Toxicon 54 (2009) 6266

Contents lists available at ScienceDirect

Toxicon
journal homepage: www.elsevier.com/locate/toxicon

Rapid extraction combined with LC-tandem mass spectrometry

(CREM-LC/MS/MS) for the determination of ciguatoxins
in ciguateric sh esh
Richard J. Lewis*, Aijun Yang, Alun Jones
Institute for Molecular Bioscience, The University of Queensland, Qld 4072, Australia

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 17 October 2008
Received in revised form 11 March 2009
Accepted 12 March 2009
Available online 19 March 2009

Ciguatera is a signicant food borne disease caused by potent polyether toxins known as
ciguatoxins, which accumulate in the esh of ciguateric sh at risk levels above 0.1 ppb.
The management of ciguatera has been hindered by the lack of analytical methods to
detect and quantify clinically relevant levels of ciguatoxin in easily prepared crude extracts
of sh. Here we report a ciguatoxin rapid extraction method (CREM) that allows the rapid
preparation of sh esh extracts for the detection and quantication of ciguatoxin by
gradient reversed-phase liquid chromatography-tandem mass spectrometry (LC/MS/MS).
CREM-LC/MS/MS delivers a linear response to P-CTX-1 spiked into sh prior to extraction.
A similar response was obtained for P-CTX-1 spiked after extraction, indicating >95%
extraction efciency was achieved overall and 85% at the limit of quantication (0.1 ppb).
Using this approach, levels 0.1 ppb P-CTX-1 could be detected and quantied from an
extract of 2 g sh esh, making it suitable as a conrmatory assay for suspect ciguateric
carnivorous sh in the Pacic Ocean. The approach is designed to simplify the extraction
and analysis of multiple samples per day.
Crown Copyright 2009 Published by Elsevier Ltd. All rights reserved.

Keywords:
Ciguatera
HPLC
Multiple reactant monitoring
Analytical detection
Solid phase extraction
Monitoring

1. Introduction
Ciguatera (sh poisoning) is a major economic and
social problem throughout tropical and sub-tropical
waters, with w 25,000 persons poisoned annually (Lewis,
2001). The disease is characterised by neurological and
gastrointestinal disorders, which typically appear from 1 to
24 h following the consumption of contaminated sh and
can last for months or longer (Gillespie et al., 1986). The
toxins involved are potent sodium channel activator toxins
known as ciguatoxins (Lewis et al., 2000) that are produced
by the benthic dinoagellate Gambierdiscus spp. (Lewis and
Holmes, 1993; Holmes and Lewis, 2002). The ciguatoxins
and structurally related brevetoxins compete at site 5 on

* Corresponding author: Tel.: 617 3346 2984; fax: 617 3346 2090.
E-mail address: r.lewis@imb.uq.edu.au (R.J. Lewis).

voltage sensitive sodium channel (Lombet et al., 1987). Two

related families of Pacic ciguatoxins (P-CTX) have been
identied in ciguateric Pacic Ocean sh (Murata et al.,
1990; Lewis et al., 1991, 1993; Satake et al., 1993). A third
family of Caribbean ciguatoxins (C-CTX) contaminate
ciguateric sh of the Caribbean Sea (Lewis et al., 1998), with
a fourth family of Indian Ocean ciguatoxins contaminating
sh of the Indian Ocean (Hamilton et al., 2002a,b). All
ciguatoxins identied to date are heat stable polyether
toxins of 10231157 Da. P-CTX-1 remains the most potent
ciguatoxin characterised (Lewis et al., 1991), often
contributing w90% of the total lethality of carnivorous
ciguateric sh capture in the western Pacic Ocean (Lewis
and Sellin, 1992), and posing a health risk at levels 0.1 ppb
(Lewis, 2001).
The traditional method of detecting the presence of
ciguatoxins in sh involves testing lipid extracts by the
mouse bioassay (Lewis and Sellin, 1993). More recently,

0041-0101/$ see front matter Crown Copyright 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2009.03.013

R.J. Lewis et al. / Toxicon 54 (2009) 6266

cytotoxicity (Manger et al., 1995), radioligand binding (Poli

et al., 1997) and antibody-based sandwich (Oguri et al.,
2003) assays have been shown to have potential as costeffective screens for the detection of ciguateric sh.
Analytical liquid chromatography-tandem mass spectrometry (LC/MS/MS) procedures have also been developed for
determining ciguatoxins in sh extracts (Lewis et al., 1999).
However, lack of a rapid extraction procedure to simplify
ciguatoxin analysis limits the usefulness of this approach. In
this report, we describe a ciguatoxin rapid extraction
method (CREM) combined with gradient reversed-phase
liquid chromatography-tandem mass spectrometry (CREMLC/MS/MS) approach for the detection and quantication of
clinically relevant levels of P-CTX-1 in sh esh.
2. Materials and methods
2.1. Extraction of sh
Coral trout (Plectropomus maculatus), a species implicated in ciguatera outbreaks in Queensland, was obtained
from commercial outlets in Queensland, diced and kept
frozen at 20  C until use. Two gram portions of the sh
were either spiked with P-CTX-1 (Lewis et al., 1991) or left
unspiked before being cooked at 70  C for 20 min in capped
50 ml Falcon tubes. Samples were then cooled before
homogenisation (IKA Ultra Turrax T25, setting 5) with 8 ml
methanol/hexane (3:1) until no lumps of sh remained
(2  30 s). The homogenate was centrifuged at 4000 rpm
for 20 min at RT, the upper hexane phase carefully removed
using a pasture pipette and discarded, and the lower
aqueous methanol phase (w5.5 ml) transferred into
a single use syringe and ltered through a 0.45-mm Millipore aqueous membrane lter (Millex@-HA) into a glass
vial. The extraction procedure is designed for use on sh
esh with normal water content and variations in water
content may inuence extraction efciency.
2.2. Solid phase extraction (SPE) of the crude extract
The crude extract was adjusted to 5055% aqueous
methanol by the addition of 2 ml H2O, before cleanup
through a C18 SPE cartridge. A number of reverse-phase
C18 cartridge types and elution conditions were trialled.
The optimised procedure used a 900 mg C18 SPE cartridge
(Alltech Prevail Maxi-Clean) pre-conditioned with 4 ml of
water before loading the w7.5 ml sample and a 0.5 ml 65%
methanol rinse of the vial. Initial studies showed that
spiked P-CTX-1 started eluting during the wash step when
methanol was 70%, while no P-CTX-1 was detected in
65% aqueous methanol washes. To maximise cleanup
without the loss of analyte, the cartridge was washed with
6 ml 65% methanol (discarded) before P-CTX-1 was eluted
with 8 ml of 80% aqueous methanol.
To reduce matrix interference and the lengthy drying
step associated with removal of water from the C18 cleanup
step, an additional orthogonal normal phase SPE cleanup
step was developed. To prepare the sample for this step, the
80% methanol fraction was collected into a 50 ml Falcon
tube and made more polar by the addition of 4.2 ml of 1 M
NaCl, before extraction with 6.7 ml chloroform with

63

vigorous shaking. The resulting two phases were separated

by centrifugation (w2000 rpm for 4 min on a bench-top
centrifuge), the upper aqueous methanol phase discarded,
and the lower chloroform phase transferred to a scintillation vial and dried under N2 and low heat to remove any
remaining methanol and water, which affect normal phase
SPE cleanup. After comparing a number of commercially
available normal phase SPE cartridges, we selected a silica
SPE cartridge (Silica Plus, Waters), pre-conditioned with
4 ml chloroform and loaded the sample in 4 ml chloroform
and a 0.5 ml chloroform rinse of the vial. The cartridge was
then washed with 4 ml chloroform before P-CTX-1 was
eluted with 8 ml chloroform/methanol (9:1). For both SPE
cleanup steps, syringe assisted positive displacement was
used to shorten the conditioning, sample loading, washing
and nal elution steps.
To validate the method, unspiked sh esh samples
were either spiked with P-CTX-1 just prior to the nal N2
drying step, or left unspiked as negative controls. All
samples were evaporated under N2 and low heat before
being redissolved in 200 ml of 50% aqueous methanol prior
to LC/MS/MS analysis. To conrm its suitability for naturally
contaminated sh, a ciguateric giant queensh (Scomberoides commersonnianus) implicated in human ciguatera
poisoning in Queensland was also extracted and analysed
by CREM-LC/MS/MS.
2.3. LC/MS/MS
The LC system comprised a C18 column (5 mm Phenomenex Luna, 2.1  250 mm) tted with a pre-column
(Phenomenex C18, 4  2.1 mm, 5 mm). Solvent A was
aqueous 2 mM ammonium formate and 0.1% formic acid,
and Solvent B was 95% acetonitrile with 2 mM ammonium
formate and 0.1% formic acid. The column was eluted at
400 ml/min with a linear gradient from 35% B to 100% B over
5 min. 100% B was held for 2 min before returning to 35% B
at 7.1 min. The column was then equilibrated for 5 min with
35% B prior to the next run, allowing a 12 min turn-around
between analyses.
Two triple-quadrupole mass spectrometers with TurboIon-Spray ionization (AB Sciex Instruments) were used for
the detection of P-CTX-1 in this study. Both mass spectrometers detected positive ions using multiple reactant
monitoring (MRM) with resolution for both Q1 and Q3 set
at low. The API 2000 was employed for the initial development work optimising the reversed-phase C18 SPE
cleanup step. However, the API 4000 (QTRAP), with its
greater sensitivity due in part to its orthogonal spray source
and design of the collision cell, was used for subsequent
analyses. MS/MS conditions were established using pure
P-CTX-1 ([M NH4] m/z 1128.7) dissolved in 50% B and
injected directly into the mass spectrometer at 10 mL/min.
MS/MS signals were optimised for the dominant product
ions originating from the [M NH4] ion. For the API 4000,
a declustering potential of 110 V and a collision energy of
32 eV were used (CUR 25, TEM 300, EP 10 and CXP 15). As
found previously using an API-III triple-quadrupole MS
(Lewis et al., 1999), three dominant fragment ions could
readily be generated from the [M NH4] ion of P-CTX-1
(m/z 1128.7 / 1093.7; 1128.7 / 1075.7; 1128.7 / 1057.7)

64

R.J. Lewis et al. / Toxicon 54 (2009) 6266

allowing the sensitive detection of P-CTX-1 in sh extracts

by LC/MS/MS. The peak height above baseline was
measured after summing the individual response data to
generate a TIC response for each analysis, with averaged
data presented as the mean  standard deviation.
To minimise the extent of matrix effects on MS response
to P-CTX-1, various injection volumes from 5 to 100 ml of the
200 ml sample after silica cleanup were analysed by LC/MS/
MS. The optimal volume was found to be 20 mL, which was
used for the analysis of all the samples and pure P-CTX-1.
2.4. Chemicals used
Chloroform (99.8% pure, Ajex Finechem) contained
ethanol, methanol or alkyl phenols stabilizers at <0.2%. All
other solvents used were HPLC grade or equivalent.
3. Results and discussion
3.1. Rapid extraction of ciguatoxins
Previous approaches to the extraction of the lipid
soluble ciguatoxins are cumbersome. Most procedures
typically extract 50100 g of sh with acetone, dry the
ltrate, remove low polarity lipids from an aqueous

methanol phase with hexane (2), remove solvent, extract

ether-solubles and P-CTX-1 from an aqueous ethanol phase
with ether (3) and solvent removal. At this point the
extract can be assayed using mice (Lewis, 2003) or run on
a silica column before LC/MS/MS analysis (Lewis et al.,
1999). To improve the efciency of ciguatoxin analysis, we
have developed an optimised ciguatoxin rapid extraction
method (CREM) that simplies the detection and quantication of P-CTX-1 by LC/MS/MS. CREM streamlines the
extraction of ciguatoxin from sh esh by (i) replacing the
initial acetone extraction and ltration step with a one-step
extraction and hexane cleanup, (ii) reducing the number
transfers and drying steps, (iii) using centrifugation to
separate phases, (iv) keeping the liquidliquid extractions
to single steps, and (v) reducing the quantity of sh esh
extracted (2 g) to allow the incorporation of two
orthogonal SPE cleanup steps. A ow diagram of CREM is
shown in Fig. 1. The initial cooking step, which is included
to denature sh esh proteins and improve extraction
efciency, can be skipped when cooked sh are analysed.
3.2. LC/MS/MS
In parallel with the development of CREM, we developed a gradient HPLC procedure that provided sufcient

Fig. 1. Flow diagram of the ciguatoxin rapid extraction method (CREM). CREM was designed to streamline the initial extraction of ciguatoxin and incorporates
two orthogonal solid phase extraction (SPE) cleanup steps prior to LC/MS/MS analysis. The ltration step uses an aqueous Millipore lter and the two evaporation
steps are performed under a stream of N2 with mild heating, avoiding the need to use a rotavapor. The SPE wash volumes indicated include 0.5 ml used to rinse
the sample vial. The conditioning and wash steps (1 and 3) are discarded, and ciguatoxin elutes at step 4. Given the similar physical and chemical properties of the
different Pacic Ocean, Caribbean Sea and Indian Ocean ciguatoxins (Lewis, 2001), CREM may have applicability to the extraction of multiple CTXs prior to
analysis.

R.J. Lewis et al. / Toxicon 54 (2009) 6266

separation between the remaining lipids and P-CTX-1 with

a relatively quick (12 min) turn-around time between
sample analyses. Finally, conditions for P-CTX-1 detection
using an API 4000 QTRAP MS were optimised. Using this
approach, pure P-CTX-1 was readily quantied by LC/MS/
MS, giving a linear response from 6 to 600 pg (Fig. 2). In
contrast, responses to P-CTX-1 were reduced by w75% after
spiking into non-toxic sh extract, presumably due to the
presence of lipids coeluting with P-CTX-1 that reduce the
efciency of the ion evaporation/ionisation process and
increase baseline noise. Despite this reduction in sensitivity, a linear response was obtained after spiking 0.26 ng
P-CTX-1 directly into 2 g of non-toxic sh esh. An equivalent response was also obtained when P-CTX-1 was spiked
into an extract from 2 g sh just prior to solvent evaporation (Fig. 2). Comparing the slopes of responses obtained
for spiking before and after extraction (see Fig. 2) gives an
estimated recovery of P-CTX-1 from sh esh of >90%,
a signicant improvement over more traditional extraction
procedures (Lewis and Sellin, 1993). The lower limit of
quantication of P-CTX-1 in 2 g sh was w20 pg using
a 20 ml injection. Comparing responses obtained for spiking
0.2 ng (0.1 ppb) P-CTX-1 before (270  62) and after
(316  112) extraction estimates recovery of 85% for P-CTX1 spiked at 0.1 ppb. Thus the approach allows the quantication of P-CTX-1 in sh esh at levels above 0.1, making it
sufciently sensitive to detect the clinically relevant levels
of P-CTX-1 (0.15 ppb) contaminating the esh of ciguateric carnivorous sh in the Pacic Ocean.
Examples of the LC/MS/MS response to 600 pg P-CTX-1
spiked into non-toxic sh esh for m/z 1128.7 / 1093.7,
1128.7 / 1075.7, and 1128.7 / 1057.7 are shown in Fig. 3A.
Also shown is a control (unspiked sample) for comparison.
These data illustrate the lack of background signal and the
clear individual MS responses obtained. These data were
summed to give the TIC traces shown in Fig. 3B which were
used to quantify P-CTX-1. Care needs to be taken when
analysing responses to pure P-CTX-1, which we found were
reduced by 95% if pure P-CTX-1 was prepared in plastic
instead of glass prior to analysis (data not shown).
However, P-CTX-1 with sh extract gave similar responses
in glass and plastics, indicating that the sh matrix
minimise ciguatoxin absorption to plastic. CREM-LC/MS/

65

Fig. 3. Gradient reversed-phase LC/MS/MS response to P-CTX-1. (A) Coeluting responses detected at m/z 1128.7 / 1093.7; 1128.7 / 1075.7600;
1128.7 / 1057.7 obtained for 600 pg P-CTX-1 spiked into non-toxic sh
esh prior to extraction. Matching responses (1075/1093/1057) obtained for
unspiked sh esh samples are also shown for comparison. (B) TIC obtained
for 20, 60, 200 and 600 pg P-CTX-1 spiked into non-toxic sh esh prior to
extraction.

MS was also used to quantify P-CTX-1 in a suspect ciguateric sh esh sample. Using this approach, 0.6  0.053 ppb
P-CTX-1 (range 0.480.74 ppb, n 4) was detected,
consistent with the sample being implicated in ciguatera
poisoning. Unfortunately, lack of a validated analytical
method for ciguatoxin precluded a direct assessment of
interfering substances that may be present in wild caught
ciguateric shes.
We also investigated removing the silica SPE cleanup
step from CREM. This truncated procedure resulted in
noticeable quantities of lipid remaining after removal of
solvent and solubilisation of extract in 200 ml of 50%
aqueous methanol was more difcult. Despite the poorer
solubility and additional matrix, both the C18 alone and
C18 silica procedures gave similar MS responses for
P-CTX-1 spiked into sh esh before cooking (data not
shown). However, increased background noise reduced the
limit of detection to w0.3 ppb for the truncated method.
Thus, while it is possible to analyse extracts after only C18
cleanup, optimisation of the LC/MS/MS method is required
to allow detection of all clinically relevant samples
contaminated with P-CTX-1 using a truncated method.
4. Conclusions

Fig. 2. Graph of LC/MS/MS total ion current (TIC) responses obtained for
different concentrations of either pure P-CTX-1, P-CTX-1 spiked into 2 g raw
sh esh prior to cooking at 70  C, or P-CTX-1 spiked into crude extracts of
2 g sh esh. Linearity (r2) was >0.98 across the three sets of data
(mean  SD, n 6). Slopes  SE (an index of sensitivity) are indicated in the
inset.

CREM-LC/MS/MS simplies the detection and quantication of clinical relevant levels of ciguatoxin in crude
extracts of sh. The method provides signicant improvements in sensitivity and is designed to allow multiple
analyses per day. Given their similar physical and chemical
properties, CREM/LC/MS/MS should be investigated to

66

R.J. Lewis et al. / Toxicon 54 (2009) 6266

determine its applicability to detect and quantify clinically

relevant levels of C-CTX-1 (Lewis et al., 1999) and I-CTX-1
(Hamilton et al., 2002b) in suspect sh from the Caribbean
Sea and Indian Ocean, respectively. As MS instrumentation
improves, it is anticipated that the limits of detection
reported here will be further lowered to allow further
miniaturisation and the detection of the full range of
ciguatoxins contributing to ciguatera. Once appropriate
standards are available, CREM-LC/MS/MS could have
applicability in environmental monitoring studies aimed at
developing a better understanding of the distribution and
factors inuencing ciguatera risk.
Acknowledgements
The work was supported by an ARC Special Research
Centre for Functional and Applied Genomics. We thank Dr.
Geoff Eaglesham for advice on optimising the MS conditions for ciguatoxins.
Conict of interest
There is no conict of interest in the reported work.
References
Gillespie, N.C., Lewis, R.J., Pearn, J.H., Bourke, A.T., Holmes, M.J., Bourke, J.B.,
Shields, W.J., 1986. Ciguatera in Australia: occurrence, clinical features,
pathophysiology and management. Med. J. Aust. 145, 585590.
Hamilton, B., Hurbungs, M., Jones, A., Lewis, R.J., 2002b. Multiple ciguatoxins present in Indian Ocean reef sh. Toxicon 40, 13471353.
Hamilton, B., Hurbungs, Mira, Vernoux, Jean-Paul, Jones, Alun,
Lewis, Richard J., 2002a. Isolation and characterisation of Indian
Ocean ciguatoxin. Toxicon 40, 685693.
Holmes, M.J., Lewis, R.J., 2002. Dinoagellate toxins. In: Menez, A. (Ed.),
Perspectives in Molecular Toxinology. John Wiley and Son, Ltd, West
Sussex, pp. 3965.
Lewis, R.J., 2001. The changing face of ciguatera. Toxicon 39, 97106.

Lewis, R.J., 2003. Detection of ciguatoxins. In: Hallegraeff (Ed.), UNESCO

Monographs on Oceanographic Methodology, Manual on Harmful
Marine Microalgae. UNESCO, pp. 253263.
Lewis, R.J., Holmes, M.J., 1993. Origin and transfer of toxins involved in
ciguatera. Comp. Biochem. Physiol. 106C, 615628.
Lewis, R.J., Sellin, M., 1992. Multiple ciguatoxins in the esh of sh. Toxicon 30, 915919.
Lewis, R.J., Sellin, M., 1993. Recovery of ciguatoxin from sh esh. Toxicon
31, 13331336.
Lewis, R.J., Sellin, M., Poli, M.A., Norton, R.S., MacLeod, J.K.,
Sheil, M.M., 1991. Purication and characterization of ciguatoxins
from moray eel (Lycodontis javanicus, Muraenidae). Toxicon 29,
11151127.
Lewis, R.J., Norton, R.S., Brereton, I.M., Eccles, C.D., 1993. Ciguatoxin-2 is
a diastereomer of ciguatoxin-3. Toxicon 31, 637643.
Lewis, R.J., Vernoux, J.-P., Brereton, I.M., 1998. Structure of Caribbean
ciguatoxin isolated from Caranx latus. J. Am. Chem. Soc. 120,
59145920.
Lewis, R.J., Jones, A., Vernoux, J.P., 1999. HPLC/tandem electrospray mass
spectrometry for the determination of sub-ppb levels of Pacic and
Caribbean ciguatoxins in crude extracts of sh. Anal Chem 71,
247250.
Lewis, R.J., Molgo, J., Adams, D.J., 2000. Pharmacology of toxins involved
in ciguatera and related sh poisonings. In: Botana, L. (Ed.), Marine
Pharmacology. Marcel Dekker, Inc., New York, pp. 419447.
Lombet, A., Bidard, J.N., Lazdunski, M., 1987. Ciguatoxin and brevetoxins
share a common receptor site on the neuronal voltage-dependent
Na channel. FEBS Lett. 219, 355359.
Manger, R.L., Leja, L.S., Lee, S.Y., Hungerford, J.M., Hokama, Y., Dickey, R.W.,
Granade, H.R., Lewis, R., Yasumoto, T., Wekell, M.M.J., 1995. Assoc. Off.
Anal. Chem. 78, 521527.
Murata, M., Legrand, A.M., Ishibashi, Y., Yasumoto, T., 1990. Structures and
congurations of ciguatoxin from the moray eel Gymnothorax javanicus and its likely precursor from the dinoagellate Gambierdiscus
toxicus. J. Am. Chem. Soc. 112, 43804386.
Oguri, H., Hirama, M., Tsumuraya, T., Fujii, I., Maruyama, M., Uehara, H.,
Nagumo, Y., 2003. Synthesis-based approach toward direct sandwich
immunoassay for ciguatoxin CTX3C. J. Am. Chem. Soc. 125,
76087612.
Poli, M.A., Lewis, R.J., Dickey, R.W., Musser, S.M., Buckner, C.A.,
Carpenter, L.G., 1997. Identication of Caribbean ciguatoxins as the
cause of an outbreak of sh poisoning among U.S. soldiers in Haiti.
Toxicon 35, 733741.
Satake, M., Murata, M., Yasumoto, T., 1993. The structure of CTX3C,
a ciguatoxin congener isolated from cultured Gambierdiscus toxicus.
Tetrahedron Lett. 34, 19751978.