier B.V. All rights reserved.
1. Introduction In the fishing industry, the expression “quality of fish” is often related to species, marketable size and commercial value. Fish considered of low quality for a fish processor could be too small or in bad condition due to incorrect processing or the presence of wounds or spots on their back, resulting in low prices and thus low profits. Very often, however, quality means freshness and good appearance, and it is related to the degree of deterioration inherent to fish [1]. To insure the quality of fish, the slaughtering and cleaning processes are very important. The processing is designed to prevent the action of digestive enzymes before evisceration, which affect parameters such as pH, total volatile nitrogenous bases, shear force, color and the sensory attributes of the fish [2]. For initial processing, the fish is immediately washed to remove mucus, which is composed of glycoproteins released by the skin glands. This washing procedure associated with hyperchlorination (10 mg L−1), also reduces the microbiota on the fish surface [3]. After slaughtering, the fish scales and guts are removed. Then, fish are washed with chlorinated drinking water to remove adhered residues. At this point, the fish are clean and could be packed and cooled or frozen for commercialization or continue to be filleted. Since fish ⁎ Corresponding author at: Av. P. H. Rolfs s/n° - Campus UFV, 36570-900, Brazil E-mail address: [email protected] (F.F. Heleno).
http://dx.doi.org/10.1016/j.microc.2017.04.019 0026-265X/© 2017 Elsevier B.V. All rights reserved.
deteriorates easily, ice must be used for cooling. Ice cubes (or any other shape) with a maximum volume of 1 cm3 are used, in the ratio ice:fish of 1:1 [4]. After filleting, another step of washing with treated water is necessary. For commercialization, the fish can be kept fresh, cooled or frozen. “Fresh” fish are consumed without having undergone additional conservation process, except for being kept on ice action. “Cooled” fish are kept properly packaged in ice and at temperatures between −0.5 and − 2 °C, and “frozen” fish are kept frozen with temperatures not to exceed −25 °C [5]. For fish stored under refrigeration, microbial proliferation has been identified as the main cause of deterioration [6]. An alternative method that has been used in washing and storing the fish is the use of chlorinated water and ice. Scherer et al. [6] observed that the use of chlorinated water and ice effectively reduces the psychotropic, mesophilic and aerobic microorganisms in grass carp meat, increasing the shelf life of the whole fish stored under refrigeration by approximately three days. However, some by-products, as trihalomethanes (THMs), may be formed during chlorination process. The chloroform (CF), dichlorobromomethane (DCBM) chlorodibromomethane (CDBM) and bromoform (BF) are the most important THMs. THMs are mutagenic, cause various types of cancer, and may take more than thirty years to degrade in the environment [7]. The World Health Organization (WHO) recommends that the highest levels of chloroform, dichlorobromomethane,
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chlorodibromomethane and bromoform allowed in drinking water are 200, 60, 100 and 100 μg L−1, respectively [8]. The maximum contaminant level allowed by Brazilian legislation for the total concentration of THMs in water is 100 μg L− 1, which corresponds to the sum of the concentrations of the four major compounds: chloroform, dichlorobromomethane, chlorodibromomethane and bromoform. Some studies from England testing several kinds of foods have found the presence of chloroform at the following concentrations: 1.4 to 33 μg kg− 1 in dairy products; 1 to 4 mg kg− 1 in meat; 2 to 10 mg kg− 1 in vegetable oils; 0.4 to 18 mg L−1 in beverages; and 2 to 18 mg kg−1 in fruits and vegetables [9]. In fish meat, the concentration of CF was from 4 to 52 μg kg− 1; in chicken meat, between 2 and 76 μg kg−1; and in pork meat, between 2 and 68 μg kg−1 of CF and 11 μg kg−1 of DCBM [10–12]. Since fish is a product with rapid deterioration and quality loss, the use of preservatives and methods to increase its shelf life, especially compared to fresh fish, is essential for the success of aquaculture activity. However, monitoring the by-products from the employed methods is very important. Thus, a headspace solid phase microextraction (HS/ SPME) with gas chromatography (GC) method was optimized and validated to simultaneously determinate the four major THMs in fish samples.
Fig. 2. The influence of temperature on the chromatographic peak areas of THMs extracted from fish samples at 1 μg kg−1. Sample mass, 2 g; extraction time, 30 min.
which were bought at the local market in Viçosa/MG. Extractions and chromatographic analysis, were carried out for different storage periods.
2. Material and methods 2.3. Chromatographic conditions 2.1. Chemicals and standard solutions All reagents used were of analytical grade. The stock solutions of trihalomethanes in methanol (Merck, Darmstad, Germany), were prepared from standard mixture obtained from Supelco, Inc. (Bellefort, PA, USA), containing chloroform, bromodichloromethane, dibromochloromethane and bromoform at a concentration of 2000 mg mL− 1. The solutions were stored at − 18 °C. The working solutions were prepared daily from the stock solutions.
A gas chromatograph Shimadzu model 2014 with an electron capture detector was used to determine the trihalomethanes. Separations were performed on a Restek RTX-5MS capillary column, with the stationary phase composed of 5% diphenyl and 95% dimethylpolysiloxane (30 m × 0.25 mm i.d. and 0.10 μm thick film). The optimized chromatographic conditions were injector temperature, 200 °C; column oven temperature, 45 °C (2 min); heating ramp of 40 °C, min−1 to 100 °C; detector temperature, 300 °C; flow of carrier gas (N2) of 1.2 mL min−1; and flow division (split) 1:10.
2.2. Samples 2.4. HS/SPME procedure For procedure optimization and method validation, fish (Nile tilapia fillets) that were slaughtered and sanitized with water (free of chlorine) was purchased from subsistence farms in Viçosa/MG. The optimized and validated method was applied to 13 samples of fish fillets from different size fish farms, small, medium and large,
Fig. 1. The influence of fiber type on the chromatographic peak areas of THMs extracted from fish samples at 1 μg kg−1. Sample mass, 2 g; extraction time, 15 min. (CF = chloroform, DCBM = dichlorobromomethane, CDBM = chlorodibromomethane and BF = bromoform).
Method optimization tests to determine trihalomethanes in fish were made with a manual holder for SPME (Supelco, Bellefort, PA, USA) equipped with a fiber. The THM free fish samples (Nile tilapia fillets) were fortified with the 4 analytes of interest (chloroform, bromodichloromethane, dibromochloromethane and bromoform) at concentrations of 1 μg kg−1. The optimized method was performed on 2.0 g samples of spiked fish in 20 mL glass flasks equipped with PVC covers and PTFE skiving
Fig. 3. The influence of exposure time on the chromatographic peak areas of THMs extracted from fish samples at 1 μg kg−1. Sample mass, 2 g; extraction temperature, 30 °C.
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2.6. Evaluation of the method for incurred samples The method for the determination of trihalomethanes was employed in samples of fresh fish (A1 and A2) and frozen fillets (A3 to A13). The samples of Nile tilapia fillets were acquired at the local market of Viçosa and the origin of the fish and the storage time were considered. Some samples were kept frozen in a freezer and were periodically analyzed (A10 to A13).
3. Results and discussion 3.1. Optimization of headspace solid phase microextraction
Fig. 4. Chromatograms obtained from the HS/SPME fish samples: (Standard) spiked with THMs (1 μg L−1) and non-spiked (Blank). Chloroform, Rt = 2.1 min; bromodichloromethane, Rt = 2.8 min; dibromochloromethane, Rt = 3.5 min and bromoform, Rt = 4.2 min.
silicone septum. The flasks were transferred to a thermostatic bath (Tecnal TE-184) with a set temperature of 30 °C. The analytes were extracted with a fiber of divinylbenzenecarboxen-polydimethylsiloxane-50/30 μm (DVB-CAR-PDMS-50/30 μm). During the sorption process, the fiber was immersed in the gaseous part of the flask (headspace) for 15 min. After that, the fiber was immediately collected and introduced to the chromatograph injector where it remained exposed until the end of the run to avoid any carry-over effects. The analytes were thermally desorbed from the fiber under the flux of the carrier gas. All analyses were performed in triplicate. 2.4.1. Parameters for HS/SPME To establish the best conditions for the extraction of the analytes from the fish, three commercially available fibers were compared. The 100 μm thick polydimethylsiloxane (PDMS) fiber, the 65 μm thick polydimethylsiloxane and divinylbenzene (PDMS-DVB) fiber and the 50/30 μm thick divinyl benzene-carboxen-polydimethylsiloxane (DVB-CARPDMS) fiber from Supelco (Bellefort, PA, USA) were tested. The temperature of the thermostatic bath (10, 20, 30, 40, 60 and 80 °C) and the exposure time of the fiber (5, 10, 20, 30, 60 and 80 min) were optimized for the selected fiber. The best extraction conditions were determined by comparing the peak areas related to the analytes of interest. 2.5. Method validation The method (HS/SPME-GC/ECD) was validated based on the recommendations of the National Health Surveillance Agency (Agência Nacional de Vigilância Sanitária - ANVISA) [13], the US Food and Drug Administration (USFDA) [14] and the Ministry of Agriculture, Livestock and Supply (Ministério da Agricultura, Pecuária e Abastecimento MAPA) [15] which establish validation criteria for analytical methods. The performance of the method was assessed by studying the selectivity, linearity, precision, accuracy and limits of detection and quantification.
The temperature, extraction time and fiber type were optimized univariately. In each test, the chromatographic peak areas for each of the analytes were considered. The appropriate fiber choice is essential for the establishment of a sensitive method for solid phase microextraction and depends on the chemical nature of the compound of interest. The normalized peak areas obtained for each fiber are presented in Fig. 1. We chose to present the results of normalized peak areas for each compound to evaluate the most suitable fiber for each compound. The DVB-CAR-PDMS fiber had the highest extraction efficiency of the volatiles among when compared to the PDMS-DVB and PDMS fibers using an analysis of variance (ANOVA) with a Turkey post hoc test (Fig. 1). Guillén et al. [16] and Iglesias and Medina [17] also reported that DVB-CAR-PDMS fiber was the best for the extraction of volatile substances from fish samples. The extraction efficiency increased in the following sequence: PDMS-100 μm b PDMS-DVB-65 μm b DVB-CAR-PDMS-50/30 μm. This observation is mainly due to the carboxen porous phase, which captures small analytes between two to twelve carbon atoms. The pore size determines which analytes will be retained. The porous phase of carboxen contains micro (2–20 Å), meso (20–500 Å) and macropores (N500 Å). The DVB-CAR-PDMS fiber has two porous phases connected to the PDMS polymer [18]. These features make it more efficient to extract compared to the other fibers. The samples were maintained at 10, 20, 30, 40, 60 and 80 °C for 30 min to evaluate the effect of temperature on fiber extraction efficiency. It was observed that at the extraction temperature of 30 °C, the results were significantly equal to the results obtained at 10 to 20 °C and better than other temperatures for all compounds when evaluated using an analysis of variance (ANOVA) with a Turkey post hoc test (Fig. 2). We chose a temperature of 30 °C because it is easiest to maintain during extraction. The influence of temperature extraction on the chromatographic response of compounds was also observed in other studies that used the HS/SPME technique for the extraction of one or more classes of compounds in samples of drinking water and soft drinks [19–21]. The effect of the exposure time of the fiber in the headspace was evaluated at 5, 10, 20, 30, 60 and 80 min at an extraction temperature of 30 °C. There was an increase in the chromatographic responses of all analytes when the extraction time increased from 5 to 10 min. After 10 min, there was no significant difference for the extraction of
Table 1 The figures of merit for the method HS/SPME-GC/MS for analysis of THMs in fish samples. Analyte
Regression equation
ra
Linear range
LODb μg kg−1
LOQc μg kg−1
Chloroform Bromodichloromethane Dibromochloromethane Bromoform
80,905× + 37,796 483,556× + 312,338 582,368× + 93,028 222,972× + 38,774
0.9879 0.9918 0.9967 0.9913
1.18–8 0.43–8 0.35–8 0.78–8
0.35 0.14 0.11 0.26
1.18 0.43 0.35 0.78
a b c
Correlation coefficient. Limit of detection. Limit of quantification.
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THMs according to the variance analysis (ANOVA) with a Tukey post hoc test, indicating that equilibrium was reached (Fig. 3). The results for variable exposure times were lower than those reported in the literature for the extraction of THMs from drinking water. San Juan et al. [22] obtained an optimal extraction time of 40 min for CHCl3, CHCl2Br, CHClBr2 and higher than 40 min for CHBr3. The difference between the results may be related to the type of matrix being evaluated since at lower temperatures, THMs tend to be poorly absorbed by meat [23], making them more available to extraction by the fiber, which causes a shorter extraction time.
Table 2 The percentage of the relative recovery of the analytes at different concentrations. Analyte
Concentration μg kg−1
% Relative recovery
Chloroform
1.2 1.5 7.8 0.78 1.5 7.8 0.78 1.5 7.8 0.78 1.5 7.8
76.5 ± 7.7 89.9 ± 6.2 78.1 ± 1.3 120 ± 4.2 114 ± 1.6 92.6 ± 4.2 102 ± 3.7 103 ± 2.9 104 ± 3.7 98.7 ± 7.7 94.6 ± 1.3 95.9 ± 3.9
Bromodichloromethane
Dibromochloromethane
Bromoform
3.2. HS/SPME-GC/ECD method validation To evaluate the selectivity, the optimized method was applied to samples of Nile tilapia fillets free of THMs acquired from properties that slaughter fish without using treated water. Subsequently, those samples were spiked with THMs to concentrations of 1 μg kg−1 and again submitted to the method of extraction and analysis. The chromatograms of these samples were compared and no interference in the retention time of the analytes of interest was observed (Fig. 4). These results indicate the selectivity of the method and confirm the efficiency of the method for determining the analytes in the presence of other compounds in the matrix. Linearity was assessed from analytical curves for the four THMs. Correlation coefficients (r) N0.98 were obtained for all analytes (Table 1). The limits of detection (LOD) and quantification (LOQ) of the method were determined using advanced statistical resources in Excel (PROJ.LIN). Fish samples spiked with the analytes of interest (THMs) in different concentrations between 0.26 and 8.00 μg kg−1 were subjected to the extraction and quantification procedure. The LOD shown in Table 1 are lower than those obtained for the methods used for the analysis of these compounds in other matrices such as soil and water [19,24]. The optimized method was capable of detecting concentrations of chloroform, bromodichloromethane, dibromochloromethane and bromoform approximately 220, 520, 530 and 317 times lower, respectively, than the maximum concentration of THMs permissible in drinking water according to the USEPA [25]. To evaluate the accuracy of the method of determination of trihalomethanes in fish samples, we spiked fish samples free from the compounds of interest (blanks) with different levels of THMs to obtain concentrations of 1.2, 1.5 and 7.8 μg kg−1 of CF and 0.78, 1.5 and 7.8 μg kg−1 for the other THMs. The results, relative recovery percentages for each analyte concentration, are presented in Table 2. The accuracy was assessed through a recovery test. The percentage of the relative recovery was calculated by the ratio of between the average concentration determined experimentally and the corresponding theoretical concentration. The results for the relative recovery were within the acceptable range of 70 to 120% [15], indicating that the proposed method has adequate accuracy for the determination of these compounds in fish. The precision of the method was evaluated by carrying out repeatability and intermediate precision tests. The method was applied to fish samples spiked with concentrations of 1.2, 1.5 and 7.8 μg kg−1 of CF and 0.78, 1.5 and 7.8 μg kg−1 for the other THMs. For the repeatability analysis, we made nine consecutive determinations of three THM concentrations in nine samples of fillets on the same day with the same equipment and by the same analyst. The intermediate precision test was performed on three different days. The results are presented in Table 3. The method is considered precise if the coefficient of variation is b15% according to the criteria adopted by the USFDA [14]. The relative standard deviations (RSD) for repeatability and intermediate precision tests were below 6.3% and 12%, respectively, which demonstrates good precision of the established method.
3.3. Analysis of the fish samples acquired in the city of Viçosa, MG The optimized and validated method was used for the determination of the trihalomethanes chloroform, bromodichloromethane, dibromochloromethane and bromoform in samples of Nile tilapia fillets destined for human consumption in the city of Viçosa/MG. Samples A1 and A2 were purchased from aquacultures that slaughter the fish with untreated water. The other samples of fish fillets were purchased in the Viçosa local market. These samples were slaughtered and sanitized with properly chlorinated water and stored on chlorinated ice. Seventy percent of the fish samples were contaminated by the trihalomethanes chloroform, bromodichloromethane, dibromochloromethane and bromoform. The maximum values for the concentration of chloroform, bromodichloromethane, dibromochloromethane and bromoform found were 7.98, 0.55, 0.72 and 2.41 μg kg−1, respectively (Table 4). The sum of the concentrations of the trihalomethanes in a sample was b 9 μg kg−1. This value is lower than the maximum contaminant level (MCL) for these compounds in potable water established by Brazilian legislation, which is 100 μg L− 1 for total THMs and very close to the MCL of 10 μg L−1 allowed in Germany and France [26]. According to the Health Surveillance's Glossary, a “Cold Chain” consists of cooling the product during production and keeping it cool until its final consumption [27]. Several cold storage operations with the product under controlled relative temperature and humidity are involved in the cold chain. The sequence of operations can be simple or much more complex, depending on the characteristics of the processed product and its commercialization process [28]. The type of equipment used for keep the stability of the temperature is an important issue for the preservation and safety of products stored at low temperatures. Equipment for domestic use, due to its lower price, is often used by small businesses instead of commercial equipment that achieves adequate and stable temperatures [29]. According to Walker et
Table 3 Repeatability and intermediate precision tests of the method for the determination of the analytes in different concentrations. Analyte
Concentration μg kg−1
Repeatability %RSD
Intermediate precision %RSD
Chloroform
1.2 1.5 7.8 0.78 1.5 7.8 0.78 1.5 7.8 0.78 1.5 7.8
5.9 5.1 1.3 2.4 1.1 3.8 3.0 2.6 3.6 6.3 1.1 3.8
10 5.3 2.7 3.4 6.4 12 2.3 5.1 4.0 5.5 2.1 3.6
Bromodichloromethane
Dibromochloromethane
Bromoform
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Table 4 Application of HS/SPME-GC/ECD for the determination of THMs in fish samples. Aquaculture size
Sample identification
Days in storage
Chloroform
Bromodichloromethane
Dibromochloromethane
Bromoform
Total THMs
Small size
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A10 A10 A11 A11 A11 A12 A12 A12 A13 A13
7
– – 7.98 2.75 3.54 1.05 bLOD 3.55 bLOD 2.16 2.27 7.87 bLOD bLOD bLOD bLOD bLOD bLOD bLOD bLOD
– – bLODa bLOD bLOD bLOD bLOD 0.42 bLOD bLOD 0.52 0.55 bLOD bLOD bLOQ bLOD 0.55 0.56 bLOQ bLOQ
– – bLOD bLOD bLOD bLOD bLOD bLOD bLOD bLOD bLOD bLOD bLOD 0.55 0.57 bLOD bLOD bLOD bLOD bLOD
–
– – 7.98 5.16 3.54 1.05 bLOQb 3.97 bLOD 2.16 3.31 8.97 bLOD 1.42 1.47 bLOD 0.55 0.56 bLOQ 1.21
Medium size
Large size a b
15 15 20 25 39 41 45 39 41 45 100 110
bLOD 2.41 bLOD bLOD bLOD bLOD bLOD bLOD 0.52 0.55 bLOD 0.87 0.90 bLOD bLOD bLOD bLOQ 1.21
Limit of detection. Limit of quantification; A1 and A2 = fresh fish; A3 to A13 = frozen fillets.
al. [29], in the United Kingdom, 60% of companies use domestic refrigerators. Generally, cold storage, transport trucks and refrigerated display counters in supermarkets do not maintain the required temperatures [28]. Due to high costs and lack of information, the cold chain is frequently broken, which presents problems for the quality and the safety of the food. According to the Regulation of Industrial and Sanitary Inspection of Animal Products (RIISPOA) from 1952, under article 439 at the third paragraph: “It is understood that frozen fish is the fish treated with suitable freezing processes, at temperatures not higher than −25 °C”. Article 440 establishes that, after freezing, the fish must be kept in cold storage at −15 °C. The Codex Alimentarius establishes that, for several species of fish, quick freezing is complete when the product has reached the stable temperature of −18 °C at thermal center. The samples used for the analysis of the applicability of the method presented in Table 4 were acquired in the city of Viçosa/MG from aquacultures of different economic sizes that have cold storage facilities more or less adequate to meet the requirements of the food. Samples from A3 to A10 were processed in small size aquacultures; samples A11 and A12, medium size; and sample A13, large size. Since the freezing slows down the physicochemical and biochemical reactions that spoil the food [30], the differences in the storage time relative to the formation of THMs may be associated with the different freezing speeds of fish in the aquacultures. 4. Conclusions A method using HS/SPME and GC/ECD was optimized and validated for the determination of THMs in samples of fish. Through optimization, a DVB-CAR-PDMS fiber, an exposure time of 15 min and an extraction temperature of 30 °C were chosen as the best conditions for the determination of THMs. The validation parameters confirmed that the method has low LOD and LOQ levels, for the four THMs and adequate accuracy and precision, thus becoming an important tool for assessing the quality of fish. Chloroform, bromoform and bromodichloromethane residues were found in 70% of the samples analyzed. Since it is a method that employs no organic solvents and offers simple procedures, the proposed analysis protocol enables routine analysis of THMs in fish intended for human consumption.
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