Determination of okadaic acid related toxins from shellfish (<i>sinonovacula constricta</i>) by high performance liquid chromatography tandem mass spectrometry

doi:10.4236/as.2013.45B001

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Vol.4, No.5B, 1-6 (2013) Agricultural Sciences

doi:10.4236/as.2013.45B001

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: [email protected]

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

ABSTRACT

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 ometric（HPLC-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

mg·kg-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

1. INTRODUCTION

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).

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. MATERIALS AND METHODS

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).

Shengsi

Daishang

Putuo

Xiangshan

Sanmen

Wenling

Dongtou

Leqing

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,

Japan).

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 60℃and 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-

MS/MS.

H.-Q. Zhang et al. / Agricultural Sciences 4 (2013) 1-6

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 30℃ and 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 [M−H]− 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. RESULTS AND DISCUSSION

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 [M−H]− 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 ion（m/z） Product ions (m/z)Declustering Potential（V） Collision Energy（V） Retention time（min）

Okadaic Acid

(OA) 803.6 255.1*

563.1 -70 -60

-68 3.38

Dinophysistoxins-1

(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

OA DTX-1

80% Methanol- water 94.2 90.6

Extract

solution 80% Acetontrile- water 96.0 92.5

40% Methanol-acetone 99.0 97.8

50% Methanol-acetone 93.2 95.2

Elution

solution

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).

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, 817.4/113.1．The 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

(mg·kg-1)

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

DTX-1

0.4 84.2 ± 4.7 5.54

Table 4. Results of the postive samples.

No.Sampling time Sampling

location

Concentration

（µg·kg-1）

1 August,2007 Shensi DTX-1：293.0

2 Spetember,2007 Shensi DTX-1：208.4

3 December,2007 Shensi OA：25.6

4 March,2008 Putuo DTX-1：155.0

5 June,2008 Putuo DTX-1：192.4

6 Spetember,2007 Xiangshan

DTX-1：246.0

7 October,2007 Xiangshan

DTX-1：168.5

8 August,2007 Shanmen OA：33.0

9 Spetember,2007 Shanmen DTX-1：257.2

10 June,2008 Wenling DTX-1：84.1

11 Spetember,2007 Leqing DTX-1：176.2

12 Spetember,2008 Leqing DTX-1：105.0

13 July,2007 Dongtou DTX-1：144.3

14 Spetember,2008 Dongtou DTX-1：97.8

4. CONCLUSIONS

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-

tra.

5. ACKNOWLEDGEMENTS

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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