Immunoaffinity Chromatography Purification and Ultrahigh

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Immunoaffinity Chromatography Purification and Ultrahigh Performance Liquid Chromatography Tandem Mass Spectrometry Determination of Tetrodotoxin in Marine Organisms Xiaojun Zhang,† Zhongyong Yan,† Ying Wang,‡ Tao Jiang,§ Jian Wang,‡ Xiumei Sun,† and Yuanming Guo*,† †

Key Lab of Sustainable Utilization of Technology Research for Fishery Resource of Zhejiang Province, Marine Fisheries Research Institute of Zhejiang, 28 Tiyu Street, Zhoushan 316021, P.R. China ‡ Jiangsu Meizheng Biotechnology Company Limited, Wuxi 214135, P.R. China § Key Laboratory of Food Safety Risk Assessment, Ministry of Health, China National Center For Food Safety Risk Assessment, NO. 7 Panjiayuan Nanli, Chaoyang District, Beijing 100021, P. R. China ABSTRACT: A highly selective and sensitive method was developed for the determination of tetrodotoxin (TTX) in marine organisms by immunoaffinity chromatography (IAC) purification coupled with ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC−MS/MS). An IAC column was prepared and used to cleanup the extracted samples. The operating conditions of the IAC column were optimized, and the capacity of new IAC column was found to be 1106 ng mL−1, which was sufficient for TTX determination. The MS/MS conditions and UPLC mobile phase were also studied to optimize the operation conditions. Fortified marine organism samples at levels of 0.3−5.0 ng g−1 were utilized, and the average recoveries were 86.5−103.6% with intra- and inter-day relative standard deviations less than 7.22 and 9.88%, respectively. The limits of detection and quantification were 0.1 and 0.3 ng g−1, respectively. The method was later successfully applied for the determination of TTX in 100 marine organism samples collected from local markets. KEYWORDS: tetrodotoxin, immunoaffinity, ultrahigh performance liquid chromatography tandem mass spectrometry

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munoassay,18 and high-performance liquid chromatography (HPLC).19 In recent years, measurements of TTX by liquid chromatography tandem mass spectrometry (LC−MS/ MS)20−23 are becoming more common due to the improved sensitivity and selectivity. The matrices of marine organism tissues are extremely complex; therefore, a comprehensive sample pretreatment procedure is required prior to the LC− MS/MS detection. In the previous studies, solid-phase extraction,24 liquid−liquid extraction,25 and ultrafiltration26 methods were used to purify the tissue extracts. However, the sensitivity and recovery of TTX were not good due to serious depression by the background interfering contaminants. Recently, efficient and robust immunoaffinity chromatography (IAC) cleanup has attracted remarkable attention because it acts as a good alternative pretreatment method for LC−MS/ MS. The significant advantage of IACs is the high specificity of imprinted antibodies to target analytes.27 The IAC technique has been extensively used in sample preparation for residue and mycotoxins analysis. An IAC method for tetrodotoxin has proved effective in analyzing urine samples in some literature.28,29 However, determination of TTX in complex organisms by IAC has rarely been studied. The main objective of this study was to develop a method for the determination of TTX in marine organisms by IAC coupled

INTRODUCTION Tetrodotoxin (TTX) is a low molecular weight marine neurotoxin1 that is extremely potent. It is a nonprotein found mainly in the liver and sex organs of puffer fish, gastropods, clams, newts, and horseshoe crab. Structurally, TTX consists of a guanidinium moiety connected to a highly oxygenated carbon skeleton possessing a 2,4-dioxaadamantane portion containing five hydroxyl groups.2 The structure of TTX is shown in Figure 1. TTX was originally discovered and isolated from puffer fish

Figure 1. Chemical structure of TTX.

in 1964.3 Later, it was found in a variety of marine organisms.4−10 TTX is one of the most potent nonprotein poisons found in nature, and specific antidote or antitoxins to TTX are not available. The toxin acts by blocking site 1 of the voltage-gated sodium channel, resulting in respiratory paralysis and often death in humans.11 Over the past few decades, numerous incidents involving TTX-food poisoning have been reported in Japan, China, and other Asian countries.12−15 Currently, methods for identification and quantification of TTX include toxicity assay in mouse,16 enzyme linked immunosorbent assay,17 gold nanoparticle probe-based im© 2015 American Chemical Society

Received: Revised: Accepted: Published: 3129

January 5, 2015 March 3, 2015 March 10, 2015 March 10, 2015 DOI: 10.1021/acs.jafc.5b00045 J. Agric. Food Chem. 2015, 63, 3129−3134

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Journal of Agricultural and Food Chemistry

immediately regenerated by PBS (50 mL) and stored in PBS (containing 0.01% sodium azide) at 4 °C for the subsequent use. LC−MS/MS Equipment and Parameters. The TTX analysis was carried out on an ACQUITY UPLC system (Waters, Milford, MA,USA) coupled with Quattro Premier XE Micromass triple− quadrupole mass spectrometer (Waters, Manchester, UK). The UPLC separation was performed on an ACQUITY UPLC BEH Amide column (50 mm × 2.1 mm I.D., 1.7 mm particle size) at 40 °C. The injection volume was 10 μL. Acetonitrile (A) and 5 mmol L−1 ammonium acetate in ultrapure water containing 0.1% formic acid (v/v) (B) were used as the mobile phases, and the flow rate was 0.3 mL min−1 throughout the analysis. The solvent gradient was increased linearly from 5−80% B in 1.5 min, kept constant for 1.5 min, and then returned to 5% B after 0.5 min, and held at this level for 1.5 min so that the system could re-equilibrate prior to the next injection. The mass spectrometer was operated in electrospray positive mode with the capillary voltage set at 3.0 kV. The source temperature was set at 110 °C, desolvation temperature at 350 °C, and cone gas flow at 50 L h−1 with desolvation gas flow at 600 L h−1. Data acquisition was in multiple reactions monitoring mode (MRM). The cone voltage and collision energy were optimized for TTX by standard infusion. The precursor/product ions monitored were 320.0 > 302.0 (cone voltage 45 V, collision energy 25 eV) and 320.0 > 161.8 (cone voltage 45 V, collision energy 35 eV). The precursor/product ion m/z 320.0 > 302.0 was used for quantification in MRM mode. Calculations of Results. Data acquisition was acquired and processed using MassLynx 4.1 and QuanLynx software. The concentrations of TTX in samples (ng g−1) were calculated directly from the area responses using a linear six-point calibration produced from 0.3−20 ng mL−1 TTX standard solutions. If the concentration of analyte exceeds the linear range, the sample solution should be diluted with mobile phase appropriately, then injected into the UPLC−MS/ MS system.

with advantageous, low-cost ultrahigh performance LC−MS/ MS (UPLC−MS/MS). This method combined the advantages of IAC specificity with the speed and sensitivity of UPLC−MS/ MS instrumentation. Further, the optimized method was applied to the detection of TTX in real samples.

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MATERIALS AND METHODS

Chemicals and Materials. TTX was purchased from Tocris Cookson (Bristol, UK) (purity ≥99.6%). HPLC grade acetonitrile, methanol, formic acid, glacial acetic acid, and ammonium acetate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was produced by a Millipore Milli-Q system (Millipore, Bedford, MA, USA). All other chemicals and solvents were of analytical grade and were obtained from Shanghai Chemical Reagent Co. (Shanghai, China). Standard Solutions and Buffers. TTX standard stock solution (100 μg mL−1) was prepared by dissolving TTX (5 mg) in ultrapure water (50 mL) containing formic acid/acetonitrile (0.1%, 5:5, v/v). The working standard solution of TTX was prepared by appropriate dilution of the stock solution using ultrapure water containing 0.1% formic acid/acetonitrile (5:5, v/v) and stored at 4 °C. Phosphate buffered saline (PBS, pH 7.4) was prepared by dissolving KH2PO4 · 2H2O (1.09 g), Na2HPO4 · 12H2O (6.45 g), and NaCl (4.25 g) in ultrapure water (500 mL). Preparation of Immunoaffinity Column. Cynogen bromide (CNBr)-activated Sepharose 4B powder (0.5 g) was added to hydrochloric acid (HCl, 1 M, 100 mL) and poured into a sintered glass funnel (40−60 mm). The gel was washed with the coupling buffer (0.1 mol L−1 NaHCO3, 0.5 mol L−1 NaCl, pH 8.3, 100 mL). Subsequently, the gel (1.5 mL) was mixed with TTX monoclonal antibody (mAb, 10 mg mL−1, 1.5 mL); the mixture was transferred into a stoppered flask and incubated on a shaker (120 rpm) at room temperature for 2 h. The resulting solution was collected to calculate the coupling efficiency and determine the antibody amount by Bradford protein assay method. The mixture was then washed with coupling buffer (30 mL) to remove the uncombined MAbs. The unreacted active groups on the sorbents were capped with the blocking buffer (0.1 mol L−1 Tris-HCl, pH 8.0) for 2 h at room temperature following which the column was washed with PBS (100 mL, 0.01 mol L−1, pH 7.3) and stored in PBS (0.01 mol L−1, pH 7.3) containing ethylmercurithiosalicylic acid (0.05%, w/v) at 2−8 °C. Determination of Column Capacity. A relatively large amount (6000 ng) of TTX was dissolved in PBS (25 mL) containing 20% methanol. The solutions were then transferred into the IAC column (preconditioned with 10 mL of PBS) with the flow rate of 1 mL min−1. The saturated column was washed with water (8 mL). Finally, methanol (4 mL) containing 0.1% acetic acid was used to elute the analytes. The eluate was evaporated to dryness under a stream of nitrogen at 60 °C. The so-obtained residue was redissolved in methanol (1 mL). Further, the solution was filtered through a 0.22 μm PTFE filter, and 10 μL of the solution was injected into the UPLC system. Sample Preparation for IAC. Muscle tissue homogenate (2 g, ± 0.01 g) was weighted into a 50 mL polypropylene centrifuge tube. Acetic acid (0.1%) in methanol (10 mL) was added to the sample and vortexed for 2 min and then ultrasonicated for 15 min. After centrifugation at 6000 × g for 50 min, 5 mL of the supernatant was decanted into a clean tube. The supernatant was diluted with PBS (20 mL, pH 7.4), and the pH was adjusted to 7−8 using 1 M NaOH solution. The IAC column was preconditioned with PBS (10 mL) prior to the analyte loading. Subsequently, the analyte solution was loaded onto the IAC at a flow rate of about 9 mL/min by gravity. The analyte was washed with water (8 mL) and eluted with 1% acetic acid in methanol (4 mL). The elute was completely dried under a stream of nitrogen at 60 °C. The residue was redissolved in LC mobile phase (1 mL, 95% A mix with 5% B) and filtered through a 0.22 μm PTFE filter into an autosampler vial for analysis. The columns, after elution, were

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RESULTS AND DISCUSSION Preparation of IAC Column. The antibody plays an important role in the potential use of the immunosorbent whether the IAC is used for a single analyte or for classselective purposes. In this study, TTX−BSA (bovine serum albumin) conjugate was synthesized, and mAb against TTX was successfully produced using the hybridoma method.30 CNBractivated Sepharose 4B was employed as a coupling matrix because it is water insoluble; however, it is hydrophilic and could be easily activated. To obtain the best binding capacity, several amounts of antibodies (3−15 mg mL−1 gel) were separately conjugated to Sepharose 4B, and the corresponding capacities were measured. Coupling efficiency of different amount of mAb combined with CNBr-activated Sepharose 4B (1 mL) are shown in Figure 2. The result showed that 6 mg of the mAb saturated in 1 mL gel exhibited a good coupling efficiency of about 97.4%. An excessive amount of antibody (>6 mg) might introduce steric hindrance to antigen/antibody recognition and thus lead to the reduction of column capacity. Optimization of IAC Conditions. Appropriate elution solvents are important not only for the efficient uncoupling of the captured analyte off the IAC, but also in preserving the column reusability.31 In this study, loading, washing, and elution conditions were developed and optimized. It is universally acknowledged that the bond between the antigen/ hapten and the antibody is due to electrostatic forces, Vander Waals forces, hydrogen bonds, and hydrophobic interactions.32 To elute the antigen from the antibody−antigen complex in the IAC, the column environment was required to be changed. In this study, pure methanol and acetonitrile were tested to elute the analyte; however, the results were not satisfying with the recoveries of only 65.6 and 75.2%, respectively. Moreover, a 3130

DOI: 10.1021/acs.jafc.5b00045 J. Agric. Food Chem. 2015, 63, 3129−3134

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Figure 2. Coupling efficiency of different amount of mAb combined with CNBr-activated Sepharose 4B (1 mL).

Figure 3. Capacity and recovery variation curves of the IAC column during ten cycles.

large amount of solvent was required for elution, and the process was time-consuming. According to the literature,29 the acidic solvents could enhance the strength of the eluent solution in IAC elution procedure. Therefore, acidic methanol and acetonitrile were tested to elute the analyte, with the volumes for elution measured to be about 1−5 mL. The recoveries corresponding to different elution conditions are listed in Table 1. The result shows that 5 mL of acidic methanol

increasing cycles of usage, in particular, during the fifth and the sixth runs. In China, the official test method for TTX in aquatic products is HPLC method with a measure range 50−500 ng g−1; and a HPLC−MS/MS confirmation is needed. When the UPLC−MS/MS method described in this paper used for confirming of TTX, the IAC column has the potential to reuse for five times with column capacity above 500 ng mL−1 to meet the HPLC method measure range. Optimization of UPLC−MS/MS Conditions. The optimization of the electrospray ionization (ESI) interface parameters was highly desirable for obtaining the maximum abundance of the molecular ions of the analyte. Acquisition parameters were determined by direct infusion of standard solution (5.0 μg mL−1) into the mass spectrometer at a flow rate of 10 μL min−1. ESI probe temperature was set at the minimum acceptable value (110 °C), and capillary voltage was kept at 3.0 kV. The predominant peak in the primary ESI spectra of TTX corresponded to the [M + H]+ ion at m/z 320.0. Then, dissociation with argon was induced, and different collision energies were tested to find the most abundant product ion. Figure 4 shows the product ion spectra of the [M

Table 1. Recoveries of TTX (400 ng) at Different Elution Conditions recovery in various fractions (%)a

elution fraction

a

fraction 1 (1 mL) fraction 2 (1 mL) fraction 3 (1 mL) fraction 4 (1 mL) fraction 5 (1 mL) total recoveryb

methanol acetonitrile 34.8 17.8 10.0 7.0 5.6 75.2

21.5 13.1 10.2 11.4 9.4 65.6

1% acetic acid in methanol

1% acetic acid in acetonitrile

89.8 5.3 2.4 0.6 0.3 98.4

68.7 7.5 5.4 2.7 2.3 86.6

a

New IAC column was preconditioned with PBS (10 mL), the analyte solution containing 400 ng of TTX was loaded, then the column washed with water (8 mL) and eluted with each elution summarized in the table. bTotal recovery is the sum of fraction 1 to fraction 5.

leads to the best recovery of 98.4% in less time, which thus indicates that organic solvents with low pH could disrupt these bonds and efficiently elute the antigen from the IAC column. Therefore, 5 mL of 1% acetic acid in methanol was selected as the optimized elution condition. Column Capacity and Reusability. In this study, the capacity was evaluated and presented as nanograms of analyte relative to IAC bed volume (ng mL−1). The column capacity of new IAC column was found to be 1106 ng mL−1, which was sufficient for TTX determination and suitable for marine organisms. The reusability of IAC depended on the concentration and activity of the immobilized antibodies and chemical stability of the support. The reusability of IAC was evaluated in 10 days at 1 day intervals. Each time after using, the IAC was stored in PBS at 4 °C. The column capacity curve is shown in Figure 3. The column capacity decreases with

Figure 4. MS/MS fragmentation spectra of TTX. 3131

DOI: 10.1021/acs.jafc.5b00045 J. Agric. Food Chem. 2015, 63, 3129−3134

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Journal of Agricultural and Food Chemistry + H]+ ion of TTX. The major fragment ions at m/z 302.0, 161.8, 284.1, 256.1, and 178.3 are observed. The daughter ion m/z 302.0 (CE 25 eV), with the highest relative abundance, was selected for quantification in MRM mode. The fragmentation pathways of TTX have been studied and reported in the literature.8 On the basis of the product ion spectra of TTX (Figure 4), the ions at m/z 302.0, 284.1, and 256.1 are assigned to [M+H−H2O]+, [M+H−2H2O]+, and [M +H−2H2O−CO]+, respectively. Notably, the ion at m/z 256.1 fragmentation pathway mentioned previously was different from that reported by Cho et al.23 The other ions at m/z 178.3 and m/z 161.8 were derived probably from [2-aminodihydroquinazoline]+ and [2-aminodihydroxyquinazoline]+, respectively, with sequential elimination of water molecules. Chromatographic conditions were optimized to obtain maximum sensitivity and best peak shape. The Acquity UPLC BEH C18, Acquity UPLC BEH HILIC, and Acquity UPLC BEH Amide columns were tested for the separation of TTX. Lack of retention was observed with reverse-phase chromatography on BEH C18 column. As expected, TTX was not retained on this stationary phase because of its high polarity. Although TTX was retained in BEH HILIC column and the column provided several advantages24 in chromatographic separation, asymmetric peak was observed in this study. UPLC BEH Amide column enabled the use of a wide range of pH values of the mobile phase, and the presence of amide group also favored both partitioning and hydrogen bonding separation mechanism, which facilitated the separation of high polarity analyte. The results revealed that BEH Amide column provided the best resolution and peak shape for the studied analyte in the shortest time of 5 min. Consequently, following the optimization of the gradient profile, the Acquity UPLC BEH Amide column was selected as the proper column for chromatographic separation. Typical LC−MS/MS chromatograms of blank sample, standard solution, spiked sample, and naturally contaminated TTX sample are shown in Figure 5. Method Validation. The proposed method was validated by assessment of its specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), recovery, and intra- and inter-day precision. The specificity of the method was determined by comparing the chromatograms of blank sample with standard-spiked sample. The TTX was detected without apparent interference from endogenous components of all marine organisms. Good linearity was obtained at concentrations of standard solutions ranging from 0.3−20 ng mL−1 for calibrating TTX in marine organisms, as demonstrated by the high correlation coefficient (r2) value above 0.9984. The LOD and LOQ for TTX in marine organisms, which were defined as signal/noise ratios of 3:1 and 10:1, respectively, were determined to be 0.1 and 0.3 ng g−1. To date, there are few methods available for the detection of TTX with LOD from 0.32−100 ng g−1.21−23 However, in this study, the method was found to be more sensitive. Precision and accuracy were assessed from replicated experiments (n = 6) using new IAC columns at three different concentrations, namely, 0.3, 1.5, and 5 ng mL−1. The values corresponding to the results for intraand inter-day precision and accuracy are listed in Table 2. The recoveries of TTX from fortified samples ranged from 86.5− 103.6% with intraday RSDs of 3.40−7.22% and interday RSDs of 4.74−9.88%. Application to Real Samples. One-hundred marine organisms samples (no. 1−100) including puffer fish, shrimps, crabs, clam, and horseshoe crab collected from Zhoushan local

Figure 5. MRM chromatogram of TTX: (A) blank puffer fish sample, (B) 1.0 ng mL−1 of standard solution, (C) spiked puffer fish sample at concentration of 1.0 ng g−1, and (D) naturally contaminated TTX puffer fish sample (24.8 ng g−1).

market were analyzed for the presence of TTX. All the samples were processed according to the IAC method described above, and the results are listed in Table 3. Nine samples were found to contain TTX at concentrations ranging from 2.22−590 ng g−1. The result shows that some puffer fish (Lagocephalus gloveri) and horseshoe crab (Carcinoscorpins rotundicauda) contain a certain amount of TTX; however, it is virtually absent in horseshoe crab (Tachpleus tridentatus). These results were consistent with the previously published study.33 One of the 20 manila clams (Ruditapes philippinarum) was found to contain TTX with a low value of 2.22 ng g−1. Although several studies22 have reported that bivalves such as Paphies australis were found to contain TTX, TTX positive Ruditapes 3132

DOI: 10.1021/acs.jafc.5b00045 J. Agric. Food Chem. 2015, 63, 3129−3134

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(3) Tsuda, K.; Ikuma, S.; Kawamura, M.; Tachikawa, R.; Sakai, K. Tetrodotoxin. VII. On the Structure of Tetrodotoxin and Its Derivatives. Chem. Pharm. Bull. (Tokyo) 1964, 12, 1357−1374. (4) Noguchi, T.; Jeon, J. K.; Arakawa, O.; Sugita, H.; Deguchi, Y.; Shida, Y.; Hashimoto, K. Occurrence of Tetrodotoxin and Anhydrotetrodotoxin in Vibrio sp. Isolated from the Intestines of a Xanthid Crab, Atergatis floridus. J. Biochem. 1986, 99, 311−314. (5) Wood, S. A.; Casas, M.; Taylor, D. I.; McNabb, P.; Salvitti, L.; Ogilvie, S.; Cary, S. C. Depuration of Tetrodotoxin and Changes in Bacterial Communities in Pleurobranchea maculata Adults and Egg Masses Maintained in Captivity. J. Chem. Ecol. 2012, 38, 1342−1350. (6) McNabb, P.; Selwood, A. I.; Munday, R.; Wood, S. A.; Taylor, D. I.; Mackenzie, L. A.; van Ginkel, R.; Rhodes, L. L.; Cornelisen, C.; Heasman, K.; Holland, P. T.; King, C. Detection of Tetrodotoxin from the Grey Side-Gilled Sea Slug, Pleurobranchea maculata, and Associated Dog Neurotoxicosis on Beaches Adjacent to the Hauraki Gulf, Auckland, New Zealand. Toxicon 2010, 56, 466−473. (7) Lin, S. J.; Hwang, D. F. Possible Source of Tetrodotoxin in the Starfish Astropecten scoparius. Toxicon 2001, 39, 573−579. (8) Bane, V.; Lehane, M.; Dikshit, M.; O’Riordan, A.; Furey, A. Tetrodotoxin: Chemistry, Toxicity, Source, Distribution, and Detection. Toxins (Basel) 2014, 6, 693−755. (9) Williams, B. L.; Hanifin, C. T.; Brodie, E. D., Jr.; Caldwell, R. L. Ontogeny of Tetrodotoxin Levels in Blue-Ringed Octopuses: Maternal Investment and Apparent Independent Production in Offspring of Hapalochlaena lunulata. J. Chem. Ecol. 2011, 37, 10−17. (10) Pratheepa, V.; Vasconcelos, V. Microbial Diversity Associated with Tetrodotoxin Production in Marine Organisms. Environ. Toxicol. Pharmacol. 2013, 36, 1046−1054. (11) Nzoughet, J. K.; Campbell, K.; Barnes, P.; Cooper, K. M.; Chevallier, O. P.; Elliott, C. T. Comparison of Sample Preparation Methods, Validation of a UPLC−MS/MS Procedure for the Quantification of Tetrodotoxin Present in Marine Gastropods and Analysis of Pufferfish. Food Chem. 2013, 136, 1584−1589. (12) Yang, C. C.; Han, K. C.; Lin, T. J.; Tsai, W. J.; Deng, J. F. An Outbreak of Tetrodotoxin Poisoning Following Gastropod Mollusc Consumption. Hum. Exp. Toxicol. 1995, 14, 446−450. (13) Botana, L. M. Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection, 2nd ed.; CRC Press: Boca Raton, FL, 2008; Vol. xvii, p 941. (14) Katikou, P.; Georgantelis, D.; Sinouris, N.; Petsi, A.; Fotaras, T. First Report on Toxicity Assessment of the Lessepsian Migrant Pufferfish Lagocephalus sceleratus (Gmelin, 1789) from European Waters (Aegean Sea, Greece). Toxicon 2009, 54, 50−55. (15) Wu, Z.; Yang, Y.; Xie, L.; Xia, G.; Hu, J.; Wang, S.; Zhang, R. Toxicity and Distribution of Tetrodotoxin-Producing Bacteria in Puffer Fish Fugu rubripes Collected from the Bohai Sea of China. Toxicon 2005, 46, 471−476. (16) Hwang, D. F.; Cheng, C. A.; Tsai, H. T.; Shih, D.; Ko, H. C.; Yang, R. Z.; Jeng, S. S. Identification of Tetrodotoxin and Paralytic Shellfish Toxins in Marine Gastropods Implicated in Food Poisoning. Fish. Sci. 1995, 61, 675−679. (17) Tao, J.; Wei, W. J.; Nan, L.; Lei, L. H.; Hui, H. C.; Fen, G. X.; Jun, L. Y.; Jing, Z.; Rong, J. Development of Competitive Indirect ELISA for the Detection of Tetrodotoxin and a Survey of the Distribution of Tetrodotoxin in the Tissues of Wild Puffer Fish in the Waters of South-East China. Food Addit. Contam., Part A 2010, 27, 1589−1597. (18) Zhou, Y.; Li, Y.; Lu, S.; Ren, H.; Li, Z.; Zhang, Y.; Pan, F.; Liu, W.; Zhang, J.; Liu, Z. Gold Nanoparticle Probe-Based Immunoassay as a New Tool for Tetrodotoxin Detection in Puffer Fish Tissues. Sens. Actuators, B 2010, 146, 368−372. (19) O’Leary, M. A.; Schneider, J. J.; Isbister, G. K. Use of High Performance Liquid Chromatography To Measure Tetrodotoxin in Serum and Urine of Poisoned Patients. Toxicon 2004, 44, 549−553. (20) Chen, X. W.; Liu, H. X.; Jin, Y. B.; Li, S. F.; Bi, X.; Chung, S.; Zhang, S. S.; Jiang, Y. Y. Separation, Identification, and Quantification of Tetrodotoxin and Its Analogs by LC−MS without Calibration of Individual Analogs. Toxicon 2011, 57, 938−943.

Table 2. Recovery, Intra- and Inter-Day Precision of TTX in Marine Organismsa species shrimps (Penaeus orientalis) puffer fish (Lagocephalus gloveri) horseshoe crab (Tachpleus tridentatus)

added (ng g−1)

average recovery (%, n = 6)

intraday RSD (%, n = 6)

interday RSD (%, n = 6)

0.3 1.5 5.0 0.3 1.5 5.0 0.3 1.5 5.0

101.8 96.7 103.6 86.5 89.1 94.7 92.0 96.9 94.4

7.22 5.42 3.94 6.31 4.82 3.40 5.36 5.95 4.54

5.33 4.74 6.07 6.74 9.88 6.03 4.86 5.15 6.63

a

TTX was determined according to section Sample Preparation for IAC using new IAC columns.

Table 3. Presence of TTX in Marine Organisms (Analysis of 100 Samples) species

sample number

puffer fish (Lagocephalus gloveri)

1−20

shrimps (Solenocera melantho, Penaeus orientalis) crabs (Portunus trituberculatus) manila clams (Ruditapes philippinarum) horseshoe crab (Carcinoscorpins rotundicauda)

21−40 41−60 61−80

horseshoe crab (Tachpleus tridentatus)

90−100

a

81−90

positive sample code

concentration of TTX (ng g−1)

3 4 14 17 18

67 83 88 90

128 103 590 146 61.6 NDa ND 2.22 162 101 24.8 ND

ND indicates “not detected”.

philippinarum found in this study has never been mentioned before.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-580-2299898. Fax: +86-580-2299882. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No.81470148) and National Key Technology R&D Program during the “12th Five-Year Plan” (2012BAD29B06). The authors would like to thank Jian Wang and Lili Lun for their valuable technical assistance.

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REFERENCES

(1) Chowdhury, F. R.; Ahasan, H. A.; Al Mamun, A.; Rashid, A. K.; Al Mahboob, A. Puffer fish (Tetrodotoxin) Poisoning: An Analysis and Outcome of Six Cases. Trop. Doct. 2007, 37, 263−264. (2) Chau, R.; Kalaitzis, J. A.; Neilan, B. A. On the Origins and Biosynthesis of Tetrodotoxin. Aquat. Toxicol. 2011, 104, 61−72. 3133

DOI: 10.1021/acs.jafc.5b00045 J. Agric. Food Chem. 2015, 63, 3129−3134

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DOI: 10.1021/acs.jafc.5b00045 J. Agric. Food Chem. 2015, 63, 3129−3134