Naomasa Oshiro

In electrospray ionization (ESI), analytes containing multiple hydroxyl or ether structures often produce multiple competing precursor ions, reducing sensitivity and complicating precursor selection. Okadaic acids (OAs) exhibited pronounced Na⁺/K⁺ adduction along with sequential dehydration. In contrast, azaspiracids, which possess a secondary amine capable of forming a stable protonated species, predominantly generate [M + H]⁺ ions. This contrast indicates that the absence of a strong charge-localization site is the dominant contributing factor under ESI conditions to ion dispersion in OAs. To address this issue, we chemically introduced a charge-localizing group via IPPAH derivatization. The resulting OAs-IPPAH derivatives exhibited substantial precursor-ion convergence in ESI(+), with up to 95% of the total ion abundance as a single [M + H]⁺ ion. Peak intensities increased by 2.6-4.7. These findings provide the first demonstration that chemical control of charge localization can suppress in-source dehydration and metal adduct formation.

The increasing spread of toxin-producing Gambierdiscus dinoflagellates has led to cases of ciguatera poisoning in previously nonendemic regions. Ciguatoxins (CTXs) are potent, heat-stable compounds responsible for ciguatera, and their rapid and reliable analysis in seafood is essential to prevent illness. This study presents the development of the first lateral flow assay (LFA) for the detection of major Pacific CTXs congeners. The assay employs previously developed monoclonal antibodies in a sandwich format: specific antibodies against CTX1B and CTX3C are immobilized on a single test line, while the reporter antibody is a single antibody recognizing major Pacific CTX congeners that is conjugated to gold nanoparticles. The LFA provides one-step visual results in 30 min, making it suitable for resource-limited settings. It exhibits a visual limit of detection of 400 pg/mL and the assay is highly specific, showing no cross-reactivity with other marine toxins. It successfully detected Pacific CTXs spiked in noncontaminated fish extracts, as well as in naturally contaminated samples as previously confirmed by LC-MS/MS, with an estimated cutoff of approximately 0.1 μg/kg. Overall, this LFA demonstrates great potential as a rapid screening tool for outbreak investigations and research programs.

シガテラ食中毒は，原因毒素シガトキシン類（CTXs）に起因する魚類による食中毒で，近年全国的な発生リスクの増加が懸念されている．魚肉に含まれるCTXsは極めて少量であるためLC-MS/MSによる高感度検出法が求められるが，現在報告されている検出法は特定の装置にのみ適用可能で，多くの試験所で対応困難な状況にある．今回，CTXsに対して広く適用可能なLC-MS/MS分析法を検討した．ギ酸および水酸化リチウムを添加した水／アセトニトリル系移動相により開裂しづらいリチウムイオン付加分子：［M＋Li］^＋が特異的に形成された．CTXs9物質を対象にmultiple reaction monitoring測定において［M＋Li］^＋>［M＋Li］^＋をモニターするLC-MS/MS分析を実施した結果，LOD 0.005～0.030 ng/mLおよびLOQ 0.010～0.061 ng/mLの検出が可能であった．魚肉5 gから試験溶液1 mLを調製した場合のLODとLOQはそれぞれ0.001～0.006 μg/kgおよび0.002～0.012 μg/kgと推算され，食用不適水準（0.175 μg/kg CTX1B当量）の検査に対応可能であることが示唆された．本法は特定の装置に依存しない普遍的な手法であることが示唆され，全国の試験所のCTXs検査体制整備に資するものと考えられた．

Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives to the mouse bioassay method. However, post-column derivatization requires a system for online derivatization reactions, and with the ion-pair LC-MS approach, it is difficult to remove residual ion-pair reagents remaining in the equipment. Moreover, HILIC-MS provides poor separation compared to reversed-phase (RP) HPLC and requires a long time to reach equilibration. Therefore, we decided to develop a TTX analytical method using pre-column derivatization and RP HPLC for the rapid assessment of outbreak samples, including food remnants. In this study, we focused on the vic-diol moiety of TTX and designed a new derivatization reagent coded as NBD-H-DAB. This NBD-H-DAB was synthesized from 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and 3-fluoro-2-formylphenylboronic acid (FFPBA) with a simple reaction system and rapidly converted to its boronate form, coded NBD-H-PBA, in an aqueous reaction solution. The NBD-H-PBA demonstrated appropriate hydrophobicity to be retained on the RP analytical column and successfully detected with a UV spectrometer. It was easily reacted with the vic-diol moiety of TTX (C6 and C11) to synthesized a boronic ester. The derivatized TTX could be detected using the RP HPLC-UV, and the limit of detection in the fish flesh samples was 0.06 mg/kg. This novel pre-column derivatization of TTX with NBD-H-PBA proves capable for the analysis of TTX.

Ciguatera poisoning (CP) is the most common type of marine biotoxin food poisoning worldwide, and it is caused by ciguatoxins (CTXs), thermostable polyether toxins produced by dinoflagellate Gambierdiscus and Fukuyoa spp. It is typically caused by the consumption of large fish high on the food chain that have accumulated CTXs in their flesh. CTXs in trace amounts are found in natural samples, and they mainly induce neurotoxic effects in consumers at concentrations as low as 0.2 µg/kg. The U.S. Food and Drug Administration has established CTX maximum permitted levels of 0.01 µg/kg for CTX1B and 0.1 µg/kg for C-CTX1 based on toxicological data. More than 20 variants of the CTX1B and CTX3C series have been identified, and the simultaneous detection of trace amounts of CTX analogs has recently been required. Previously published works using LC-MS/MS achieved the safety levels by monitoring the sodium adduct ions of CTXs ([M+Na]⁺ > [M+Na]⁺). In this study, we optimized a highly sensitive method for the detection of CTXs using the sodium or lithium adducts, [M+Na]⁺ or [M+Li]⁺, by adding alkali metals such as Na⁺ or Li⁺ to the mobile phase. This work demonstrates that CTXs can be successfully detected at the low concentrations recommended by the FDA with good chromatographic separation using LC-MS/MS. It also reports on the method’s new analytical conditions and accuracy using [M+Li]⁺.

Ciguatoxins (CTXs) stand as the primary toxins causing ciguatera fish poisoning (CFP) and are essential compounds distinguished by their characteristic polycyclic ether structure. In a previous report, we identified the structures of product ions generated via homolytic fragmentation by assuming three charge sites in the mass spectrometry (MS)/MS spectrum of ciguatoxin-3C (CTX3C) using LC-MS. This study aims to elucidate the homolytic fragmentation of a ciguatoxin-3C congener. We assigned detailed structures of the product ions in the MS/MS spectrum of a naturally occurring ciguatoxin-3C congener, 51-hydroxyciguatoxin-3C (51-hydoxyCTX3C), employing liquid chromatography/quadrupole time-of-flight mass spectrometry with an atmospheric pressure chemical ionization (APCI) source. The introduction of a hydroxy substituent on C51 induced different fragmentation pathways, including a novel cleavage mechanism of the M ring involving the elimination of 51-OH and the formation of enol ether. Consequently, new cleavage patterns generated product ions at m/z 979 (C₅₅H₇₉O₁₅), 439 (C₂₄H₃₉O₇), 149 (C₁₀H₁₃O), 135 (C₉H₁₁O), and 115 (C₆H₁₁O₂). Additionally, characteristic product ions were observed at m/z 509 (C₂₈H₄₅O₈), 491 (C₂₈H₄₃O₇), 481 (C₂₆H₄₁O₈), 463 (C₂₆H₃₉O₇), 439 (C₂₄H₃₉O₇), 421 (C₂₄H₃₇O₆), 171 (C₉H₁₅O₃), 153 (C₉H₁₃O₂), 141 (C₈H₁₃O₂), and 123 (C₈H₁₁O).

Okadaic acids (OAs) are causative agents of diarrhetic shellfish poisoning, produced by the dinoflagellates Dinophysis spp. and Prorocentrum spp. Microcystins (MCs) are cyclic heptapeptide hepatotoxins produced by some cyanobacteria genera, including Microcystis spp. Traditionally, toxicity detection and quantification of these natural toxins were performed using a mouse bioassay (MBA); however, this is no longer widely employed owing to its lack of accuracy, sensitivity, and with regard to animal welfare. Therefore, alternative toxicity analyses have been developed based on MCs’ and OAs’ specific inhibition of protein phosphatase 2A (PP2A), using p-nitrophenylphosphate (p-NPP) as a substrate. The assay is simple, inexpensive, ready for use on site, and can be applied to several samples at once. For OA detection, this assay method is appropriate for widespread application as a substitute for MBA, as evidenced by its alignment with the oral toxicity of MBA. In this review, we summarize the structure and function of PP2A, the inhibitory activities of OAs and MCs against PP2A, and the practical applications of the PP2A assay, with the aim of improving understanding of the PP2A assay as an OAs and MCs detection and quantification method, as well as its suitability for screening before confirmatory chemical analysis.

Marine biotoxins refer to bioactive natural products primarily produced by microalgae and bacteria and may affect aquatic organisms and human health

Ciguatera fish poisoning (CFP) refers to an illness caused by ingesting fish that have accumulated ciguatoxins (CTXs). CFP frequently occurs in the tropical and subtropical Indo-Pacific Ocean and the Caribbean Sea. In Japan, CFP occurs sporadically but constantly in Okinawa and the Amami Islands. The grouper Variola albimarginata is regarded to be safe for consumption. To assess the real risk of V. albimarginata, we analyzed 133 specimens of the fish in Okinawa using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Ciguatoxin-1B, 54-deoxyciguatoxin-1B, and 52-epi-54-deoxyciguatoxin-1B were detected in 28 specimens (21%). In 11 of these specimens (8%), the CTX levels exceeded the US FDA guidance level (0.01 µg/kg CTX1B equivalent). However, only one fish (V. albimarginata in Okinawa and Yaeyama Islands was 4909 kg or 13,636 fish. As many as 1227 fish had levels over the US FDA guidance level, but only 136 fish had levels above the Japanese recommendation. Risk management based on the Japanese recommendation level seems to be effective in protecting public health and enabling appropriate exploitation of fishery resources.

シガテラ魚類食中毒は，シガトキシン類（CTXs）を含有する魚類による動物性自然毒食中毒で，主に熱帯・亜熱帯で発生している．日本では，沖縄県を中心に発生報告があるが，日本本土の太平洋岸産魚類による食中毒事例も散発的に発生し，原因魚種のほとんどがイシガキダイOplegnathus punctatus の老成個体である．イシガキダイにおけるCTXsの含有状況を調査するために，本州，四国，九州，奄美，沖縄，および小笠原の沿岸産176個体（標準体長：13.1～60.0 cm,体重：100～6,350 g,年齢：0～11歳）を収集し，LC-MS/MSによる分析を実施した．そのうち沖縄産2個体（全試料の1.1％）からCTXsが検出され，沖縄産14個体に限定した検出率は14％であった．CTXsが検出された2個体の魚肉中の総CTX含量は，0.014 μg/kgおよび0.040 μg/kgであり，いずれも米国FDAの推奨値0.01 μg CTX1B equivalent/kg以上であったが，ヒトの最小発症量（10 MU, CTX1B換算で70 ng）に達するには，1.5 kg以上の摂食が必要であるため，シガテラ魚類食中毒発症のリスクは高くないと考えられる．沖縄産イシガキダイのCTXs組成はCTX1B系列のみで，バラフエダイやバラハタなどの肉食魚が含有するCTX1Bおよび52-epi-54-deoxyCTX1Bに加えて，渦鞭毛藻が産生するCTX4AおよびCTX4BがCTX1Bと同程度のレベルで検出された．なお，CTX3C系列は検出されなかった．