Development and validation of a specific and sensitive liquid chromatography tandem mass spectrometry method for determination of tetrodotoxin in human urine and its pharmacokinetic study.

Tetrodotoxin (TTX) exhibits the therapeutic potential in blocking pain and in low doses can safely relieve severe pain. The urinary excretion profiles of TTX in humans have not been reported due to the extremely low lethal dose. In this study, a rapid and specific method based on protein precipitation coupled to liquid chromatography tandem mass spectrometry was developed to determine the level of TTX in human urine samples. 11-Deoxytetrodotoxin was used as an internal standard (IS). Multiple reaction monitoring mode was used for quantification using target fragment ions m/z 320.0 → 162.1 for TTX and m/z 304.0 → 176.0 for 11-deoxyTTX. The separation of analytes was achieved on a hydrophilic interaction liquid chromatography column (250 × 4.6 mm, 5.0 µm). The mobile phase consisted of 5 mM ammonium formate in water (pH = 4.50) and 5 mM ammonium formate in acetonitrile (pH = 4.50). The flow rate was set at 0.80 mL/min in a gradient condition. Calibration plots were linear throughout the range 0.986-98.6 ng/mL of TTX in human urine. The intra-assay accuracies and precisions were within the acceptable range. The method was successfully applied to a urinary excretion study after intravenous administration of TTX to healthy volunteers. The developed method will be helpful for future pharmacological studies of TTX.

A simple, versatile, and automated pulse-diffusion-focusing strategy for sensitive milliliter-level-injection HILIC-MS/MS analysis of hydrophilic toxins.

The measurement of trace hydrophilic toxins in complex aqueous-rich matrices is a daunting challenge. To address this analytical bottleneck, pulse diffusion focusing (PDF), a novel sample injection technique for hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS), was developed. Theoretical and experimental investigations of the mechanism and key parameters revealed that the pulse-injection approach, assisted by solvent diffusion, efficiently solved the volume overload problem. This milliliter-level-injection HILIC-MS/MS technique was reported for the first time herein, and provided a significant enhancement in sensitivity compared to the conventional injection method, in addition to being an efficient approach to address the solvent incompatibility of off-line sample preparation and HILIC. The automated PDF-HILIC-MS/MS system was obtained by slightly modifying a commercial LC-MS/MS instrument in an easy and economical manner. The efficiency of the system was demonstrated through the detection of trace tetrodotoxin contents in plasma and urine samples. Low limits of detection (i.e., 0.65 and 2.2 ng·mL-1) were obtained using the simplified sample preparation method. The recoveries were in the range 91-113.3 % with intra-day and inter-day precisions of ≤9.6 %. Further experimental results proved the method to be versatile for various hydrophilic toxins.

Rapid screening and multi-toxin profile confirmation of tetrodotoxins and analogues in human body fluids derived from a puffer fish poisoning incident in New Caledonia.

In August 2014, a puffer fish poisoning incidence resulting in one fatality was reported in New Caledonia. Although tetrodotoxin (TTX) intoxication was established from the patients' signs and symptoms, the determination of TTX in the patient's urine, serum or plasma is essential to confirm the clinical diagnosis. To provide a simple cost-effective rapid screening tool for clinical analysis, a maleimide-based enzyme-linked immunosorbent assay (mELISA) adapted for the determination of TTX contents in human body fluids was assessed. The mELISA was applied to the analysis of urine samples from two patients and a response for the presence of TTX and/or structurally similar analogues was detected in all samples. The analysis by LC-MS/MS confirmed the presence of TTX but also TTX analogues (4-epiTTX, 4,9-anhydroTTX and 5,6,11-trideoxyTTX) in the urine. A change in the multi-toxin profile in the urine based on time following consumption was observed. LC-MS/MS analysis of serum and plasma samples also revealed the presence of TTX (32.9 ng/mL) and 5,6,11-trideoxyTTX (374.6 ng/mL) in the post-mortem plasma. The results provide for the first time the TTX multi-toxin profile of human samples from a puffer fish intoxication and clearly demonstrate the implication of TTX as the causative agent of the reported intoxication case.

RETRACTED: Facilely self-assembled magnetic nanoparticles/aptamer/carbon dots nanocomposites for highly sensitive up-conversion fluorescence turn-on detection of tetrodotoxin.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor following concerns raised by a reader. The article uses two electron micrographs which have been used in other publications as well, denoting different samples. Fig. 1A is identical to Fig. 3E in RSC Adv., 2013,3, 20959-20969 doi: 10.1039/C3RA43120G despite describing different samples. Fig. 2A is identical to Fig. 1B in Sensors & Actuators B: Chemical, vol 245, pp 386-394 https://doi.org/10.1016/j.snb.2017.01.166 and Fig. 1A in Materials Letters vol 195 pp 131-135 https://doi.org/10.1016/j.matlet.2017.02.119 despite describing different samples. These problems with the data presented cast doubt on all the data, and accordingly also the conclusions based on that data, in this publication. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process., http://dx.doi.org/10.1016/j.talanta.2017.08.043.

Urinary Excretion of Tetrodotoxin Modeled in a Porcine Renal Proximal Tubule Epithelial Cell Line, LLC-PK1.

This study examined the urinary excretion of tetrodotoxin (TTX) modeled in a porcine renal proximal tubule epithelial cell line, LLC-PK1. Time course profiles of TTX excretion and reabsorption across the cell monolayers at 37 °C showed that the amount of TTX transported increased linearly for 60 min. However, at 4 °C, the amount of TTX transported was approximately 20% of the value at 37 °C. These results indicate that TTX transport is both a transcellular and carrier-mediated process. Using a transport inhibition assay in which cell monolayers were incubated with 50 µM TTX and 5 mM of a transport inhibitor at 37 °C for 30 min, urinary excretion was significantly reduced by probenecid, tetraethylammonium (TEA), l-carnitine, and cimetidine, slightly reduced by p-aminohippuric acid (PAH), and unaffected by 1-methyl-4-phenylpyridinium (MPP+), oxaliplatin, and cefalexin. Renal reabsorption was significantly reduced by PAH, but was unaffected by probenecid, TEA and l-carnitine. These findings indicate that TTX is primarily excreted by organic cation transporters (OCTs) and organic cation/carnitine transporters (OCTNs), partially transported by organic anion transporters (OATs) and multidrug resistance-associated proteins (MRPs), and negligibly transported by multidrug and toxic compound extrusion transporters (MATEs).

A Study of 11-[³H]-Tetrodotoxin Absorption, Distribution, Metabolism and Excretion (ADME) in Adult Sprague-Dawley Rats.

Tetrodotoxin (TTX) is a powerful sodium channel blocker that in low doses can safely relieve severe pain. Studying the absorption, distribution, metabolism and excretion (ADME) of TTX is challenging given the extremely low lethal dose. We conducted radiolabeled ADME studies in Sprague-Dawley rats. After a single dose of 6 µg/(16 µCi/kg) 11-[³H]TTX, pharmacokinetics of plasma total radioactivity were similar in male and female rats. Maximum radioactivity (5.56 ng Eq./mL) was reached in 10 min. [³H]TTX was below detection in plasma after 24 h. The area under the curve from 0 to 8 h was 5.89 h·ng Eq./mL; mean residence time was 1.62 h and t½ was 2.31 h. Bile secretion accounted for 0.43% and approximately 51% of the dose was recovered in the urine, the predominant route of elimination. Approximately 69% was recovered, suggesting that hydrogen tritium exchange in rats produced tritiated water excreted in breath and saliva. Average total radioactivity in the stomach, lungs, kidney and intestines was higher than plasma concentrations. Metabolite analysis of plasma, urine and feces samples demonstrated oxidized TTX, the only identified metabolite. In conclusion, TTX was rapidly absorbed and excreted in rats, a standard preclinical model used to guide the design of clinical trials.

[Tetrodotoxin (TTX) Monitoring of Biological Specimens and Toxin Profile in a Food Poisoning Case Caused by the Scavenging Gastropod Nassarius (Alectrion) glans "Kinshibai"].

In November 2015, a patient presented with symptoms of toxicity after eating whole boiled samples of the scavenging gastropod Nassarius (Alectrion) glans "Kinshibai" in Nagasaki. This food poisoning case was the third recorded in Japan. The case was investigated by evaluation of the toxin profile of the gastropod, and monitoring of tetrodotoxin (TTX) levels in serum and urine sampled from the affected individual. One gastropod contained a harmful dose of TTX (2.5 mg/ individual in food residue sample 2). In biological samples, maximum TTX concentrations were 42.8 ng/mL in serum on the day after onset of symptoms. TTX urinary excretion was calculated to be 2.4 mg. From the measured TTX concentrations, it was estimated that a lethal dose had been ingested in this case. Moreover, it was found by LC-QqQ-MS/MS analysis and mouse bioassay that the toxicity of "Kinshibai" was not solely due to TTX. The remaining toxicity was thought to be due to 11-oxoTTX. As in previous poisoning cases, it was concluded that ingestion of this gastropod poses a high risk of food poisoning.

Development and validation of a high-throughput online solid phase extraction - Liquid chromatography - Tandem mass spectrometry method for the detection of tetrodotoxin in human urine.

Tetrodotoxin (TTX) is an extremely potent paralytic toxin responsible for yearly illness and death around the world. A clinical measurement is necessary to confirm exposure because symptoms of TTX intoxication cannot be distinguished from other paralytic toxins. Our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography (HILIC) method for the analysis of TTX in human urine with tandem mass spectrometry. The reportable range for the method was 2.80 - 249 ng/mL in urine with precision and accuracy within 15% as determined for all quality control samples. No isotopically-labeled internal standard is available for TTX; thus a surrogate internal standard, voglibose, was investigated to compensate for matrix effects and ionization suppression. However, upon evaluation, voglibose was ineffective for this purpose. This new online method rapidly identifies TTX, facilitating the work of public health authorities and providing support to monitoring programs worldwide.

Acute paralysis after seafood ingestion.

The first European case of tetrodotoxin intoxication is reported in a patient who ingested a trumpet shellfish from the Atlantic Ocean in Southern Europe. He suffered general acute paralysis with respiratory failure necessitating ventilation. Early neurophysiologic studies showed complete peripheral nerve inexcitability, with no recordable sensory or motor responses, and normal electroencephalography. Tetrodotoxin was detected in high quantities in the patient's blood and urine through high performance liquid chromatography-mass spectrometry analysis. Seventy-two hours after admission the patient recovered normal strength, reflexes and sensation.

Analytical challenges: determination of tetrodotoxin in human urine and plasma by LC-MS/MS.

Tetrodotoxin (TTX) is a powerful sodium channel blocker found in puffer fish and some marine animals. Cases of TTX poisoning most often result from puffer fish ingestion. Diagnosis is mainly from patient's signs and symptoms or the detection of TTX in the leftover food. If leftover food is unavailable, the determination of TTX in the patient's urine and/or plasma is essential to confirm the diagnosis. Although various methods for the determination of TTX have been published, most of them are for food tissue samples. Dealing with human urine and blood samples is much more challenging. Unlike in food, the amount of toxin in the urine and blood of a patient is generally extremely low; therefore a very sensitive method is required to detect it. In this regard, mass spectrometry (MS) methods are the best choice. Since TTX is a very polar compound, there will be lack of retention on conventional reverse-phase columns; use of ion pair reagent or hydrophilic interaction liquid chromatography (HILIC) can help solve this problem. The problem of ion suppression is another challenge in analyzing polar compound in biological samples. This review will discuss different MS methods and their pros and cons.

Development and validation of a high-throughput double solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma.

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL(-1) for urine and 20 ng mL(-1) for plasma. The limit of detection and limit of quantification were 0.13 ng mL(-1) and 2.5 ng mL(-1), respectively, for both biological matrices. Recoveries were in the range of 75-81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL(-1), but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng µmol(-1) creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.

[Determination of TTX in biological samples by LC-MS/MS].

OBJECTIVE: To propose a method for determination of tetrodotoxin (TTX) in human blood, urine and liver by liquid chromatography with tandem mass spectrometry (LC-MS/MS). METHODS: Solid phase extraction is used after the samples are precipitated, then the samples will be analyzed by LC-MS/MS. RESULTS: The limits of detection were 2 ng/mL in blood and urine and 4 ng/g in liver for TIX respectively, the linear correlation coefficients were not less than 0.9973, both of the intra-day and inter-day precisions were less than 12.80%, the recoveries for all kinds of specimens were more than 47.2%. CONCLUSION: This method is efficient, sensitive and accurate and was successfully validated for implementation in forensic toxicological analysis.

Rapid screening of tetrodotoxin in urine and plasma of patients with puffer fish poisoning by HPLC with creatinine correction.

A rapid and simple detection method for tetrodotoxin (TTX) in urine and plasma of patients with puffer fish poisoning was developed using commercially pre-packed solid-phase extraction (SPE) cartridges (C18 and weak cation exchange columns) and subsequent analyses by HPLC with UV detection. The detection limit of the standard TTX, TTX-spiked urine and plasma samples were all 10 ng/ml and the average TTX recovery in urine and plasma samples after SPE were 90.3 +/- 4.0 and 87.1 +/- 2.9%, respectively. It was noticed that the creatinine-adjusted urinary TTX levels obtained within the first 24 h of presentation apparently correlated much better with the severity of poisoning than the urinary TTX concentration without adjusting for variations in concomitant creatinine excretion.

Determination of TTX in biological samples by LC-MS/MS / 法医学杂志

OBJECTIVE@#To propose a method for determination of tetrodotoxin (TTX) in human blood, urine and liver by liquid chromatography with tandem mass spectrometry (LC-MS/MS).@*METHODS@#Solid phase extraction is used after the samples are precipitated, then the samples will be analyzed by LC-MS/MS.@*RESULTS@#The limits of detection were 2 ng/mL in blood and urine and 4 ng/g in liver for TIX respectively, the linear correlation coefficients were not less than 0.9973, both of the intra-day and inter-day precisions were less than 12.80%, the recoveries for all kinds of specimens were more than 47.2%.@*CONCLUSION@#This method is efficient, sensitive and accurate and was successfully validated for implementation in forensic toxicological analysis.

[Determination of tetrodotoxin in puffer-fish tissues, and in serum and urine of intoxicated humans by liquid chromatography with tandem mass spectrometry].

A simple and rapid method was developed for the analysis of tetrodotoxin in puffer-fish tissues, and in serum and urine of humans poisoned after consuming puffer-fish, by means of high-performance liquid chromatography with tandem mass spectrometry (LC/MS/MS). Tetrodotoxin was extracted with 2% acetic acid. The extracted solution from puffer-fish tissues was diluted with water, and the extracted solution from human serum and urine was cleaned up by LC/MS/MS with a methacrylate-styrenedivinylbenzene cartridge. The LC separation was performed on a C18 column (50 mm x 2.1 mm i.d.) using 10 mmol/L IPCC-MS7-methanol (65 : 35) as the mobile phase at a flow rate of 0.2 mL/min. The mass spectral acquisition was done in the positive ion mode by applying selected reaction monitoring (SRM). The recoveries of tetrodotoxin were 79-90% from puffer-fish tissues fortified at 0.1 microg/g and 1 microg/g, and 93-101% from human serum and urine fortified at 0.5 ng/mL and 5 ng/mL. The detection limits of tetrodotoxin were 0.01 microg/g in puffer-fish tissues and 0.1 ng/mL in human serum and urine. Thirty samples of puffer-fish from wholesale markets, and 7 serum and 5 urine samples of humans poisoned after consuming puffer-fish were analyzed by this method. Tetrodotoxin was detected in all puffer-fish tissues, and all serum and urine samples at the levels of 0.04-140 microg/g, 0.9-1.8 ng/mL and 15-150 ng/mL, respectively.

Determination of tetrodotoxin in human urine and blood using C18 cartridge column, ultrafiltration and LC-MS.

Six fishermen were victims (including one death) of food poisoning from unknown fish on their boat in central Taiwan Strait, in April 2001. The symptoms were like those of tetrodotoxin (TTX) poisoning. As there was no remaining fish, a new protocol was developed to determine TTX in the urine and blood of the victims. The urine and blood samples were cleansed using a C18 Sep-Pak cartridge column, and the toxin was extracted by methanol. The eluate was filtered through a microcentrifuge filter. The filtrate was freeze-dried, dissolved in distilled water, and determined by LC-MS. The recovery was more than 88.9%. The detection limit was 15.6 nM. A linear relationship between response and concentration was obtained between 93.75 and 9375 nM of TTX. It was shown that the urine and blood of the victims contained TTX. The range of TTX was 4.5-40.6 nM in blood and 47-344 nM in urine. Judging from the symptoms of the victims and the experimental data, the causative agent of the food poisoning was identified as TTX.

Acute tetrodotoxin-induced neurotoxicity after ingestion of puffer fish.

This study documents the effects of puffer-fish poisoning on peripheral nerve. Excitability measurements investigated membrane properties of sensory and motor axons in four patients. The median nerve was stimulated at the wrist, with compound muscle potentials recorded from abductor pollicis brevis and compound sensory potentials from digit 2. Stimulus-responses, strength-duration time constant (tau(SD)), threshold electrotonus, and current-threshold relations were recorded. The urine of each patient tested positive for tetrodotoxin. Compared with controls, axons were of higher threshold, compound muscle action potentials and compound sensory nerve action potentials were reduced in amplitude, latency was prolonged, and tau(SD) was reduced. In recovery cycles, refractoriness, superexcitability, and late subexcitability were decreased. Threshold electrotonus of motor axons exhibited distinctive abnormalities with less threshold decline than normal on depolarization and greater threshold increase on hyperpolarization (p < 0.0005 for each patient). The changes in excitability were reproduced in a mathematical model by reducing sodium (Na(+)) permeabilities by a factor of two. This study confirms that the neurotoxic effects of puffer-fish poisoning can be explained by tetrodotoxin blockade of Na(+) channels. It demonstrates the ability of noninvasive nerve excitability studies to detect Na(+) channel blockade in vivo and also the utility of mathematical modeling to aid interpretation of altered excitability properties in disease.

Use of high performance liquid chromatography to measure tetrodotoxin in serum and urine of poisoned patients.

Tetrodotoxin (TTX) poisoning is an infrequent but important problem in South-eastern Asia. Despite it being a considerable public health issue in some countries and its potential lethality there continues to be no readily available method for measuring TTX in urine or serum. Previously published methods have used immunoaffinity chromatography, or the conversion of TTX to its C9-base derivative for measurement by mass spectrometry. A simple and reproducible method was developed using solid phase extraction cartridges to clean up serum and urine samples from TTX-poisoned patients, and the subsequent analysis of the samples by high performance liquid chromatography with post-column derivatisation and fluorescence detection. Minimum quantifiable concentrations of TTX were 5 and 20 ng/ml for serum and urine, respectively. Precision and accuracy of the assay were 13 and 15%, respectively. The standard curves were linear in the range of 20-300 ng/ml for urine and 5-20 ng/ml for serum. TTX was quantified in six samples of urine and six samples of serum from seven patients who ingested common toadfish and had clinical effects consistent with TTX poisoning. TTX was detected in all urine samples but in only one serum sample. Using this method confirmation of TTX poisoning will be far simpler and readily available. A 24 h urine collection in the period immediately following poisoning is likely to be the most sensitive test for TTX poisoning. With appropriately collected and timed serum and urine specimens it will be possible to properly define the pharmacokinetics of TTX in humans.

Application of immunoaffinity chromatography for detection of tetrodotoxin from urine samples of poisoned patients.

Immunoaffinity chromatography using the monoclonal antibody (Tl-1) specific for tetrodotoxin (TTX) has been developed for isolating TTX from urine samples. By combining immunoaffinity chromatography with fluorometric high performance liquid chromatography (HPLC), it has become possible to detect a small amount of TTX in urine samples. The detection limit of TTX in urine was 2 ng/ml. By this combined method, TTX was detected in all the urine samples that were collected from poisoned patients during the week following TTX ingestion. The combination of immunoaffinity chromatography with HPLC was very useful in detecting TTX from the urine samples of poisoned patients for diagnosis of TTX-food poisoning.