Toxins 2015, 7, 1324-1340; doi:10.3390/ oxins7041324
oxins
ISSN 2072-6651
www.mdpi.com/jou nal/ oxins
A icle
In luence o Di e en Shell ish Ma ices on he Sepa a ion o
PSP Toxins Using a Pos column Oxida ion Liquid
Ch oma og aphy Me hod
Ve ónica Rey 1, Ampa o Al onso 2, Luis M. Bo ana 2,* and Ana M. Bo ana 1,*
1 Depa men o Analy ical Chemis y, Science Facul y, Uni e si y o San iago de Compos ela,
Lugo 27002, Spain; E-Mail: [email p o ec ed]
2 Depa men o Pha macology, Ve e ina y Facul y, Uni e si y o San iago de Compos ela,
Lugo 27002, Spain; E-Mail: [email p o ec ed]
* Au ho s o whom co espondence should be add essed;
E-Mails: [email p o ec ed] (L.M.B.); [email p o ec ed] (A.M.B.);
Tel.: +34-982-822233 (L.M.B.); +34-982-824071 (A.M.B.).
Academic Edi o : G eg Boye
Recei ed: 3 Ma ch 2015 / Accep ed: 3 Ap il 2015 / Published: 15 Ap il 2015
Abs ac : The sepa a ion o PSP oxins using liquid ch oma og aphy wi h a pos -column
oxida ion luo escence de ec ion me hod was pe o med wi h di e en ma ices. The
sepa a ion o PSP oxins depends on se e al ac o s, and i is c ucial o ake in o accoun
he p esence o in e e ing ma ix peaks o p oduce a good sepa a ion. The ma ix peaks a e
no always he same, which is a signi ican issue when i comes o p oducing good, eliable
esul s ega ding esolu ion and oxici y in o ma ion. Di e en eal shell ish ma ices
(mussel, scallop, clam and oys e ) we e s udied, and i was seen ha he in e e ence is no
he same o each indi idual ma ix. I also depends on he species, sampling loca ion and
he da e o collec ion. I was p oposed ha sepa a ion should be accomplished aking in o
accoun he ype o ma ix, as well as he concen a ion o hep ane sul ona e in bo h
sol en s, since he mobile phase a ies ega ding he ma ix. Scallop and oys e ma ices
needed a dec ease in he concen a ion o hep ane sul ona e o sepa a e GTX4 om ma ix
peaks, as well as dcGTX3 o oys e s, wi h a concen a ion o 6.5 mM o sol en A and
6.25 mM o sol en B. Fo mussel and clam ma ices, in e e ing peaks a e no as la ge as
hey a e in he o he g oup, and he hep ane sul ona e concen a ion was 8.25 mM o bo h
sol en s. Also, o scallops and oys e s, ma ix in e e ences depend no only on he
OPEN ACCESS
Toxins 2015, 7 1325
sampling si e bu also on he da e o collec ion as well as he species; o mussels and clams,
di e ences a e no ed only when he sampling si e a ies.
Keywo ds: pa aly ic shell ish poisoning; oxins; pos column oxida ion me hod; in e e ing
ma ix peaks
1. In oduc ion
Pa aly ic shell ish oxins a e po en compounds p oduced by se e al species o dino lagella es, such
as Alexand ium ama ense, Gymnodinium ca ena um and Py odinium bahamense, and inges ion o
con amina ed shell ish wi h PSP oxins can cause se ious li e- h ea ening in oxica ions [1]. The highly
oxic and unp edic able na u e o hese bio oxin blooms means ha he oxin con en o shell ish in
a ec ed a eas is moni o ed [2]. PSP moni o ing p og ams ely on ela i ely in ensi e sampling and
analysis p o ocols ha equi e apid, sensi i e, accu a e, and p ecise analy ical echniques o he
analysis o PSP oxins [3].
The mouse bioassay [4] is he me hod adi ionally used o de e mine he p esence o PSP oxins in
shell ish. Howe e , e hical conside a ions ega ding he use o mammals in assays led o a sea ch o
o he me hods, and high-pe o mance liquid ch oma og aphy (HPLC) wi h luo ime ic de ec ion was
chosen [5,6].
High-pe o mance liquid ch oma og aphy me hods a e widely used o iden i y and quan i y PSP
oxins p esen in sea ood, and hey can also es ablish he oxin's p o ile, which can be e y di e en
depending on he geog aphical a ea, he phy oplank on species and he shell ish species [7,8].
HPLC-FLD wi h a p ecolumn oxida ion me hod, also called he Law ence me hod, was alida ed by
he Associa ion o O icial Analy ical Chemis s h ough a collabo a i e s udy [9] and was in eg a ed
in o Eu opean Di ec i es o ac as a legal al e na i e o he mouse bioassay [10] o he de e mina ion
o PSP oxins.
In he las ew yea s, conside able p og ess has been made in he de elopmen o hese me hods as
an al e na i e o bioassay [11–13]. In 2012, Tu ne e al. [14] p oposed a e inemen o AOAC me hod
2005.06 o he de e mina ion o PSP oxins in oys e s, because in his ma ix he oxin eco e y and
sensi i i y was poo . The e ined LC-FLD me hod imp o ed pe o mance cha ac e is ics o he
de e mina ion o PSP oxins in whole king and queen scallops. Howe e , he lack o s anda ds
(C3, C4, GTX6, dcGTX1, dcGTX4), o he co ec iden i ica ion and quan i a ion o PSP oxins was a
handicap, as was he inabili y o dis inguish be ween ce ain analogues wi h di e en oxici y; all his
c ea es p oblems wi h he adequa e iden i ica ion and quan i ica ion o se e al PSP oxins [8].
Along wi h he p ecolumn me hod, a pos column oxida ion HPLC-FLD me hod p oposed by
Oshima [15,16] has been used o yea s o de ec and quan i y PSP oxins. This pos column oxida ion
(PCOX) me hod has unde gone se e al modi ica ions [17], and was used success ully in a
collabo a i e s udy [18], which g ea ly inc eased i s ele ance; i hen became an o icial AOAC
me hod [19], and was p oposed o eplace he cu en AOAC mouse bioassay as well, as an al e na i e
o he p ecolumn me hod. Ne e heless, he e a e some s udies which show ha he analysis o PSP
oxins is di e en ega ding he sample ma ix; Tu ne e al. [12] epo ed he possibili y o
Toxins 2015, 7 1326
luo escence enhancemen o PSP oxins in he oys e ma ix, al hough he e is no e idence o ma ix
componen s alsely enhancing he oxin signals. They poin ed ou ha i is possible ha a ia ions in
luo escence enhancemen o supp ession p e iously obse ed be ween di e en shell ish species may
also occu be ween di e en samples o he same species wi h di e en spa ial and empo al o igins.
The e is some e idence o he po en ial e ec o high concen a ions o zinc and manganese in
oys e esul ing in a supp ession o he MBA (mouse bioassay) esponse [14]; mo eo e , i is no ed
ha whils high concen a ions o zinc do no a ec he p ecolumn oxida ion me hod, he e is a
po en ial o me al ions o in e e e wi h he ion-pai ing-based pos column oxida ion me hod [20].
All his shows ha he e a e di e ences be ween di e en ma ices and also be ween ei he p e- o
pos -column me hods. Wi h his wo k, se e al samples om di e en shell ish ma ices we e analyzed
using he alida ed pos column me hod. The di e ences in sepa a ion ega ding he sea ood ma ix
we e s udied. I is shown ha no all he ma ices ha e he same p o ile; i was necessa y o change he
condi ions o he me hod when he sample ma ix was changed.
2. Resul s
The ini ial PCOX me hod unde wen se e al changes: pos column condi ions we e changed
(oxidan low, acid low and acid concen a ion) o each a pH ou low o 5–7 [19].
The eac ion empe a u e was es ed om 65 °C o 85 °C in he wa e ba h [16]. I has been seen ha ,
up o 80 °C, he highe he empe a u e, he bigge he peak a ea signal in he ch oma og ams, bu om
85 °C some peaks we e lowe . The e o e, he wo king eac ion empe a u e was 80 °C.
The shell ish issue ex ac used o dilu ing GTXs and STXs was p epa ed wi h mino
modi ica ions. I was ound ha when adding 35 µL o 1 M NaOH o each pH = 3, as ecommended
a e dep o eina ion, GTX1, GTX4 and NEO deg aded a e successi e injec ions. Gene ally, he PSP
oxins a e s able unde acidic condi ions, al hough he s abili y depends on he chemical s uc u e;
GTX1, GTX4 and NEO a e less s able a acidic pH han GTX2, GTX3 and STX [21]. The e o e,
pH alues o 4 and 5 we e es ed obse ing ha a pH = 4, oxins we e mo e s able and he signal
was g ea e .
The age/s a us o he LC columns has a la ge impac on he esolu ion, and he pH o he mobile
phase as well as he concen a ion o he eagen s in i a e also c ucial. The mos impo an componen
is he ion-pai eagen hep ane sul ona e; in ou labo a o y a be e sepa a ion was ob ained when he
concen a ion o hep ane sul ona e was adjus ed o 8.25 mM [22]. Wi h hese condi ions i was
possible o sepa a e GTX5 om dcGTX2, as i is shown in Figu e 1a (11 mM hep ane sul ona e) and
Figu e 1b (8.25 mM hep ane sul ona e). Figu e 2 shows he ch oma og ams o he wo wo king
s anda d solu ions a e checking how new condi ions wo k o all he s anda ds, whe e GTXs and
STXs a e sepa a ed in mussel issue (Figu e 2a) and Cs a e sepa a ed in deionized wa e (DIW)
(Figu e 2b).
Toxins 2015, 7 1327
Figu e 1. Ch oma og aphic sepa a ion o dcGTX3-GTX5-dcGTX2, (a) wi h 11 mM
hep ane sul ona e in mobile phase; (b) wi h 8.25 mM hep ane sul ona e in mobile phase.
Figu e 2. Ch oma og ams o he wo wo king s anda d solu ions. (a) Sepa a ion o
he gonyau oxins and saxi oxins in mussel issue using 8.25 mM hep ane sul ona e;
(b) sepa a ion o C- oxins in DIW.
Toxins 2015, 7 1328
Rou ine analysis o samples equi e he use o ma ix- o i ied s anda ds o diminish he e ec
ha he ma ix has on he e en ion ime o he ea ly elu ing oxins; howe e , some imes hese ma ix
peaks elu e wi h he oxins and so i is impo an o ge op imal elu ion condi ions o isola e hese
in e e ing peaks a he maximum and achie e a good esolu ion. The e o e, he e ec o di e en
oxin- ee ma ices on he sepa a ion o PSP oxins was s udied. Mussel, clam, scallop and oys e
ma ices we e used. Comme cial shell ish samples we e used in all cases and no oxin- ee
homogena es like NRCC-CRM-Ze o-Mus [18], because he beha io o eal ma ices could be obse ed.
I was ound ha he beha io o PSP oxins in mussels and clams is simila . The same condi ions
we e used o hese wo shell ish. As men ioned, he concen a ion o hep ane sul ona e o bo h sol en s
in o de o ge a good esolu ion was 8.25 mM in all cases. Howe e , one scallop ma ix peak coelu ed
wi h GTX4; o sepa a e hem hep ane sul ona e concen a ion was changed in sol en A, wi h 6.5 mM
being he app op ia e concen a ion. When oys e was s udied he same p oblem was ound as in he
case o he scallop, namely ha GTX4 coelu ed wi h a ma ix peak and also ano he peak coelu ed wi h
dcGTX3. The e o e, he hep ane sul ona e concen a ion was modi ied o 6.5 mM in sol en A and o
6.25 mM in sol en B, al hough he sepa a ion be ween dcGTX3 and he ma ix peak was no op imal.
Figu e 3 shows he ch oma og ams o (a) a oxin- ee scallop issue, whe e i is possible o see he
ma ix peak o a 11 mM hep ane sul ona e concen a ion; (b) he o e lapping o GTX4 and ha peak
a ha concen a ion; and inally, (c) he sepa a ion o bo h peaks, when he concen a ion o hep ane
sul ona e was changed. The ch oma og am in (d) shows he sepa a ion ob ained o oys e .
Figu e 3. (a) Scallop PSP oxin- ee wi h 11 mM hep ane sul ona e in mobile phase;
(b) GTX4 and GTX1 in scallop issue wi h 11 mM hep ane sul ona e in mobile phase;
(c) PSP oxins s anda ds in scallop issue wi h 6.5 mM hep ane sul ona e in sol en A and
6.25 mM hep ane sul ona e in sol en B; (d) PSP oxins s anda ds in oys e issue wi h
6.5 mM hep ane sul ona e in sol en A and 6.25 mM hep ane sul ona e in sol en B.

Toxins 2015, 7 1329
A e op imiza ion o sepa a ion condi ions, he linea i y, epea abili y, LODs and LOQs we e s udied.
The linea i y esul s a e summa ized in Table 1, and all he linea co ela ion coe icien s a e highe
han 0.98, showing an excellen co ela ion. The lowes alue o mussels was 0.0002 mg/kg and he
highes alue was 5.2756 mg/kg; o clams hey we e 0.0026 mg/kg and 0.5284 mg/kg, espec i ely;
o scallops hey we e 0.0006 mg/kg and 0.8220 mg/kg; and o oys e s, he lowes and highes alues
we e 0.0171 mg/kg and 4.1170 mg/kg, espec i ely.
Table 1. Linea i y o he ins umen esponse o each ma ix- o i ied PSP s anda d (mg/kg).
Toxin Ma ix Linea i y ange Calib a ion cu e
Equa ion Co ela ion (R2)
GTX4
Mussel 0.0004–0.1341 y = 472166x − 1281 0.9823
Clam 0.0042–0.2681 y = 407086x + 218418 0.9851
Scallop 0.0010–0.2681 y = 175936x − 2349 0.9904
Oys e 0.0495–0.7922 y = 225692x − 914 0.9989
GTX1
Mussel 0.0064–1.6442 y = 275537x + 29094 0.9905
Clam 0.0128–0.4110 y = 104377x + 32765 0.9855
Scallop 0.0032–0.8220 y = 266520x − 10630 0.9929
Oys e 0.1518–1.2141 y = 142355x + 1477 0.9978
dcGTX3
Mussel 0.0002–0.2255 y = 3 × 106x + 37075 0.9917
Clam 0.0046–0.1480 y = 2 × 106x − 14452 0.9779
Scallop 0.0006–0.1480 y = 2 × 106x − 19014 0.9927
Oys e 0.0185–0.2980 y = 737742x + 6381 0.9879
GTX5
Mussel 0.0004–1.0620 y = 105632x − 2213 0.9983
Clam 0.0083–0.2655 y = 295206x − 94 0.9816
Scallop 0.0021–0.5310 y = 510734x − 14907 0.9938
Oys e 0.0576–0.9222 y = 213243x + 5600 0.9949
dcGTX2
Mussel 0.0008–0.8032 y = 3 × 106x + 18524 0.9976
Clam 0.0165–0.5284 y = 1 × 106x − 80684 0.9800
Scallop 0.0021–0.5284 y = 2 × 106x − 44732 0.9947
Oys e 0.0332–2.1289 y = 424621x + 5576 0.9866
GTX3
Mussel 0.0002–0.3421 y = 3 × 106x + 69830 0.9879
Clam 0.0027–0.0428 y = 1 × 106x + 5344 0.9904
Scallop 0.0007–0.1711 y = 4 × 106x − 27177 0.9960
Oys e 0.0171–1.0941 y = 758162x − 4082 0.9989
GTX2
Mussel 0.0005–1.0296 y = 2 × 106x + 74858 0.9943
Clam 0.0080–0.2574 y = 823267x − 20717 0.9801
Scallop 0.0020–0.5148 y = 2 × 106x − 26429 0.9969
Oys e 0.0514–1.6464 y = 497495x − 3098 0.9877
NEO
Mussel 0.0032–0.8186 y = 157597x − 4730 0.9972
Clam 0.0064–0.1023 y = 57166x + 1369 0.9892
Scallop 0.0016–0.4093 y = 169005x − 6014 0.9907
Oys e 0.2573–4.1170 y = 54360x + 1380 0.9981
Toxins 2015, 7 1330
Table 1. Con .
Toxin Ma ix Linea i y ange Calib a ion cu e
Equa ion Co ela ion (R2)
dcSTX
Mussel 0.0013–0.3281 y = 412156x − 4413 0.9970
Clam 0.0026–0.0820 y = 462063x − 2023 0.9918
Scallop 0.0006–0.0820 y = 522829x − 3457 0.9913
Oys e 0.0390–2.4978 y = 127331x + 429 0.9943
STX
Mussel 0.0018–0.1183 y = 700921x + 339 0.9953
Clam 0.0037–0.1183 y = 467605x − 3866 0.9850
Scallop 0.0009–0.2367 y = 856619x − 9104 0.9964
Oys e 0.0267–0.8543 y = 265150x − 1806 0.9957
C1 Mussel 0.0103–5.2756 y = 2 × 106x − 50438 0.9947
C2 Mussel 0.0316–1.6197 y = 3 × 106x − 40838 0.9964
Repea abili y was s udied, wi h 5 daily injec ions o e 3 days (n = 15), a an in e media e
concen a ion (linea calib a ion in e al) o each ma ix. Da a o le els o concen a ion and %RSD
a e shown in Table 2; he %RSD o he oxins in each ma ix is wi hin he accep able ange (in mos
cases below 7%), and so he epea abili y o all oxins in all ma ixes appea s o be consis en .
Table 2. Me hod epea abili y, %RSD.
Toxin µM mg STX eq/kg Mussels Clams Scallops Oys e s
GTX4 0.0815 0.02202 6.55 7.39 6.68 4.63
GTX1 0.2498 0.09242 6.34 7.20 6.65 3.88
dcGTX3 0.0400 0.00561 3.88 7.89 8.78 9.52
GTX5 0.1425 0.00816 3.94 5.76 5.46 2.36
dcGTX2 0.1749 0.00419 3.46 5.81 3.52 5.53
GTX3 0.0541 0.01284 4.20 3.64 2.65 3.96
GTX2 0.1627 0.02175 0.25 4.69 3.28 5.83
NEO 0.1623 0.05584 1.89 6.32 7.79 3.22
dcSTX 0.0800 0.01528 1.28 6.89 5.66 10.47
STX 0.0795 0.02959 2.23 7.55 8.02 12.48
C1 0.3135 0.00070 6.18
C2 0.0962 0.00345 5.19
LOD (limi o de ec ion) and LOQ (limi o quan i a ion) alues we e bo h calcula ed o each
ma ix analyzing 5 eplica e ex ac s o a blank ma ix, epea ed o e 6 days (n = 30). The baseline
signal- o-noise a io a he app oxima e e en ion ime o all oxins was calcula ed. The LOD alue
was ob ained, aking he noise esponse (a ea uni s) mul iplied by 3, con e ed o µmol and exp essed
as mg STX eq/kg o each oxin, and he LOQ was calcula ed as LOD × 3 [23]. Table 3 summa izes
LODs and LOQs o each oxin in each ma ix. The esul s show ha sensi i i y is di e en o he
di e en ma ices, and he bes esul s in gene al a e hose ob ained o mussels.
Toxins 2015, 7 1331
Table 3. LODs and LOQs (mg STX·diHCl eq/kg) o he me hod.
Toxin Mussels Clams Scallops Oys e s
LOD LOQ LOD LOQ LOD LOQ LOD LOQ
GTX4 0.0183 0.0549 0.0201 0.0603 0.0221 0.0663 0.048 0.144
GTX1 0.0642 0.1926 0.0320 0.0960 0.0488 0.1464 0.057 0.171
dcGTX3 0.0071 0.0213 0.0096 0.0288 0.0097 0.0291 0.003 0.009
GTX5 0.0011 0.0033 0.0088 0.0264 0.0298 0.0894 0.025 0.075
dcGTX2 0.0210 0.0630 0.0181 0.0543 0.0244 0.0732 0.030 0.090
GTX3 0.0055 0.0165 0.0072 0.0216 0.0073 0.0219 0.011 0.033
GTX2 0.0099 0.0297 0.0306 0.0918 0.0199 0.0597 0.023 0.069
NEO 0.0239 0.0717 0.0329 0.0987 0.0375 0.1125 0.018 0.054
dcSTX 0.0047 0.0141 0.0172 0.0516 0.0074 0.0222 0.0005 0.001
STX 0.0100 0.0300 0.0394 0.1182 0.0111 0.0333 0.009 0.027
C1 0.0001 0.0003
C2 0.0011 0.0033
In he pos -column me hod, he s anda ds we e dilu ed i s ly in DIW (pH = 5) o Cs oxins and in
HCl 0.003 M o GTXs and STXs. New dilu ions ei he in DIW (pH = 5) o Cs oxins o in shell ish
ex ac o GTXs and STXs we e p epa ed; he issue used a his s age should be he ma ix being
analyzed a he momen in he labo a o y, because he condi ions (hep ane sul ona e concen a ion in
mobile phase) o each ma ix a e di e en , as men ioned.
The nex s ep was he analysis o PSP oxins in se e al samples using he pos -column me hod o
e i y ha he changes made we e e ec i e in eal samples o di e en species, di e en o igins and
ha es ing da e. Toxin p o iles a e illus a ed in Figu e 4 o sample PSP3 (clam), as an example, and
he esul s ob ained o he quan i ica ion o each PSP oxin in he samples a e summa ized in Table 4.
Figu e 4. PSP oxin p o ile o sample PSP3. (a) C oxins and (b) GTX and STX oxins.
Toxins 2015, 7 1332
Table 4. PSP oxin concen a ions (µg/kg) in he samples ob ained using HPLC wi h he
pos -column me hod.
µg/kg GTX 1 GTX4 dcGTX2 dcGTX3 GTX5 GTX2 GTX3 NEO dcSTX STX C1 C2 C3 C4
PSP5 73.6 245.5 2.9 14.6 14.5 - -
EXT.2 65.5 43.9 NQ * 10.6
PSP6 148 237.4 4.1 NQ * 3.3 20.1 24.6 - -
EXT.7 13.5 515.2 4.4 NQ * 14.7 14
PSP4 624.7 176.5 4.1 2.9 15.2 11.8 35.9 19.4 - -
EXT.1 34.5 NQ * 4.1 2.5 0.01 12 134.9 47.3
PSP2 33.3 10.3 59.1 44.5 - -
PSP3 82.3 45.3 55.2 58.9 22.2 34.8 175.7 73.1 - -
PSP1 68.9 ND * 54.4 56.3 43.9 - -
EXT.3 45.4 56.2 5.7 5.4 11.6 10.4 149.2 122.6
EXT.6 157.7 264.1 NQ * NQ * 12.3 16.4 139.1 53.7
EXT.8 72.93 156.2 4.2 NQ * 17.6 12.9
EXT.9
PSP7 122.7 44.2 121.9 51.2 309.6 - -
PSP8 162.8 NQ * 917.4 482.6 103.9 47.4 627.5 17.7
PSP11 57.6 184.9 4.3 17.9 1.4 - -
PSP12 93.1 ND * 422.9 193.5 64.2 14.6
PSP10 167.4 187.2 234.3 213.6 203.5 781.7 11.2 3.4 - -
EXT.4 131.4 165.5 147.6 161.8 169.2 386.3 57.3 32.3
PSP 10H 1934.2 4265.3 343.4 30 441.9 469.5 282.3 14.4 5.5
PSP 10G 92.3 215.7 150.2 72.7 59.9 377.5 - -
PSP 10B 66.64 158.64 94.49 140.23 - -
PSP 10M 59.44 63.87 120.18 26.35 51.67 185.3 - -
PSP 10Ma 37.83 87.3 148.89 135.49 - -
PSP 13 30.70 2.57 NQ * 14.97 3.96 23.04 35.86 27.36 5.62 17.55 8.10 - -
PSP 14 26.44
PSP 15 6.31
* ND: no de ec ed; NQ: no quan i a ed.
3. Discussion
This wo k was done o iden i y he PSP oxin p o ile o se e al samples wi h di e en geog aphical
loca ions ecei ed om 2007 o 2013, using he pos -column oxida ion me hod. This me hod, alida ed
by an de Rie e al. [18], imp o ed on Oshima’s [16], which ini ially became qui e popula because i
could ch oma og aphically sepa a e all PSP oxins, al hough i s main d awback is ha i is a
ime-consuming me hod. Wi h Van de Rie ’s imp o emen , all indi idual PSP analogues a e
de e mined, excep some me aboli es, in a sho e ime.
GTX1 and GTX4 a e wo key oxins in he me hod [18]. The ac ha hey a e he ea lies elu ing
analy es inc eases he chance ha he p esence o sample ma ix peaks will al e he e en ion ime o
he compounds compa ed o wha is seen in ma ix- ee s anda d solu ions. The use o ma ix-ma ched
calib a ion solu ions was implemen ed in an e o o comba his shi and o make he da a
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