Table 1.
Recently developed methods for the analysis of TTX and its analogues, described following the approach used in the EFSA opinion [16].
| Method Type | Method Principle | Matrix | LOD 1 | LOQ 2 | Method Remarks | Reference |
|---|---|---|---|---|---|---|
| In vitro bioassays | ||||||
| Cell-based assay | Sensitivity to neuroblastoma neuro-2a (N2a) cell line | Shellfish | 20 µg TTX/kg | n.a. 3 | Potential false positive results if PSP toxins are present in the sample, even at low levels | [29] |
| Immunoassay | Competitive inhibition enzymatic immunoassay (mELISA) | Mussels | 30 μg TTX/kg | n.a. | No cross-reactivity of the antibody with co-occurring PSP toxins | [30] |
| Oysters | 20 μg TTX/kg | |||||
| Electrochemical magnetic bead-based immunosensing | Pufferfish tissue | 1.2 ng/mL | n.a. | Cross-reactivity with TTX analogues related to concentration and not to toxicity | [31] | |
| Surface Plasmon Resonance (SPR) | Nanoarray planar waveguide biosensor | Pufferfish tissue | 0.4 mg/kg | n.a. | Cross-reactivity with some of the known TTX analogues | [32] |
| Spectrophotometric methods | ||||||
| Spectrofluorimetric | Up-conversion fluorescence using a microfluidic aptasensor | TTX-spiked human biological fluids (gastric juice, human serum and urine) | 0.06 ng/mL | 1 ng/mL | High detection recoveries (96.0–104.2%), indicating limited interference by other toxins, biomolecules and anions | [33] |
| Chemical-analytical methods | ||||||
| Liquid Chromatography Mass Spectrometry (LC-MS) - Single analyte group | Hydrophilic Interaction LC - tandem MS (HILIC-MS/MS) | Shellfish (mussels, Pacific oysters) | ~0.25 μg/kg | ~ 0.8 μg/kg | Matrix-matched calibration. Inclusion of 5 TTX analogues (4-epiTTX; 5,6,11-trideoxyTTX; 11-norTTX-6-ol; 5-deoxyTTX; and 4,9-anhydroTTX). Recovery in mussels: 70%–77% Recovery in oysters: 76%–84%. Expanded uncertainty (k = 2) for TTX: mussels 36% and oysters 51%. LOD, LOQ, and within-laboratory reproducibility satisfactory also for 11-norTTX-6-ol, 5-deoxyTTX, and 4,9-anhydroTTX. Combined acquisition of PSP toxins possible |
[34] |
| HILIC-MS/MS | Shellfish (mussels, oysters) | Mussels: 0.31 ± 0.12 μg/kg Oysters: 0.36 ± 0.11 μg/kg |
Mussels: 1.03 ± 0.34 μg/kg Oysters: 0.86 ± 0.13 μg/kg |
Matrix-matched calibration. Inclusion of 6 TTX analogues (4-epiTTX; 5,6,11-trideoxyTTX; 11-norTTX-6-ol; 5-deoxyTTX; 11-deoxyTTX; and 4,9-anhydroTTX). Recovery in mussels: 91.6%–109.5% Recovery in oysters: 69.3%–79.6% |
[35] | |
| Ultra Performance LC-MS/MS (UPLC-MS/MS) | Shellfish (mussels, oysters) | 3 μg/kg | 20 μg/kg | Matrix-matched calibration. Inclusion of 6 TTX analogues (4-epiTTX; 5,6,11-trideoxyTTX; 11-norTTX-6-ol; 5-deoxyTTX; 11-deoxyTTX; and 4,9-anhydroTTX). | [29] | |
| LC-MS - Multi-analyte toxin group | UPLC-MS/MS | Mussels | 0.47 μg/Kg | 1.56 μg/Kg | Simultaneous detection and quantification of all EU-regulated marine toxins (both lipophilic and hydrophilic) and TTX (parent and 4,9-anhydroTTX). | [36] |
| LC-MS - Screening for multiple toxin groups (marine and freshwater) | LC-MS coupled to Fourier transform mass spectrometer (FTMS) - High Resolution LC-MS/MS (LC-HRMS/MS) | Shellfish (mussels, oysters) | n.r. 4 | n.r. | Broad screening including approx. 800 marine and freshwater toxins, parent TTX and 30 TTX analogues | [29] |
| LC-MS - Screening and quantitation for TTX and known analogues | LC-HRMS/MS | Puffer fish (Lagocephalus sceleratus) shellfish (Charonia lampas) | 0.041 mg/kg in TTX-spiked mackerel extracts | 0.136 mg/kg in TTX-spiked mackerel extracts | Screening for parent TTX and 30 known TTX analogues. Mean recovery (in TTX-spiked mackerel extracts) = 61.17% (57.33–65.00%) | [37] |
| LC-HRMS/MS | Puffer fish (L. sceleratus) | 0.05 mg/kg | 0.1 mg/kg | Multi-toxin analysis including TTX all analogues previously described for the species and other analogues described in the literature | [38] | |
| Capillary electrophoresis - mass spectrometry | Capillary electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) | Puffer fish, mussels, sea slug (Pleurobranchaea maculata) | Mussels: 0.0052 mg/kg | n.r. | Multiclass analysis of polar marine toxins (paralytic shellfish toxins, tetrodotoxins and domoic acid). Inclusion of parent TTX, 4-epiTTX and 4,9-anhydroTTX (separation evaluated in standard solution) | [39] |
1 LOD = Limit of detection; 2 LOQ = Limit of quantification; 3 n.a. = not applicable; 4 n.r. = not reported.