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Determination of fenobucarb residues in animal and aquatic food products using liquid chromatography-tandem mass spectrometry coupled with a QuEChERS extraction method
Journal of Chromatography B 1058 (2017) 1–7
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Determination of fenobucarb residues in animal and aquatic food products using liquid chromatography-tandem mass spectrometry coupled with a QuEChERS extraction method
MARK
⁎⁎
Weijia Zhenga,1, Jin-A Parka,1, Dan Zhanga, A.M. Abd El-Atya,b, , Seong-Kwan Kima, Sang-Hyun Choa, Jeong-Min Choia, Jae-Han Shimc, Byung-Joon Changd, Jin-Suk Kima, ⁎ Ho-Chul Shina, a
Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Republic of Korea Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, 12211, Giza, Egypt Natural Products Chemistry Laboratory, College of Agriculture and Life Sciences, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Republic of Korea d Department of Veterinary Anatomy, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Republic of Korea b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Fenobucarb Porcine muscle Egg Milk Fish Flatfish Shrimp Residues LC–MS/MS
A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method coupled with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI+/MS-MS) was developed for quantification of fenobucarb residues in animal food products, such as porcine muscle, egg, and whole milk, and aquatic food products, such as eel, flatfish, and shrimp. Acetonitrile with the addition of 0.1% trifluoroacetic acid was employed as an extraction solvent and was compared with acetonitrile alone and 0.1% formic acid in acetonitrile. All extracted samples were purified using C18 sorbent. The best extraction efficiencies, expressed as recovery at two spiking levels equivalent to 1- and 2-times the limit of quantification (LOQ = 2 μg/kg) were achieved using 0.1% trifluoroacetic acid in acetonitrile and ranged from 61.38 to 102.21% in all matrices, with relative standard deviations (RSDs) < 13% (except for the low spiking of porcine muscle and the high spiking of whole milk, for which the RSDs were > 20%). Six-point matrix-matched calibration was used for quantification and the determination coefficients were good (R2 ≥ 0.9865). The method was verified by application to samples purchased from local markets and none of the samples tested positive. In conclusion, the developed method is simple and versatile and can be used for the routine detection of fenobucarb in different animal food products having varying protein and fat contents with satisfactory accuracy and precision.
1. Introduction Pesticides play an essential role in pest and disease control for a diverse range of agricultural products. Although the use of these compounds brings enormous benefits, the spraying methods employed can lead to contamination and present a severe risk of bioaccumulation in food products [1,2]. The potential hazards this poses to human health, including dysfunction of the nervous and reproductive systems, have aroused a great deal of public concern worldwide [2]. Fenobucarb (Fig. 1) is a widely used carbamate insecticide derived from carbamic acid that inhibits cholinesterase enzymes (competitively) so as to affect nerve impulse transmission [3]. Carbamate pesticides are widely
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applied on grassland and farmland to protect crops from plagues. However, its careless use may give rise to the contamination of plants and by grazing on these contaminated crops, or by direct oral or injected administration, animals such as swine, cattle, and chickens may accumulate fenobucarb residues in their muscle tissues, milk, and eggs [4,5]. In recent years, rapid population expansion has led to a massive increase in food consumption throughout the world, especially aquatic food products as they are rich in proteins and other nutrient elements essential for both minors and adults. Drugs excreted in feces and urine as mixtures of unchanged parent compounds and their metabolites can enter aquatic environments through treated and untreated waste water. Consequently, these compounds may accumu-
Corresponding author at: Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, Seoul, 143-701, Republic of Korea. Corresponding author at: Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, Seoul, 143-701, Republic of Korea. E-mail addresses: [email protected], [email protected] (A.M. Abd El-Aty), [email protected] (H.-C. Shin). 1 The first two authors contributed equally to this article. ⁎⁎
http://dx.doi.org/10.1016/j.jchromb.2017.05.008 Received 14 March 2017; Received in revised form 7 May 2017; Accepted 9 May 2017 Available online 10 May 2017 1570-0232/ © 2017 Elsevier B.V. All rights reserved.
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Korea) for preparing the mobile phase. 2.2. Standard solutions A 1000 μg/mL standard stock solution of fenobucarb was made by accurately weighing 10 mg of the solid using an AG 285 analytical balance (METTLER TOLEDO, Seoul, Republic of Korea) and transferring it to a 15-mL high-clarity polypropylene conical tube (Falcon, Corning Science Mexico S. A. de C.V., Tamaulipas, Mexico), then dissolving it in 10 mL methanol. The intermediate (100 μg/kg) and working solutions were prepared by further dilution in 0.2% formic acid + 10 mM ammonium formate in methanol (mobile phase B), yielding the various concentrations (2, 4, 6, 8, 10, and 12 μg/kg) that were used for constructing the calibration curves. All solutions were stored at −20 °C in the dark and analyzed within a week.
Fig. 1. Chemical structure of fenobucarb.
late in the edible tissues of aquatic species such eels, flatfish, and shrimps. Subsequent exposure presents a severe problem in terms of consumer safety and public health. Therefore, the accurate monitoring of pesticides is of extreme importance [2]. In the screening and detection of pesticide residues of animal origin, solid-liquid extraction (SLE) and solid-phase extraction (SPE) are widely applied for sample pretreatment. However, these techniques suffer from complicated handling, the formation of emulsions, and time consuming operation [6]. Owing to the complexity and diversity of biological matrices, as well as the low limit of quantification (< 10 μg/ kg) stipulated by the Korean Ministry of Food and Drug Safety (MFDS) for drugs with no predefined maximum residue limits (MRL), these routine analytical procedures no longer satisfy the requirements of sample preparation [7]. Notably, the Japanese Ministry of Health, Labor, and Welfare (MHLW) is the only organizations, which prescribed the MRL of fenobucarb in porcine muscle and milk (20 μg/kg) and no MRL in egg, eel, flatfish, and shrimp has yet been established by other regulatory agencies [8–11]. In 2003, Anastassiades and his colleagues developed the concept of ‘quick, easy, cheap, effective, rugged, and safe (QuEChERS) sample preparation, an approach that is now used in the treatment of samples for multi-residue pesticide analysis [12]. Due to its simplicity, automatability, time efficiency, and convenience, QuEChERS methodology has been rapidly and widely adopted for separating target analytes from lipids and proteins in pesticide residue analysis [13]. Several analytical methods have been applied for the determination of carbamate residues in foods, including biosensors [14], gas chromatography with electron capture detection (GC-ECD) [4], and gas chromatography-mass spectrometry (GC–MS) [15]. However, liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) has proved to be the most significant quantitative analytical technique in recent years, because it can be employed for the direct detection of target analytes and products susceptible to degradation in various matrices, and presents numerous advantages such as high selectivity, high sensitivity, reliable identification, and rapid cleanup procedures [16–18]. To the best of our knowledge, there are no published methods based on QuEChERS extraction followed by LC-electrospray ionization (ESI) MS/MS for analyzing fenobucarb residues, except for one 2013 report concerning residues in beef muscles [13]. Consequently, the aim of the study presented here was to develop an effective approach for the quantification of fenobucarb residues in porcine muscle, milk, egg, eel, flatfish, and shrimp using QuEChERS extraction and purification followed by LC–MS/MS sample analysis.
2.3. Sample preparation Samples for detection and quantification were purchased from local markets in Seoul, Republic of Korea. The extraction and cleanup steps were carried out on the basis of EN QuEChERS methodology [19] with some improvements. Chopped porcine muscle (5 g), eel (5 g), shrimp (5 g), homogenized whole egg (without shell) (5 mL), and homogenized whole milk (5 mL) were transferred to 50-mL polypropylene conical tubes (Falcon, Corning Science Mexico S. A. de C.V., Tamaulipas, Mexico). After being spiked with working standard solution (0.5 mL) to a concentration of 20 μg/kg, the samples were left undisturbed for 10 min. The analyte was then extracted with 20 mL 0.1% TFA in acetonitrile, shaken vigorously for 5 min, and added to reagent kits (4 g magnesium sulfate, 1 g sodium chloride, 1 g sodium citrate tribasic dihydrate (SCTD), 0.5 g sodium citrate dibasic sesquihydrate (SCDS)), followed by vortex mixing (BenchMixer™ Multi-Tube Vortexer, Benchmark Scientific, NJ, USA) for 5 min. The tubes were then centrifuged at 2600g (Union 32 R Plus, Hanil Science Industrial Co., Ltd., Incheon, Republic of Korea) at 4 °C for 15 min. The supernatants were then transferred to 50-mL conical tubes and placed under gentle nitrogen gas flow at 50 °C (TurboVap®RV, Caliper Life Sciences, Hopkinton, USA) for evaporation until the volume reduced to 10 mL. The solutions obtained were transferred to 15-mL centrifuge tubes containing 150 mg C18 and 900 mg MgSO4 (Agilent Bond Elut, Agilent Technologies, CA, USA) and then vortex mixed sufficiently prior to further centrifugation at 2600g and 4 °C for 15 min. The upper layer was transferred and concentrated to dryness under nitrogen gas as previously stated. The residue (approximately ∼0.3 mL) was reconstituted in a 1:1 (v/v) mixture of mobile phases A and B to 1 mL, and the mixtures obtained were filtered through a 0.45-μm syringe filter prior to analysis. 2.4. LC–MS/MS analysis LC was performed using an Agilent series 1100 HPLC system (Agilent Technologies, CA, USA) equipped with a G1311A Quart pump, a G1313A autosampler, a G1322A degasser, a G1316A column oven, and an API 2000TM LC–MS/MS detector (Applied Biosystems, NY, USA). Chromatographic separation was performed using a Waters XBridge™ C18 reversed-phase analytical column (2.1 × 100 mm; 3.5 μm particle size; Waters, Milford, CT, USA) maintained at 35 °C. A binary mobile phase system, consisting of (A) 0.2% formic acid with 10 mM ammonium formate in distilled water and (B) 0.2% formic acid with 10 mM ammonium formate in methanol (1:1, v/v) was employed in gradient pump mode with an injection volume of 10 μL. The linear mobile phase gradient at a flow rate of 0.3 mL/min was started at 5% B (0–1 min), increased to 95% B (1–2 min), maintained for 8 min (2–10 min), decreased to 5% B (10–11 min), and maintained to the end (11–15 min). Triple quadrupole tandem mass spectrometric (MS/MS) analysis was employed under an ESI source in positive (ESI+) and negative
2. Materials and methods 2.1. Chemicals and regents Fenobucarb (CAS Number: 3766-81-2), analytical-grade formic acid (98%), ammonium formate (97%), and trifluoroacetic acid (TFA, 99%) were provided by Sigma-Aldrich Corporation (St. Louis, MO, USA). HPLC-grade methanol (99%) and acetonitrile (100%) were supplied by J. T. Baker Chemicals (Phillipsburg, NJ, USA). GH polypro (GHP) membranes and syringe filters (0.45 μm) were purchased from Pall (Michigan, USA). Ultrahigh purity water was obtained from an aqua MAX™ water purification system (Young Wha, Seoul, Republic of 2
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Table 1 Multiple reaction monitoring data acquisition parameters for fenobucarb. Compound
Chemical structure
Molecular weight
Mode
RT (min)
Precursor Ion (m/z)
Product Ion (m/z)
DP (V)
CE (V)
CEP (V)
Fenobucarb
C12H17NO2
207.27
Positive
6.73
208
95 152 77
31 31 31
17 11 51
12 12 12
DP: declustering potential, CE: collision energy, CEP: cell exit potential.
Fig. 2. LC–MS/MS chromatograms of fenobucarb in (top) blank samples, (middle) market samples, and (bottom) spiked samples (4 μg/kg) for (A) porcine muscle, and (B) milk samples.
above. The high- and low-intensity transitions were used for quantification and confirmation, respectively. The specific results are summarized in Table 1.
(ESI-) modes. Data collection was implemented in multiple reaction monitoring (MRM) mode with ABI software (version 1.4.2) employed for administration. An ion spray voltage of 5.5 kV and a capillary temperature of 350 °C were employed. The pressure was held at 50 psi in order to provide good conditions for ion source gas 1 (GS1) and ion source gas 2 (GS2). Optimization of precursor ions, product ions, declustering potential, and collision energy was performed by injecting the working standard solution directly into the MS unit. Precursor ions in the form of fragment [M + H]+ were selected under the conditions
2.5. Validation The method developed herein was verified according to the criteria stipulated by the MFDS (2015) [11], including requisite values for linearity, accuracy, precision, and limits of detection (LODs) and 3
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Fig. 3. LC–MS/MS chromatograms of fenobucarb in (top) blank samples, (middle) market samples, and (bottom) spiked samples (4 μg/kg) for (C) egg, and (D) eel samples.
[20]. A brief shaking step was added to ensure sufficient combination of the matrices with the working standard solutions. In the deproteinization and pH adjustment step, organic solvents including acetonitrile, ethanol, and methanol are usually applied with the addition of an acidic buffer [21] to improve extraction efficiency. In the present study, acetonitrile was initially employed to extract the analytes. However, the supernatant was not as clear as we expected when acetonitrile with no additives was used. Therefore, 0.1% trifluoroacetic acid or 0.1% formic acid was added to the acetonitrile for comparison. The addition of 0.1% trifluoroacetic acid resulted in the most transparent solution and the highest recovery. Furthermore, to avoid insufficient extraction and vortex mixing, the ratio of sample to organic solvent had to be optimized. Ultimately, a ratio of 1:4 was found to be optimal. The selection of QuEChERS reagents depends on the constituents of the diverse matrices. For instance, C18 is widely used for removing fats, sterols, and non-polar interfering substances; primary-secondary amine (PSA) is used for elimination of different sugars, fatty acids, and other organic acids; and magnesium sulfate is used to remove excess water
quantification (LOQs). The linearity was assessed from matrix-fortified calibration measured at six concentration levels. The calibration curve was prepared by plotting the response factor as a function of analyte concentration. Recovery and repeatability (i.e., intraday precision) were assessed at spiking concentrations of 1 × LOQ and 2 × LOQ (MFDS) (n = 3) in a single day. In addition, the results were calculated by comparing the spiked matrices with blank ones. For assessment of interday precision (i.e., reproducibility), the same concentration levels were tested (n = 3) over three consecutive days. The LOD and LOQ were calculated at three and ten times the signal-to-noise ratio, respectively. 3. Results and discussion 3.1. Optimization of extraction and cleanup procedures Instead of homogenization, solid samples were finely chopped in a blender as a fundamental procedure throughout the experimental work 4
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Fig. 4. LC–MS/MS chromatograms of fenobucarb in (top) blank samples, (middle) market samples, and (bottom) spiked samples (4 μg/kg) for (E) flatfish, and (F) shrimp samples.
After comparison with two other analytical columns (Phenomenex Kinetex 2.6 u C18 and Agilent Eclipse XDB-C18) a Waters Xbridge C18 column was employed because it afforded sharp peaks with a smooth baseline and better chromatographic separation of the target analytes [20]. The composition of the mobile phase also has a profound influence on ionization in LC–MS/MS. In the present study, mobile phases consisting of 0.1% formic acid, 0.1% formic acid in acetonitrile, 0.2% formic acid, 0.2% formic acid in acetonitrile, 0.2% formic acid + 10 mM ammonium formate, 0.2% formic acid + 10 mM ammonium formate in acetonitrile, 0.2% formic acid + 10 mM ammonium formate, and 0.2% formic acid + 10 mM ammonium formate in methanol were assayed to obtain the optimal separation, and the mobile phase consisting of methanol and both formic acid and ammonium formate produced the highest intensity signals. In addition, filtration of the extracts through a membrane filter and reconstitution in a mobile phase mixture (1:1 v/v) prior to LC–MS/MS analysis are necessary for the protection of the instrument and column
[22]. The data collected regarding the constituents of the samples obtained from local markets indicated that they are mainly composed of water, protein, and a small amount of fat. On account of the low level of carbohydrate contained, an EN QuEChERS method [19], which employs 900 mg magnesium sulfate and 150 mg C18 without PSA, was selected for the cleanup procedure. In addition, reagent kits containing 4 g magnesium sulfate, 1 g sodium chloride, 1 g SCTD, and 0.5 g SCDS were added in order to induce transfer of the analytes to the organic phase and stabilize the pH. Furthermore, a brief evaporation step was utilized to achieve a suitable solution volume for the QuEChERS process. The vortexing and centrifuging steps are indispensable for sufficient reaction and cleanup. 3.2. Optimization of LC conditions Both positive and negative ESI modes were assessed for quantification, and positive ion mode was selected for determination using an m/z 100–400 full spectrum scan. 5
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Table 2 Method performance of fenobucarb in different matrices. Analyte
Matrix
Spiking levels (μg/kg)
Precision
Intra-day (n = 3) Recovery (%) Fenobucarb
Porcine muscle Milk Egg Eel Flatfish Shrimp
2 4 2 4 2 4 2 4 2 4 2 4
75.33 64.39 61.38 69.87 61.72 63.54 90.80 93.31 83.48 92.68 97.97 92.99
± ± ± ± ± ± ± ± ± ± ± ±
17.87 8.20 4.96 17.04 2.70 4.46 2.00 3.82 2.45 3.43 1.65 2.22
Calibration curve (μg/kg)
R2
Linear range (μg/ kg)
LOD (μg/kg)
LOQ (μg/kg)
y = 4 × 106 × + 821.67
0.9865
2–12
0.7
2
y = 4 × 106 × + 313.33
0.9991
y = 4 × 106 × −1376.7
0.9902
6
y = 5 × 10 × +1338.3
0.9869
y = 6 × 106 × −893.33
0.9965
y = 5 × 106 × +2076.7
0.9936
Inter-day (n = 3) RSD (%)
Recovery (%)
RSD (%)
23.72 12.74 8.08 24.40 4.37 7.02 2.21 4.10 2.93 3.70 1.69 2.39
85.84 ± 11.01 63.35 ± 1.20 61.65 ± 1.19 71.65 ± 5.43 69.41 ± 7.56 63.39 ± 0.80 87.91 ± 3.01 91.73 ± 1.38 83.03 ± 1.20 93.49 ± 1.95 102.21 ± 3.67 94.04 ± 1.05
12.83 1.89 1.92 7.57 10.89 1.26 3.42 1.51 1.44 2.08 3.59 1.12
[23].
3.4. Method application
3.3. Validation
The applicability of the present method was assessed by screening three sorts of animal products for each matrix reported here. All samples were purchased from local markets, and handled according to the procedures outlined in Section 2.3. Notably, none of the samples contained the target analyte.
3.3.1. Specificity Specificity was assessed by means of comparison with blank samples consisting of porcine muscle, whole milk, egg, eel, flatfish, and shrimp matrices (n = 3) to identify potential interference from endogenous components. As shown in Figs. 2–4 , no interfering peaks are observed around the retention times of the analytes.
4. Conclusions A QuEChERS-based extraction coupled with an LC–MS/MS method was developed and optimized for the rapid determination of fenobucarb in porcine muscle, milk, egg, eel, flatfish, and shrimp. An extracting solvent of 0.1% TFA in acetonitrile and an EN QuEChERS cleanup step using C18 were utilized for removal of matrix proteins and fats. The acceptable recoveries indicated that the method is appropriate for multiple matrices with satisfactory accuracy. The LOQ of 2 μg/kg, is much lower than the MRL (20 μg/kg) set by the MHLW, Japan.
3.3.2. Linearity According to the MFDS guidelines, a matrix-matched calibration curve was constructed by determining the peak area of fenobucarb at specific concentrations, i.e., 2, 4, 6, 8, 10, and 12 μg/kg, which are equivalent to 1 × LOQ to 6 × LOQ. The correlation coefficient (R2) of ≥0.9865 is in excess of 0.95, the minimum requirement according to Codex Alimentarius Commission (1993) [24].
Conflict of interests 3.3.3. Recovery Recoveries ranging from 61.38 to 102.21% for three replicates using different samples were accomplished at two concentrations (2 and 4 μg/kg) with intra-and interday relative standard deviations (RSDs) between 1.12 and 12.83%, which are satisfactory based on the accepted criteria provided by the Codex Alimentarius Commission (spiking concentration: > 1–≤10 μg/kg; recovery range: 60–120%; inter-laboratory repeatability: 30%) [24]. Overall, the RSDs were around 13%, except for two values (> 20%) for porcine muscle and whole milk, which contain a much higher fat content compared to the other samples, and thus might not be sufficiently purified, however, they are still within the acceptable criteria set by Codex Alimentarius Commission as stated above [24]. The precision values for the matrices are summarized in Table 2 and are within limits set by ruling 2002/657/EC [25]. Therefore, the validation results indicate that the method is reliable and precise.
The authors declare no conflict of interests. Acknowledgement This research was supported by a grant (14162MFDS886) from the Ministry of Food and Drug Safety Administration, Republic of Korea in 2014. References [1] G.-F. Pang, Y.-Z. Cao, J.-J. Zhang, C.-L. Fan, Y.-M. Liu, X.-M. Li, G.-Q. Jia, Z.-Y. Li, Y.-Q. Shi, Y.-P. Wu, Validation study on 660 pesticide residues in animal tissues by gel permeation chromatography clean up/gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry, J. Chromatogr. A 1125 (2006) 1–30. [2] G.-F. Pang, C.-L. Fan, Y.-M. Liu, Y.-Z. Cao, J.-J. Zhang, X.-M. Li, Z.-Y. Li, Y.-P. Wu, T.-T. Guo, Determination of residues of 446 pesticides in fruits and vegetables by three-cartridge solid-phase extraction gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, J. AOAC Int. 89 (2006) 740–771. [3] T.R. Fukuto, Mechanism of action of organophosphorus and carbamate insecticides, Environ. Health Perspect 87 (1990) 245. [4] R. Fagnani, V. Beloti, A.P.P. Battaglini, K.d.S. Dunga, R. Tamanini, Organophosphorus and carbamates residues in milk and feed stuff supplied to dairy cattle, Pesquisa Veterinária Brasileira 31 (2011) 598–602. [5] S. Wang, H. Mu, Y. Bai, Y. Zhang, H. Liu, Multiresidue determination of fluoroquinolones, organophosphorus and N-methyl carbamates simultaneously in porcine tissue using MSPD and HPLC–DAD, J. Chromatogr. B 877 (2009)
3.3.4. Limits of detection and quantitation Limits of detection (LOD) and quantification (LOQ) were calculated according to the RSDs of the response and the slope. Ultimately, an LOD of 0.7 μg/kg and an LOQ of 2 μg/kg were obtained. Notably, the LOQs obtained for porcine muscle and milk are much lower than the MRL (20 μg/kg) prescribed by the Japanese Ministry of Health, Labor, and Welfare (MHLW) in 2007 [10], which indicates that this analytical method is adequately sensitive. 6
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Determination of fenobucarb residues in animal and aquatic food products using liquid chromatography-tandem mass spectrometry coupled with a QuEChERS extraction method
MARK
⁎⁎
Weijia Zhenga,1, Jin-A Parka,1, Dan Zhanga, A.M. Abd El-Atya,b, , Seong-Kwan Kima, Sang-Hyun Choa, Jeong-Min Choia, Jae-Han Shimc, Byung-Joon Changd, Jin-Suk Kima, ⁎ Ho-Chul Shina, a
Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Republic of Korea Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, 12211, Giza, Egypt Natural Products Chemistry Laboratory, College of Agriculture and Life Sciences, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Republic of Korea d Department of Veterinary Anatomy, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Republic of Korea b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Fenobucarb Porcine muscle Egg Milk Fish Flatfish Shrimp Residues LC–MS/MS
A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method coupled with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI+/MS-MS) was developed for quantification of fenobucarb residues in animal food products, such as porcine muscle, egg, and whole milk, and aquatic food products, such as eel, flatfish, and shrimp. Acetonitrile with the addition of 0.1% trifluoroacetic acid was employed as an extraction solvent and was compared with acetonitrile alone and 0.1% formic acid in acetonitrile. All extracted samples were purified using C18 sorbent. The best extraction efficiencies, expressed as recovery at two spiking levels equivalent to 1- and 2-times the limit of quantification (LOQ = 2 μg/kg) were achieved using 0.1% trifluoroacetic acid in acetonitrile and ranged from 61.38 to 102.21% in all matrices, with relative standard deviations (RSDs) < 13% (except for the low spiking of porcine muscle and the high spiking of whole milk, for which the RSDs were > 20%). Six-point matrix-matched calibration was used for quantification and the determination coefficients were good (R2 ≥ 0.9865). The method was verified by application to samples purchased from local markets and none of the samples tested positive. In conclusion, the developed method is simple and versatile and can be used for the routine detection of fenobucarb in different animal food products having varying protein and fat contents with satisfactory accuracy and precision.
1. Introduction Pesticides play an essential role in pest and disease control for a diverse range of agricultural products. Although the use of these compounds brings enormous benefits, the spraying methods employed can lead to contamination and present a severe risk of bioaccumulation in food products [1,2]. The potential hazards this poses to human health, including dysfunction of the nervous and reproductive systems, have aroused a great deal of public concern worldwide [2]. Fenobucarb (Fig. 1) is a widely used carbamate insecticide derived from carbamic acid that inhibits cholinesterase enzymes (competitively) so as to affect nerve impulse transmission [3]. Carbamate pesticides are widely
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applied on grassland and farmland to protect crops from plagues. However, its careless use may give rise to the contamination of plants and by grazing on these contaminated crops, or by direct oral or injected administration, animals such as swine, cattle, and chickens may accumulate fenobucarb residues in their muscle tissues, milk, and eggs [4,5]. In recent years, rapid population expansion has led to a massive increase in food consumption throughout the world, especially aquatic food products as they are rich in proteins and other nutrient elements essential for both minors and adults. Drugs excreted in feces and urine as mixtures of unchanged parent compounds and their metabolites can enter aquatic environments through treated and untreated waste water. Consequently, these compounds may accumu-
Corresponding author at: Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, Seoul, 143-701, Republic of Korea. Corresponding author at: Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, Seoul, 143-701, Republic of Korea. E-mail addresses: [email protected], [email protected] (A.M. Abd El-Aty), [email protected] (H.-C. Shin). 1 The first two authors contributed equally to this article. ⁎⁎
http://dx.doi.org/10.1016/j.jchromb.2017.05.008 Received 14 March 2017; Received in revised form 7 May 2017; Accepted 9 May 2017 Available online 10 May 2017 1570-0232/ © 2017 Elsevier B.V. All rights reserved.
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Korea) for preparing the mobile phase. 2.2. Standard solutions A 1000 μg/mL standard stock solution of fenobucarb was made by accurately weighing 10 mg of the solid using an AG 285 analytical balance (METTLER TOLEDO, Seoul, Republic of Korea) and transferring it to a 15-mL high-clarity polypropylene conical tube (Falcon, Corning Science Mexico S. A. de C.V., Tamaulipas, Mexico), then dissolving it in 10 mL methanol. The intermediate (100 μg/kg) and working solutions were prepared by further dilution in 0.2% formic acid + 10 mM ammonium formate in methanol (mobile phase B), yielding the various concentrations (2, 4, 6, 8, 10, and 12 μg/kg) that were used for constructing the calibration curves. All solutions were stored at −20 °C in the dark and analyzed within a week.
Fig. 1. Chemical structure of fenobucarb.
late in the edible tissues of aquatic species such eels, flatfish, and shrimps. Subsequent exposure presents a severe problem in terms of consumer safety and public health. Therefore, the accurate monitoring of pesticides is of extreme importance [2]. In the screening and detection of pesticide residues of animal origin, solid-liquid extraction (SLE) and solid-phase extraction (SPE) are widely applied for sample pretreatment. However, these techniques suffer from complicated handling, the formation of emulsions, and time consuming operation [6]. Owing to the complexity and diversity of biological matrices, as well as the low limit of quantification (< 10 μg/ kg) stipulated by the Korean Ministry of Food and Drug Safety (MFDS) for drugs with no predefined maximum residue limits (MRL), these routine analytical procedures no longer satisfy the requirements of sample preparation [7]. Notably, the Japanese Ministry of Health, Labor, and Welfare (MHLW) is the only organizations, which prescribed the MRL of fenobucarb in porcine muscle and milk (20 μg/kg) and no MRL in egg, eel, flatfish, and shrimp has yet been established by other regulatory agencies [8–11]. In 2003, Anastassiades and his colleagues developed the concept of ‘quick, easy, cheap, effective, rugged, and safe (QuEChERS) sample preparation, an approach that is now used in the treatment of samples for multi-residue pesticide analysis [12]. Due to its simplicity, automatability, time efficiency, and convenience, QuEChERS methodology has been rapidly and widely adopted for separating target analytes from lipids and proteins in pesticide residue analysis [13]. Several analytical methods have been applied for the determination of carbamate residues in foods, including biosensors [14], gas chromatography with electron capture detection (GC-ECD) [4], and gas chromatography-mass spectrometry (GC–MS) [15]. However, liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) has proved to be the most significant quantitative analytical technique in recent years, because it can be employed for the direct detection of target analytes and products susceptible to degradation in various matrices, and presents numerous advantages such as high selectivity, high sensitivity, reliable identification, and rapid cleanup procedures [16–18]. To the best of our knowledge, there are no published methods based on QuEChERS extraction followed by LC-electrospray ionization (ESI) MS/MS for analyzing fenobucarb residues, except for one 2013 report concerning residues in beef muscles [13]. Consequently, the aim of the study presented here was to develop an effective approach for the quantification of fenobucarb residues in porcine muscle, milk, egg, eel, flatfish, and shrimp using QuEChERS extraction and purification followed by LC–MS/MS sample analysis.
2.3. Sample preparation Samples for detection and quantification were purchased from local markets in Seoul, Republic of Korea. The extraction and cleanup steps were carried out on the basis of EN QuEChERS methodology [19] with some improvements. Chopped porcine muscle (5 g), eel (5 g), shrimp (5 g), homogenized whole egg (without shell) (5 mL), and homogenized whole milk (5 mL) were transferred to 50-mL polypropylene conical tubes (Falcon, Corning Science Mexico S. A. de C.V., Tamaulipas, Mexico). After being spiked with working standard solution (0.5 mL) to a concentration of 20 μg/kg, the samples were left undisturbed for 10 min. The analyte was then extracted with 20 mL 0.1% TFA in acetonitrile, shaken vigorously for 5 min, and added to reagent kits (4 g magnesium sulfate, 1 g sodium chloride, 1 g sodium citrate tribasic dihydrate (SCTD), 0.5 g sodium citrate dibasic sesquihydrate (SCDS)), followed by vortex mixing (BenchMixer™ Multi-Tube Vortexer, Benchmark Scientific, NJ, USA) for 5 min. The tubes were then centrifuged at 2600g (Union 32 R Plus, Hanil Science Industrial Co., Ltd., Incheon, Republic of Korea) at 4 °C for 15 min. The supernatants were then transferred to 50-mL conical tubes and placed under gentle nitrogen gas flow at 50 °C (TurboVap®RV, Caliper Life Sciences, Hopkinton, USA) for evaporation until the volume reduced to 10 mL. The solutions obtained were transferred to 15-mL centrifuge tubes containing 150 mg C18 and 900 mg MgSO4 (Agilent Bond Elut, Agilent Technologies, CA, USA) and then vortex mixed sufficiently prior to further centrifugation at 2600g and 4 °C for 15 min. The upper layer was transferred and concentrated to dryness under nitrogen gas as previously stated. The residue (approximately ∼0.3 mL) was reconstituted in a 1:1 (v/v) mixture of mobile phases A and B to 1 mL, and the mixtures obtained were filtered through a 0.45-μm syringe filter prior to analysis. 2.4. LC–MS/MS analysis LC was performed using an Agilent series 1100 HPLC system (Agilent Technologies, CA, USA) equipped with a G1311A Quart pump, a G1313A autosampler, a G1322A degasser, a G1316A column oven, and an API 2000TM LC–MS/MS detector (Applied Biosystems, NY, USA). Chromatographic separation was performed using a Waters XBridge™ C18 reversed-phase analytical column (2.1 × 100 mm; 3.5 μm particle size; Waters, Milford, CT, USA) maintained at 35 °C. A binary mobile phase system, consisting of (A) 0.2% formic acid with 10 mM ammonium formate in distilled water and (B) 0.2% formic acid with 10 mM ammonium formate in methanol (1:1, v/v) was employed in gradient pump mode with an injection volume of 10 μL. The linear mobile phase gradient at a flow rate of 0.3 mL/min was started at 5% B (0–1 min), increased to 95% B (1–2 min), maintained for 8 min (2–10 min), decreased to 5% B (10–11 min), and maintained to the end (11–15 min). Triple quadrupole tandem mass spectrometric (MS/MS) analysis was employed under an ESI source in positive (ESI+) and negative
2. Materials and methods 2.1. Chemicals and regents Fenobucarb (CAS Number: 3766-81-2), analytical-grade formic acid (98%), ammonium formate (97%), and trifluoroacetic acid (TFA, 99%) were provided by Sigma-Aldrich Corporation (St. Louis, MO, USA). HPLC-grade methanol (99%) and acetonitrile (100%) were supplied by J. T. Baker Chemicals (Phillipsburg, NJ, USA). GH polypro (GHP) membranes and syringe filters (0.45 μm) were purchased from Pall (Michigan, USA). Ultrahigh purity water was obtained from an aqua MAX™ water purification system (Young Wha, Seoul, Republic of 2
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Table 1 Multiple reaction monitoring data acquisition parameters for fenobucarb. Compound
Chemical structure
Molecular weight
Mode
RT (min)
Precursor Ion (m/z)
Product Ion (m/z)
DP (V)
CE (V)
CEP (V)
Fenobucarb
C12H17NO2
207.27
Positive
6.73
208
95 152 77
31 31 31
17 11 51
12 12 12
DP: declustering potential, CE: collision energy, CEP: cell exit potential.
Fig. 2. LC–MS/MS chromatograms of fenobucarb in (top) blank samples, (middle) market samples, and (bottom) spiked samples (4 μg/kg) for (A) porcine muscle, and (B) milk samples.
above. The high- and low-intensity transitions were used for quantification and confirmation, respectively. The specific results are summarized in Table 1.
(ESI-) modes. Data collection was implemented in multiple reaction monitoring (MRM) mode with ABI software (version 1.4.2) employed for administration. An ion spray voltage of 5.5 kV and a capillary temperature of 350 °C were employed. The pressure was held at 50 psi in order to provide good conditions for ion source gas 1 (GS1) and ion source gas 2 (GS2). Optimization of precursor ions, product ions, declustering potential, and collision energy was performed by injecting the working standard solution directly into the MS unit. Precursor ions in the form of fragment [M + H]+ were selected under the conditions
2.5. Validation The method developed herein was verified according to the criteria stipulated by the MFDS (2015) [11], including requisite values for linearity, accuracy, precision, and limits of detection (LODs) and 3
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Fig. 3. LC–MS/MS chromatograms of fenobucarb in (top) blank samples, (middle) market samples, and (bottom) spiked samples (4 μg/kg) for (C) egg, and (D) eel samples.
[20]. A brief shaking step was added to ensure sufficient combination of the matrices with the working standard solutions. In the deproteinization and pH adjustment step, organic solvents including acetonitrile, ethanol, and methanol are usually applied with the addition of an acidic buffer [21] to improve extraction efficiency. In the present study, acetonitrile was initially employed to extract the analytes. However, the supernatant was not as clear as we expected when acetonitrile with no additives was used. Therefore, 0.1% trifluoroacetic acid or 0.1% formic acid was added to the acetonitrile for comparison. The addition of 0.1% trifluoroacetic acid resulted in the most transparent solution and the highest recovery. Furthermore, to avoid insufficient extraction and vortex mixing, the ratio of sample to organic solvent had to be optimized. Ultimately, a ratio of 1:4 was found to be optimal. The selection of QuEChERS reagents depends on the constituents of the diverse matrices. For instance, C18 is widely used for removing fats, sterols, and non-polar interfering substances; primary-secondary amine (PSA) is used for elimination of different sugars, fatty acids, and other organic acids; and magnesium sulfate is used to remove excess water
quantification (LOQs). The linearity was assessed from matrix-fortified calibration measured at six concentration levels. The calibration curve was prepared by plotting the response factor as a function of analyte concentration. Recovery and repeatability (i.e., intraday precision) were assessed at spiking concentrations of 1 × LOQ and 2 × LOQ (MFDS) (n = 3) in a single day. In addition, the results were calculated by comparing the spiked matrices with blank ones. For assessment of interday precision (i.e., reproducibility), the same concentration levels were tested (n = 3) over three consecutive days. The LOD and LOQ were calculated at three and ten times the signal-to-noise ratio, respectively. 3. Results and discussion 3.1. Optimization of extraction and cleanup procedures Instead of homogenization, solid samples were finely chopped in a blender as a fundamental procedure throughout the experimental work 4
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Fig. 4. LC–MS/MS chromatograms of fenobucarb in (top) blank samples, (middle) market samples, and (bottom) spiked samples (4 μg/kg) for (E) flatfish, and (F) shrimp samples.
After comparison with two other analytical columns (Phenomenex Kinetex 2.6 u C18 and Agilent Eclipse XDB-C18) a Waters Xbridge C18 column was employed because it afforded sharp peaks with a smooth baseline and better chromatographic separation of the target analytes [20]. The composition of the mobile phase also has a profound influence on ionization in LC–MS/MS. In the present study, mobile phases consisting of 0.1% formic acid, 0.1% formic acid in acetonitrile, 0.2% formic acid, 0.2% formic acid in acetonitrile, 0.2% formic acid + 10 mM ammonium formate, 0.2% formic acid + 10 mM ammonium formate in acetonitrile, 0.2% formic acid + 10 mM ammonium formate, and 0.2% formic acid + 10 mM ammonium formate in methanol were assayed to obtain the optimal separation, and the mobile phase consisting of methanol and both formic acid and ammonium formate produced the highest intensity signals. In addition, filtration of the extracts through a membrane filter and reconstitution in a mobile phase mixture (1:1 v/v) prior to LC–MS/MS analysis are necessary for the protection of the instrument and column
[22]. The data collected regarding the constituents of the samples obtained from local markets indicated that they are mainly composed of water, protein, and a small amount of fat. On account of the low level of carbohydrate contained, an EN QuEChERS method [19], which employs 900 mg magnesium sulfate and 150 mg C18 without PSA, was selected for the cleanup procedure. In addition, reagent kits containing 4 g magnesium sulfate, 1 g sodium chloride, 1 g SCTD, and 0.5 g SCDS were added in order to induce transfer of the analytes to the organic phase and stabilize the pH. Furthermore, a brief evaporation step was utilized to achieve a suitable solution volume for the QuEChERS process. The vortexing and centrifuging steps are indispensable for sufficient reaction and cleanup. 3.2. Optimization of LC conditions Both positive and negative ESI modes were assessed for quantification, and positive ion mode was selected for determination using an m/z 100–400 full spectrum scan. 5
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Table 2 Method performance of fenobucarb in different matrices. Analyte
Matrix
Spiking levels (μg/kg)
Precision
Intra-day (n = 3) Recovery (%) Fenobucarb
Porcine muscle Milk Egg Eel Flatfish Shrimp
2 4 2 4 2 4 2 4 2 4 2 4
75.33 64.39 61.38 69.87 61.72 63.54 90.80 93.31 83.48 92.68 97.97 92.99
± ± ± ± ± ± ± ± ± ± ± ±
17.87 8.20 4.96 17.04 2.70 4.46 2.00 3.82 2.45 3.43 1.65 2.22
Calibration curve (μg/kg)
R2
Linear range (μg/ kg)
LOD (μg/kg)
LOQ (μg/kg)
y = 4 × 106 × + 821.67
0.9865
2–12
0.7
2
y = 4 × 106 × + 313.33
0.9991
y = 4 × 106 × −1376.7
0.9902
6
y = 5 × 10 × +1338.3
0.9869
y = 6 × 106 × −893.33
0.9965
y = 5 × 106 × +2076.7
0.9936
Inter-day (n = 3) RSD (%)
Recovery (%)
RSD (%)
23.72 12.74 8.08 24.40 4.37 7.02 2.21 4.10 2.93 3.70 1.69 2.39
85.84 ± 11.01 63.35 ± 1.20 61.65 ± 1.19 71.65 ± 5.43 69.41 ± 7.56 63.39 ± 0.80 87.91 ± 3.01 91.73 ± 1.38 83.03 ± 1.20 93.49 ± 1.95 102.21 ± 3.67 94.04 ± 1.05
12.83 1.89 1.92 7.57 10.89 1.26 3.42 1.51 1.44 2.08 3.59 1.12
[23].
3.4. Method application
3.3. Validation
The applicability of the present method was assessed by screening three sorts of animal products for each matrix reported here. All samples were purchased from local markets, and handled according to the procedures outlined in Section 2.3. Notably, none of the samples contained the target analyte.
3.3.1. Specificity Specificity was assessed by means of comparison with blank samples consisting of porcine muscle, whole milk, egg, eel, flatfish, and shrimp matrices (n = 3) to identify potential interference from endogenous components. As shown in Figs. 2–4 , no interfering peaks are observed around the retention times of the analytes.
4. Conclusions A QuEChERS-based extraction coupled with an LC–MS/MS method was developed and optimized for the rapid determination of fenobucarb in porcine muscle, milk, egg, eel, flatfish, and shrimp. An extracting solvent of 0.1% TFA in acetonitrile and an EN QuEChERS cleanup step using C18 were utilized for removal of matrix proteins and fats. The acceptable recoveries indicated that the method is appropriate for multiple matrices with satisfactory accuracy. The LOQ of 2 μg/kg, is much lower than the MRL (20 μg/kg) set by the MHLW, Japan.
3.3.2. Linearity According to the MFDS guidelines, a matrix-matched calibration curve was constructed by determining the peak area of fenobucarb at specific concentrations, i.e., 2, 4, 6, 8, 10, and 12 μg/kg, which are equivalent to 1 × LOQ to 6 × LOQ. The correlation coefficient (R2) of ≥0.9865 is in excess of 0.95, the minimum requirement according to Codex Alimentarius Commission (1993) [24].
Conflict of interests 3.3.3. Recovery Recoveries ranging from 61.38 to 102.21% for three replicates using different samples were accomplished at two concentrations (2 and 4 μg/kg) with intra-and interday relative standard deviations (RSDs) between 1.12 and 12.83%, which are satisfactory based on the accepted criteria provided by the Codex Alimentarius Commission (spiking concentration: > 1–≤10 μg/kg; recovery range: 60–120%; inter-laboratory repeatability: 30%) [24]. Overall, the RSDs were around 13%, except for two values (> 20%) for porcine muscle and whole milk, which contain a much higher fat content compared to the other samples, and thus might not be sufficiently purified, however, they are still within the acceptable criteria set by Codex Alimentarius Commission as stated above [24]. The precision values for the matrices are summarized in Table 2 and are within limits set by ruling 2002/657/EC [25]. Therefore, the validation results indicate that the method is reliable and precise.
The authors declare no conflict of interests. Acknowledgement This research was supported by a grant (14162MFDS886) from the Ministry of Food and Drug Safety Administration, Republic of Korea in 2014. References [1] G.-F. Pang, Y.-Z. Cao, J.-J. Zhang, C.-L. Fan, Y.-M. Liu, X.-M. Li, G.-Q. Jia, Z.-Y. Li, Y.-Q. Shi, Y.-P. Wu, Validation study on 660 pesticide residues in animal tissues by gel permeation chromatography clean up/gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry, J. Chromatogr. A 1125 (2006) 1–30. [2] G.-F. Pang, C.-L. Fan, Y.-M. Liu, Y.-Z. Cao, J.-J. Zhang, X.-M. Li, Z.-Y. Li, Y.-P. Wu, T.-T. Guo, Determination of residues of 446 pesticides in fruits and vegetables by three-cartridge solid-phase extraction gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, J. AOAC Int. 89 (2006) 740–771. [3] T.R. Fukuto, Mechanism of action of organophosphorus and carbamate insecticides, Environ. Health Perspect 87 (1990) 245. [4] R. Fagnani, V. Beloti, A.P.P. Battaglini, K.d.S. Dunga, R. Tamanini, Organophosphorus and carbamates residues in milk and feed stuff supplied to dairy cattle, Pesquisa Veterinária Brasileira 31 (2011) 598–602. [5] S. Wang, H. Mu, Y. Bai, Y. Zhang, H. Liu, Multiresidue determination of fluoroquinolones, organophosphorus and N-methyl carbamates simultaneously in porcine tissue using MSPD and HPLC–DAD, J. Chromatogr. B 877 (2009)
3.3.4. Limits of detection and quantitation Limits of detection (LOD) and quantification (LOQ) were calculated according to the RSDs of the response and the slope. Ultimately, an LOD of 0.7 μg/kg and an LOQ of 2 μg/kg were obtained. Notably, the LOQs obtained for porcine muscle and milk are much lower than the MRL (20 μg/kg) prescribed by the Japanese Ministry of Health, Labor, and Welfare (MHLW) in 2007 [10], which indicates that this analytical method is adequately sensitive. 6
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