UHPLC-HR-Orbitrap mass spe ctrom etry fo r quantitative analysis o f
lip o ph ilic marine to x in s in shellfish
Orellana G abriel12, Julie Vanden Bussche', Lieven Van M eulebroek1, M ichiei Vandegehuchte2,
Colin R. Janssen2 and Lynn Vanhaecke1
1 Laboratory o f Chemical Analysis, Faculty o f V eterinary Medicine, Ghent University,
Salisburylaan IBB, B-9820 Merelbeke, Belgium
E-mail: Gabriel.Qrellana@UGent.be
2 Laboratory o f Environm ental T o xico lo g y and A quatic Ecology, Faculty o f Bioscience engineering,
Ghent University, Jozef Plateaustraat 22, B-9000 Ghent, Belgium
Up to now, analytical m ethods used fo r the detection o f marine to x in s have generally been
focussing on the analysis o f 13 lip o p h ilic to xins. However, to date more than 200 lip o p h ilic marine
to xin s have been described (Gerssen e t al., 2011). Edible shellfish can bioaccum ulate marine to xin s
when grazing on to x ic marine m icroorganism s, particula rly marine microalgae such as certain
species o f dinoflagellates and diatom s, creating a potential food safety risk to humans. D uring the
last years, many analytical m ethods based on liquid chrom atography coupled to tandem mass
spectrom etry (LC-MS/MS) have been consolidated by inter-la bo ra to ry validations. However, the main
drawback o f LC-MS/MS m ethods remains the lim ited num ber o f com pounds th a t can be analysed in
a single run. Moreover, due to the targeted nature o f LC-MS/MS only known to x in s fo r which
m ethods have been previously o ptim ize d w ill be detected. Therefore in th is study, a m ethod based
on ultra high-perform ance liquid chrom atography (UHPLC) coupled to high resolution (HR) O rbitrap
mass spectrom etry was developed and its quantitative perform ance evaluated fo r co nfirm a to ry
analysis o f regulated lip o p h ilic m arine to x in s in shellfish flesh (M ytilus edulis, C rassostrea gigas,
C erastoderm a edule, and Ensis ensis) according to Com m ission Decision 2 00 2/6 5 7/E C (CD) and EU
Reference Laboratory fo r Marine Biotoxins SOP 2011. Okadaic acid (OA), dinophysistoxin-1 (DTX-1),
pectenotoxin-2 (PTX-2), azaspiracid-1 (AZA-1), yessotoxin (YTX) and 13-desm ethyl spirolide C (SPX1) were q uantified using m atrix-m atched calibration curves. The obtained results fo r recovery,
repeatability, w ith in -la b o ra to ry re p ro d u c ib ility (RSDr), decision lim it , line arity and ruggedness were
co m p lia nt w ith CD recom m endations. The linearity was evaluated by preparing 8 -p oin t calibration
curves in m atrix and correlation coefficients (R2) obtained fo r each com pound were > 0.99. For all
com pounds the RSDr ranged fro m 2.9% to 4.9%, repeatability from 2.9% to 4.8% and recovery from
90% to 112% fo r three spiked levels. In a dd ition, a firs t co n firm a to ry ide ntifica tion o f the
com pounds was perform ed by detecting the [M+H]+ or [M-H] ion w ith th e ir specific retention tim es
and accuracies. Secondly, the nC /l2C diagnostic isotopic ratio was selected fo r co nfirm a tion o f a
co m p ou nd ’s identity. In conclusion, UHPLC-HR-Orbitrap MS allowed a more accurate and faster (less
than 4m in.) detection o f the ta rg et to x in s than previously described LC-MS/MS m ethods.
Furtherm ore, HRMS allows to retrospectively screen fo r many to x in analogues and m etabolites
using its fu ll scan capabilities but also untargeted screening th ro u g h the use o f m etabolom ics
software.
References
Gerssen A., P.P.J. M ulder And J. De Boer. 2011. Screening o f lip o p h ilic marine to x in s in shellfish and
algae: Developm ent o f a library using liquid ch rom atography coupled to O rbitrap mass
spectrom etry. Analytica Chim ica Acta 685:176-185.
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