Vol.4, No.5B, 1-6 (2013) Agricultural Sciences
Determination of okadaic acid related toxins from
shellfish (sinonovacula constricta) by high
performance liquid chromatography tandem
mass spectrometry
Hai-qi Zhang1,2*, Weicheng Liu3, Xin He4, Li-jun Liang1, Wenyong Ding3, Zhong-yang He1
1Zhejiang Fisheries Quality Testing Centre, Hangzhou, China; *Corresponding Author: zmk407@126.com
2College of life science, Zhejiang University, Hangzhou, China
3College of Food Science and Biotechnology Engineering, Zhejiang Gongshang University, Hangzhou, China
4Zhejiang Mariculture Research Institute, Wenzhou, China
Received 2013
Consumption of shellfish contaminated with algal
toxins produced by marine dinoflagellates can
lead to diarrhetic shellfish poisoning (DSP). It
was therefore essential that there are analytical
techniques to iden tify and quanti fy DSP toxins i n
shellfish. This new methodology could facilitate
DSP monitoring and create a means of rapidly
responding to incidents threatening public health.
In the last years there were different analytical
methods for DSP, such as mouse bioassay and
LC-FLD. With the development of instrument,
Liquid chromatography-mass spectrometry was
substituted for other analytical methods with its
good sensitivity and selectivity and without de-
rivatization for the determination of DSP. In this
report, a high performance liquid chromatogra-
phy-t andem mass spectr ometricHPLC-MS/MS
method was developed for the simultaneous
determination of okadaic acid (OA) and dino-
physistoxins(DTX-1) in Sinonovacula constricta.
Optimization of pretreatment experiment was
carried out to maximize recoveries and the ef-
fectiveness. The analytes were determined un-
der multi-reactions monitoring (MRM) scan type
with tandem mass analyzer using negative ion
electrospray ionization (-ESI) mode .Finally, the
detection and identification of OA and DTX-1
were based upon their retention times (RT) and
the fragmentation patterns of their mass spectra.
The method of LOQ for the t wo poisons was 0.02
mkg-1.The real sample test showed that this
method could be used for sensitive, fast, and
accurate determination of the two diarrheic
shellfish poisons in shellfish.
Keywords: Sinonovacula Con stri cta; High
Performance Liquid Ch romatogra phy-Ta ndem Mass
Spectrometry; Okadaic Acid; Dinoph ys istoxins-1
Among the phycotoxin-related toxic phenomena, Di-
arrhetic Shellfish poisoning (DSP) is a severe gastroin-
testinal illness caused by consumption of shellfish con-
taminated with toxigenic dinoflagellates. Toxins respon-
sible for DSP intoxication belong to the group of the
lipophilic marine biotoxins. The main cause of world-
wide DSP syndrome (Yasomotor et al., 1993) are oka-
daic acid (OA) and its derivatives named dinophysistox-
ins (DTXs). These toxins have been shown to be potent
phosphatase inhibitors, a property which can cause in-
flammation of the intestinal tract and diarrhea. OA and
its 35-methyl derivative named dinophysistoxin-1 (DTX-1)
(Figure 1) have also been shown to have tumors-pro-
moting activity (Sylvaine et al., 2002). In order to pre-
vent human intoxication, many monitoring program of
shellfish toxicity have been established in many devel-
oped countries. According to the current regulation with
respect to this issue, the maximum permitted level for
marketable shellfish is 0.16 μg OA equivalent/g shellfish
meat (Emilia et al., 2008).
Figure 1. Structure of Okadaic acid (OA) and Dinophysistox-
ins-1 (DTX-1).
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H.-Q. Zhang et al. / Agricultural Sciences 4 (2013) 1-6
Although, routine monitoring of shellfish for DSP tox-
ins is generally carried out using mouse bioassay (Pam-
ela et al., 1997; Nuria et al., 2007), this approach suffers
from poor reproducibility, low sensitivity, and interfere-
ences from certain endogenous compounds. Thus, in-
strumental methods offer the possibility of precise, sensi-
tive and automated determination of the individual DSP
toxins. The most of the previous studies on determination
of DSP toxin profiles employed methods that targeted
acidic polyether toxins. In fact, such toxins can be deri-
vatized by using fluorometric derivatization reagents, which
namely dramatization 9-anthryldiazomethane (ADAM)
(Kevin et al., 1997), 4-bromo-methyl-7-methoxycoumarin
(Br-Mmc) (Shen et al., 1997), 1-bromoacetylpyrene (BAP)
(Jose´C et al., 2000), 3-bromomethyl-6,7-dimethoxy-1-
methyl-2(1H)-quinoxalinone (BrDMEQ) and 9-chloro-
methylanthracene (CA) (Nogueiras et al., 2003), then
followed by quantification using liquid chromatography
with fluorimetric detection(LC-FLD).The major disad-
vantage of this method is that toxins lacking the carboxylic
acid functionality cannot be revealed. Although these
were obvious improvements of the LC methods based on
fluorometric derivatization reagents, they were unstable
and not always available.
In the latest decade, liquid chromatography coupled
with mass spectrometry (LC–MS) using atmospheric-
pressure ionization (API) has proven to be the most
valuable instrumental tool for direct determination of
toxins without derivatization (Rosa et al., 1995; Toshi-
yuki and Takeshi, 2000; Shinya and Katsuo, 2001; Pa-
trizia et al., 2006). It has both high sensitivity and selec-
tivity, which makes it possible to determine DSP by di-
rect injection. Meanwhile, Liquid chromatography–tan-
dem mass spectrometry (LC–MS/MS) (Lincoln Mackenzie
et al., 2002; Patricia et al., 2004; Suzuki et al., 2004;
Beatriz et al., 2007) has been shown to be a valuable
analytical tool for identifying and quantifying the shell-
fish poisons and their metabolites. Moreover, it is a par-
ticularly useful method for handing very small samples
with low analytic concentrations.
The primary aim of this work was to develop a rapid
and sensitive method for the simultaneous determination
and confirmation of OA, DTX-1 in shellfish at low levels
by means of high performance liquid chromatography
tandem mass spectrometry (HPLC–MS/MS).
2.1. Materials and Reagents
Shellfish used as the negative control and for spiking
and for test were collected from the southeastern coast,
Zhejiang, China in June 2009. The adductor muscle and
digestive glands were separated from other tissues, ho-
mogenized and kept frozen at -20 until used.72 sam-
ples collected from 8 different areas were tested (Figure 2).
Figure 2. Origins of Sinonovacula constricta.
HPLC grade solvents (acetonitrile, methanol) and ana-
lytical grade solvents (n-hexane, chloroform, acetone,
acetic acid) were purchased from Tedia (Ohio, USA).
Distilled water was passed through a Milli-Q water puri-
fication system (Millipore, France). Sep-Pak silica plus
cartridge columns (500 mg, 3 mL) were purchased from
Supelco (Milford, MA, USA). Purified OA standard (
95%) was purchased from Sigma–Aldridge (Dublin, Ire-
land), DTX-1(90%) was purchased from Wako(Osaka,
2.2. Sample Extraction and Purification
The Homogenized shellfish hepatopancreas (2 g) was
mixed with 10 mL 80% methanol for 1min in a 50 mL
polypropylene tube, after ultrasonic extraction during 5
min, then centrifuged for 10 min at 4000 rmin-1. An ali-
quot (5 mL) of the supernatant was transferred to another
15 mL tube, washed with 5 mL hexane. The hexane layer
was aspirated to waste and 1 mL water was added to the
residual solution, and then was extracted with 6 mL
chloroform. The water layer was transferred to another
15 mL tube and extracted with chloroform (2 mL×3 mL)
again. The chloroform extracts were combined and
evaporated to dryness under nitrogen at 60and recon-
stituted in 1ml 20% hexane-acetone.
A Sep-Pak silica cartridge was conditioned sequen-
tially with 10mL acetone, 10 mL methanol and 10 mL
20% hexane/acetone. Then the column was load with the
1mL extract sample and washed with 1ml 20% hex-
ane/acetone followed by 10 mL 3% methanol/acetone.
After drying, the remaining toxins were eluted with 10
mL 40% methanol/acetone and evaporated to dryness
under nitrogen at 45.Then the residue was dissolved in
1mL 80% methanol. Finally the extract was filtrated
through 0.22 μm organic filter and analyzed by HPLC-
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H.-Q. Zhang et al. / Agricultural Sciences 4 (2013) 1-6
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2.3. HPLC-MS/MS Analysis
HPLC-MS/MS was performed on an HP 1100 series
liquid chromatograph (Agilent, Palo Alto,CA, USA),
coupled to an API 3000 triple quadrupole mass spec-
trometer (Applied Biosystem) with an atmospheric pres-
sure ionization source and an electrospray ionization
(ESI) interface. The instrumentation was controlled using
Analyst v.1.2 software.
Chromatographic separations of OA and DTX-1 were
carried out under the following combinations of column
and mobile phases: Zorbax XDB C18 (2.1 mm × 150
mm, 5 μm, Agilent) with the mobile phase, acetonitrile -
0.1% acetic acid(70:30,v/v). The column temperature
and flow-rate were kept at 30and 0.25 mL·min-1, re-
spectively. 10 μL of sample were injected onto the col-
umn at the room temperature.
The mass spectrometer was operated by electrospray
in negative ion mode (ESI-) with multiple reaction moni-
toring (MRM) for the detection of OA, DTX-1. The
monitored ions were the [MH] precursor ions at m/z of
803.6 (OA), 817.4 (DTX-1), respectively and the most
abundant product ion observed for each toxin. The MS
parameters were optimized for the ionization of standard
toxins using flow injection analysis. Two different prod-
uctions were used to verify the selectivity for determi-
nation of OA and DTX-1 as shown in Table1. The opti-
mized MRM experiment was established for the concur-
rent determination of the aforementioned toxins using the
following conditions: Ionspray Voltage -4500 V, Auxil-
iary Gas Speed 7 L·min-1, Turbo Ionspray Source Tem-
perature 500, Nebulizer Gas 9 psi, Curtain Gas 8 psi,
Collision Gas 8 psi, Focusing Potential -260 V, Entrance
Potential -9 V, and Cell Exit Potential -13 V. All gases in
the MRM experiment were high-purity nitrogen gas. Other
optimization of MS conditions as shown in Table 1.
2.4. HPLC-MS/MS Ass ay
Stock solutions (10 mg·L-1) of individual shellfish
toxin standards (OA, DTX-1) were prepared by dissolving
in methanol. A mixed stock solution (1 mg·L-1) contain-
ing two standards was prepared from stock solutions of
individual standards by mixing and diluting with metha-
nol. Different calibration standards (20, 50, 100, 200, 500,
800 μg·L-1) were prepared by appropriate dilution of the
mixed stock solution with methanol. The standards were
injected directly into the HPLC-MS/MS system. The
calibration curve was obtained by the peak area (y-axis)
plotted against the concentration of toxins standard
(χ-axis). The qualitative analysis of OA and DTX-1 of
the experimental samples were performed based on the
retention time and the ion ration of standard solution.
2.5. The Experiment of Recovery, Precision
and Accuracy
Homogenized negative shellfish hepatopancreas (2 g),
which spiked with 0.02, 0.1, 0.2 and 0.4 mg·kg-1 mixed
standard solution of OA and DTX-1 respectively, were
pretreated as section 2.2 and then analyzed by HPLC-
MS/MS. Three replicate samples at each concentration
were analyzed on the same day. The percentage of re-
covery was calculated by comparing the concentration
obtained according to the calibration curve with the ac-
tual spiked concentration of standard solution (OA,
DTX-1). The precision was evaluated by coefficients of
variation (CV %) and the accuracy was estimated based
on the average percentage of recovery.
3.1. HPLC-MS/MS Condition Analysis
The mass spectra of each compound were measured in
the positive and the negative ion modes for the precursor
ion full-scan of toxins standard (1 mg·L-1). It was found
that the detection sensitivity for the toxins studied was
better in negative rather than in positive mode with the
precursor ion [MH] at m/z 803.6 for OA, m/z 817.4 for
DTX-1, respectively. The fragmentation of the target
toxins was optimized to efficiently generate several
product ions from each precursor ion by collision-in-
duced dissociation (CID) and shown in Figure 3. Select-
ing two precursor/product ion combinations (Q1/Q3
pairs) as monitor ions to verify the selectivity and deter-
mination of toxins, which were m/z 803.6/255.0,
803.6/563.4 for OA, m/z 817.4/255.0 and 817.4/113.1 for
DTX-1 and shown in table 1. To achieve optimum sensi-
tivity and selectivity, MRM was implemented and the
optimization of MS conditions as shown in the afore-
mentioned experiment.
Table 1. Optimization of the partial MS condition.
Analyte Precursor ionm/z Product ions (m/z)Declustering PotentialV Collision EnergyV Retention timemin
Okadaic Acid
(OA) 803.6 255.1*
563.1 -70 -60
-68 3.38
(DTX-1) 817.4 255.1*
113.1 -110 -68
-94 5.70
*Quantificational ion.
H.-Q. Zhang et al. / Agricultural Sciences 4 (2013) 1-6
The polar solvent (methanol, acetonitrile) usually is used
as mobile phase for reverse phase column C18. It was
found that the efficiency of ionization with 70% metha-
nol was inferior to that obtained in 70% acetonitriler;
meanwhile ionization efficiency would be intensified
with 0.1% acetic acid (Toshiyuki and Takeshi, 2000). So
70% acetonitrile containing 0.1% acetic acid was se-
lected as the mobile phase of LC-MS/MS.
3.2. Sample Extraction and Purification
Ta b l e 2 listed the recovery of the preliminary extrac-
tion and elution with different solutions at the spiked
level of 0.1 mg·kg-1 from standard toxins. The recovery
of toxins from 80% methanol extracts were 94.2% for
OA and 90.6% for DTX-1, slightly lower than 80% ace-
tontril extracts. Besides the 90% methanol (Hirofumi et
al., 2001) gave least residue than other organic solvent
(methanol, acetone) as extractant. Considering that the
toxicity of acetontrile was more harmful than methanol
and wasted more time during evaporation under nitrogen.
And there is no obvious difference for the recovery be-
tween 80% and 90% for methanol-water. Thus, experi-
ment chose 80% methanol as extractant insuring against
good recovery.
Figure 3. Product ions full-scan negative-ion ESI mass spec-
trums of OA (up) and DTX-1(down).
Purification of extractant (OA and DTX-1) from the
shellfish hepatopancreas was carried out by Sep-pak sil-
ica cartridge as previously described (Patrizia et al., 2006)
due to the significant suppression of ionization by con-
taminants. In test of elution for the toxins using three
different rates of acetone–methanol, it shows that the
recovery in 40% methanol/acetone was the highest (99%
for OA, 97.8% for DTX-1) than others.
3.3. Method Evaluation (Recovery, Precision
and Accuracy)
Figure 4 showed the chromatogram of the toxins (OA,
DTX-1) standard with 200 μg·L-1. From which it could
Table 2. Comparison of extract and elution from different solu-
tions at the spiked level of 0.1 mg·kg-1.
Recovery (%, n = 6)
SolutionSolution component
80% Methanol- water 94.2 90.6
solution 80% Acetontrile- water 96.0 92.5
40% Methanol-acetone 99.0 97.8
50% Methanol-acetone 93.2 95.2
60% Methanol-acetone 91.5 84.4
Figure 4. Chromatograms of the OA and DTX-1 standard
solution (200 μg·l-1)(up) and the negative spiked sample
(0.1 mg· kg-1) (down).
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H.-Q. Zhang et al. / Agricultural Sciences 4 (2013) 1-6 5
be seen there was a single and symmetric peak and the
retention time is 3.4 min for OA with the monitor ion
pairs m/z 803.6/255.0, 803.6/563.4 and 5.7 min for
DTX-1 with m/z 817.4/255.0, 817.4/113.1The down of
Figure 4 showed the chromatogram of negative sample
was spiked with 0.1mg·kg-1. And it could be seen there
was no obvious interferential peaks when the sample was
pretreated as described above to the section 2.2 experi-
ment and detected with HPLC-MS/MS. The good linear-
ity of the peak area plotted against concentrations for the
toxins with the linear (OA:у = 20-241, r = 0.9995;
DTX-1: у = 141χ-85, r = 0.9997) over concentration
ranging from 20 μg·L-1 to 800 μg·L-1.
Table 3 gives the recovery and coefficients of varia-
tion (CV %) data corresponding to negative samples that
were spiked each with 0.02,0.1,0.2 and 0.4 mg OA and
DTX-1 per 1kg of the hepatopancreas . The average re-
covery of OA and DTX-1 were decreased with the spiked
level from 0.02 to 0.4 mg·kg-1 because of the matrix ef-
fect. This suggested that interferential compounds of the
matrix reduced ionization efficiency of toxins. It is ac-
ceptant that the mean recoveries were within the range
from 79.0% to 92.2%, and the CV% was lower than
11.6%. The signal to noise(S/N) was calculated from the
ratio between analyte peak signal to base line and
peak-to- peak noise signal. The S/N was above 10 for the
mixed standard solution (20 μg·L-1). The LOQ of the
method at an S/N ratio of 10 were estimated to 0.02
mg·kg-1 for the both of OA and DTX-1.
3.4. Method Application
The developed method was applied to the analysis of
72 batches of that were collected from 8 different areas
in Zhejiang province. Ta b le 4 showed the results of the
14 postive samples. 2 samples were found OA, with the
concentration of 25.6 µg·kg-1 and 33.0 µg·kg-1, repec-
tively. 12 samples were found DTX-1, with the concen-
tration from 84.1 µg·kg-1 to 293.0 µg·kg-1.
Table 3. Recovery and CV% for the OA and DTX-1.
Analyte Spiked level
Recovery (%,n = 6)
(mean ± S.D) CV (%)
0.02 89.4 ± 7.9 8.87
0.1 87.9 ± 10.2 11.6
0.2 84.7 ± 7.3 8.57
0.4 79.0 ± 8.5 10.8
0.02 92.2 ± 3.8 4.09
0.1 90.8 ± 4.1 4.51
0.2 89.9 ± 6.3 6.98
0.4 84.2 ± 4.7 5.54
Table 4. Results of the postive samples.
No.Sampling time Sampling
1 August,2007 Shensi DTX-1293.0
2 Spetember,2007 Shensi DTX-1208.4
3 December,2007 Shensi OA25.6
4 March,2008 Putuo DTX-1155.0
5 June,2008 Putuo DTX-1192.4
6 Spetember,2007 Xiangshan
7 October,2007 Xiangshan
8 August,2007 Shanmen OA33.0
9 Spetember,2007 Shanmen DTX-1257.2
10 June,2008 Wenling DTX-184.1
11 Spetember,2007 Leqing DTX-1176.2
12 Spetember,2008 Leqing DTX-1105.0
13 July,2007 Dongtou DTX-1144.3
14 Spetember,2008 Dongtou DTX-197.8
In conclusion, the proposed method, which offers a
rather newly and rapid developed extraction, clean-up of
the sample, was found to have acceptable reproducibility,
high specificity and sensitivity and a detection capability
which allows the detection of the diarrheic shellfish poi-
sons (OA and DTX-1) by means of HPLC-MS/MS under
multi-reactions monitoring (MRM) scan with tandem
mass analyzer using negative ion electrospray ionization
(-ESI) mode and identification based upon their retention
times and the fragmentation patterns of their mass spec-
We are grateful to the financial support from the Key Scientific Re-
search Projects of Zhejiang Province (No.2007C23081).We also thank
all the members of the Zhejiang Fisheries Quality Testing Center and
Center of Analysis and Measurement(Zhejiang University) for their
helpful discussions.
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