1 INTRODUCTION

Tetrodotoxin (TTX) is a kind of low-molecularweight non-protein neurotoxin. It is one of the most potent neurotoxins found in nature, and is found in pufferfish and various marine biota, including mollusks, newts, octopuses, flatworms, and gastropods (Chau et al., 2011; Salvitti et al., 2015), and has also 1 INTRODUCTION Tetrodotoxin (TTX) is a kind of low-molecularweight non-protein neurotoxin. It is one of the most potent neurotoxins found in nature, and is found in pufferfish and various marine biota, including mollusks, newts, octopuses, flatworms, and gastropods (Chau et al., 2011; Salvitti et al., 2015), and has also * Supported by the National Natural Science Foundation of China (No. 41106109) and the China National Food Safety Standards Development Project (No. ZHENGHE-2015-356) ** Corresponding author: tanzj@ysfri.ac.cn884 CHIN. J. OCEANOL. LIMNOL., 35(4), 2017 Vol. 35 recently been found in the bivalve shellfish, such as Paphies australis clams, pacific oysters and mussels (McNabb et al., 2014; Turner et al., 2015a; Vlamis et al., 2015). TTX is a powerful and specific Na⁺ channel blocker that prevents nerve conduction, causing nerve palsy, and may even result in death (Lombet et al., 1982). Cases of TTX poisoning are relatively common in Asia, due to its high toxicity (human median lethal dose 8.7 ng/g), unpredictability, and lack of antidote (Chen et al., 2011). Most of these cases of poisoning were caused by improper preparation of pufferfish and pufferfish-based products (Chowdhury et al., 2007; Chua and Chew, 2009).

Most of the tissues of pufferfish, including the skin, liver, and testes, are considered to be delicacies in parts of Asia, and there is a potential risk of TTX intoxication for consumers of these products. Previous studies have shown that TTX may be present in many pufferfish tissues, especially the ovaries and liver, followed by the intestine and skin (Itoi et al., 2012; Okita et al., 2013). Some cases of poisoning have been reported due to the ingestion of pufferfish skin (Laobhripatr et al., 1990; Mahmud et al., 2000) and liver (Bentur et al., 2008). Even pufferfish muscles contain high levels of TTX in some species, such as Lagocephalus lunaris and Chelonodon patoca (Noguchi and Arakawa, 2008). Further, pufferfish is sometimes adulterated into other highly valued fish such as Plecoglossus altivelis, Epinephelus malabaricus, and Trachurus japonicus, during the manufacturing of dry-dressed fish fillet (Hsieh et al., 2010). It is very difficult for consumers to distinguish between the toxic pufferfish and dry-dressed fish fillet products, which brings huge potential health risks to consumers, and cases of TTX-associated food poisoning resulting from pufferfish-based products have been reported (Chen et al., 2002). Thus, a TTX monitoring program is necessary to protect consumers from food poisoning by accidental consumption of improperly prepared pufferfish tissues and pufferfishbased products. Specifically, a simple, reliable, and sensitive method for the determination of TTX in pufferfish samples with a complicated matrix is needed.

Currently, a few methods can determine TTX in pufferfish samples, including GC-MS (Man et al., 2010), HPLC with post-column derivatization using fluorescence detection (O'Leary et al., 2004), and the ELISA (Ling et al., 2015). Among these methods, the GC-MS method entails complex sample preparation, while the HPLC method is generally limited in terms of its sensitivity, and requires special equipment for alkaline hydrolysis. The ELISA method has relatively high sensitivity, but is inferior at positive identification. In recent years, LC-MS/MS has been found to be a better choice, with high confidence and efficiency, and has become more available for routine qualitative and quantitative analysis of TTX (Huang et al., 2008; Rodríguez et al., 2012; Nzoughet et al., 2013).

However, the previously reported methods of LCMS/MS for TTX determination have some limitations, including the obvious matrix interferences, ion suppression and the unstable chromatograph retention time caused by the complicated matrix in biological samples (Zhang et al., 2014). Thus, a matrix-matched standard curve needs to be used to correct for recovery against the matrix inhibition effects. However, it is difficult to obtain different matrix blanks as they adversely affect accurate quantification in pufferfish tissues and pufferfish-based products. To accurately detect TTX in biological samples using LC-MS/MS, any impurities need to be removed, and ion suppression needs to be eliminated. On the other hand, LC-MS/MS methods generally rely on mass spectrometer selectivity and specificity through multiple reaction monitoring (MRM) and matching retention times of sample components with the TTX standard for positive confirmation of TTX. The selection of non-specific transitions that are the same as that of an interfering matrix compound or the weak response of the second MRM transition at low intensity can lead to false positive or negative identification (Zhang et al., 2012). When there are obvious matrix interferences or low concentrations of TTX in the complicated matrices of pufferfish tissues and pufferfish-based products, the resulting secondary mass spectrograms may have insufficient characteristic fragments or their relative ion intensity ratio deviations may be larger.

In this study, we tried to combine the advantages of the specificity of immunoaffinity columns (IACs) with those of the identification ability and sensitivity of liquid chromatography/quadrupole-linear ion trap mass spectrometry (LC-QqLIT-MS) to develop a method for the determination of pufferfish samples with complicated matrices. By using the IACs, the matrix effect was eliminated, and the stability of the retention time improved substantially. In terms of the high-lipid pufferfish tissues, freezing lipid precipitation (FLP) easily removed the large amounts of lipids from the extracts, and was effectively combined with the purification by the IACs. For an added level of TTX confirmation in the complicated matrices of pufferfish samples, the informationdependent acquisition (IDA) procedure and a single enhanced product ion (EPI) mass spectra library were developed using LC-QqLIT-MS. Further, we used this method to determine TTX in pufferfish tissues (skin, muscle, liver, gonads, and intestine) and pufferfish-based products, and the results show that our method has the advantages of good precision, simple operation, and high specificity.

2 MATERIAL AND METHOD 2.1 Chemicals and materials

TTX standards (purity, ≥99%) were purchased from Sigma Corp. (St. Louis, MO, USA). The IACs were obtained from Meizheng Biological Technology Corporation (Jiangsu, China), and the columns were connected in tandem using Supelco SPE Tube Adaptors (57020-U; Sigma-Aldrich Ltd., Gillingham, UK). Methanol, acetonitrile (ACN), and formic acid were of HPLC grade and were obtained from Merck (Darmstadt, Germany). Water was prepared using the Milli-Q water purification system (EMD Millipore, Billerica, MA, USA). Phosphate-buffered saline (PBS) was prepared at a concentration of 0.5 mol/L and pH of 7.5. All other reagents used in the experiment were of analytical grade and were used without further purification.

2.2 Test samples

The effectiveness of the method was evaluated by testing 206 samples covering different pufferfish matrices. Eighteen tissues of skin, muscle, and liver were obtained from wild pufferfish (Takifugu rubripes and T. xanthopterus) caught in the Yellow Sea of China in May 2015. Additionally, 160 tissues of muscle, liver, skin, gonads (ovaries or testes), and intestine from cultivated fresh pufferfish (T. rubripes, T. xanthopterus, T. flavidus and T. obscurus) were collected from farming areas in Shandong, Liaoning, Hebei, and Jiangsu Provinces of China in June 2015. Lastly, 28 commercial dry-dressed fish fillets and roasted pufferfish bars were purchased from markets in Qingdao, China, in June 2015. All samples (20– 100 g) were homogenized in a T25 Ultra Turrax mixer at 12 000 r/min (IKA Works, Wilmington, NC, USA) and stored at -22℃ until analysis.

2.3 Sample preparation and delipidation

Samples (2.0 g) were weighed out in centrifuge tubes and vortexed with 8 mL of 1% acetic acid in methanol (v/v) for 2 min, and then extracted using ultrasonic extraction for 15 min at 50℃. The samples were centrifuged at 6 000×g for 5 min, and the supernatants were transferred into 10 mL colorimetric tubes, which were then filled up to 10 mL with 1% acetic acid in methanol (v/v). For the high-lipid tissues, FLP was used to remove the lipids extracted in the next step, that is, the 10 mL extract solution was transferred to a new centrifuge tube and stored at -22℃ for 30 min to freeze the lipids, and then immediately centrifuged at 8 000×g at 4℃ for 2 min. A 5 mL aliquot of the supernatant was then diluted with 20 mL PBS. The pH values of the final extracts were adjusted to 7–8 using 1 mol/L NaOH.

2.4 IAC purification

The diluted extracts were loaded on the IACs (connected in tandem as described above) at a flow rate of one drop/sec, and the columns were washed with 10 mL water. Finally, the TTX was eluted twice from the columns with 2 mL of 2% acetic acid in methanol (v/v). The extracts were then evaporated to dryness under nitrogen at 45℃; the residue was dissolved in 1 mL ACN: 0.1% formic acid in water (50:50, v/v), and then filtered through a 0.2-μm filter for LC-QqLIT-MS analysis.

2.5 LC-QqLIT-MS analysis

Liquid chromatography was conducted on an ultrafast liquid chromatography system (Prominence LC-20ADXR; Shimadzu Corp., Kyoto, Japan), and separation was performed on a TSK gel Amide-80 column (150 mm×2.0 mm, 5 μm; Tosoh Bioscience, Tokyo, Japan) maintained at 40℃ with 10 μL injection. The mobile phase consisted of 0.1% formic acid (v/v) containing 5 mmol/L ammonium acetate in water (solvent A) and ACN (solvent B), and the flow rate was 0.30 mL/min. The initial proportion of solvent A was 10%, which was kept constant for 2 min and ramped to 90% over 1 min, held for 3 min, and reduced to 10% over 0.1 min. The total run time for the analysis was 8 min for one run.

A hybrid triple quadrupole-linear ion trap mass spectrometer (5500 QTRAP MS system; AB Sciex, Framingham, MA, USA) equipped with a turbo V source and an ESI probe was used for MS/MS detection. The turbo ion spray source settings were as follows: ion-spray voltage (positive polarity), 5 500 V; curtain gas, 20 psi; GS1 and GS2, 50 psi; ion source temperature, 500℃; collisionactivated dissociation (CAD), high; optimal declustering potential (DP), 70 V; collision cell exit potentials (CXP), 10 V; and entrance potential (EP), 10 V. Nitrogen served as the nebulizer gas and collision gas in both modes. The precursor/product ions monitored were 320.2 > 302.2 (collision energy, 25 eV) and 320.2 > 162.0 (collision energy, 39 eV). Data were acquired and processed with Analyst software version 1.5.2 (AB Sciex).

2.5.1 MRM parameters

For quantitation, the MS system was operated in MRM mode. A scheduled MRM mode with an MRM detection window of 60 s and a target scan time of 1 s was used. Optimization of the MS parameters, DP and EP for the precursor ions, and collision energy (CE) and CXP for the product ions was conducted via flow injection analysis of 1 mg/L of TTX.

2.5.2 Information-dependent acquisition conditions

For further confirmation of the presence of the toxin, the MS system was used in an IDA experiment. The scan intensity threshold was set to 1 000 cps. The dependent EPI scans were acquired using four standardized CEs of 20, 35, 50, and 35 eV with a collision energy spread (CES) of 15 eV (35±15 eV) to ensure a characteristic MS/MS pattern that was independent of the efficiency of the TTX fragmentation. MS/MS spectra were searched against the EPI library to compare the positive sample data with a standard spectrum.

2.6 Method validation

The TTX standard solution was prepared in ACN: 0.1% formic acid in water (50:50, v/v) for the validation studies. A dilution series of TTX with a concentration range of 1 to 1 000 μg/L was used for establishing linearity. Six replicates of each sample with three spiked levels (1, 10, and 100 μg/kg) were treated following the methods described in Sections 2.3 and 2.4 to validate the accuracy and precision of the method. The LOD and LOQ were calculated using standard solutions prepared in purified extracts. The LOD was determined as the concentration that yielded a signal-to-noise (S/N) ratio (S/N) ≥ 3, and the LOQ was determined as the concentration that yielded an S/N ratio (S/N) ≥ 10.

3 RESULT AND DISCUSSION 3.1 Optimization of chromatographic conditions

The strong polar characteristic of TTX makes it extremely difficult for it to be retained in a C18 reversed-phase column. Therefore, we used a hydrophilic interaction liquid chromatography (HILIC) column, which offers superior retention for strongly polar compounds. Moreover, considering that the molecular structure of TTX contains guanidine, and thus, protonates easily under acidic conditions, ACN-0.1% formic acid (v/v) in water was selected as the mobile phase. In this study, the Atlantis HILIC Silica and TSK gel Amide-80 column were tested for the separation of TTX using the same mobile phase. The results revealed that the TSK gel Amide-80 column provided better separation and resolution for TTX analysis than the Atlantis HILIC Silica column, without any shoulders or interference peaks close to the target. The addition of ammonium acetate was also tested since it can improve the ionization efficiency and peak shape of analyte (Xu et al., 2013). Excellent results were obtained using ACN-0.1% formic acid (v/v) in water containing 5 mmol/L ammonium acetate as the mobile phase, which enabled a symmetrical shape and good sensitivity. The ammonium acetate acted as a volatile electrolyte, and improved the response for TTX by two-fold. Consequently, following the optimization of the elution program described in Section 2.5, the TSK gel Amide-80 column was selected for chromatographic separation using ACN-0.1% formic acid (v/v) in water containing 5 mmol/L ammonium acetate as the mobile phase, providing superior chromatographic resolution, higher sensitivity, and effective separation of impurities.

3.2 Extraction

Aqueous acetic acid solution or acetic acid methanol solution are usually chosen as the extraction solvents in TTX determination, since TTX contains a guanidine group that makes it unstable under alkaline conditions (Rodríguez et al., 2012; Wu et al., 2014b). In this experiment, we compared and tested the ability of 0.1%, 0.3%, and 1% aqueous acetic acid and the same series of acetic acid methanol solutions to extract TTX from pufferfish liver samples (Fig. 1).

Interestingly, the results demonstrated opposing tendencies of TTX recovery with increasing concentrations of acetic acid in water and in methanol. The ability of acetic acid in water to extract TTX seemed to be decreased with increasing concentration, since the recovery of TTX decreased from 60% in 0.1% to 50% in 0.3% aqueous acetic acid solution. As for 1% acetic acid in water, the extracts were too turbid and stringy to proceed to the next IAC purification step. Therefore, these results show that acetic acid in water is not suitable for extracting TTX from pufferfish liver samples; the most likely reason is an increase in matrix interference due to protein, lipid, and/or other co-extracts in the crude liver extract, which has a negative effect on the recovery of TTX. Conversely, the acetic acid methanol solution demonstrated perfect extraction, which increased with increasing acetic acid concentration from 0.1% to 1%; 1% acetic acid methanol solution resulted in a mean recovery of 92.6% for TTX at 100 μg/kg. This is likely due to protein precipitation by the methanol, which provides acceptable cleaning extract and decreases ion suppression for the LC-MS/MS analysis. Therefore, 1% acetic acid in methanol was chosen as the final extraction solvent.

3.3 Delipidation

The lipid contents of most pufferfish liver and ovaries are approximately > 40% and 10% of the net mass of the tissues, respectively (Xu et al., 2014). The presence of lipids in the extracts tends to affect the active surface of the stationary phase, degrades the resolving power of the column, and contaminates the mass spectrometric system. Moreover, the high levels of fat in the high-lipid extracts makes SPE using IACs difficult. Thus, the lipids in the extracts must be eliminated or reduced as much as possible in order to extend the column lifetime and to improve detection limits (Pensado et al., 2005).

FLP entails solidifying lipids in an organic solvent between -18℃ and -25℃ (Yoon et al., 2015), and was effectively applied to remove the lipids from the high-lipid samples in this study. During the FLP procedure, lipids in the extracts were frozen at -22℃, and the frozen lipids were then precipitated as a frozen fat lump (Fig. 2).

Finally, the cold extracts were immediately centrifuged at 8 000×g at 4℃ for 2 min to remove the lipids and precipitate as much of the solids as possible. This delipidation method is simple and can greatly reduce the amount of organic solvent required, as well as time and effort. In addition, the removal of lipids from the extracts made it easier to load the samples on the IACs and to carry out the next IAC purification step.

3.4 IAC purification

The qualitation and quantitation of TTX may still be affected by the ion suppression effects caused by the complicated matrix components in pufferfish tissues, even if the samples were extracted well. To achieve good results, many researchers have focused on methods for removing impurities and reducing ion suppression. To our knowledge, SPE (Jen et al., 2008), QuEChERS (Cao et al., 2014), and ultrafiltration (Tsai et al., 2006) are the methods that are most often used for purifying TTX from biological samples. However, these methods have some disadvantages, such as high background interference, ion suppression, and low sensitivity and accuracy. In the present study, we tried using IACs, which contain antibodies specific for TTX, thus allowing selective solation of TTX from biological samples. IACs have been shown to be able to purify matrix components from the urine, plasma, and muscle tissues (Kawatsu et al., 1999; Zhang et al., 2014; Zhang et al., 2015). However, to our knowledge, there have been no reports of the ability of IACs to determine TTX in other tissues with more complex matrices, such as the liver and gonads (ovaries or testes) and pufferfishbased products, which contain much higher concentrations of TTX than the muscle. Although it is well recognized that IAC clean-up produces extracts that are free of any matrix interference (Hoyos Ossa et al., 2015), we conducted an experiment to compare the influence of the matrix interference effect in purified extracts of pufferfish liver using different clean-up methods. Briefly, 1 mL of TTX standard (40 μg/L) was evaporated to dryness under nitrogen at 45℃. Then, the TTX-free pufferfish liver matrix solution was prepared using the same preparation method described in Section 2.3 and different purification methods, namely C18 SPE (Jen et al., 2008), QuEChERS (Cao et al., 2014), and our method described in Section 2.4, and then respectively added to the TTX standards up to 1 mL, with a final theoretical TTX concentration of 20 ng/g for each sample. The TTX concentrations of the above samples were corrected with calibration curves constructed using TTX standards in solvent. The quantitative results are shown in Fig. 3, and the chromatograms and mass spectrums are shown in Fig. 4.

Significant ion suppression was observed in samples purified by nonspecific approaches (C18 SPE and QuEChERS) in this study, and the concentrations of TTX were only 16.1%–38.5% of the theoretical value. However, the concentrations of TTX were almost the same as the theoretical value when the samples were purified by our method. Using our method, the ion suppression for the target analyte caused by co-eluted endogenous matrix components could be eliminated using the optimized experimental conditions of our method, which demonstrates the suitability of using standards in solvent when constructing calibration curves. The S/N ratio in Figure 4 shows that the sensitivity of our method for the detection of TTX was greatly improved compared with the other purification methods.

3.5 TTX confirmation using IDA

The optimized MRM transitions and the EPI scan were run for the same injection to develop a method for determining and confirming the presence of TTX. The IDA methods consist of a single MRM transition as the survey scan and an EPI scan as the threshold dependent scan. If the signal in an MRM transition reaches the defined threshold, an EPI scan is rapidly acquired on the Q1 ion of the MRM transition, and the instrument then returns to screening the MRM transitions included in the method, and the EPI spectra acquired as part of a run can then be searched against the EPI library to confirm the identity of the substance (McCarron et al., 2014). The spectral library search information comprises the fit, reverse fit, and purity fit values. The fit values indicate the similarity between the reference and the unknown spectra, the reverse fit values compares in the opposite manner, indicating the similarity between the unknown and the reference spectra, and the purity fit value is a combination of the two. Purity values > 70 were set as appropriate thresholds for positive identification (Wu et al., 2014a).

3.6 Method validation

The TTX standards were diluted to make a series of working solutions with a concentration gradient, and were determined respectively under the conditions described in Section 2.5. The external TTX standard solution calibration curves showed good linearity over the calibration ranges from 1 to 1 000 μg/L (R²=0.999 6), and the matrix effects of the method was evaluated by comparing the calibration curve for TTX standard solutions with the fitting curve for standard-spiked samples (Fig. 5). The results show that the matrix effects were substantially reduced due to the high specificity of the IACs, and thus, relatively accurate quantitative results were achieved with external standard calibration curves using standards prepared in the mobile phase.

The LOD (S/N≥3) was 0.3 ng/g, and the LOQ (S/ N≥10) was 1 ng/g in both pufferfish tissues and pufferfish-based products. To date, there are few LCMS/MS methods in complex organisms available for the determination of TTX with LOD from 0.7 to 100 ng/g (Rodríguez et al., 2012; Liang et al., 2015; Turner et al., 2015b). This method was found to have a relatively high sensitivity.

The accuracy and precision of our method were determined using TTX-free pufferfish tissue and drydressed fish fillet with three different concentrations of TTX standards added, respectively (Table 1). The recoveries were in the range of 75.8%–107%, with a RSD less than 15%. These results confirm the applicability of our method for extracting TTX from different pufferfish samples with different matrices.

3.7 Application of the method

The optimized method was carefully evaluated by using it to detect TTX in a total of 206 samples representing different pufferfish matrices. According to the results, 48 samples were found to be contaminated with TTX, with concentrations between < LOQ~720 ng/g (Table 2). The results show that the wild pufferfish (T. rubripes and T. xanthopterus) contained a certain amount of TTX; whereas the cultivated pufferfish (T. rubripes, T. flavidus, and T. obscurus) were non-toxic or weakly toxic. In the cultivated pufferfish, the toxicity in the ovaries was higher than that in the other tissues (skin, muscle, intestine, and liver), followed by the liver and skin, successively; the ovaries were weakly toxic, while the testes were not toxic. The TTX finding of these tissues were consistent with the previously published study (Azman et al., 2014; Kosker et al., 2016), but they especially found higher levels of TTX in muscle and skin tissues of wild pufferfish compared to our study. First the numbers of wild pufferfish investigated by this study was too small, and another reason was that the environmental conditions and geographical differences. Further, the four processed dry-dressed fish fillets and fish bars were found to contain TTX at concentrations ranging from 7.41 to 312 ng/g, which shows that these types of processed dry-dressed fish products may be mislabeled or adulterated fish products that could result in potential health risks after consumption. The IDA-EPI experiments were conducted in a single run for positive identification (Fig. 6). All of the positive samples were searched for in an EPI library, and the fit, reverse fit, and purity fit values were > 85%, > 83%, and > 78%, respectively.

4 CONCLUSION

In the present study, IACs and LC-QqLIT-MS were combined to simultaneously identify and quantify TTX in fresh pufferfish and pufferfish-based products. A chromatogram with stable retention time and symmetrical shape was obtained by optimization of the chromatographic conditions. FLP was successfully used to efficiently remove the lipids extracted from the high-lipid samples before IAC purification, and our experiment showed that this method can be used to conveniently and easily load the extracts on the IACs. Further, the purification steps for the IACs effectively removed impurities and reduced ion suppression, which makes it a better method compared with the tedious clean-up procedure required in traditional methods, along with the absence of background interference, high sensitivity, and satisfactory precision. Thus, the matrix-matched calibration and expensive isotope-labeled internal standards required in traditional methods are unnecessary in TTX analysis methods using LC-MS/ MS with IAC clean-up. In addition, the use of LCQqLIT-MS allowed for positive peak confirmation with high reliability and the quantification of trace amounts of TTX. As demonstrated by our application of this method to real samples, the developed method is an effective and highly selective technique for the routine determination and confirmation of TTX in a variety of pufferfish tissues and pufferfish-based products.