**2. Isolation of TTX crystals by column chromatography**

Of the TTX-bearing animals, our specimens of ribbon worms ("himomushi" in Japanese) adherent to the cultured oyster *Crassostrea gigas* hanging onto floating culture rafts, were found to be extremely toxic and to contain tetrodotoxin, during surveillance of the toxicity of various marine fouling organisms in Hiroshima Bay, Hiroshima Prefecture, which is one of the largest oyster culture areas in Japan [23]. In these analyses, the toxicity was examined on each ribbon worm specimen by the standard bioassay method for TTX. Ribbon worm specimens were collected in Hiroshima Bay between November and May from 1998 to 2005 approximately every two weeks during the harvest time for cultured oysters. A total of 764 specimens were collected, and assayed for toxicity. All specimens that were assayed throughout the season covered found to be toxic, and the toxicity scores ranged from 169 to 25,590 MU/g (Figures 4 and 5). The ratio of strongly toxic (more than 1,000 MU/g) specimens to the total number of specimens was 80%. Furthermore, the percentage of specimens possessing toxicity scores higher than 2,000 MU/g to the total was high (48%). The highest toxicity detected was 25,590 MU/g from a specimen collected on June25 (1999). The total toxicity for this sample was approximately calculated to be 5,631 MU, which is approximately equivalent to half of the minimum lethal dose of TTX in humans, which is reported to be 10,000 MU. The specimens of ribbon worms (390 g) obtained during the survey were semi-defrosted and homogenized with three volumes of 1% AcOH in 80% MeOH for 3 min, then centrifuged. This operation was repeated two more times. The supernatants (total toxicity; 2,897,000 MU) were combined, concentrated under reduced pressure, and defatted by shaking gently with approximately the same volume of chloroform several times. The aqueous layer (2,750,000 MU) was applied to an activated charcoal column and the adsorbed toxin was eluted with 1% AcOH in 20% EtOH after washing the column with dist. H2O. The water eluate (Fr.I) and the eluate with 1% AcOH in 20% EtOH (Fr.II) was isolated (Figure 6). The main toxic fraction (Fr.II) was evaporated to dryness *in vacuo*. The resulting residue (total toxicity 2,433,000 MU; specific toxicity 99 MU/mg) was dissolved in a small amount of water, and the pH was adjusted to 5.5 with 1N NaOH. This solution was applied to a Bio-Gel P-2 column (φ 3.5 × 100cm). The column was washed with 3000 ml of water and then developed with 2,000 ml of 0.03 M AcOH. The toxicity was detected exclusively in the 0.03 M AcOH fraction. This fraction was concentrated to dryness under reduced pressure and the residue (3,300 MU/mg) was dissolved in a small volume of water. The resulting solution was chromatographed on a Bio-Rex 70 (H+ form, φ1.0 x 100cm) column using a linear gradient of H2O and 0.03 M AcOH at the flow rate of 0.5 ml /min. The toxic fractions were monitored for TTX via the mouse bioassay and high performance liquid chromatography (HPLC) as described later. The main toxic fractions (fr. 85–100; Fr.I) and minor fractions (fr.50-84) were obtained, and rechromatographed in the same manner (Figure 7). The toxic fraction (Fr.I) thus obtained were freeze-dried, and then dissolved in 0.5 ml of 1% AcOH. Approximately 2.0 ml of MeOH and 5.0 ml of diethylether were added to this solution, and the mixture was stored in the refrigerator overnight. During storage, stratified plate-like crystals appeared (Figure. 8). The crystals were isolated by decantation, and recrystallized by method same as that described above. Approximately, Bio-Gel P-2 column chromatography was very effective, as the specific toxicity sharply increased from 99 to 3,300 MU/mg. After recrystallization, the specific toxicity of this toxin ultimately increased to 3,520 MU/mg. From the combined homogenates with the toxicity of roughly 7,400 MU/g, approximately 25 mg of the stratified plate-like crystalline TTX was obtained. Generally, the ribbon worm has a simple structure. Since pure crystals of TTX could be obtained from this highly toxic ribbon worm efficiently by the above-described series of chromatographies, the ribbon worm is a promising source of TTX for use as a reagent in the fields of medicine and pharmacology. Previously, authentic specimens of TTX were typically prepared from pufferfish ovaries for use as reference standards, as reported in [4].

250 Chromatography – The Most Versatile Method of Chemical Analysis

(A) Hemilactal type, (B) Lacton type, (C) 4,9-Anhydro type **Figure 3.** The structure of three types of TTX analogues

**2. Isolation of TTX crystals by column chromatography** 

Of the TTX-bearing animals, our specimens of ribbon worms ("himomushi" in Japanese) adherent to the cultured oyster *Crassostrea gigas* hanging onto floating culture rafts, were found to be extremely toxic and to contain tetrodotoxin, during surveillance of the toxicity of various marine fouling organisms in Hiroshima Bay, Hiroshima Prefecture, which is one of the largest oyster culture areas in Japan [23]. In these analyses, the toxicity was examined on each ribbon worm specimen by the standard bioassay method for TTX. Ribbon worm specimens were collected in Hiroshima Bay between November and May from 1998 to 2005 approximately every two weeks during the harvest time for cultured oysters. A total of 764 specimens were collected, and assayed for toxicity. All specimens that were assayed throughout the season covered found to be toxic, and the toxicity scores ranged from 169 to 25,590 MU/g (Figures 4 and 5). The ratio of strongly toxic (more than 1,000 MU/g) specimens to the total number of specimens was 80%. Furthermore, the percentage of specimens possessing toxicity scores higher than 2,000 MU/g to the total was high (48%). The highest toxicity detected was 25,590 MU/g from a specimen collected on June25 (1999). The total toxicity for this sample was approximately calculated to be 5,631 MU, which is approximately equivalent to half of the minimum lethal dose of TTX in humans, which is reported to be 10,000 MU. The specimens of ribbon worms (390 g) obtained during the survey were semi-defrosted and homogenized with three volumes of 1% AcOH in 80% MeOH for 3 min, then centrifuged. This operation was repeated two more times. The supernatants (total toxicity; 2,897,000 MU) were combined, concentrated under reduced pressure, and defatted by shaking gently with approximately the same volume of chloroform several times. The aqueous layer (2,750,000 MU) was applied to an activated charcoal column and the

(A): Arrow indicates the ribbon worm (scale bar = 10 cm) on the surface of the cultured oyster shell. (B): Ribbon worm removed from the cultured oyster shell

**Figure 4.** Ribbon worms *Cephalothrix* sp. ("himomushi" in Japanese) adherent to the cultured oyster *Crassostrea gigas* hanging onto floating culture rafts in Hiroshima Bay.

Instrumental Analysis of Tetrodotoxin 253

identified by comparing their retention times with those of authentic TTXs. For the quantitative analysis by HPLC, the detection limit of TTX is approximately 0.03μg, which is satisfactory for practical applications. To date, several continuous improvements have been made to detect TTX and its analogs under different HPLC conditions, and a number of advances in understanding the biochemistry of TTXs are the outcomes of these developments. Briefly, a few promising methodologies are described as follows. In the early 1980's, a fluorometric continuous TTX analyzer was constructed by combining HPLC and a post-column reaction with NaOH to monitor potentially harmful puffer toxins [24]. In this system, the toxin was first separated from contaminants on a column composed of a weak cation exchange gel with a 0.06 M citrate buffer solution (pH 4.0), and toxin concentrations above 8 MU/g were detected. However, because of the poor performance of the original system in separating and detecting TTX analogs, an improved analyzer was later constructed. Using HPLC-FLD, naturally occurring TTX analogs, 4-*epi*TTX, 4,9-anhydroTTX [2,3,4,25] 6-*epi*TTX [26], 11-deoxyTTX [26], 11-oxoTTX [27], 11-*nor*TTX-6(*R*)-ol [28], 11 *nor*TTX-6(*S*)-ol [29], 1-hydroxy-5,11-dideoxyTTX [30], 5,6,11-trideoxyTTX [31], 5-deoxyTTX [32], 4,9-anhydro-6-*epi*TTX, 4-*epi*-11-deoxy-TTX, and 4,9-anhydro-11-deoxyTTX isolated from puffer and newt specimens and/or frogs [33]. The separation of TTX and 6-*epi*TTX is considered as a major achievement for this improved analyzer. In addition to this, attempts were made to apply a post-column fluorescent-HPLC system for quantitative assay of TTX and its analog 6-*epi*TTX in newts from southern Germany [34]. A reversed-phase ion-pairing HPLC method, in which HAS is used as a counter ion has also been the system preferred by many researchers for fastest and most efficient analysis of TTX and its analogs. In this method, the detection reagent for TTX and related substances does not react with any PSP component if present in the contaminant sample. A reversed - phase HPLC system (Table 2) with slight modification in the method proposed by of Nagashima *et al.*[35] is commonly used to analyze toxin compositions of extremely toxic Japanese ribbon worms, the xanthid crab *Demania cultripes* from Cebu Island, Philippines(Figure 6,7 and 9) and TTX-producing

bacteria[23, 36, 37].

Mobile phase

HPLC control system JASCO - BORWIN/HSS-2000

Column temperature 30oC CO-2067 plus (JASCO)

60mM ammomium phosphate buffer (pH5.0) containing 10mM HAS and 2% CH3CN

Reaction temperature 110oC 860 CO (JASCO)

Detection Excitation 384nm, emission 505nm JASCO FP-2025 plus

(LiChrospher 100 RP-18e, 5μm) Column size: 4 x 250 mm

PU-2080 plus (JASCO) 0.5 ml/min

MINICHEMI PUMP SP-D-2502 (Nihonseimitsukagaku) 0.5 ml/min

Column LiChroCART 250-4 (Merck)

**Table 2.** Operating conditions of HPLC system for the analysis of TTX

Reagent 3 M NaOH

(A): Relationship between toxicity (MU/g) and body weight (g) (B): Relationship between toxicity (MU/specimen) and body weight (g)

**Figure 5.** Toxicity of ribbon worm *Cephalothrix* sp. from Hiroshima Bay (1998-2005)

## **3. HPLC – Fluorescence detection**

Rapid progress in TTX research, especially in intoxication mechanism of TTX-bearing organisms, is due to recent advancements in instrumental analysis. In particular, postcolumn-HPLC fluorescence detection (HPLC-FLD) methods expected to replace the conventional mouse bioassay, have been explored by many researchers for both qualitative and quantitative analysis of TTX and its analogs. HPLC techniques allow the separation and sensitive detection of individual TTX and its analogs irrespective of their number and group. Therefore, HPLC methods have opened up a new dimension in TTX analysis. However, the results obtained have to be comparable to those of the mouse bioassay. Additionally, accurate HPLC determination of the various TTX components in the samples is a necessity. Using these methods, the toxic principles produced peaks identical to those of authentic TTX and its derivatives. The HPLC- FLD method utilizes a computer controlled by a high pressure pump with a syringe loading sample injector or an autosampler system, a stainless steel column, a reaction pump for delivering reagents, and a fluoromonitor and chromato-recorder for calculation of the peak area. In this method, a strong alkali treatment is applied to TTX which produces a fluorescent compound with excitation and emission wavelengths of 384 and 505 nm, respectively. In this system, first, toxins are separated from the contaminants by a buffer solution on a reversed-phase column packed with C18 resin with an ion-pair reagent (sodium 1-heptanesulfonate; HSA). Then, the isolated toxins are mixed with NaOH, which converts them into fluorescent compounds that are then passed through a stainless steel tube (φ 0.25mm × 100cm) placed in an oven. Eventually, when the fluorescent compounds are passed through a fluoromonitor equipped with a lamp, the retention time of the toxin and fluorescence intensity are recorded. The treated toxins are identified by comparing their retention times with those of authentic TTXs. For the quantitative analysis by HPLC, the detection limit of TTX is approximately 0.03μg, which is satisfactory for practical applications. To date, several continuous improvements have been made to detect TTX and its analogs under different HPLC conditions, and a number of advances in understanding the biochemistry of TTXs are the outcomes of these developments. Briefly, a few promising methodologies are described as follows. In the early 1980's, a fluorometric continuous TTX analyzer was constructed by combining HPLC and a post-column reaction with NaOH to monitor potentially harmful puffer toxins [24]. In this system, the toxin was first separated from contaminants on a column composed of a weak cation exchange gel with a 0.06 M citrate buffer solution (pH 4.0), and toxin concentrations above 8 MU/g were detected. However, because of the poor performance of the original system in separating and detecting TTX analogs, an improved analyzer was later constructed. Using HPLC-FLD, naturally occurring TTX analogs, 4-*epi*TTX, 4,9-anhydroTTX [2,3,4,25] 6-*epi*TTX [26], 11-deoxyTTX [26], 11-oxoTTX [27], 11-*nor*TTX-6(*R*)-ol [28], 11 *nor*TTX-6(*S*)-ol [29], 1-hydroxy-5,11-dideoxyTTX [30], 5,6,11-trideoxyTTX [31], 5-deoxyTTX [32], 4,9-anhydro-6-*epi*TTX, 4-*epi*-11-deoxy-TTX, and 4,9-anhydro-11-deoxyTTX isolated from puffer and newt specimens and/or frogs [33]. The separation of TTX and 6-*epi*TTX is considered as a major achievement for this improved analyzer. In addition to this, attempts were made to apply a post-column fluorescent-HPLC system for quantitative assay of TTX and its analog 6-*epi*TTX in newts from southern Germany [34]. A reversed-phase ion-pairing HPLC method, in which HAS is used as a counter ion has also been the system preferred by many researchers for fastest and most efficient analysis of TTX and its analogs. In this method, the detection reagent for TTX and related substances does not react with any PSP component if present in the contaminant sample. A reversed - phase HPLC system (Table 2) with slight modification in the method proposed by of Nagashima *et al.*[35] is commonly used to analyze toxin compositions of extremely toxic Japanese ribbon worms, the xanthid crab *Demania cultripes* from Cebu Island, Philippines(Figure 6,7 and 9) and TTX-producing bacteria[23, 36, 37].

252 Chromatography – The Most Versatile Method of Chemical Analysis

(A): Relationship between toxicity (MU/g) and body weight (g) (B): Relationship between toxicity (MU/specimen) and body weight (g)

**3. HPLC – Fluorescence detection** 

**Figure 5.** Toxicity of ribbon worm *Cephalothrix* sp. from Hiroshima Bay (1998-2005)

Rapid progress in TTX research, especially in intoxication mechanism of TTX-bearing organisms, is due to recent advancements in instrumental analysis. In particular, postcolumn-HPLC fluorescence detection (HPLC-FLD) methods expected to replace the conventional mouse bioassay, have been explored by many researchers for both qualitative and quantitative analysis of TTX and its analogs. HPLC techniques allow the separation and sensitive detection of individual TTX and its analogs irrespective of their number and group. Therefore, HPLC methods have opened up a new dimension in TTX analysis. However, the results obtained have to be comparable to those of the mouse bioassay. Additionally, accurate HPLC determination of the various TTX components in the samples is a necessity. Using these methods, the toxic principles produced peaks identical to those of authentic TTX and its derivatives. The HPLC- FLD method utilizes a computer controlled by a high pressure pump with a syringe loading sample injector or an autosampler system, a stainless steel column, a reaction pump for delivering reagents, and a fluoromonitor and chromato-recorder for calculation of the peak area. In this method, a strong alkali treatment is applied to TTX which produces a fluorescent compound with excitation and emission wavelengths of 384 and 505 nm, respectively. In this system, first, toxins are separated from the contaminants by a buffer solution on a reversed-phase column packed with C18 resin with an ion-pair reagent (sodium 1-heptanesulfonate; HSA). Then, the isolated toxins are mixed with NaOH, which converts them into fluorescent compounds that are then passed through a stainless steel tube (φ 0.25mm × 100cm) placed in an oven. Eventually, when the fluorescent compounds are passed through a fluoromonitor equipped with a lamp, the retention time of the toxin and fluorescence intensity are recorded. The treated toxins are


**Table 2.** Operating conditions of HPLC system for the analysis of TTX

Instrumental Analysis of Tetrodotoxin 255

(A): Fraction II bound on activated charcoal column

(B): Fraction I unbound on activated charcoal column

(C): TTX standards; TDA (tetrodonic acid), TTX (tetrodotoxin), 4-*epi*TTX (4*epi*tetrodotoxin), 4, 9-anhyTTX (4,9 anhydrotetrodotoxin)

\*HPLC-FLD: high performance liquid chromatography**-**fluorescence detection (HPLC-FLD)

**Figure 6.** HPLC-FLD \* patterns (top) of fractions from the toxins contained in the ribbon worm *Cephalothrix* sp. in an activated charcoal column chromatography. The bottom patterns represent the distribution of toxicity in HPLC chromatograms, as estimated by mouse bioassay.

(A): Elution diagram

fr.85(left), TTX standards(right)

gradient from 0 to 0.03M AcOH

(B): Toxin compositions contained in fraction fr. 85 by HPLC-FLD analysis

**Figure 7.** Elution profile of the ribbon worm *Cephalothrix* sp. toxin from a Bio-Rex 70 column with linear

(A): Elution diagram

254 Chromatography – The Most Versatile Method of Chemical Analysis

(A): Fraction II bound on activated charcoal column (B): Fraction I unbound on activated charcoal column

anhydrotetrodotoxin)

(C): TTX standards; TDA (tetrodonic acid), TTX (tetrodotoxin), 4-*epi*TTX (4*epi*tetrodotoxin), 4, 9-anhyTTX (4,9-

**Figure 6.** HPLC-FLD \* patterns (top) of fractions from the toxins contained in the ribbon worm *Cephalothrix* sp. in an activated charcoal column chromatography. The bottom patterns represent the

\*HPLC-FLD: high performance liquid chromatography**-**fluorescence detection (HPLC-FLD)

distribution of toxicity in HPLC chromatograms, as estimated by mouse bioassay.

(B): Toxin compositions contained in fraction fr. 85 by HPLC-FLD analysis fr.85(left), TTX standards(right)

**Figure 7.** Elution profile of the ribbon worm *Cephalothrix* sp. toxin from a Bio-Rex 70 column with linear gradient from 0 to 0.03M AcOH

Instrumental Analysis of Tetrodotoxin 257

**4. Mass spectrometry** 

**4.1 Gas-Chromatography-Mass Spectrometry** 

extraction and GC-MS was reported [42].

**4.2. Fast atom bombardment mass spectrometry** 

Fast atom bombardment mass spectrometry (FAB-MS) is a direct method for the qualitative confirmation of TTX. The analysis was performed on a JEOL JMX DX-300 mass spectrometer [43]. Xenon is used to provide the primary beam of atoms, the acceleration voltage of the primary ion being 3 kV. Scanning is repeated within a mass range of *m/z* = 100-500. In this analysis, approximately 0.1 mg of TTX and glycerol are placed as the matrix on the sample

Gas chromatography (GC) and mass spectrometry (MS) form an effective combination for chemical analysis. GC-MS analysis is an indirect method to detect TTX in a crude extract which is difficult to purify in other advanced analysis methods [33]. In this method, TTX and its derivatives are dissolved in 2 ml of 3 M NaOH and heated in a boiling water bath for 30 min. After cooling to room temperature, the alkaline solution of decomposed compounds is adjusted to pH 4.0 with 1N HCl and the resulting mixture is chromatographed on a Sep-Pak C18 cartridge (Waters). After washing with H2O first and then 10% MeOH, 100% MeOH fraction were collected and evaporated to dryness *in vacuo*. To the resulting residue, a mixture of *N*, *O*-bis acetamide, trimethylchlorosilane and pyridine (2: 1: 1) is added to generate trimethylsilyl (TMS) ''C9-base'' compounds (Figure 10). The derivatives are then placed in a Hewlett Packard gas chromatograph (HP-5890-II) equipped with a mass spectrometer (AutoSpec, Micromass Inc., UK). A column (φ 0.25 mm × 250 cm) of UB-5 (GL Sci., Japan) is used, and the column temperature is increased from 180 to 250°C at the rate of 5 or 8°C/min. The flow rate of inlet helium carrier gas is maintained at 20 ml/min. The ionizing voltage is generally maintained at 70 eV with the ion source temperature at 200°C. Scanning was performed in the mass range of *m*/*z* 40–600 at 3s intervals. The total ion chromatogram (TIC) and the fragment ion chromatogram (FIC) were selectively monitored. The TMS derivative of 2-amino-6-hydroxymethyl-8-hydroxyquinazoline (C9 base), prepared using gastropods and ribbon worms from Hiroshima Bay by the procedure described previously, was analyzed by GC-MS [18, 38, 39]. Recently, the isolation and characterization of bacteria from the copepod *Pseudocaligus fugu* ectoparasitie on the panther puffer T*akifugu pardalis* with an emphasis on TTX was reported [40]. The mass spectrum of the peak showed typical ions at *m/z* = 407 and 392, which correspond to M+ and (M-CH3)+ of C9-base-(TMS)3, respectively. Sharp fragment ions appear at *m*/*z* = 407 (parent peak), 392 (base peak) and 376, indicating the presence of quinazoline skeleton in the toxin (Figure 11). It is noteworthy that each peak of selected ion monitored at *m*/*z* = 376, 392 and 407 appears at the same retention time. In the selected ion-monitored mass chromatogram of the TMS derivatives of alkali-hydrolyzed from crystals prepared from ribbon worm in Hiroshima Bay, mass fragment ion peaks at *m*/*z* 376, 392 and 407, which are characteristic of the quinazoline skeleton (C9 base), appeared at retention times (8.33 and 8.34 min.), almost the same as those from the TMS-C9 base derived from authentic TTX (Figure 12). Screening of tetrodotoxin in pufferfish using GC-MS was reported [41]. Sensitive analysis of TTX in human plasma by solid-phase

scale bar = 5 μm

**Figure 8.** Scanning electron micrograph of crystalline toxin isolated from the ribbon worm *Cephalothrix* sp. from Hiroshima Bay.

(A): *Demania cultripes* 

(B): HPLC-FID analysis of the toxin contained in the viscera of *D.cultripes*  (C): TTX standards

**Figure 9.** HPLC-FLD analysis of TTX in the viscera of toxic crab *Demania cultripes* from Cebu Island, in the Philippines

## **4. Mass spectrometry**

256 Chromatography – The Most Versatile Method of Chemical Analysis

**Figure 8.** Scanning electron micrograph of crystalline toxin isolated from the ribbon worm *Cephalothrix*

**Figure 9.** HPLC-FLD analysis of TTX in the viscera of toxic crab *Demania cultripes* from Cebu Island, in

scale bar = 5 μm

sp. from Hiroshima Bay.

(A): *Demania cultripes* 

(C): TTX standards

the Philippines

(B): HPLC-FID analysis of the toxin contained in the viscera of *D.cultripes* 

## **4.1 Gas-Chromatography-Mass Spectrometry**

Gas chromatography (GC) and mass spectrometry (MS) form an effective combination for chemical analysis. GC-MS analysis is an indirect method to detect TTX in a crude extract which is difficult to purify in other advanced analysis methods [33]. In this method, TTX and its derivatives are dissolved in 2 ml of 3 M NaOH and heated in a boiling water bath for 30 min. After cooling to room temperature, the alkaline solution of decomposed compounds is adjusted to pH 4.0 with 1N HCl and the resulting mixture is chromatographed on a Sep-Pak C18 cartridge (Waters). After washing with H2O first and then 10% MeOH, 100% MeOH fraction were collected and evaporated to dryness *in vacuo*. To the resulting residue, a mixture of *N*, *O*-bis acetamide, trimethylchlorosilane and pyridine (2: 1: 1) is added to generate trimethylsilyl (TMS) ''C9-base'' compounds (Figure 10). The derivatives are then placed in a Hewlett Packard gas chromatograph (HP-5890-II) equipped with a mass spectrometer (AutoSpec, Micromass Inc., UK). A column (φ 0.25 mm × 250 cm) of UB-5 (GL Sci., Japan) is used, and the column temperature is increased from 180 to 250°C at the rate of 5 or 8°C/min. The flow rate of inlet helium carrier gas is maintained at 20 ml/min. The ionizing voltage is generally maintained at 70 eV with the ion source temperature at 200°C. Scanning was performed in the mass range of *m*/*z* 40–600 at 3s intervals. The total ion chromatogram (TIC) and the fragment ion chromatogram (FIC) were selectively monitored. The TMS derivative of 2-amino-6-hydroxymethyl-8-hydroxyquinazoline (C9 base), prepared using gastropods and ribbon worms from Hiroshima Bay by the procedure described previously, was analyzed by GC-MS [18, 38, 39]. Recently, the isolation and characterization of bacteria from the copepod *Pseudocaligus fugu* ectoparasitie on the panther puffer T*akifugu pardalis* with an emphasis on TTX was reported [40]. The mass spectrum of the peak showed typical ions at *m/z* = 407 and 392, which correspond to M+ and (M-CH3)+ of C9-base-(TMS)3, respectively. Sharp fragment ions appear at *m*/*z* = 407 (parent peak), 392 (base peak) and 376, indicating the presence of quinazoline skeleton in the toxin (Figure 11). It is noteworthy that each peak of selected ion monitored at *m*/*z* = 376, 392 and 407 appears at the same retention time. In the selected ion-monitored mass chromatogram of the TMS derivatives of alkali-hydrolyzed from crystals prepared from ribbon worm in Hiroshima Bay, mass fragment ion peaks at *m*/*z* 376, 392 and 407, which are characteristic of the quinazoline skeleton (C9 base), appeared at retention times (8.33 and 8.34 min.), almost the same as those from the TMS-C9 base derived from authentic TTX (Figure 12). Screening of tetrodotoxin in pufferfish using GC-MS was reported [41]. Sensitive analysis of TTX in human plasma by solid-phase extraction and GC-MS was reported [42].

#### **4.2. Fast atom bombardment mass spectrometry**

Fast atom bombardment mass spectrometry (FAB-MS) is a direct method for the qualitative confirmation of TTX. The analysis was performed on a JEOL JMX DX-300 mass spectrometer [43]. Xenon is used to provide the primary beam of atoms, the acceleration voltage of the primary ion being 3 kV. Scanning is repeated within a mass range of *m/z* = 100-500. In this analysis, approximately 0.1 mg of TTX and glycerol are placed as the matrix on the sample

258 Chromatography – The Most Versatile Method of Chemical Analysis

Instrumental Analysis of Tetrodotoxin 259

(A) *m/z* = 392 (B) *m/z* = 407 (C) *m/z* = 376

mass spectrum, and an (M-H)-

**Figure 12.** Selected ion-monitored (SIM) mass chromatograms of the trimethylsilyl (TMS) derivatives of

stage of the mass spectrometer, mixed well, and placed in the ion chamber of the spectrometer. Then, both positive and negative mass spectra of TTX are measured. TTX shows (M+H)+ and (M+H-H2O)+ ion peaks at *m/z* 320 and 302, respectively, in the positive

Secondary ion mass spectrometry (SIMS) performed with a Hitachi M-80B mass spectrometer gave essentially the same result as that obtained by FABMS. An extensively purified sample is required for the successful application of this method. Nagashima *et al*. developed a method to detect TTX by TLC-FAB-MS, in which the limit for detection TTX was approximately 0.1 μgTTX. [44]. TTX was also detected clearly by cellulose acetate membrane electrophoresis/FAB-MS, along with selected ion-monitored chromatograms of a

peak at *m/z* 318 in the negative spectrum (Figure 13).

alkali-hydrolyzed toxin from the ribbon worm *Cephalothrix* sp. from Hiroshima Bay.

mixture of TTX, anhydroTTX, and tetrodonic acid (TDA).

**Figure 10.** Reaction pathways from TTX to C9-base-TMS

**Figure 11.** Mass spectrum of the trimethylsilyl (TMS) derivatives of alkali-hydrolyzed toxin from the ribbon worm *Cephalothrix* sp. from Hiroshima Bay.

(A) *m/z* = 392 (B) *m/z* = 407 (C) *m/z* = 376

258 Chromatography – The Most Versatile Method of Chemical Analysis

**Figure 10.** Reaction pathways from TTX to C9-base-TMS

ribbon worm *Cephalothrix* sp. from Hiroshima Bay.

**Figure 11.** Mass spectrum of the trimethylsilyl (TMS) derivatives of alkali-hydrolyzed toxin from the

**Figure 12.** Selected ion-monitored (SIM) mass chromatograms of the trimethylsilyl (TMS) derivatives of alkali-hydrolyzed toxin from the ribbon worm *Cephalothrix* sp. from Hiroshima Bay.

stage of the mass spectrometer, mixed well, and placed in the ion chamber of the spectrometer. Then, both positive and negative mass spectra of TTX are measured. TTX shows (M+H)+ and (M+H-H2O)+ ion peaks at *m/z* 320 and 302, respectively, in the positive mass spectrum, and an (M-H) peak at *m/z* 318 in the negative spectrum (Figure 13). Secondary ion mass spectrometry (SIMS) performed with a Hitachi M-80B mass spectrometer gave essentially the same result as that obtained by FABMS. An extensively purified sample is required for the successful application of this method. Nagashima *et al*. developed a method to detect TTX by TLC-FAB-MS, in which the limit for detection TTX was approximately 0.1 μgTTX. [44]. TTX was also detected clearly by cellulose acetate membrane electrophoresis/FAB-MS, along with selected ion-monitored chromatograms of a mixture of TTX, anhydroTTX, and tetrodonic acid (TDA).

Instrumental Analysis of Tetrodotoxin 261

the protonated molecular ion peak (M + H)+ appeared at *m/z* = 320.1103, suggesting the molecular weight of the toxin to be 319.1025 which agrees well with that of authentic TTX

**Figure 14.** LC-MS of the toxin from the ribbon worm *Cephalothrix* sp. from Hiroshima Bay.

**Figure 15.** ESI-TOF/MS analysis of a frog *Polypedates* sp. toxin [46].

(C11H17N3O8 = 319.1016).

(A): Mass chromatogram of the ribbon worm toxin (B): Mass spectrum of the ribbon worm toxin

**Figure 13.** Positive (right) and negative (left) FAB mass spectra of TTX [41].

### **4.3. Liquid chromatography mass spectrometry**

Liquid chromatography-mass spectrometry (LC-MS) is developed to detect TTX with considerable accuracy [45]. The major disadvantage of LC-FLD is the large difference in the structure-dependent fluorescence intensities of the analogs. In particular, the fluorescence intensities of 5-deoxyTTX and 11-deoxyTTX are approximately 1/20 and less than 1/100 of that of TTX, respectively, while those of 6-*epi*TTX and 11-norTTX-6(*R*)-ol are approximately 20-fold and 10-fold greater than that of TTX, respectively [46]. LC-MS could solve this problem, if sufficient separation and high ionization intensities could be achieved. In this method, combined HPLC-MS is performed using a Hitachi M-1000 system coupled to a mass spectrometer. The HPLC system is equipped with an ODS (φ 1.5 × 150 mm) column. MeOH or acetonitrile (50%, flow rate 70 μl/min) is used as the mobile phase. The effluent from the column is split to provide flow to the ion-spray interface. Brackish water puffer toxins were analyzed by LC-MS [10, 11]. An example of LC-MS of a toxin purified from ribbon worms from Hiroshima Bay is shown in Figure 14. In the MS, a protonated molecular ion peak (M+H)+ appeared at *m/z* = 320 showing a molecular weight for the toxin(319) in good accordance with that of TTX. Tsuruda *et al.*, detected TTX, 4-*epi*TTX, 4, 9-anhydroTTX, 6-*epi*TTX and 4, 9-anhydro-6-*epi*TTX from toxin secreted by newts *Cynops pyrrhogaster* on being subjected to "handling stimulus" [47].

### **4.4. Electrospray ionization – Time of flight – Mass spectrometry**

Electrospray ionization time of flight mass spectrometry (ESI-TOF-MS) is applicable to many fields including the analysis of proteins, natural extracts, synthetic mixtures and medical drugs. ESI-TOF-MS is a valuable technique for identification of TTX, although it is not widely used to date in marine toxin determinations. In this analysis, a portion of purified TTX (less than 0.05 mg) is dissolved in a small amount of 1% AcOH, and the resulting solution is added to 50% aqueous MeOH. ESI-TOF-MS is run on a Micromass Q-TOF mass spectrometer. TTX in a tree frog *Polypedates* sp. extract has been successfully evaluated by ESI-TOF-MS analysis [48]. As shown in the spectrum of the toxin (Figure 15), the protonated molecular ion peak (M + H)+ appeared at *m/z* = 320.1103, suggesting the molecular weight of the toxin to be 319.1025 which agrees well with that of authentic TTX (C11H17N3O8 = 319.1016).

(A): Mass chromatogram of the ribbon worm toxin (B): Mass spectrum of the ribbon worm toxin

260 Chromatography – The Most Versatile Method of Chemical Analysis

**Figure 13.** Positive (right) and negative (left) FAB mass spectra of TTX [41].

Liquid chromatography-mass spectrometry (LC-MS) is developed to detect TTX with considerable accuracy [45]. The major disadvantage of LC-FLD is the large difference in the structure-dependent fluorescence intensities of the analogs. In particular, the fluorescence intensities of 5-deoxyTTX and 11-deoxyTTX are approximately 1/20 and less than 1/100 of that of TTX, respectively, while those of 6-*epi*TTX and 11-norTTX-6(*R*)-ol are approximately 20-fold and 10-fold greater than that of TTX, respectively [46]. LC-MS could solve this problem, if sufficient separation and high ionization intensities could be achieved. In this method, combined HPLC-MS is performed using a Hitachi M-1000 system coupled to a mass spectrometer. The HPLC system is equipped with an ODS (φ 1.5 × 150 mm) column. MeOH or acetonitrile (50%, flow rate 70 μl/min) is used as the mobile phase. The effluent from the column is split to provide flow to the ion-spray interface. Brackish water puffer toxins were analyzed by LC-MS [10, 11]. An example of LC-MS of a toxin purified from ribbon worms from Hiroshima Bay is shown in Figure 14. In the MS, a protonated molecular ion peak (M+H)+ appeared at *m/z* = 320 showing a molecular weight for the toxin(319) in good accordance with that of TTX. Tsuruda *et al.*, detected TTX, 4-*epi*TTX, 4, 9-anhydroTTX, 6-*epi*TTX and 4, 9-anhydro-6-*epi*TTX from toxin secreted by newts *Cynops pyrrhogaster* on

**4.3. Liquid chromatography mass spectrometry** 

being subjected to "handling stimulus" [47].

**4.4. Electrospray ionization – Time of flight – Mass spectrometry** 

Electrospray ionization time of flight mass spectrometry (ESI-TOF-MS) is applicable to many fields including the analysis of proteins, natural extracts, synthetic mixtures and medical drugs. ESI-TOF-MS is a valuable technique for identification of TTX, although it is not widely used to date in marine toxin determinations. In this analysis, a portion of purified TTX (less than 0.05 mg) is dissolved in a small amount of 1% AcOH, and the resulting solution is added to 50% aqueous MeOH. ESI-TOF-MS is run on a Micromass Q-TOF mass spectrometer. TTX in a tree frog *Polypedates* sp. extract has been successfully evaluated by ESI-TOF-MS analysis [48]. As shown in the spectrum of the toxin (Figure 15),

**Figure 14.** LC-MS of the toxin from the ribbon worm *Cephalothrix* sp. from Hiroshima Bay.

**Figure 15.** ESI-TOF/MS analysis of a frog *Polypedates* sp. toxin [46].

### **5. Infrared (IR) spectrometry**

IR spectrometry is the analytical technique for the determination of functional groups in TTX. Although the IR spectrum is presumed to be complex, it is a helpful tool to identify TTX. IR-spectra of KBr pellet were acquired using IR spectrophotometer, which was used by Onoue *et al.* for determination the IR spectrum of pufferfish toxin [49]. On the other hand, Tsuda *et al*. reported the IR spectrum of a TTX-HCl salt by the "Nujol" method [2]. Here we introduce another method as mentioned below. A part of TTX crystals purified from the specimens of ribbon worms were placed on a small KBr plate, and the IR spectrum was acquired using a FT-IR spectrometer (Perkin Elmer, Spectrum 2000) equipped with FT-IR microscope. As shown in Figure 16, absorption bands at 3353, 3235, 1666, 1612 and 1076 cm-1 were observed in the spectrum of this crystal. This spectrum was indistinguishable from that of TTX reported previously, showing characteristic absorptions for the functional groups OH, guanidium, and COO- [2]. The absorption near 2400 cm-1 was derived from the existence of CO2 in the air. The absorption around 1800 cm-1 and in the range of 3,600 – 4,000 cm-1 was derived from H2O in the air. Although the spectrum appears to be complex, it is a helpful tool for identification of TTX.

Instrumental Analysis of Tetrodotoxin 263

**6. Ultraviolet (UV) spectroscopy** 

derivatives in toxic and nontoxic pufferfish [50].

(A): TTX fraction from non toxic species of pufferfish *L. wheeleri* 

pufferfish ("shirosabafugu" in Japanese) *Lagocephalus wheeleri* [48].

(B): Authentic TTX

In UV spectroscopy, TTX is generally determined by irradiating a crude toxin with UV light. A small amount of TTX is dissolved in 2 ml of 2 M NaOH and heated in a boiling water bath for 45 min. After cooling to room temperature, the UV spectrum of the solution is examined for characteristic absorptions, associated with C9-base, 2-amino-6-hydroxymethyl-8 hydroxyquinazoline, possibly formed from TTX and/or related substances, if present. In the analysis, the UVspectrum of the alkali decomposed compounds of TTX appears as a shoulder at near 276 nm, indicating the formation of C9-base specific to TTX or related substances (Figure 17). Saito *et al.* used this method in experiments analyzing TTX and its

**Figure 17.** UV absorption spectra of the alkaline hydrolyzates of TTX fraction from non toxic species of

1H-NMR has played an important role as a complementary method to determine the absolute configuration of TTX. To date, many derivatives of TTX have been isolated, and their 1H-NMR data have been reported by various investigators. In a typical 1H-NMR

**7. Proton nuclear magnetic resonance (1H-NMR) spectrometry** 

IR spectrum was taken on a FT-IR spectrometer (Perkin Elmer, Spectrum 2000) equipped with FT-IR microscope. **Figure 16.** IR spectrum of the toxin isolated from the ribbon worm *Cephalothrix* sp. from Hiroshima Bay.

## **6. Ultraviolet (UV) spectroscopy**

262 Chromatography – The Most Versatile Method of Chemical Analysis

IR spectrometry is the analytical technique for the determination of functional groups in TTX. Although the IR spectrum is presumed to be complex, it is a helpful tool to identify TTX. IR-spectra of KBr pellet were acquired using IR spectrophotometer, which was used by Onoue *et al.* for determination the IR spectrum of pufferfish toxin [49]. On the other hand, Tsuda *et al*. reported the IR spectrum of a TTX-HCl salt by the "Nujol" method [2]. Here we introduce another method as mentioned below. A part of TTX crystals purified from the specimens of ribbon worms were placed on a small KBr plate, and the IR spectrum was acquired using a FT-IR spectrometer (Perkin Elmer, Spectrum 2000) equipped with FT-IR microscope. As shown in Figure 16, absorption bands at 3353, 3235, 1666, 1612 and 1076 cm-1 were observed in the spectrum of this crystal. This spectrum was indistinguishable from that of TTX reported previously, showing characteristic absorptions for the functional groups OH, guanidium, and COO- [2]. The absorption near 2400 cm-1 was derived from the existence of CO2 in the air. The absorption around 1800 cm-1 and in the range of 3,600 – 4,000 cm-1 was derived from H2O in the air. Although the spectrum appears to be complex, it is a

IR spectrum was taken on a FT-IR spectrometer (Perkin Elmer, Spectrum 2000) equipped with FT-IR microscope. **Figure 16.** IR spectrum of the toxin isolated from the ribbon worm *Cephalothrix* sp. from Hiroshima Bay.

**5. Infrared (IR) spectrometry** 

helpful tool for identification of TTX.

In UV spectroscopy, TTX is generally determined by irradiating a crude toxin with UV light. A small amount of TTX is dissolved in 2 ml of 2 M NaOH and heated in a boiling water bath for 45 min. After cooling to room temperature, the UV spectrum of the solution is examined for characteristic absorptions, associated with C9-base, 2-amino-6-hydroxymethyl-8 hydroxyquinazoline, possibly formed from TTX and/or related substances, if present. In the analysis, the UVspectrum of the alkali decomposed compounds of TTX appears as a shoulder at near 276 nm, indicating the formation of C9-base specific to TTX or related substances (Figure 17). Saito *et al.* used this method in experiments analyzing TTX and its derivatives in toxic and nontoxic pufferfish [50].

(A): TTX fraction from non toxic species of pufferfish *L. wheeleri*  (B): Authentic TTX

**Figure 17.** UV absorption spectra of the alkaline hydrolyzates of TTX fraction from non toxic species of pufferfish ("shirosabafugu" in Japanese) *Lagocephalus wheeleri* [48].

## **7. Proton nuclear magnetic resonance (1H-NMR) spectrometry**

1H-NMR has played an important role as a complementary method to determine the absolute configuration of TTX. To date, many derivatives of TTX have been isolated, and their 1H-NMR data have been reported by various investigators. In a typical 1H-NMR

analysis, 5 mg of TTX crystals have been dissolved in 0.5 ml of 1% CD3COOD in D2O, and placed in a test tube. Figure 18 shows the 1H-NMR spectrum obtained with a 500 MHz JEOL JNM-500 spectrometer, using the methyl group protons of acetone as the internal standard [39]. The 1H-NMR spectrum exhibited a singlet at 2.20 ppm (CH3COCH3), a doublet centered at 2.33 ppm (J =10.0 Hz), a large proton peak at 4.76 ppm (HDO) and a doublet centered at 5.48 ppm (J=10.0 Hz). The pair of doublets around 2.33 and 5.48 ppm, which are the hallmarks of TTX and are assigned to H-4a and H-4, respectively, have been confirmed to be coupled with each other by double irradiation (Figure 19). These results agree well with the corresponding data of TTX. The signals at 4.24, 4.06, 4.28, 3.94, 4.00 and 4.02 ppm are assigned toH-5, H-7, H-8, H-9 and H-11, respectively (Figure 20). A toxin isolated from the horseshoe crab *Carcinoscorpius rotundicauda* from Bangladesh was analyzed by HPLC-FLD, TLC, electrophoresis and 1H-NMR, and was identified as TTX [51]. Identification of a neurotoxin from the blue-ringed octopus, brackish water pufferfish, marine pufferfish and so on as TTX via this method were reported [9, 10, 11, 17].

Instrumental Analysis of Tetrodotoxin 265

Five milligrams of the ribbon worm toxin was dissolved in 0.5ml of 1% CD3COOD in D2O and measured for 1H-NMR

spectrum measured on a JEOL JNM-500NMR spectrometer, using acetone as the internal standard.

Hiroshima Bay by means of irradiation at C4-H.

Hiroshima Bay, along with the structure of TTX.

**Figure 19.** 1H-NMR spectrum of the toxin isolated from the ribbon worm *Cephalothrix* sp. from

**Figure 20.** 1H-NMR spectral data of the toxin isolated from the ribbon worm *Cephalothrix* sp. from

Five milligrams of the ribbon worm toxin was dissolved in 0.5ml of 1% CD3COOD in D2O and measured for 1H-NMR spectrum measured on a JEOL JNM-500NMR spectrometer, using acetone as the internal standard.

**Figure 18.** 1H-NMR spectrum of the toxin isolated from the ribbon worm *Cephalothrix* sp. from Hiroshima Bay.

so on as TTX via this method were reported [9, 10, 11, 17].

analysis, 5 mg of TTX crystals have been dissolved in 0.5 ml of 1% CD3COOD in D2O, and placed in a test tube. Figure 18 shows the 1H-NMR spectrum obtained with a 500 MHz JEOL JNM-500 spectrometer, using the methyl group protons of acetone as the internal standard [39]. The 1H-NMR spectrum exhibited a singlet at 2.20 ppm (CH3COCH3), a doublet centered at 2.33 ppm (J =10.0 Hz), a large proton peak at 4.76 ppm (HDO) and a doublet centered at 5.48 ppm (J=10.0 Hz). The pair of doublets around 2.33 and 5.48 ppm, which are the hallmarks of TTX and are assigned to H-4a and H-4, respectively, have been confirmed to be coupled with each other by double irradiation (Figure 19). These results agree well with the corresponding data of TTX. The signals at 4.24, 4.06, 4.28, 3.94, 4.00 and 4.02 ppm are assigned toH-5, H-7, H-8, H-9 and H-11, respectively (Figure 20). A toxin isolated from the horseshoe crab *Carcinoscorpius rotundicauda* from Bangladesh was analyzed by HPLC-FLD, TLC, electrophoresis and 1H-NMR, and was identified as TTX [51]. Identification of a neurotoxin from the blue-ringed octopus, brackish water pufferfish, marine pufferfish and

Five milligrams of the ribbon worm toxin was dissolved in 0.5ml of 1% CD3COOD in D2O and measured for 1H-NMR

spectrum measured on a JEOL JNM-500NMR spectrometer, using acetone as the internal standard.

Hiroshima Bay.

**Figure 18.** 1H-NMR spectrum of the toxin isolated from the ribbon worm *Cephalothrix* sp. from

Five milligrams of the ribbon worm toxin was dissolved in 0.5ml of 1% CD3COOD in D2O and measured for 1H-NMR spectrum measured on a JEOL JNM-500NMR spectrometer, using acetone as the internal standard.

**Figure 19.** 1H-NMR spectrum of the toxin isolated from the ribbon worm *Cephalothrix* sp. from Hiroshima Bay by means of irradiation at C4-H.


**Figure 20.** 1H-NMR spectral data of the toxin isolated from the ribbon worm *Cephalothrix* sp. from Hiroshima Bay, along with the structure of TTX.
