Food Chemistry 129 (2011) 533–540

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Food Chemistry

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Analytical Methods

Automated on-line solid-phase extraction coupled to liquid chromatography–tandem mass spectrometry for determination of lipophilic marine toxins in shellfish

Jorge Regueiro a,⇑, Araceli E. Rossignoli a, Gonzalo Álvarez a,b, Juan Blanco a

a Centro de Investigacións Mariñas (Xunta de Galicia), Apartado 13, E-36620 Vilanova de Arousa, Pontevedra, Spainb Facultad de Ciencias del Mar, Departamento de Acuicultura, Universidad Católica del Norte, Larrondo 1281, Coquimbo, Chile

a r t i c l e i n f o a b s t r a c t

Article history:Received 26 November 2010Received in revised form 12 March 2011Accepted 19 April 2011Available online 4 May 2011

Keywords:Lipophilic marine toxinsShellfish poisoningMusselOn-line solid-phase extractionColumn switchingLiquid chromatography–tandem massspectrometry

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.04.054

⇑ Corresponding author. Tel.: +34 886 20 63 44.E-mail address: regueiro.jorge@cimacoron.org (J. R

Automated on-line solid-phase extraction (SPE) coupled to liquid chromatography–tandem mass spec-trometry (LC–MS/MS) has been developed for fast determination of lipophilic marine toxins in shellfishsamples. The direct coupling of an on-line SPE column to LC–MS/MS was accomplished using columnswitching techniques. Suitable chromatographic separation was performed on a reversed-phase C18 col-umn under alkaline conditions (pH 11).

The selected reversed-phase C18 SPE column allowed rapid and efficient on-line desalting of hydroly-sed shellfish samples, avoiding signal suppression during mass spectrometry detection. Furthermore, theon-line SPE procedure allowed reducing matrix effects observed in raw and hydrolysed shellfish extracts.

The proposed method was evaluated in terms of linearity, precision, accuracy and limits of detection(LODs). Quantitative recovery (97–102%) and satisfactory inter-day precision (RSD < 8%) were achievedfor all target compounds. LODs in the sub-lg kg�1 level (0.37–0.68 lg kg�1) were obtained for all toxinsexcept for okadaic acid, which showed a value of 2.75 lg kg�1.

Several mussel samples from North-western Spain were finally analysed in order to demonstrate theapplicability of the method. Okadaic acid was the predominant toxin in all samples, although other lipo-philic toxins were also detected.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Lipophilic marine toxins, produced by several microalgae spe-cies, are bioaccumulated in filter-feeding molluscan shellfish suchas mussels, scallops, oysters, cockles and clams. During a harmfulalgal bloom, the concentrations of these toxins in shellfish can reachtoxic levels, responsible for different kind of shellfish poisoning inconsumers. This is a major concern for public health authorities aswell as for the shellfish industry, since the closure of harvesting sitesdue to these toxic episodes has also an important economic impact(Cabado & Vieites, 2008; Morgan, Larkin, & Adams, 2009).

On the basis of their chemical structure, several major groups oflipophilic toxins can be discerned: the okadaic acid (OA) group, theyessotoxin (YTX) group, the azaspiracid (AZA) group, the pecteno-toxin (PTX) group, the brevetoxin (BTX) group and the cyclic iminesgroup, mainly composed by spirolides (SPXs) gymnodimines(GYMs) (Quilliam, 2003).

Among the intoxications caused by consumption of shellfishcontaminated with marine toxins, diarrheic shellfish poisoning(DSP) is one of most common syndromes all around the world.

ll rights reserved.

egueiro).

DSP results in adverse effects such as gastrointestinal disorder,diarrhoea, abdominal cramps, nausea and vomiting (FAO, 2004).Toxins responsible for this intoxication belong to the OA group,composed by OA, dinophysistoxin-1 (DTX-1) and dinophysistox-in-2 (DTX-2), as well as their esterified forms.

In order to protect shellfish consumers, EU regulation has estab-lished maximum levels of several of these toxins that should not beexceeded when placing shellfish products on the market. Thepermitted levels for the sum of OA, DTXs, and PTXs is set at160 lg OA-equivalents kg�1, the sum of relevant YTXs is set at atotal of 1 mg YTX-equivalents kg�1 and the sum of relevant AZAsat 160 lg AZA-1 equivalents kg�1 edible shellfish (European Com-mission, 2004). Regulatory limits for brevetoxin and cyclic iminegroups have not yet been established.

Currently, the EU reference method for detection of lipophilictoxins in shellfish is the mousse bioassay (MBA), but there is grow-ing interest in the development of alternative non-animal basedmethods. Furthermore, MBA presents some drawbacks such aslow sensitivity and reproducibility, and lack of specificity andinformation on toxin composition (Fernández, Richard, & Cembella,2003; Hess, 2010).

Liquid chromatography coupled to electrospray ionisationtandem mass spectrometry (LC–ESI-MS/MS) is arising as the most

534 J. Regueiro et al. / Food Chemistry 129 (2011) 533–540

promising alternative for detection and quantification of marinetoxins in shellfish (Chapela, Reboreda, Vieites, & Cabado, 2008;Fux, McMillan, Bire, & Hess, 2007; Stobo et al., 2005; These, Scholz,& Preiss-Weigert, 2009). This technique exhibits enormous potentialfor the replacement of MBA due to its high selectivity and sensitivity,and the possibility to detect multiple shellfish toxin groups in asingle analysis. Its main limitation is derived from the endogenousmatrix components that may co-elute with target analytes andaffect the efficiency of the ionisation process, causing the so-calledmatrix effects, i.e. signal suppression or enhancement (Fux, Rode,Bire, & Hess, 2008). This problem may be increased for the analysisof toxins belonging to the OA group, as long as their determinationgenerally implies a previous alkaline hydrolysis to transform theminto the corresponding free acid forms. As a result, high salt concen-trations are obtained, which usually leads to signal suppression inthe ionisation source.

To overcome matrix effects, different strategies can be performedincluding an improved sample cleanup or/and a more efficientchromatographic separation. Recently, alkaline chromatographicconditions have been used to increase sensitivity of OA group toxinsand to improve chromatographic separation of main lipophilicmarine toxins (Gerssen, Mulder, McElhinney, & de Boer, 2009).Furthermore, reduced matrix effects were observed when these con-ditions were applied in combination with SPE using Stratra-X poly-meric sorbent (Gerssen et al., 2009). Kilcoyne and Fux (2010) havealso reported the elimination of matrix effects for OA and azaspir-acid-1 (AZA-1) in shellfish when using the same alkaline conditionswithout any sample cleanup.

In the recent years, on-line SPE has been shown as a powerfuland reliable tool for sample treatment of complex matrices, whichallows reducing most problems associated with off-line samplepreparation, such as time-consumption, cost, contamination,procedural errors and risk of low recoveries. This approach hasbeen, very recently, applied by Kilcoyne and Fux (2010) for theevaluation of matrix effects in the analysis of OA and azaspir-acid-1 (AZA-1). The use of OASIS HLB polymeric sorbent underacidic chromatographic conditions, was proved to significantlyreduce matrix effects associated with OA analysis in raw extractsof different shellfish species.

The aim of the present study was to develop a fast and sensitivemethod based on the use of on-line SPE coupled to LC–ESI-MS/MSfor the determination of lipophilic marine toxins in shellfishsamples. To the best of our knowledge, an on-line SPE procedureis proposed for the first time for the simultaneous extraction andcleanup of all main lipophilic toxins, including all of thosecurrently regulated by EU legislation. Chromatographic separationwas accomplished under alkaline conditions in order to improvechromatographic efficiency.

Aiming to increase sample throughput, the potential of theproposed methodology for the on-line desalting of hydrolysedshellfish extracts was also evaluated.

Method performance was assessed in terms of recovery,precision and LODs. Matrix effects were studied in both raw andhydrolysed mussel extracts. Finally, several mussel samples fromNorth-western Spain were analysed by the proposed method.

2. Materials and methods

2.1. Reagents, standards and materials

Lipophilic marine toxins were purchased from the NationalResearch Council (Halifax, Canada) as certified reference materi-als in methanol at the following concentrations: OA 24.1 ±0.8 lg mL�1, YTX 5.5 ± 0.3 lg mL�1, AZA-1 1.24 ± 0.07 lg mL�1,GYM 5.0 ± 0.2 lg mL�1, 13-desmethyl spirolide C (SPX-1)

7.0 ± 0.4 lg mL�1 and pectenotoxin-2 (PTX-2) 8.6 ± 0.3 lg mL�1.DTX-1 (>95%) was obtained from Wako Pure Chemical Industries(Osaka, Japan) and DTX-2 was purchased from CIFGA (Lugo,Spain) as a standard in methanol at 2.48 lM.

Ammonium hydroxide (NH4OH, 25%), sodium hydroxide(NaOH P 99%) and hydrochloric acid (HCl, 37%), all of analyticalgrade, were obtained from Merck (Barcelona, Spain).

Acetonitrile (MeCN) and methanol (MeOH) of HPLC grade werepurchased from Rathburn (Walkerburn, Scotland) and Lab-scan(Dublin, Ireland), respectively. Ultrapure water was obtained froma Milli-Q Gradient water purification system (Millipore Iberica,Madrid, Spain).

Individual stock solutions of each toxin and a mixture of themwere prepared in methanol. Different working standard solutionswere made by appropriate dilution in methanol and then storedin amber glass vials at �20 �C.

2.2. Samples and sample preparation

Mussels Mytilus galloprovincialis were collected from raftcultures placed at different locations of the Galician Rías (NWSpain) during 2009–2010. Sample collection was carried out bythe Galician organisation responsible for the monitoring andcontrol programme for marine biotoxins (INTECMAR).

Soft tissues were removed from the shells and the digestiveglands were separated, homogenised with a blender and storedat �80 �C until processed. Samples were extracted using anUltra-Turrax T25 high performance dispersing system (IKA, Stau-fen, Germany). Two grams aliquots of homogenised tissues wereaccurately weighed into the extraction tubes and extracted twicewith 8 mL 80% MeOH in water (v/v) at 11,000 rpm for 3 min. Aftereach extraction, the resulting slurry was centrifuged at 20,000g for5 min and both supernatants were then combined in a 20 mLvolumetric flask and filled up to the mark with 80% MeOH.Analiquot of shellfish extract was filtered by 0.20 lm and analysedby LC–ESI-MS/MS.

In order to determine the amount of OA group toxins present inesterified forms, an aliquot of each extract was submitted to alka-line hydrolysis prior to the analysis. Hydrolysis was carried out byadding 125 lL of aqueous NaOH 2.5 M to 1 mL of methanolicextract, homogenising in a vortex mixer for 0.5 min and heatingthe mixture at 76 �C for 40 min. After cooling down to room tem-perature, the extract was neutralised by adding 125 lL of HCl2.5 M and homogenising in vortex for 0.5 min. The resultingextracts were filtered by 0.20 lm and analysed by LC–ESI-MS/MS. The concentration of toxins in esterified forms was calculatedfrom the difference between the concentration of free toxins in theextracts before and after the hydrolysis process.

2.3. On-line SPE–HPLC system

The on-line SPE–HPLC system consisted of an Accela autosam-pler, and a HPLC quaternary pump from Thermo Fisher Scientific(San Jose, CA, USA), an isocratic HPLC pump Jasco PU 2080 Plus(Jasco, Madrid, Spain) and an electronically controlled 2-position,6-port switching valve MXT715-000 from Rheodyne (Cotati, CA,USA). Xcalibur 2.1 software (Thermo Fisher Scientific) was usedto programme and control all components except for the isocraticpump, which was run independently. A schematic diagram of thedeveloped system is presented in Fig. 1.

Chromatographic separation was performed on a reversed-phasecolumn Gemini-NX C18 (100 � 2.0 mm, 3 lm) from Phenomenex(Torrance, California, USA), maintained at 40 �C. A security guardcolumn Geminni-NX C18 (4.0 � 2.0 mm) also from Phenomenexwas employed as trap column for the on-line SPE. Polyetherether-

2

3

4

5

6

1

AS

Waste

Pump 1

Analytical column

Pump 2

SPE column

ESI-MS/MS

A)

2

3

4

5

6

1

AS

Waste

Pump 1

Analytical column

Pump 2

SPE column

ESI-MS/MS

B)

Fig. 1. Schematic diagram of the proposed on-line SPE–HPLC system: (A) loading/washing position and (B) elution position.

Table 1Specific MRM conditions for determination of lipophilic marine toxins.

Toxin tR

(min)Ionisation Parent ion MS/MS

transitionCE(eV)

T1/T2 ± tol.b

OA 3.24 ESI� [M�H]� 803.5 > 255.2a 48 6.5 ± 1.3803.5 > 563.5 43

DTX-2

3.40 ESI� [M�H]� 803.5 > 255.2a 48 6.9 ± 1.4803.5 > 563.5 43

YTX 3.46 ESI� [M�2H]2� 570.4 > 467.4a 30 3.8 ± 0.8570.4 > 396.4 30

DTX-1

3.72 ESI� [M�H]� 817.5 > 255.2a 48 5.5 ± 1.10817.5 > 563.5 43

AZA-1

4.55 ESI+ [M+H]+ 842.5 > 824.5a 28 16.3 ± 3.3842.5 > 654.4 44

GYM 6.33 ESI+ [M+H]+ 508.3 > 490.2a 22 25.9 ± 5.2508.3 > 162.2 42

SPX-1

6.85 ESI+ [M+H]+ 692.5 > 674.4a 27 2.0 ± 0.4692.5 > 164.3 42

PTX-2

6.92 ESI+ [M+NH4]+ 876.5 > 823.5a 23 2.3 ± 0.5876.5 > 805.5 21

a Quantifier MS/MS transition.b Quantifier-to-qualifier transition ratios and tolerances for positive identifica-

tion (European Commission, 2002).

J. Regueiro et al. / Food Chemistry 129 (2011) 533–540 535

ketone (PEEK) capillary tubing (1/16 in. O.D. � 0.005 in. I.D., ThermoScientific) was used to minimise the void volume of the system.

The on-line SPE-HPLC procedure involved three steps accord-ing to the switching valve positions. Initially, with the switchingvalve in the loading/washing position (Fig. 1A), 10 lL of samplewas injected by the autosampler (AS) and loaded onto the SPEcolumn by the isocratic pump (pump 1 in Fig. 1) at a flow of0.6 mL min�1. The loading mobile phase consisted of MeCN/water(10/90, v/v) containing 6.7 mM NH4OH. The non-retained matrixcomponents were directly eluted to waste, whereas the analyteswere trapped in the column. The analytical column wasmeanwhile equilibrated to the initial conditions of the chromato-graphic separation.

At 1.5 min the valve was switched into the elution position(Fig. 1B) and the gradient pump (pump 2 in Fig. 1) back-flushedthe SPE column at a flow of 0.4 mL min�1, eluting the retained ana-lytes onto the analytical column. Elution mobile phases A and Bwere, respectively, water and MeCN/water (90/10, v/v), bothphases containing 6.7 mM NH4OH. The following linear gradientwas used to achieve the elution from the SPE column and the sub-sequent chromatographic separation of the toxins: hold at 25%B for1.5 min, increased from 25%B to 95%B over 6 min, hold at 95%B for2 min, then returned to initial conditions over 3 min and re-equil-ibrated for 1.5 min.

At 5.5 min the valve was switched back into the starting posi-tion and the SPE column was re-equilibrated with the loadingsolution prior to the next injection. The total analysis run timewas 13.5 min.

2.4. Mass spectrometry

The on-line SPE-HPLC system was coupled to a triple stagequadrupole mass spectrometer TSQ Quantum Access MAX (Ther-mo Fisher Scientific, San Jose, CA, USA) equipped with a heatedelectrospray ionisation source HESI-II.

536 J. Regueiro et al. / Food Chemistry 129 (2011) 533–540

The ion transfer tube temperature and the HESI vapouriser tem-perature were set at 360 �C and 110 �C, respectively. Nitrogen(>99.98%) was employed as sheath gas and auxiliary gas at pres-sures of 60 and 10 arbitrary TSQ Quantum units, respectively. HESIwas operated in both polarity modes at spray voltages of 3500 Vfor negative ionisation and 3800 V for positive ionisation.

During method development, when the on-line SPE was notemployed, the LC eluate was diverted to waste during the first1.5 min of the chromatographic run. The detection was accom-plished in the multiple reaction monitoring (MRM) mode using ar-gon (>99.999%) as collision-induced-dissociation (CID) gas at1.5 mTorr. Selected ionisation polarities, optimised MS/MS iontransitions and the corresponding transition confirmation detailsfor each compound are detailed in Table 1. Instrument controland data acquisition were performed with Xcalibur 2.1 software(Thermo Fisher Scientific).

3. Results and discussion

3.1. Chromatographic analysis

Preliminary experiments were conducted to optimise the chro-matographic separation of lipophilic marine toxins as well as theirMS/MS detection.

Chromatographic conditions were based on those reported byGerssen et al. (2009) for LC–ESI-MS/MS analysis of lipophilic mar-ine toxins. These conditions imply the use of alkaline mobilephases (pH 11), which were demonstrated to improve the separa-tion of these toxins as well as the ionisation efficiency for the OAgroup toxins. A further study found that matrix effects in the anal-ysis of lipophilic toxins were significantly reduced when these con-ditions were combined with SPE on polymeric sorbents (Gerssenet al., 2009). Very recently, Kilcoyne and Fux (2010) have reportedthe elimination of matrix effects for OA and AZA-1 in differentshellfish matrices without any sample cleanup when using an alka-line chromatographic separation coupled to a triple stage quadru-pole mass spectrometer.

Several linear gradients using water and MeCN/water (90/10, v/v), both phases containing 6.7 mM NH4OH, were tested in order tooptimise the chromatographic separation on a reversed-phase col-umn Gemini-NX C18 (100 � 2.0 mm, 3 lm). The stationary phaseof this column incorporates cross-linked ethane groups, which al-low an extended working pH range from 1 to 12. Special attentionwas paid to the separation between OA and its isomer DTX-2 be-cause of their coincident MS/MS transitions (Table 1).

The optimised LC conditions, which are detailed in Section 2.3,enabled a suitable separation of target compounds in a total anal-ysis time of 13.5 min, including column re-equilibration. Baseline

0

20

40

60

80

100

Without SPE SPE

OA solution

Nor

mal

ised

resp

onse

(%)

Fig. 2. Normalised responses obtained for a hydroly

separation (Rs P 1.5) between OA and DTX-2 was achieved underthese conditions.

Optimisation of MRM conditions was carried out by post-col-umn infusion of standard solutions. ESI provided single chargedprecursor ions for all toxins except for YTX, which yielded a pre-dominant double negatively charged precursor ion ([M�2H]2�) atm/z 570.4. Two MS/MS ion transitions were selected for each com-pound (Table 1). The most sensitive transition was used for quan-tification, while the other one was employed for identification.Confirmation was accomplished by comparing the quantifier-to-qualifier transition ratios to those of the calibration standardswithin a 20% tolerance range (European Commission, 2002).

3.2. On-line SPE optimisation

On-line SPE was performed by column switching using a 2-po-sition, 6-port automated switching valve. During the sample injec-tion the valve is switched into the loading/washing position andthe analytes are retained on the SPE column (Fig. 1A).

Sorbent type, mobile phase compositions, and timing for load-ing/washing and elution are critical parameters for the optimisa-tion of an on-line SPE procedure. Due to the lipophilic nature ofthe target compounds, a reversed-phase Gemini-NX C18 securityguard cartridge (4 � 2.0 mm) was selected as SPE column. In orderto reduce matrix interferents that might affect sensitivity andselectivity, as well as to obtain quantitative recovery of analytesduring the SPE step, the composition of the loading/washing mo-bile phase was optimised. The best result was obtained withMeCN/water (10/90, v/v) containing 6.7 mM NH4OH at a flow rateof 0.6 mL min�1.

The extraction efficiency of the SPE procedure was evaluated atloading times ranging from 1 to 6 min. All experiments were per-formed using a real mussel extract spiked with a mixture of lipo-philic toxins at 50 ng mL�1. Under these loading conditions, nobreakthrough was observed for any compound for at least 6 min,which indicated a high extraction efficiency of the SPE system.

As commented in Section 1, signal suppression is usually ob-served for the LC–ESI-MS analysis of total content of OA group tox-ins, due to the high saline concentration (ca. 0.25 M NaCl) resultingfrom the alkaline hydrolysis process. During the loading/washingstep, the SPE column is washed with the selected mobile phase,so salts and un-retained matrix components are eluted to waste.

To characterise the desalting efficiency of the on-line SPE proce-dure, a 20 ng mL�1 OA standard solution was submitted to thehydrolysis process and the resulting saline solution was analysedwith loading/washing times of 1.5 and 4 min, respectively. Thesame saline solution was also analysed without SPE and, in thiscase, the LC eluate was diverted to waste during the first 1.5 minof the chromatographic run prior to MS detection. The correspond-

1.5 min SPE 4 min

Hydrolysed OA solution

sed OA solution with and without on-line SPE.

J. Regueiro et al. / Food Chemistry 129 (2011) 533–540 537

ing responses, normalised to that of the original OA standard solu-tion without on-line SPE cleanup, are depicted in Fig. 2.

When the on-line SPE was carried out, similar responses wereobtained in both the original and the hydrolysed solution. Further-more, no differences were observed when the loading time wasincreased from 1.5 to 4 min, which indicated that the salts respon-sible for OA signal suppression are discharged to waste within thefirst 1.5 min of the loading/washing step. It is probably due to thereduced volume of the SPE column (ca. 13 lL), which enables thatat least 70 column volumes of mobile phase are passed throughduring this time, ensuring an efficient removal of salts in a veryshort time. Therefore, a loading/washing time of 1.5 min wasselected in order to shorten the total analysis time.

In the absence of the on-line SPE procedure, it was observed thatthe OA response in the saline solution decreased by about 25% inrelation to the original solution. Furthermore, the precision of theanalysis was highly reduced showing a relative standard deviation(RSD, n = 6) close to 17%, whereas it was below 5% for the on-lineSPE analysis. Therefore, the use of the divert valve of the mass spec-trometer to reduce signal suppression was demonstrated as a lessefficient strategy, mainly due to the different bed volumes of boththe analytical and the SPE columns. So, when the divert valve ofthe mass spectrometer is used to discharge the salty eluate to waste

2.5 3.0 3.5 4.0 4.5 5

Time (min

0

50

100

0

50

100

0

50

100

0

50

100

Rel

ativ

e A

bund

ance

(%)

0

50

100

0

50

100

0

50

100OA

DTX-1

AZA

DTX-2

YTX

Fig. 3. MRM chromatograms for the quantification MS/MS transitions obtained for a mu

during the first 1.5 min of the chromatographic run, only 2 columnvolumes of mobile phase are passed through the analytical columndue to its higher volume (ca. 315 lL). Consequently, a much lowerdegree of desalting is achieved by this technique.

After the loading/washing step, the valve was switched into theelution position and the SPE column was back-flushed by the ana-lytical mobile phase (Fig. 1B). One of the most common limitationsof on-line SPE is compromised HPLC resolution, which can be cru-cial for the correct quantification of OA and DTX-2. The reducedvolume of the selected SPE column as well as the use of a back-flush elution, contributed to minimise peak broadening of analytes.

Different elution times (1, 2, 3, 4 and 5 min) were evaluated inorder to select the minimum time necessary for complete elutionof the retained compounds. Experiments were performed using astandard mixture solution of toxins at 20 ng mL�1.

Under the described gradient conditions (Section 2.3), it wasfound that only OA and DTX-2 were completely eluted from theSPE column within the first 2 min. For complete elution of YTX andDTX-1 it was necessary 3 min, whereas for the rest of consideredtoxins, elution was only quantitative after 4 min. Consequently, anelution time of 4 min was selected for the on-line SPE procedure.

After this time, the valve was switched back into the loading po-sition in order to re-equilibrate the SPE column with the loading

.0 5.5 6.0 6.5 7.0

)

803.5>255.2

570.4>467.4

842.5>824.5

508.2>490.2

692.5>674.4

876.5>823.5

817.5>255.2

-1

GYM

SPX-1

PTX-2

ssel extract spiked with the lipophilic toxins (concentrations are given in the text).

538 J. Regueiro et al. / Food Chemistry 129 (2011) 533–540

mobile phase. Meanwhile, chromatographic separation is contin-ued to take place through the analytical column.

Fig. 3 shows the MRM chromatograms for the quantificationMS/MS transitions obtained for a mussel sample spiked with thelipophilic toxins (OA at 5 ng mL�1, DTX-2 at 4 ng mL�1, DTX-1 at5 ng mL�1, YTX at 0.60 ng mL�1, AZA-1 at 0.63 ng mL�1, GYM at0.89 ng mL�1, SPX-1 at 0.93 ng mL�1 and PTX-2 at 0.79 ng mL�1).Under these conditions, no significant reduction of chromato-graphic performance was observed and baseline separation be-tween OA and DTX-2 was still maintained.

3.3. Method validation

In order to assess the performance of the proposed method, themain analytical quality parameters were thoroughly evaluated.Table 2 summarises the method validation data.

The recovery of the on-line SPE procedure was determined bycomparing the responses obtained for two standard mixture solu-tions (10 and 200 ng mL�1) with those obtained by direct injec-tions. In all cases, recoveries ranged from 97% to 102% with RSDsbelow 6%, which demonstrated the high extraction efficiency ofthe developed procedure.

Matrix effects were evaluated using the post-extraction addi-tion method, which is based on the comparison of the responsesobtained for a spiked extract with those obtained for a standardsolution at the same concentration. As proposed by Chambers,Wagrowski-Diehl, Lu, and Mazzeo (2007), the per cent absolutematrix effect (%ME) was calculated as (Rse/Rs � 1) � 100, whereRse extract is the response of the analyte in the spiked extractand Rs is the corresponding response in the standard solution. Inthis context, a negative result indicates ionisation suppression,whereas a positive result indicates signal enhancement. Matrix ef-fects were studied without (crude) and with on-line SPE using bothraw and hydrolysed extracts spiked at two concentration levels (10and 200 ng mL�1).

As shown in Table 2, no significant matrix effects(�6% < %ME < 5%) were observed without cleanup for OA, YTX andAZA-1 in the raw extracts under these chromatographic conditions.Minor signal suppression (%ME > �10%) was obtained for GYM andSPX-1 with RSDs below 7% at both concentration levels. Higher sig-nificant suppression, %ME between �19% and �14%, was observedfor PTX-2 when analysed in raw extracts without sample cleanup.

For hydrolysed extracts, important signal suppression(�34 < %ME < �21%) was obtained for the first eluting compounds,

Table 2Performance and validation data of the proposed method.

Toxin % SPE Recovery (%RSD,n = 3)

% Matrix effect (%RSD, n = 3)

10 ng mL�1 200 ng mL�1 Crude On-line SPE

10 ng mL�1 200 ng mL�1 10 ng mL�1

Raw Hydrol. Raw Hydrol. Raw Hy

OA 98.7 (3.2) 99.4 (2.6) 3.1(4.2)

�33(20)

�1.8(4.7) �24(16)

4.3 (5.1) �3(4

YTX 102.2 (4.8) 98.3 (3.7) �5.4(4.7)

�25(14)

4.8 (5.5) �18(12)

3.2 (4.6) 1.(5

AZA-1

101.4 (4.1) 98.1 (5.0) 3.3(5.6)

�16(13)

4.6 (4.3) �20(10)

�1.5(4.2)

4.(5

GYM 99.8 (3.9) 102.4 (3.9) �9.2(6.3)

�12(8.8)

�8.2(5.4)

�10.3(7.6)

5.6 (5.8) 3.(3

SPX-1

98.5 (4.2) 100.4 (4.9) �7.5(6.4)

�9.1(5.9)

�4.1(5.7)

�7.9(7.0)

�2.8(5.7) 0.(5

PTX-2

97.2 (3.8) 98.5 (5.6) �19(7.8)

�15(8.3)

�14 (7.3) �16(10)

1.3 (4.8) 3.(5

Hidrol.: hydrolysed extracts.

OA, YTX and AZA-1, probably due to the presence of remainingsalts from the hydrolysis process. Poor repeatability, with RSDs be-tween 20% and 10%, was achieved. These results were in agreementwith those described in Section 3.2 for the analysis of a hydrolysedOA standard solution. Significant signal suppression (%ME > �17%)was also observed for GYM, SPX-1 and PTX-2, showing RSDs valuesbelow 11%.

The on-line SPE cleanup allowed reducing matrix effects(�5% < %ME < 6%) in both raw and hydrolysed extracts (Table 2).For GYM, SPX-1 and PTX-2 signal suppression in raw extracts de-creased to below 5% with RSDs below 6%. Regarding hydrolysed ex-tracts, ionisation suppression was also reduced to values below 4%for all studied compounds, which can be considered negligible. Sat-isfactory repeatability was achieved for these extracts, showingRSDs below 6% at both concentration levels.

Therefore, the proposed on-line SPE procedure was shown as anefficient approach to minimise matrix effects in mussel extractsunder basic chromatographic conditions on the selected TSQinstrument.

The precision of the method was assed using a raw extractspiked at 20 ng mL�1. The intra-day precision was determinedfrom the analysis of five replicates on the same day, whereas theinter-day precision was calculated from analyses performed over5 consecutive days, with 3 replicates per day. As shown in Table2, both intra- and inter-day precision studies presented satisfac-tory results for all compounds, with RSDs below 6% and 8%,respectively.

Since no matrix effects were observed, even for hydrolysedsamples, quantification could be performed by external calibration.The linearity of the method was tested using standard solutions atseven concentration levels from 0.5 ng mL�1 to 1000 ng mL�1 forall toxins except for OA, which was assessed from 2 to1000 ng mL�1. Calibration curves were found to be linear in thestudied range with determination coefficients (R2) P 0.996 andRSD(n = 3) 6 5%. Due to the lack of the corresponding certifiedstandards and assuming equimolar responses, the calibration curveconstructed for OA was also used for the quantification of DTX-1and DTX-2.

Limits of detection were calculated as the average concentra-tion of analyte producing a signal-to-noise ratio (S/N) of 3 usingthe less sensitive MS/MS transition, i.e. the one permitting theunambiguous identification of toxins. Values ranged from 0.37 to0.68 lg kg�1 for all toxins except for OA, which showed a LOD of2.75 lg kg�1 (Table 2).

Intra-dayprecision (%RSD,n = 5)

Inter-day precision(%RSD, 5 days, n = 3)

LOD(lgkg�1)

200 ng mL�1

drol. Raw Hydrol.

.4.2)

1.2 (3.1) 2.0(4.3)

5.2 6.5 2.75

7.4)

5.7 (2.4) �1.6(3.5)

5.7 5.9 0.68

2.7)

2.4 (4.7) 0.48(3.9)

4.8 5.7 0.37

0.5)

�2.7(2.9) �1.9(5.1)

4.9 6.0 0.56

81.9)

4.3 (6.0) 2.5(4.7)

5.3 5.8 0.41

3.5)

�4.9(3.8) �2.9(5.2)

5.9 7.3 0.50

Table 3Analysis of lipophilic toxins in mussels Mytilus galloprovincialis from NW Spain.

Samples Concentration ± SD (n = 3) (lg kg�1)

OA DTX-2 YTX DTX-1 AZA-1 GYM SPX-1 PTX-2

1 24.6 ± 1.4 n.d. n.d. n.d. n.d. n.d. n.d. n.q.2 32.7 ± 1.5 n.d. n.d. n.d. n.d. n.d. n.d. n.d.3 1232 ± 75 n.d. 17.88 ± 0.82 n.d. n.d. n.d. n.d. n.d.4 60.3 ± 3.2 n.d. n.d. n.d. n.d. n.d. 4.28 ± 0.20 n.d.5 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.6 170.6 ± 6.7 135.2 ± 6.2 n.d. n.d. n.d. n.d. n.d. n.d.7 63.1 ± 3.7 n.d. n.d. n.d. n.d. n.d. 6.41 ± 0.35 n.d.8 199 ± 11 n.d. n.d. n.d. n.d. n.d. 9.72 ± 0.28 n.d.9 2853 ± 83 n.d. 27.5 ± 1.5 n.d. n.d. n.d. n.d. n.d.

10 306 ± 12 n.d. n.d. n.d. n.d. n.d. n.d. n.d.11 272 ± 12 n.d. n.d. n.d. n.d. n.d. 4.91 ± 0.24 6.64 ± 0.4112 1589 ± 78 n.d. n.d. n.d. n.d. n.d. n.d. n.d.13 20.5 ± 1.2 n.d. n.d. n.d. n.d. n.d. n.d. n.d.14 n.q. n.d. n.d. n.d. n.d. n.d. n.d. n.d.15 11.80 ± 0.73 n.d. n.d. n.d. n.d. n.d. n.d. n.d.

n.d.: not detected (<LOD).n.q.: not quantified (<LOQ).SD: standard deviation.

J. Regueiro et al. / Food Chemistry 129 (2011) 533–540 539

Sample to sample carry-over is a major concern in any on-lineSPE method. Analyses of blank samples were performed for everybatch of samples to assess carry-over and background contamina-tion. No significant signals were observed in these blank sampleseven after highly concentrated samples (>2000 ng mL�1).

Deterioration of the SPE column, which resulted in increasedbackpressure and peak broadening, was not observed up toapproximately 600 injections of both raw and hydrolysed musselextracts.

3.4. Application to real samples

Several mussel samples were analysed by the proposed method.Analyses were performed at least by triplicate and results areshown in Table 3, where data for OA, DTX-2 and DTX-1 areexpressed as total concentration after hydrolysis process, i.e. freetoxin plus esterified forms. According to these results, 7 of the 15samples were contaminated with lipophilic toxins at levels abovethe European regulatory limits (European Commission, 2004).The present study confirmed the results obtained by MBA withinthe monitoring programme, which led to the closure of the corre-sponding harvesting areas.

The toxins found mainly belonged to the OA group, althoughlow levels of YTX, SPX-1 and PTX-2 were also detected below thecurrent regulatory limits. No traces of DTX-1, AZA-1 and GYM weredetected in any sample.

OA was the predominant lipophilic toxin in most of samples,with concentrations ranging from 11.80 lg kg�1 to 2852 lg kg�1.DTX-2 was found in only one sample at a concentration of135.2 lg kg�1. The percentage of OA/DTX-2 esters to the total toxincontent ranged from 25% to 63% which is in agreement withreported ester profiles for M. galloprovincialis (Blanco, Mariño,Martín, & Acosta, 2007; Villar-González, Rodríguez-Velasco,Ben-Gigirey, & Botana, 2007).

Two samples presented YTX at concentrations of 17.88 lg kg�1

and 27.5, whereas SPX-1 was detected in 4 samples with valuesranging from 4.28 lg kg�1 to 9.72 lg kg�1. PTX-2 was found intwo samples, although only one of them was above the limit ofquantification.

The lipophilic toxin profiles found in this study are mostlyrelated to the presence in plankton of Dinophysis species such asD. acuminata and D. acuta, but other dinoflagellate species withcapability of producing SPXs (Alexandrium) and YTXs (Lingulodini-um, Prorocentrum or Gonyaulax) are also probably involved.

4. Conclusions

Application of on-line SPE coupled to LC–ESI-MS/MS resulted ina simple, rapid and robust method for the determination oflipophilic marine toxins in shellfish samples. To the best of ourknowledge, this approach is for the first time applied to the deter-mination of all main lipophilic marine toxin groups, including all ofthose currently regulated by EU legislation.

The developed procedure allowed increasing sample through-put, while minimising sample preparation and solvent consump-tion. Under final optimised conditions, the total analysis timewas below 14 min, including sample injection, on-line cleanup,chromatographic separation and column re-equilibration. Baselineseparation between OA and its isomer DTX-2 was achieved, whichmade possible their correct quantification.

On-line SPE using a C18 guard column was also demonstratedas an efficient and rapid desalting tool for hydrolysed shellfishextracts, enabling their direct ESI-MS/MS analysis without signifi-cant signal suppression (%ME > �5%). Furthermore, this approachallowed reducing those matrix effects observed in raw musselextracts for GYM, SPX-1 and PTX-2 on the selected TSQ instrument.

The SPE column was proved to have a long lifetime (ca. 600sample injections), which allowed reducing costs with regards tooff-line SPE procedures. Performance of the method was studiedin terms of linearity, accuracy, precision and LODs. Quantitativerecoveries (97–102%) were obtained and method precision wasalso satisfactory (RSD < 8%). LODs ranged from 0.37 lg kg�1 forAZA-1 to 2.75 lg kg�1 for OA, showing the high sensitivity of theproposed method when using the selected TSQ instrument.

The applicability of the proposed method was demonstratedthrough the analysis of several mussel samples from NW Spain.OA was the predominant toxin in all samples, although low levelsof YTX, SPX-1 and PTX-2 were also detected below the currentregulatory limits. Obtained results were in good agreement withthose provided by MBA within the monitoring programme, whichled to the close of the corresponding harvesting areas.

Acknowledgements

This work has been carried out in the framework of the collab-oration agreement EPITOX, funded by the Consellería de Economíae Industria, Xunta de Galicia. We also thank the Department ofBiotoxins of INTECMAR (Instituto Tecnolóxico para o control doMedio Mariño de Galicia) for supplying the mussel samples used

540 J. Regueiro et al. / Food Chemistry 129 (2011) 533–540

in this study. The work of Araceli Escudeiro Rossignoli was fundedby a grant of the Consellería Mar, Xunta de Galicia, in the frame-work of the Programa de Recursos Humanos of the PGDIT 2006–2010. Gonzalo Álvarez was funded by a MAEC-AECID grant. Wethank Carmen Mariño and David Fernández for their technicalassistance.

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