As a test for tetrodotoxin, a biological method using mice is long being used for testing fi sh tissues; but it is not suitable for blood and urine specimens of poisoned patients.. In th
Trang 1© Springer-Verlag Berlin Heidelberg 2005
by Sunao Fukushima and Yukio Ohtsuka
Introduction
Japanese people are very fond of eating fugu (puff er) fi shes (especially Takifugu rubripes) as a
feast especially in the winter season However, the fi sh contains highly poisonous toxin tetro-dotoxin (TTX) especially in its liver and ovary Th erefore, the number of fugu (puff er) fi 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
Th e origins of tetrodotoxin, the presence of its analogs (> Figure 3.1) and its mechanisms
of action have been being clarifi ed [1,2] However, for the tetrodotoxin analogs (> Figure 3.1),
their presence, toxicokinetics and toxicities especially in human puff er fi sh poisoning cases have not been studied Th 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 fi 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
The number
of fatality
Mortality ratio (%)
1980–1984 143 242 47 19.4
1985–1989 147 256 29 11.3
1990–1994 138 221 13 5.9
1995–1999 126 193 17 8.8
Total 554 912 106 11.6
Structures of tetrodotoxin (TTX) and its analogs.
⊡ Figure 3.1
Trang 2482 Tetrodotoxin
GC/MS analysis
TTX is soluble only in acidic alcohols; it cannot be analyzed it by GC/MS in its unchanged form TTX should be treated with alkali to form 2-amino-6-hydroxymethyl-8-hydroxyquinazoline (so-called “ C9 base”, M.W 191), which can be trimethylsilylated [C9 base-(TMS)3, M.W 407] for
GC/MS analysis (> Figure 3.2).
Reagents and their preparation
i Reagents
TTXb, diethylamine, trifl uoroacetic acid (TFA), β-estradiol (BD) and
N,O-bis(trimethylsilyl)tri-fl uoroacetamide (BSTFA) can be purchased from Sigma (St Louis, MO, USA); SylonBFT (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 Aft 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 Th 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
Trang 3duced pressure; the resulting yellow residue (C9 base) is dissolved in 10 mL of ethanol contain-ing 2 % acetic acid (C9 base standard solution, 100 µg/mL)
GC/MS conditions
GC columnc: a DB-5 fused silica capillary column (30 m × 0.25 mm i.d., fi lm thickness 5 µm, J&W Scientifi c, Folsom, CA, USA)
Conditions; instrument: a quadrupole GC/MS instrumentd; column (oven) temperature:
220 °C → 5 °C/min → 250 °C; injection temperature: 250 °C; detector temperature: 280 °C; carrier gas: He; its fl ow rate: 1.0 mL/min; injection mode: splitless
Procedures
i Procedure 1 (body fluid and tissue specimens) [4]
i A 2-mL volume of a body fl uid specimen (or well-homogenized suspension of a 2-g tissue specimen) is extracted with 30 mL methanol containing 2 % acetic acid 2–3 times with refl uxing for 10 min each
ii Th e extracts are combined, passed through a fi lter paper and evaporated to dryness under reduced pressure
iii If necessary, the residue is degreased with diethyl ethere, dissolved in 5 mL of 0.5 % acetic acid solution and neutralized with 5 % KOH solution
iv Th e above solution is passed through the fi rst Sep-Pak C18 cartridgef (Waters, Milford, MA, USA), which had been pretreated by passing 5 mL methanol and 10 mL distilled water A 3-mL volume of distilled water is also passed through it; both fi ltrates are combined
v Th e fi ltrate solution is mixed with 20 % KOH solution to adjust its pH to 9–10, and
heat-ed at 100 °C for 20 ming
vi Aft er cooling to room temperature, the above solution is neutralized with concentrated HCl solution, and poured into the 2nd Sep-Pak C18 cartridgeh, followed by washing with
2 mL water and 2 mL of methanol/distilled water (2:8); the target compound is eluted with 5 mL methanol
vii Th 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 Tefl on cap, and again evaporated to dryness under reduced pressurei
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 Aft er cooling to room tempera-ture, 10 µL diethylamine is added to the mixture to neutralize it A 1-µL aliquot of the
fi nal solution is injected into GC/MSj,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 refl uxing in a water bath with heating
ii Th e mixture is centrifuged at 3,000 rpm for 5 min, and the resulting supernatant solution
is evaporated to dryness under reduced pressure
iii Th e residue is shaken with 10 mL of 0.1 % acetic acid aqueous solution/chloroform (1:1)
Trang 4484 Tetrodotoxin
iv It is centrifuged at 3,000 rpm for 5 min; the aqueous phase is passed through the fi rst Sep-Pak PS-2 cartridge m (Waters)
v Th e fi ltrate is mixed with a half volume of 3 M KOH aqueous solution and heated for
15 min in a boiling water bath; aft er cooling to room temperature, the solution is neutral-ized with 2 M HCl solution
vi Th e above solution is mixed with 17 µL TFA and 10 mL of 0.1 M phosphate buff er (pH 7.0) and poured into the 2nd Sep-Pak PS-2 cartridgem
vii Just before the completion of the fl ow through the cartridge, the fl ow is stopped and left for 60 min n; then the cartridge is washed with 10 mL of purifi ed water and dried by pass-ing air through it
viii Th e C9 base is eluted from the cartridge with 5 mL of acetonitrile containing 0.5 % acetic acid and evaporated to dryness under a stream of nitrogen
ix Th e residue is dissolved in a small amount of acetonitrile containing 0.5 % acetic acid and transferred to a small glass vial with a Tefl on cap, and again evaporated to dryness in it
x Th e residue is dissolved in 19 µL dimethylformamide containing 0.2 % BD, mixed with 22 µL BSTFA and 1 µL TMCS, capped airtightly and heated at 95 °C for 10 min for derivatization
x Aft er cooling to room temperature, the above solution is neutralized with diethylamine;
a 2-µL aliquot of the fi nal solution is injected into GC/MS
Assessment of the methods
i Procedure 1
By GC/MS analysis of C9 base-(TMS)3, a mass spectrum is obtained as shown in > Figure 3.3 Peaks appear at m/z 407, 392 and 376 [6] Th e mass spectral profi le can be used for identifi ca-tion Th e base peak at m/z 392 is used for quantitation with its peak areas by the external
cali-bration method Th e detection limit by this method is about 1 ng/mL
ii Procedure 2
Th e three ions are used for qualitative analysis Th 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] Th e detection limit by this method is 0.5 ng/mL l in blood plasma
EI mass spectrum of the C base-(TMS)
⊡ Figure 3.3
Trang 5HPLC analysis
Yasumoto et al [7, 8] developed a TTX analyzer by combining an HPLC instrument with a
fl 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 Th 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 Aft er separation, TTX is decomposed to a fl uorescent compound (a stable intermediate in the middle of reaction into the C9 base) in a reaction box by alkaline solution, which is supplied
by Pump B; the fl uorescent compound is measured with the fl uorescence detector aft er cooling the reaction solution to room temperature
To obtain the best conditions of this system, the concentration of the alkaline solution, re-action temperature, and excitation and emission wavelengths should be optimized
Reagents and their preparation
Th ey are almost the same as described in the GC/MS section Sodium dodecyl sulfate can be obtained from Sigma
HPLC conditions
HPLC column: Inertsil ODS-2 (250 × 4.6 mm i d., GL Sciences, Tokyo, Japan) or other columns of similar qualityo
Conditions; pumps: LC-10AD for both; fl uorescence detector: RF-550; reaction box: CRB-6A; reaction coil in the box: stainless steel tube to be used for HPLC (15 m × 0.25 mm i.d.) (all obtained from Shimadzu Corp., Kyoto, Japan); mobile phase for Pump A (fl ow rate):
50 mM sodium potassium phosphate buff er containing 2 mM sodium dodecyl sulfate (pH 6.8)
Schema of an HPLC system for analysis of TTX.
⊡ Figure 3.4
Trang 6486 Tetrodotoxin
(0.4 mL/min); mobile phase for Pump B (fl ow rate): 4 M NaOH (0.4 mL/min)p; reaction box temperature: 120 °C; exitation wavelength: 400 nm; emission wavelength: 495 nmq
Procedure
i A 2-mL volume of a urine specimen is mixed with 0.5 mL of 0.5 % acetic acid solution, and poured into a Bond Elut SCX (Varian, Harbor City, CA, USA) cartridge, which had been equilibrated with 5 mL purifi ed water, 5 mL methanol and 5 mL of 0.1 % acetic acid solu-tion
ii Th e cartridge is washed with 2 mL of 0.1 % acetic acid solution, 2 mL methanol and 4 mL purifi ed water
iii TTX is eluted from the cartridge with 4 mL of 0.1 % sodium potassium phosphate buff er (pH 7.0); 20-µL of the eluate is injected into HPLC
iv Th is method is applicable only to urine specimens at the present time
Assessment of the method
Aft er addition of a fi 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
Th 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 aft 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
Th 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,
Trang 7the victim cooked Fugu niphobles in a large amount, was poisoned by eating its liver and died
just aft er arrival at a hospital In Case No 3, the victim was found dead in a ship on ocean navigation In Case No 4, the victim was found dead in an automobile in a public park Th e cases Nos 3 and 4 were treated as unnatural death In other cases, all patients recovered aft er being admitted in hospitals In Cases Nos 2, 5, 9 and 11, the subjects landed fugu fi shes by themselves and ate them aft er cooking to be poisoned; they were treated as self-negligence cases In Case No 6, the victim bought the skin of a fugu fi sh at a store and was poisoned; in Cases Nos 7, 8, 10 and 13, the victims ate fugu dishes containing the organs (the liver and/or skin) of the fi sh at restaurants All of these cases were treated as negligent homicide incidents; the store and restaurants were ordered to suspend their business Th e accidents occurred due
to erroneous knowledges on cooking methods of fugu fi shes
> Figure 3.5 shows the plots of blood TTX concentrations on a calibration curve; its
con-centrations in the fatal cases are higher than those in the survived cases It can be estimated that borderline blood concentrations between the fatal and survived cases seem to be about
100 ng/mL, which is in accordance with that reported by Suenaga et al [6] At 25–100 ng/mL
of TTX in blood, paresthesia, verbal paralysis, disappearance of various refl exes and fi nally respiratory paralysis appear as poisoning symptoms; however, in these cases, by early emer-gency treatments, they could survive
⊡ Table 3.3
TTX concentrations in human specimens in its poisoning
Victim (age/sex) Specimen Concentration
(ng/mL)
Outcome
No 1 (93/F) blood
urine
93.0 650
dead
No 2 (62/M) blood 36.3 recovered
No 3 (34/M) blood 175 dead
No 4 (65/M) blood 320 dead
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
15.3 245
recovered
Trang 8488 Tetrodotoxin
Periods for TTX excretion into urine
Urine is a very advantageous specimen, because it can be obtained noninvasively and contains relatively high concentrations of poisons and relatively low contents of impurities; these advan-tages make its analysis simple and rapid As shown in > Table 3.3, among 13 victims, urine
specimens were available for 8 victims; only one was fatal among the 8 victims with her urinary concentration being 650 ng/mL Th e TTX excretion into urine was monitored as a function of time aft er ingestion > Figure 3.6 shows the time course of TTX excretion into urine obtained
by sampling stockpiled urine every 12 h up to 3 days for Cases 6 and 9 listed in > Table 3.3
For both subjects, the excretion was highest in the period from 12 to 24 h; TTX was detectable from urine even on the 3rd day Unfortunately, urine specimens could not be obtained aft er
3 days; therefore, it is not clear how long the urinary TTX is detectable aft er ingestion
Calibration curve for blood TTX.
⊡ Figure 3.5
Urinary excretion of TTX as a function of time after ingestion.
⊡ Figure 3.6
Trang 9a) Th ese fi gures were obtained from data collected by Bureau of Medical Drugs, Ministry of Health, Labour and Welfare of Japan
b) TTX is not commercially available as pure crystals; TTX powder for a biochemical use (about 99 %) is available
c) Packed columns (5 % SE-52, 1 m × 3 mm i.d.) or wide-bore capillary columns ( DB-17,
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) Th e extent and times of washings of the residue with diethyl ether are diff erent in diff erent specimens (impurities or lipid contents); some skillfulness based on experience is required for the technique
f) Th e fi 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 C9 base is completed in about
10 min; aft er 30 min of heating, the recovery becomes much lower
h) Th e 2nd Sep-Pak C18 cartridge is used for extraction of the C9 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) Th e column should be fi 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; aft er cooling to room tempera-ture, the mixture is neutralized with 10 µL diethylamine [C9 base-(TMS)3 derivative mix-ture, total volume 300 µL] and 3-µL of the solution is injected into GC/MS
Formation rates of the C base as a function of time of heating TTX at 100 °C.
⊡ Figure 3.7
Trang 10490 Tetrodotoxin
l) Th e procedure 2 gives higher sensitivity than the procedure 1 However, for actual
mea-surements of poisoning specimens (> Table 3.3), the procedure 1 had been used.
m) For activation of Sep-Pak PS-2 cartridges, methanol, purifi ed water, acetonitrile containing 0.5 % acetic acid, purifi ed water, 1.68 % (50 mM) EDTA aqueous solution and purifi ed water, 3 mL each, were passed though them For the 2nd cartridge, 3 mL of 0.1 M sodium potassium phosphate buff er (pH 7.0) was added at the end for activation Like the above Sep-Pak C18 cartridges, the fi rst Sep-Pak PS-2 cartridge is used only for removal of hydro-phobic impurities and the 2nd one for extraction of the C9 base produced by alkali treat-ment from TTX
n) To secure the complete adsorption of the neutral C9 base to the cartridge, the 60 min inter-val is necessary
o) For the TTX analyzer, Hitachi gel 3011C (400 × 5 mm i d.) (GL Sciences) is being used p) Since the concentration of NaOH is very high, the whole line system should be washed completely aft er use
q) Th e ranges of various conditions used for preliminary optimization experiments were: NaOH concentration: 1–5 M; reaction box temperature: 100–140 °C; excitation wave-length: 390–410 nm; emission wavewave-length: 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)