Tetrodotoxin detection in body fluids from a puffer fish poisoning incidence

Transcription

1 Tetrodotoxin detection in body fluids from a puffer fish poisoning incidence Maria Rambla-Alegre Sandra Leonardo, Yann Barguil, Mònica Campàs, Jorge Diogène Sant Carles de la Ràpita, 4 th June 2018

2 A poisoning incidence reported by the Territorial Hospital of New Caledonia in August 2014, resulting from the ingestion of an Arothron nigropunctatus puffer fish, is addressed in this work to demonstrate the implication of in this intoxication. The detection of s in clinical samples of the poisoned patients can be essential to confirm intoxication. Mechanism of action: a very potent neurotoxin that acts as a sodium channel blockers 1 F. Rafael Nieto et al. (2012) Mar. Drugs, 10(2), In Japan, a value of 2 mg equiv./kg of edible puffer fish flesh has been used as a criterion to judge the acceptability of puffer fish as food,

3 Clinical case At around 6 pm on the 27 th of August, four men aged 33, 34, 37 and 38 years old, with no known medical history, consumed a boiled fish that the oldest man had captured whilst fishing. The older fisherman ate most of the liver and the gonads in their totality. One of the other men consumed, the flesh in great quantity and, the other man consumed, a small portion of the liver. On the morning of the 28 th of August, the fisherman was found deceased and the two men that presented with neurological signs were hospitalised. Pt #2 Small portion of liver a small part of the flesh Pt #1 Flesh in great quantity Fisherman Most of the liver and gonads in their totality

4 Human body fluid samples collected Date Time Samples 28/08/14 11:25h Urine, serum and heparinized plasma Pt#1 29/08/14 07:35h Heparinized plasma 29/08/14 12:h Heparinized plasma 29/08/14 15:15h Urine and serum Pt#2 28/08/14 17:00h Heparinized plasma 29/08/14 13:00h Urine and serum Fisherman 28/08/14 11:30h Post-mortem plasma (with fluoride)

5 Urine by melisa Colorimetric maleimide-based enzyme-linked immunosorbent assay Urine Pure ½ dilution ¼ dilution recovery (%) 27 ± 3 76 ± ± 2 ¼ dilution of urine in buffer is an effective way to overcome urine matrix effects, avoiding sample pre-treatment Serum and plasma High non-specific adsorption!

6 Sample preparation for LC-MS/MS analysis Solid-phase extraction (SPE) clean-up was used adapting the protocol described by Boundy et al (2015). Graphitized polymer carbon ENVI-carb cartridges were conditioned with 3 ml of ACN/H 2 O/AA (20:80:1, v:v:v), followed by 3 ml of H 2 O/NH 4 OH (1000:1, v:v). 400 µl of sample was loaded onto the conditioned cartridges and washed with 700 µl of deionized H 2 O. the retained s were eluted with 2 ml of ACN/H 2 O/AA (20:80:1, v:v:v). The eluent was diluted by transferring 100 µl to an insert and adding 100 µl of ACN. Sample vials could be stored at -20 C until analysis,

7 Brief intro to the Thermo TSQ HPLC : High Pressure Liquid Chromatography Chromatography separates compounds according to their polarity Mass Spectrometry separates ions according to their mass-to-charge rate ratio (m/z) Mobile phase Pumps Injector MS/MS: Mass Spectrometry in tandem Column (Stationary phase) Ionization source Triple Quadrupole

8 Structure of main s s R 1 R 2 R 3 R 4 MH + (m/z) in pufferfishes 1,2 Hemiacetal type Tetrodotoxin () H OH OH CH 2 OH epi OH H OH CH 2 OH deoxy H OH OH CH nor-6(S)-ol H OH OH H nor-6(R)-ol H OH H OH ,11-dideoxy H OH H CH Lactone type 5-deoxy OH CH 2 OH ,6,11-trideoxy H CH ,9-Anhydride type 4,9-anhydro P. Rodríguez et al. (2012) Food Chemistry 132, P. Katikou et al. (2009), Toxicon, 54(1): -55.

9 Product ion Collision energy > > > > > Optimization of -MS/MS detection Intensity, cps 1.2e6 1.1e6 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 162 : [M+H] m/z e e e e m/z, amu Product Ion Spectrum of from the [M+H] m/z CE = ev)

10 Pt#2 Urine by LC-MS/MS analysis epi / (MRM1) 4,9-anhydro (MRM1) 5,6,11-trideoxy (MRM1) Time (min) 4-epi / (MRM2) 4,9-anhydro (MRM2) 5,6,11-trideoxy (MRM2) NL: 1.23E5 TIC MS NL: 2.34E5 [320, ] NL: 9.73E4 [320, ] NL: 1.65E3 [302, ] NL: 1.33E4 [ ] NL: 7.21E4 [272, ] NL: 5.E3 [272, ]

11 Fisherman Plasma by LC-MS/MS analysis 100 (MRM1) (MRM2) 5,6,11-trideoxy (MRM1) NL: 1.E4 TIC MS NL: 1.00E4 [ ] NL: 1.00E4 [ ] NL: 8.00E4 [ ] 0 5,6,11-trideoxy (MRM2) Time (min) NL: 1.00E4 [ ]

12 Analysis of urine samples TEF: Toxicity equivalency factors CRFs: Cross-reactivity factors LC-MS/MS (ng/ml) melisa Hours after ingestion 4-epi 4,9- anhydro 5,6,11- trideoxy equiv. (applying TEFs) equiv. (applying CRFs) equiv. Pt# Pt# Pt# Pt#1 Urine multi-toxin profile Pt#1 Pt#2 LC-MS/MS LOD (Urine): 12 ng/ml LOD (plasma): 25 ng/ml LOD (sèrum): 22 ng/ml melisa LOD (urine): 3,6 ng/ml ~17 h after ingestion ~45 h after ingestion ~42 h after ingestion 4-epi 4,9-anydro 4,9-anhydroTT 5,6,11-trideoxy

13 Analysis of urine samples LC-MS/MS (ng/ml) TEF: Toxicity equivalency factors CRFs: Cross-reactivity factors melisa Hours after ingestion 4-epi 4,9- anhydro 5,6,11- trideoxy equiv. (applying TEFs) equiv. (applying CRFs) equiv. Pt# Pt# Pt# Urine multi-toxin profile Pt#1 ~17 h after ingestion Pt#1 ~45 h after ingestion Pt#2 ~42 h after ingestion 4-epi 4,9-anydro 4,9-anhydro 5,6,11-trideoxy

14 Analysis of urine samples TEF: Toxicity equivalency factors CRFs: Cross-reactivity factors Pt# 1 Pt# 1 Pt# 2 Hours after ing, 4- epitt X 4,9- anhydrott X (ng/ml) UC- (ng/µmol creatinine) LC-MS/MS melisa Creatinine LC-MS/MS melisa (µmol/l) 5,6,11- trideoxy equiv. (applying TEFs) equiv. (applying CRFs) equiv. equiv. (applying TEFs) equiv. (applying CRFs) equiv , , n.d.* , Analysis of post-mortem plasma 5,6,11-trideoxy 32.9 ng/ml ng/ml Note: 9 ng/ml suggested potentially lethal (Islam et al., 2011)

15 Conclusions The implication of as the responsible agent of the reported intoxication case in New Caledonia following consumption of puffer fish has been proven. The applicability of the melisa to the analysis of urine samples has been demonstrated, confirming the presence of equiv. in all urine samples from the hospitalised patients. LC-MS/MS analysis revealed a multi-toxin profile, with the presence of and three analogues (4 epi, 4,9-anhydro and 5,6,11-trideoxy). and 5,6,11-trideoxy were detected in the post-mortem plasma of the fisherman at 37.0 ng/ml, after applying the TEFs, concentration which has been described to be lethal. The strategy used within this research work could be a valuable tool for future food safety monitoring: Analysis of urine samples by melisa is a reliable, simple and cost-effective strategy to estimate the degree of poisoning in humans The complementary analysis by LC-MS/MS performed in parallel confirms the causative intoxication agent and provide a full characterisation of the multitoxin profile of the sample. FUTURE WORK

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17 Thanks