6.3
6.3
© Springer-Verlag Berlin Heidelberg 2005
II.6.3 Tetrodotoxin
by Sunao Fukushima and Yukio Ohtsuka
Introduction
Japanese people are very fond of eating fugu (pu er)  shes (especially Takifugu rubripes) as a
feast especially in the winter season. However, the  sh contains highly poisonous toxin tetro-
dotoxin (TTX) especially in its liver and ovary.  erefore, the number of fugu (pu er)  sh
poisoning incidents in Japan were 554, in which 912 people were poisoned during 20 years
in 1980–1999; among the 912 people, 106 subjects were fatal (mortality rate, 11.6 %)
(
> Table 3.1)
a
. However, in recent 10 years, its incidence has been reduced to about half due
to the improvement of early life-saving systems in emergency medicine.
 e origins of tetrodotoxin, the presence of its analogs (
> Figure 3.1) and its mechanisms
of action have been being clari ed [1,2]. However, for the tetrodotoxin analogs (
> Figure 3.1),
their presence, toxicokinetics and toxicities especially in human pu er  sh poisoning cases have
not been studied.  erefore, in this chapter, the target is limited to tetrodotoxin only. As a test
for tetrodotoxin, a biological method using mice is long being used for testing  sh tissues; but it
is not suitable for blood and urine specimens of poisoned patients. In this chapter, the methods
of analysis of tetrodotoxin in human specimens by GC/MS and HPLC are described [3].
⊡ Table 3.1
Incidence of fugu (puffer) fish poisoning cases in Japan
Year The number
of incidents
The number
of patients

(BSTFA/TMCS, 99:1) from Supelco (Bellefonte, PA, USA); trimethylchlorosilane (TMCS)
from Pierce (Rockford, IL, USA). Other common chemicals used were of the highest purity
commercially available.
ii. TTX standard solution
A 1-mg aliquot of TTX is dissolved in 10 µL acetic acid and diluted with distilled water to
prepare the standard 10 mL solution (100 µg/mL).
iii. C
9
base standard solution
A 1-mg aliquot of TTX is dissolved in 0.5 mL of 5 % KOH solution and heated at 100 °C for
30 min. A er cooling it to room temperature, the solution is neutralized with concentrated
HCl solution, saturated with KCl (by addition of KCl if necessary) and extracted with 5 mL of
n-butanol three times.  e combined n-butanol extracts are evaporated to dryness under re-
Pretreatment and derivatization procedures for TTX for its GC/MS analysis.
⊡ Figure 3.2
483
duced pressure; the resulting yellow residue (C
9
base) is dissolved in 10 mL of ethanol contain-
ing 2 % acetic acid (C
9
base standard solution, 100 µg/mL).
GC/MS conditions
GC column
c
: a DB-5 fused silica capillary column (30 m × 0.25 mm i.d.,  lm thickness 5 µm,
J&W Scienti c, Folsom, CA, USA).
Conditions; instrument: a quadrupole GC/MS instrument
d
; column (oven) temperature:

2 mL water and 2 mL of methanol/distilled water (2:8); the target compound is eluted
with 5 mL methanol.
vii.  e eluate is evaporated to dryness under reduced pressure, and the residue is dissolved
in a small amount of methanol containing 0.5 % acetic acid and transferred to a small
glass vial with a Te on cap, and again evaporated to dryness under reduced pressure
i
.
viii. A 10-µL aliquot of dimethylformamide and 30 µL of SylonBFT are placed in the above
vial, capped airtightly and heated at 100 °C for 10 min. A er cooling to room tempera-
ture, 10 µL diethylamine is added to the mixture to neutralize it. A 1-µL aliquot of the
 nal solution is injected into GC/MS
j,k
.
ii. Procedure 2
l
(blood plasma) [5]
i. A 2-mL volume of a plasma specimen is mixed with 30 mL of methanol containing 2 %
acetic acid and extracted with re uxing in a water bath with heating.
ii.  e mixture is centrifuged at 3,000 rpm for 5 min, and the resulting supernatant solution
is evaporated to dryness under reduced pressure.
iii.  e residue is shaken with 10 mL of 0.1 % acetic acid aqueous solution/chloroform (1:1).
GC/MS analysis
484 Tetrodotoxin
iv. It is centrifuged at 3,000 rpm for 5 min; the aqueous phase is passed through the  rst
Sep-Pak PS-2 cartridge
m
(Waters).
v.  e  ltrate is mixed with a half volume of 3 M KOH aqueous solution and heated for
15 min in a boiling water bath; a er cooling to room temperature, the solution is neutral-
ized with 2 M HCl solution.

ii. Procedure 2
 e three ions are used for qualitative analysis.  e quantitation is performed using the peak
area ratios of the ion at m/z 392 to that at m/z 285 [the base peak of BD-(TMS)
2
].  e detection
limit by this method is 0.5 ng/mL
l
in blood plasma.
EI mass spectrum of the C
9
base-(TMS)
3
.
⊡ Figure 3.3
485
HPLC analysis
Yasumoto et al. [7, 8] developed a TTX analyzer by combining an HPLC instrument with a
 uorophotometer; it enables separation of TTX from crude biological matrices and its quanti-
tation. Fuchi et al. [9] determined TTX in sea foods by a similar method.  e authors [10] also
tried to measure the compound in urine of poisoned patients; the scheme of the system is
shown in
> Figure 3.4.
A specimen mixed with a mobile phase enters a separation column by the action of Pump
A. A er separation, TTX is decomposed to a  uorescent compound (a stable intermediate in
the middle of reaction into the C
9
base) in a reaction box by alkaline solution, which is supplied
by Pump B; the  uorescent compound is measured with the  uorescence detector a er cooling
the reaction solution to room temperature.
To obtain the best conditions of this system, the concentration of the alkaline solution, re-

iii. TTX is eluted from the cartridge with 4 mL of 0.1 % sodium potassium phosphate bu er
(pH 7.0); 20-µL of the eluate is injected into HPLC.
iv.  is method is applicable only to urine specimens at the present time.
Assessment of the method
A er addition of a  xed amount of TTX to urine, its concentration is measured by HPLC and
conventional GC/MS (Procedure 1); the values obtained by the methods were almost the same.
 erefore, it was concluded that the quantitation of TTX can be made even by HPLC [10].
For urine specimens obtained from TTX poisoning cases, the quantitation was made by
both methods as shown in
> Table 3.2; each case showed very similar values obtained by both
methods [11]. Except for Case No. 1, the three patients listed in
> Table 3.2 showed poison-
ing symptoms of intermediate severity, but recovered a er treatments.
⊡ Table 3.2
Comparison of measurements of tetrodotoxin (TTX) in urine by HPLC with those by GC/MS in
actual poisoning cases
Victim (age/sex) TTX concentration (ng/mL)
HPLC GC/MS
No. 1 (22/M) 2,550 2,480
No. 2 (51/F) 146 132
No. 3 (52/M) 152 135
No. 4 (39/M) 301 288
Poisoning cases and toxic concentrations
Blood TTX concentrations and fatal levels
 e authors also experienced the analysis of TTX in blood and/or urine of 13 subjects in TTX
poisoning. As shown in
> Table 3.3, the number of blood specimens was 12 in 13 cases.
Among the poisoned subjects, 3 subjects were fatal and 10 recovered. In Case No. 1 of the table,
487
the victim cooked Fugu niphobles in a large amount, was poisoned by eating its liver and died

No. 5 (39/M) blood
urine
12.0
295
recovered
No. 6 (57/M) blood
urine
35.5
78.5
recovered
No. 7 (50/M) blood
urine
6.5
443
recovered
No. 8 (?/M) blood
urine
2.5
105
recovered
No. 9 (35/M) blood
urine
63.9
27.2
recovered
No. 10 (39/M) urine 68.5 recovered
No. 11 (51/M) blood 37.9 recovered
No. 12 (?/M) blood 16.1 recovered
No. 13 (49/M) blood
urine

15 m × 0.53 mm i.d.) can be also used. However, in view of separation ability and con-
tamination, medium-bore capillary columns are preferable.
d) For example, Shimadzu QP5050A, Shimadzu 1100EX or HP5971A can be used.
e)  e extent and times of washings of the residue with diethyl ether are di erent in di erent
specimens (impurities or lipid contents); some skillfulness based on experience is required
for the technique.
f)  e  rst Sep-Pak cartridge is not used for extraction of TTX, but used only for removal of
hydrophobic impurities being contained in TTX specimens.
g) As shown in
> Figure 3.7, the conversion of TTX into C
9
base is completed in about
10 min; a er 30 min of heating, the recovery becomes much lower.
h)  e 2nd Sep-Pak C
18
cartridge is used for extraction of the C
9
base produced by the alkali
treatment.
i) It is essential to dry it up completely for silylation; it is sometimes dried up under reduced
pressure in the presence of phosphorus pentaoxide.
j)  e column should be  lled with the silylating reagent gas. When the silylating reagent
only is injected into GC/MS between the injections of sample extracts, reproducibility of
the assay may be enhanced.
k) When a packed column is used for GC/MS, the residue is mixed with 80 µL DMF, 200 µL
BSTFA and 10 µL TMCS and heated at 100 °C for 10 min; a er cooling to room tempera-
ture, the mixture is neutralized with 10 µL diethylamine [C
9
base-(TMS)
3

NaOH concentration: 1–5 M; reaction box temperature: 100–140 °C; excitation wave-
length: 390–410 nm; emission wavelength: 485–505 nm.
References
1) Hashimoto C (ed) (1988) Recent Advance of Fugu Toxin Studies. Koseisha-koseikaku, Tokyo (in Japanese)
2) Noguchi T, Arakawa O, Hashimoto C (1989) Fugu poison: its origins and the mechanisms of toxigenicity. Jpn J
Food Hyg Assoc 30:281–288 (in Japanese with an English abstract)
3) Matsui T, Ohtsuka Y, Sakai J (2000) Recent advance of studies on fugu toxin. Yakugaku Zasshi 10:825–837 (in
Japanese with an English abstract)
4) Fukushima S (1992) Analysis of tetrodotoxin in body fluids and tissues. Reports of Studies by the 9th Trainees
of Forensic Science Training Institute, Forensic Science Training Institute, Tokyo, pp 285–295 (in Japanese)
5) Ohtsuka Y, Tokunaga H, Fukushima S et al. (1997) Sensitive determination of tetrodotoxin in serum by GC/MS.
Proceedings of TIAFT XXXV Annual Meeting, Padova, Italy, pp 614–618
6) Suenaga K, Kotoku S (1980) Detection of tetrodotoxin in autopsy material by gas chromatography. Arch Toxicol
44:291–297
7) Yasumoto K (1989) Analysis and applications of marine toxins. Chemistry and Biology 27:401–406 (in
Japanese)
8) Yasumoto T, Mitishita T (1985) Fluorometric determination of tetrodotoxin by high performance liquid chroma-
tography. Agr Biol Chem 49:3077–3080
9) Fuchi Y, Morisaki S, Nagata T et al. (1988) Determination of tetrodotoxin in sea foods by high-performance liquid
chromatography. Jpn J Food Hyg Assoc 5:306–312 (in Japanese with an English abstract)
10) Fukushima S (1996) Trace analysis of tetrodotoxin in human specimens. Simple determination by HPLC with
fluorescence detection. Abstracts of the 116th Annual Meeting of the Pharmaceutical Society of Japan, p 188
(in Japanese)
11) Fukushima S (1996) Analysis of tetrodotoxin in specimens of poisoned victims. Jpn J Toxicol 9:473–474 (in
Japanese)