Depa amen o de Química
Más e Uni e si a io en Oceanog a ía
As axan hin de e mina ion in ma ine
biological samples: an o e iew
Las u o as:
Zo aida Sosa Fe e a Mª Es he To es Pad ón
Lidia Robaina Robaina
La alumna:
Tania Da ias He nández
Las Palmas de G an Cana ia, 4 de diciemb e de 2013.
Index
Abs ac ............................................................................................................................3
1.In oduc ion...................................................................................................................5
2. Analy ical Me hods....................................................................................................14
2.1. Sample P epa a ion and ex ac ion me hods in mic oalgae...................16
2.2. Sample P epa a ion and ex ac ion me hods in sh imps.......................20
2.3. Sample P epa a ion and ex ac ion me hods in c abs...........................21
2.4. Sample P epa a ion and ex ac ion me hods in ishes............................21
3. De e mina ion............................................................................................................24
3.1 HPLC............................................................................................................24
3.2 UHPLC.........................................................................................................30
3.3 MS o iden i ica ion and quan i ica ion...................................................30
4. Conclusions and u u e ends..................................................................................34
5. Acknowledgmen s......................................................................................................34
6. Re e ences...................................................................................................................35
3
Abs ac
As axan hin (AX) (3,3’dyhyd oxy-β,β- ca o ene-4,4´dione) is a pigmen ha
belongs o he amily o he xan hophylls, he oxygena ed de i a i es o ca o enoids
whose syn hesis in plan s de i es om lycopene. AX, which may no be syn hesized de
no o by animals, is one o he main pigmen s in ma ine ecosys ems ound in c us acean,
and many ish species. In pelagic ma ine ood webs, copepods a e he main p oduce s o
AX, being also he p incipal componen s o An a c ic K ill pigmen . Thus, in he
aquacul u e indus y AX is p esen in eed o mula ions o salmonids and o he a med
ish species, whe e ep esen an impo an cos o he eeds.
Depending in hei o igin, AX can be ound in associa ion wi h o he
compounds. I may be s e i ied in one o bo h hyd oxyl g oups wi h di e en a y acids
such as palmi ic, oleic, es ea ic, o linoleic. I may also be ound ee, ha is, wi h he
hyd oxyl g oups wi hou s e i ica ion; o else, o ming a chemical complex wi h
p o eins (ca o enop o eins) o lipop o eins (ca o enolipop o eins). Syn he ic AX is no
s e i ied while ound in algae is always s e i ied.
AX has an eno mous comme cial and indus ial p ospec . The e o e, due he
g owing demand o na u al oods has been s imula ed he sea ch o na u al sou ces o
AX wi h po en ial o indus ializa ion like mic oalgae, sh imp, k ill, c ab and
langos illa be ween hem. In he aquacul u e con ex , he use o AX o he eeding
indus y is impo an no only om he s andpoin o pigmen a ion o inc ease consume
accep ance bu also as a necessa y nu ien o adequa e g ow h and ep oduc ion o
comme cially aluable species.
The e o e, he accu a e de e mina ion o AX o ms in his kind o biological
ma ices is necessa y which in ol es ex ac ion p io o hei de e mina ion. In his
wo k, a e iew o he epo ed me hods o he analysis o AX in ma ine o ganisms
implied in he aquacul u e indus y (mic oalgae, sh imps, c abs and ishes) which a e
always based on LC coupled o di e en de ec o s like diode a ay (DAD) and mass
spec ome y (MS) was done, and he di e en ex ac ion and clean-up echniques
cu en ly employed discussed.

4
Abb e ia ions
APCI: A mosphe ic p essu e pho oioniza ion; APPI:A mosphe ic p essu e
chemical ioniza ion; ASAP: A mosphe ic p essu e solids analysis; AX: As axan hin;
BHT: Bu yl-hid oxy oluen; CO2: Ca bon dioxide; ESI: Elec osp ay; FAB :Fas a om
bomba dmen ; GC: Gas ch oma og aphy; HPLC: High pe o mance liquid
Ch oma og aphy; EI :Elec on impac ; H. plu ialis: Haema ococcus plu ialis; IL-SI:
Ionic liquid based silicas; LC: Liquid ch oma og aphy; MALDI: ma ix-assis ed lase
deso p ion/ioniza ion; MS:De ec ion mass; MTEB;:me h yl e - Bu yl-E he ; PLE:
p essu ized liquid ex ac ion; SC-C O2: supe c i ical ca bon dioxide; SFE-CO2:
supe c i ical ca bon dioxide luid ex ac ion; SJ: Saccaha ina Japanica; SPE: Solid
phase ex ac ion; SUPRAS: Sup amolecula Sol en ; UHPLC: Ul a High- pe o mance
liquid ch oma og aphy; UV-VIS: Ul a iole isible.
Más e Uni e si a io en Oceanog a ía Tania Da ias He nández
5
1. INTRODUCTION
Pigmen s a e chemical compounds ha abso b ligh in he wa eleng h ange o
he isible egion. P oduced colo is due o a molecule-speci ic s uc u e
(ch omopho e).
They can be classi ied by hei o igin. Na u al pigmen s a e p oduced by li ing
o ganisms such as plan s, animals, ungi, and mic oo ganisms. Na u al and syn he ic
pigmen s a e o ganic and ino ganic compounds.
Mo eo e , na u al pigmen s can be classi ied by hei s uc u al cha ac e is ics
as:
(a) Te apy ole de i a i es: chlo ophylls and heme colo s.
(b) N-he e ocyclic compounds di e en om e apy oles: pu ines, p e ins,
la ins, phenazines, phenoxazines, and be alains.
(c) Benzopy an de i a i es (oxygena ed he e ocyclic compounds): an hocyanins
and o he la onoid pigmen s.
(d) Quinones: benzoquinone, naph hoquinone, an h aquinone.
(e) Melanins.
( ) Isop enoid de i a i es: ca o enoids and i idoids.
Wi hin o isop enoid de i a i es, ca o enoids a e na u al pigmen s syn he ized
by plan s and some mic oo ganisms. They a e eadily soluble in non-pola o ganic
sol en s such as ace one, die hyl e he , chlo o o m and hexane, whe eas hei
oxygena ed de i a i es, xan hophylls, dissol e bes in pola sol en s such as alcohols.
Tania Da ias He nández Más e Uni e si a io en Oceanog a ía
6
Ca o enoids a e known o be indispensable cellula componen s in
mic oo ganisms, ungi, algae, highe plan s, animals, and humans and a e one o he
mos impo an na u al ma ine pigmen g oups. They a e also one o he main na u al
ood colo an s wi h widesp ead use (Goodwin, 1986).
Rema kable p og ess has been made in esea ch ega ding he s uc u e and
chemis y o ca o enoids. The chemical s uc u es o app oxima ely 600 ca o enoids,
which a e syn hesized de no o in highe
plan s, mosses, algae, bac e ia and ungi
ha e been well known, al hough hei
unc ions in li ing cells emain unknown
(Ka naukho , 1990).
Ca o enoids a e isop enoid polyenes
o med by joining o eigh C5-isop ene
uni s (Figu e 1) in a egula head- o- ail
manne excep in he cen e o he molecule whe e he o de is ail o ail and he
molecule is symme ical. They a e a anged in such a way ha he wo cen al me hyl
g oups a e in he 1,6 posi ion, while he emaining me hyl g oups a e in he 1,5 posi ion
ela i e o each o he . A se ies o conjuga ed double bonds cons i u es a ch oma opho e
o a iable leng h, esul ing in cha ac e is ic yellow o ed colo s (Nelis, 1989).
All ca o enoids can be conside ed as lycopene (C40H56).de i a i es (Figu e 2) by
eac ions in ol ing: (1) hyd ogena ion, (2) dehyd ogena ion, (3) cycliza ion, (4) oxygen
inse ion, (5) double bond mig a ion, (6) me hyl mig a ion, (7) chain elonga ion, (8)
chain sho ening (Goodwin, 1980).
Figu e 1. Isop ene s uc u e
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7
Ca o enoids ha e been classi ied as:
(a) p ima y ca o enoids: compounds equi ed by plan s in pho osyn hesis like β-
ca o ene, iolaxan hin, and neoxan hin and;
(b) seconda y ca o enoids: localized in ui s and lowe s like α-c yp oxan hin,
zeaxan hin, an he axan hin, capsan hin, capso ubin,... (Lich enhale , 1987).
Also, ca o enoids a e classi ied by hei chemical s uc u e, as shown in Figu e
3:
(1) ca o enes ha a e cons i u ed by ca bon and hyd ogen, such as, β-ca o ene
and lycopene;
(2) oxyca o enoids o xan hophylls ha ha e ca bon, hyd ogen and,
addi ionally, oxygen. In his g oup, oxygen can be p esen as OH g oups, as in
zeaxan hin o as oxi-g oups, as in can haxan hin o in a combina ion o bo h, as
in as axan hin.
Figu e 2. S uc u e o ca o enoid lycopene wi h common numbe ing sys em (Nami ha
e al. 2010)
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Un o unely, he analysis me hods o AX con en a e no s anda dized al hough
FDA has accep ed a me hod by liquid ch oma og aphy (LC) analysis (me hod 21 CFR
73.185) and i is used by di e en in e na ional agencies. In any case, his de e mina ion
necessa ily in ol es he ex ac ion o AX om he ma ine o ganisms p io o hei
de e mina ion.
In his wo k, an in ense e iew o he epo ed me hods used o he analysis o
AX in ma ine o ganisms implied in he aquacul u e indus y (mic oalgae, yea s, c abs
and ishes) which a e always based on LC coupled o di e en de ec o s like diode
a ay (DAD) and mass spec ome y (MS) was done, and he di e en ex ac ion and
clean-up echniques cu en ly employed discussed.
2. ANALYTICAL PROCEDURES
An impo an aspec o be conside ed when dealing wi h he de e mina ion o
o ganic compounds om na u al complex ma ices, an ex ac ion s ep p io o hei
de e mina ion is equi ed. In gene al, he ex ac ion o ca o enoids mus be ca ied ou
e y quickly, a oiding exposu e o ligh , oxygen, high empe a u es and o p ooxidan
me als, such as i on o coppe , in o de o minimize au ooxida ion and isome iza ion. To
a oid oxida ion o AX, se e al me hods a e usually employed o imp o e he s abili y
o his pigmen in he ma ix o s udy. The addi ion o an ioxidan s, such as asco bic
acid, py ogallol has been ecommended o p e en ca o enoid losses du ing he
ex ac ion p ocedu e.
In his sense, he i s p oblem a ises in he s anda d p epa a ion. I is necessa y
o p epa e i in a non pola dissol en , like chlo o o m, sonica e in a ul asonic ba h
du ing 15 min and add bu yl-hid oxy- oluen (BHT) like an ioxidan eac i e. An aliquo
(10 mL) o his solu ion is ans e ed in o a 100 mL olume ic lask and combined
wi h isohexane un il 100 mL. This is he s ock AX solu ion o measu emen a 470 nm
on a spec opho ome e and o injec ion on o he HPLC. This AX s anda d mus be
p epa ed e e y 6 weeks. The concen a ion o AX s ock is calcula ed acco ding o he
ollowing equa ion (L. Robaina and F. S achan, pe sonal communica ion):

Más e Uni e si a io en Oceanog a ía Tania Da ias He nández
15
C = abso p ion x 10000/ 2100 (mg/L).
Al hough i is necessa y o de elop app opia e, as , cos -e ec i e and
en i onmen ally iendly ex ac ion p ocesses o isola e he compounds o in e es ,
sampling and s o age a e also impo an s eps p io o sample p epa a ion (Rod iguez e
al., 2006).
Respec he sampling and s o age, mic oalgae cul u es ha e o g ow in 8- cm-
wide glass eac o s con aining 1 L o modi ied Bold’s basal medium supplemen ed
wi h KNO3 and subjec ed o con inuous s i ing by bubbling ai h ough he mix u e a
a cons an low a e. Pu e CO2 has o supply e e y 30 s a 10-min in e als o he ai
s eam o p o ide ino ganic ca bon and o main ain he pH a 8. Reac o s a e main ained
in a cul u e chambe a 24±2 °C, wi h a 16:8 h ligh :da k pho ope iod using luo escen
ligh a a pho osyn he ic pho on lux densi y o 400 μmol pho ons pe squa e me e pe
second. A e he cells had eached he la e exponen ial phase, biomass was ha es ed
by cen i uga ion (7000 pm o 5 min a 10 °C), ozen a −20 °C, eeze d ied a −40°C
o 48 h, and s o ed unde d y and da k condi ions un il u he use (Cas o-Puyana e
al., 2013).
Sh imps a e ha es ed and anspo ed on ice o keep hem ozen o do no lose
hei p ope ies. Some au ho s used was e shell (ca apace) o sh imps because hey a e a
ich sou ce o AX. López-Ce an es e al. (2006) and o he as Sanches-Sil a e al.
(2012) used aw and cooked sh imps. These a e peeled and he head, exoskele on and
mea (peeled sh imp) we e analyzed independen ly. Sachind a e al. (2005) p ocess he
samples by emo ing he head and body shell and he yield o mea , head and ca apace
a e de e mined by weighing.
Samples o ma ine c ab a e anspo ed om he landing cen es and collec ed
and anspo ed o he labo a o y unde iced condi ion (-20ºC) un il analysis (Co al-
Hinos oza e al., 2001; Vilasoa-Ma íınez e al., 2008; Sachind a, 2005). C abs a e
p ocessed by sepa a ing he mea om body and he claws. The gills, isce a e c., we e
disca ded and he yield o mea and shell is de e mined by weighing (Sachind a, 2005).
Tania Da ias He nández Más e Uni e si a io en Oceanog a ía
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The de os ing can be pe o med in di e en ways. The sample was hawed in a wa e
ba h empe a u e a 60-70°C. Then, he shells a e d ied in an o en a 55°C un il a inal
d y weigh s cons an . Pul e ized samples we e ob ained, homogenized and s o ed in
bo les o opaz and s o ed in he eeze (Vilasoa e .al., 2008).
Howe e , Felix-Valenzuela e al., (2001) d ied he samples di ec ly a 75ºC
du ing 4 h in a con ec ion o en un il cons an weigh is ob ained and s o ed hem in a
polye hylene bag a oom empe a u e un il he AX ex ac ion.
Wi h espec o he ishes, hey a e collec ed o allow he animals o go h ough
igo mo is. They a e hen weighed, selec ed and packed indi idually in plas ic bags o
educe he isk o physical changes. The indi idual ish a e cu in h ee pieces (head,
middle and ail) o equal leng h, o he analysis o AX. The middle po ion ( ille ) is cu
in u n as shown in Figu e 5 wi h o wi hou skin and s o ed in cold (RØnshold e al.,
2001) A e hawed s eaks, hey a e g ound and mixed homogeneously and s o ed in
sealed plas ic con aine s a -70º/-80°C un il analyzing (Johs on e al., 2006).
2.1 P epa a ion and ex ac ion me hods in mic oalgae.
Mic oalgae cons i u e a complex and he e ogeneous g oup o o ganisms
cha ac e ized by being pho osyn he ic o ganisms ha possess simple ep oduc i e
s uc u es (He e o e al., 2012). The huge di e si y o mic oalgae makes hem an
almos unlimi ed esou ce o disco e y o bioac i e compounds. Mic oalgal
Figu e 5. Cu s in a ille salmon
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bio echnology has ad anced conside ably, and i is possible o comme cially p oduce
some ca o enoids h ough aquacul u e. Fo ins ance, Dunaliella salina is able o
accumula e high amoun s o β-ca o ene when subjec ed o pa icula g ow h condi ions,
including high salini y (Zhu e al., 2008), whe eas Haema ococcus plu ialis is he majo
p oduce o AX unde en i onmen al s ess, being able o selec i ely accumula e his
ca o enoid up o 5 % o i s d y weigh (Yuan e al., 2002).
AX occu s in algae like H. plu ialis in h ee main o ms: ee (5%), monoes e s
(70%) and dies e s (25%). Monoes e s and dies e s could be hyd olyzed o ee AX by
saponi ica ion (Yuan e al., 2002). The e o e, o ganic sol en s we e used o ex ac AX
and saponi ica ion was included o inc ease he con en o ee AX.
To op imize he saponi ica ion p ocess, addi ion o NaOH was necessa y o he
hyd olysis o AX es e s, bu i could esul in deg ada ion o AX because AX is an
alkali-labile ca o enoid (López-Ce an es e al., 2006). Yuan e al., (1998) indica ed ha
he high empe a u e a o ed he a e o hyd olysis o AX es e s, bu on he con as , he
deg ada ion o AX was also p omo ed a he same ime. Acco ding hei esul s, he
op imal saponi ica ion o AX and i s es e s we e hyd olyzed by 0.075 mol/L
NaOH/me hanol solu ion a 40ºC o 30 min.
T adi ionally, na u al compounds ha e been ob ained by me hod o con en ional
ex ac ion wi h o ganic sol en s. In his sense, Zou e al. (2011) applied con en ional
sol en ex ac ion and saponi ica ion o ob ain ee AX om Saccha ina japonica (SJ),
a species o ma ine algae ex ensi ely cul i a ed in Eas Asia. In his s udy, g inding
wi h mo al and a pes le was chosen o b eak SJ cell wall. Du ing he ex ac ion
p ocesses, d y SJ powde was ex ac ed by di e en sol en s (me hanol, wa e , e hyl
ace a e, e hanol, n-hexane, ace one and dichlo ome hane) a oom empe a u e. A e 12
h o imme sion and s i ing, me hanol and dichlo ome hane we e p o ed o be be e
han he o he sol en s. High solubili y o AX in dichlo ome hane (app oxima ely 30
g/L) migh acili a e he ex ac ion o AX, bu dichlo ome hane solu ion could no be
comple ely sepa a ed om he SJ cell deb is by cen i uga ion in his s udy. Howe e ,
comple e sepa a ion could be achie ed when dichlo ome hane was mixed wi h
me hanol. The e o e, he ex ac ed amoun o AX wi h di e en a io o
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me hanol/dichlo ome hane was in es iga ed and he maximum ex ac ed amoun (29.3
μg/g) was ob ained a he a io o 25/75 (me hanol/dichlo ome hane, / ) a e 5 h
ex ac ion.
Impo an s ep a e ex ac ion and p e ious o he de e mina ion is he solid-
phase ex ac ion (SPE). SPE has eme ged as a powe ul ool o chemical isola ion and
i can be used as ex ac ion me hods o liquid samples and/o as clean-up s ep p e ious
o he de e mina ion. SPE could be pe o med using con en ional so ben s o ionic
liquid based silicas (IL-Si). They ha e excellen chemical and physical p ope ies and
ha e ecen ly been used as SPE so ben s (Li e al., 2005) Acco ding o he chemical
s uc u e o AX (Yuan e al., 2002), he hyd ophobic, dipole–dipole, and π–π
in e ac ions be ween AX and IL-Si exhibi some excellen e ec s o inc ease he
sepa a ion e iciency o SPE. Fo ha , 10 mL AX solu ion we e selec ed o load on o
he SPE ca idges con aining Silp EMIm like IL-Si so ben s wi hou a leak and hen
allowed o equilib a e o 6 h. Washing and elu ion we e in es iga ed o op imize
selec i e ex ac ion. Ini ially, washing sol en s o di e en pola i ies (wa e , e hanol,
ace oni ile, and n-hexane) we e in es iga ed. Wa e was ound o be he mos sui able.
Mos in e e ences we e elimina ed wi h 3 mL o wa e wi hou loss o a ge
compounds. E hanol was he bes o elu e AX om he s a iona y phase (Zou e al.,
2011).
Ano he ex ac ion me hod is he p essu ized liquid ex ac ion (PLE). I uses
sol en s ca ego ized as gene ally ecognized as sa e o ex ac ca o enoids om
di e en mic oalgae such as H. plu ialis, D. salina, Chlo ella ulga is, and Spi ulina
pla ensis has been demons a ed (Jaime e al., 2010; Plaza e al., 2012). This ex ac ion
echnique is based on he ex ac ion using empe a u e and p essu e ha main ain he
ex ac ion sol en in he liquid s a e du ing he ex ac ion p ocess (Mendiola e al.,
2007). High p essu e o ces he sol en in o he ma ix, acili a ing he ex ac ion,
whe eas high empe a u e p omo es highe analy e solubili y (by inc easing he
solubili y and mass ans e a e) and dec eases he iscosi y and he su ace ension o
he sol en s, imp o ing he ex ac ion a e (Kau man e al., 2002). In he liquid s a e, i
is possible o ob ain highe ex ac ion yields in a sho e ime using signi ican ly smalle
olumes o ex ac ion sol en s han in con en ional ex ac ion me hods.
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Cas o-Puyana e al. (2013) op imized he ex ac ion o ca o enoids, including
AX, om Neochlo is oleoabundans using PLE wi h ood-g ade sol en s such as e hanol
and limonene. Expe imen al ac o s, including he ex ac ion empe a u e and he
sol en composi ion, we e op imized using a ac o ial design.
The i s s ep used o achie e e icien ex ac ion om he mic oalgae was o
b eak he cell wall o induce he lysis and his can hinde he ex ac ion and a ailabili y
o compounds and ob ain he g ea es quan i y o ca o enoids (Sa ada e al. 2006; Ce ón
e al., 2008). Among di e en me hods used, he highes yield o d y weigh mass om
N. oleoabundans (calcula ed as d y weigh /ini ial weigh exp essed as a pe cen age) was
ob ained using eezing– hawing and c yogenic g inding as he p e ea men p io o
PLE. The op imum condi ions p edic ed by he model we e 112 °C as he ex ac ion
empe a u e and 100 % e hanol as he ex ac ion sol en (Cas o-Puyana e al., 2013).
Ne e heless, possible changes in he physicochemical p ope ies o ex ac s can
al e hei unc ionali y, so he ex ac ion p ocesses should be pe o med a sui able and
mild condi ions. Supe c i ical ca bon dioxide luid ex ac ion (SFE-CO2) is an ad anced
echnology ha has a low en i onmen al impac because i en ails no esidue o ha m ul
sol en s being non oxic and nonco osi e and ha e easy sepa a ion om ex ac s
(Macías-Sánchez e al., 2005). Wi h an ope a ing empe a u e and p essu e ha a e
abo e he c i ical alues, supe c i ical luids ha e hei own special physicochemical
p ope ies be ween hose o a liquid and gas, such as high di usi i y, high
comp essibili y, low iscosi y, and low su ace ension (Felix-Valenzuela e al., 2001).
This allows he luid o di use easily h ough he na u al solid ma ix, and hus achie e
be e quali y ex ac ion o he na u al compounds compa ed o he con en ional liquid
sol en s. Fu he mo e, since CO2 has a low c i ical empe a u e and p essu e (31.1ºC
and 1084.86 psi), he ex ac s would no su e he mal deg ada ion du ing he p ocess,
making i an ideal sol en o bioac i e cons i uen s (Macmudah e al., 2006).
Pan e al. (2012) used SFE-CO2 o ex ac AX om H. plu ialis and
demons a ed ha he addi ion o e hanol o SC-CO2 was he mos e ec i e in e ms o
solubili y and ex ac ion yield wi h ega d o SC-CO2 luid (Macmudah e al., 2006; de
la Fuen e e al., 2006). The ba ch ex ac ion yield dec eased wi h inc eases in he

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amoun o H. plu ialis, low a e, s a ic ex ac ion ime, ex ac ion p essu e and
empe a u e (abo e 66.0ºC), due o he abso p ion e ec o he e hanol modi ie and
he mal deg ada ion o AX. The bes condi ions de e mined om his wo k we e H.
plu ialis weigh 21.67 g/LCO2- low a e 6.0 NL/min, ex ac ion ime 20 min, ex ac ion
p essu e 4500 psi, olume o e hanol modi ie added 60 mL, ex ac ion empe a u e
50ºC and modi ie composi ion 99.5%.
2.2. Sample p epa a ion and ex ac ion me hods in sh imps
In gene al, con en ional ex ac ion me hods a e used o ex ac AX om sh imps
samples. Howe e , each au ho used di e en o ganic sol en s o pe o m he
ex ac ion. The mos o he me hods epo ed o ex ac AX om shell ma ix employ
edible oils, hyd ochlo ic acid o o ganic sol en (Sachind a e al., 2005a).
In his sense, he ex ac ion conduc ed by Simpson e al. (1985) consis ed in he
addi ion o ace one o he homogenised samples o ex ac he pigmen . The ace one
ex ac s we e combined and sepa a ed in phase wi h pe oleum e he (40-60°C). The
pe oleum e he ex ac was washed se e al imes wi h 0.1M o NaCl solu ion o
emo e aces o ace one, hen d ied wi h sodium sulpha e, il e ed and washed again
and e apo a ed wi h ni ogen gas. The esul ing concen a e was collec ed in pe oleum
e he .
López-Ce an es e al. (2006) measu ed he AX con en in samples o lipid
ac ions he sh imps. An aliquo o 5 mL o di e en sol en s (me hanol, n-hexane,
e hanol, ace oni ile and me hyle hylce one) was added and immedia ely shaken o 20
s. Then, he samples we e sonica ed o 5 min o comple e ex ac ion, ollowed by
cen i uga ion o 15 min a 425 pm. Bes esul s we e ob ained wi h n-hexane al hough
hey do no use i because i is incompa ible wi h he mobile phase. Me hanol was
selec ed in his s udy.
Sánchez-Sil a (2012) p epa ed he sh imps samples acco ding o he me hod
desc ibed by López- Ce an es e al. (2006) wi h ligh modi ica ions. In his case, abou
0.1–0.25 g o sh imp by-p oduc s we e weigh ed, 5 mL o me hanol was added and i
Más e Uni e si a io en Oceanog a ía Tania Da ias He nández
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was ho oughly mixed in a o ex o 1 min. A e wa ds, samples we e kep in an
ul asonic ba h o 10 min o allow comple e ex ac ion, ollowed by il a ion h ough
125mm diame e il e pape om Wha man, and hen il e ed h ough a 0.2 mm po e
size PTFE sy inge il e .
2.3. Sample P epa a ion and ex ac ion me hods in c abs.
In he same sense, con en ional ex ac ion me hods a e used o ex ac AX om
c ab samples wi h di e en o ganic sol en s o pe o m he ex ac ion al hough Felix-
Valenzuela e al. (2000) used supe c i ical CO2/e hanol ex ac ion o ex ac AX om
c ab samples ob aining good esul s.
In gene al, he ex ac ion p ocedu e is ca ied ou wi h ace one. Sachind a e al.
(2005b) and López e al. (2006) ob ained ca o enoids, including AX, om 25 g o
samples which we e ex ac ed using ace one and phases sepa a ed wi h pe oleum e he .
The pe oleum e he ex ac is d ied wi h sodium sulpha e, il e ed, lushed wi h
ni ogen and hen e apo a ed unde acuum a 40º C using a o a y lash e apo a o .
The esul ing ca o enoid concen a e was aken up in pe oleum e he
.
Ano he au ho s made some changes using o ganic sol en s oo (Vilasoa –
Ma ínes e al., 2008). In his case, one g am o d y sample wi h h ee po ions o 5 mL
o ace one was cen i uged a 2000 pm o 5 min. O ganic phase was sepa a ed and he
esul ing solu ion ha con aining he ca o enoids, including AX, is e apo a ed o
d yness on a o a y e apo a o sys em. Ob ained ex ac is dissol ed in 1mL o
me hanol-dichlo ome hane-hexane (50:25:25), il e ed and injec ed o analysis.
2.4. Sample P epa a ion and ex ac ion me hods in ishes.
Fo ex ac ion o he pigmen s in he sample ishes, di e en au ho s ha e used
con en ional solid-liquid ex ac ion, using o ganic sol en s. Sheehan e al. (1998) and
Hanne e al. (1998) made he p ocess wi h ace one. Th ee g ams o each sample we e
homogenised wi h an equal weigh o anhyd ous sodium sulpha e and ex ac ed wi h
h ee 10 mL aliquo s o ace one. The combined ex ac s we e il e ed, he sol en
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e apo a ed unde ni ogen (< 50°C) and he esidue edissol ed in 5 mL n-hep ane. This
solu ion was hen ans e ed on o a silica S-Pak ca idge like clean-up SPE s ep. The
ca idge was elu ed wi h l0m1 o 20% die hyl e he in n- hep ane and 10 mL e hanol.
The e hanol elua e was e apo a ed unde ni ogen and he esidue was edissol ed in 1
mL o me hanol.
Rønshold e al. (2001) used en g ams o sample and we e mixed wi h 14 mL
o demine alized wa e in a 250 mL cen i uge bo le (high-densi y p opylene). Fi y
millili es o me hanol we e added and he sample was mixed a 2200 pm o 30 s.
Then, 25 mL o CHCl3 was added and he sample was mixed o 30 s. Finally, 25 mL
o demine alized wa e was added and mixed o 30 s. The sample was subsequen ly
cen i uged a 2900 pm o 10 min a 103ºC in a cooling cen i uge. A e
cen i uga ion, he me hanol phase ( op) was sucked ou , he p o ein laye was gen ly
il ed and he chlo o o m phase (bo om) con aining he oil and ca o enoids was
decan ed and was p o ec ed om ligh by w apping in aluminium oil. Pe cen age
eco e y o ca o enoids was de e mined by adding 2 mL AX s anda d solu ion (40
mg/L) .
Ba ua (2001) p esen ed an ex ac ion ha is ca ied ou wi h e hyl ace a e:
me hanol (1:1), ollowed by e hyl ace a e, and inally, hexane. Pooled supe na an s we e
e apo a ed o d yness unde ni ogen a mosphe e, edissol ing he ca o enoid esidue in
a olume o hexane.
Bike land e al. (2004) did simila ex ac ion ha ano he au ho s. The sample
was ex ac ed wi h chlo o o m using an ul a- u ax mace a o (13000 pm) o 1 min.
A e se ling o 10 min in he da k, he sample was cen i uged. The wa e -phase was
decan ed o , and he ex ac ion p ocedu e was epea ed wice. In an e o o assu e
comple e ex ac ion o AX om he p o ein ex ac s, he chlo o o m phase was
subjec ed o a hi d ex ac ion. The ex ac was il e ed in o an HPLC sample ial
h ough a 0.45-mm sy inge il e .
O he au ho s u ilized 10-20 g ams o ish muscle c umbles in o a beake and
swam 5 g o hyd a ed magnesium sul a e. A e , 40 mL o ace one is added and mixed
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23
using a homogenize . Then, he mix u e is il e ed and he emaining esidue was
esuspended in 40 mL o ace one and ollowed by homogenizing. This p ocess is
epea ed se e al imes, un il i becomes colo less. To emo e excess wa e , 20 mL o
e hanol is e apo a ed, and he emaining oil was dissol ed in mobile phase (86:14
hexane / ace one) and analyzed (Johns on e al., 2006).
Repo ed ex ac ion me hods use la ge olumes ypically 150–170 mL (Johns on
e al., 2006; Rønshold e al., 2001;) o oxic, lammable and en i onmen ally damaging
o ganic sol en s, hey a e labo ious and ime-consuming and in a iably in ol e mul i-
s ep ex ac ions. Fu he mo e, some imes sol en e apo a ion is equi ed (Johns on e
al., 2006) and he ish a s in e e e in he pho ome ic de e mina ion and hei
con ibu ion o he abso bance measu emen s mus be sub ac ed o ob ain accu a e
esul s (Rønshold e al., 2001).
An al e na i e o o ganic sol en s is he use o sup amolecula sol en s
(SUPRASs) o he e ec i e, apid and inexpensi e ex ac ion o AX om a med
salmonid ishes ob aining accep able esul s (Caballo e al., 2012). SUPRASs is made
up o biosu ac an , e ahyd o u ane (THF) and wa e and hey a e wa e immiscible
liquids made up o su ac an agg ega es dispe sed in a con inuous phase (usually
wa e ), which a e p oduced by wo well-de ined sel -assembly p ocesses occu ing on
wo scales, molecula and nano. Fi s , su ac an s agg ega e abo e a c i ical
concen a ion and hen, he gene a ed nanos uc u es sel -assemble unde he ac ion o a
ex e nal s imuli (e.g. empe a u e, pH, elec oly e, a non-sol en o he su ac an
agg ega e) and sepa a e as an immiscible liquid in equilib ium wi h a su ac an -lean
phase. These sol en s cons i u e an ad an ageous al e na i e o he ex ac ion o
o ganic compounds in a wide pola i y ange om bo h liquid (e.g., Ga cía-Fonseca,
Balles e os-Gómez, Rubio and Pé ez-Bendi o, 2008) and solid samples (e.g., Mo al e
al, 2009). In his case, p ecision o he me hod was 3.3%, and eco e ies we e nea o
100%.
Tania Da ias He nández Más e Uni e si a io en Oceanog a ía
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3.2 UHPLC
UHPLC is a p omising ool o ca o enoid analysis, including AX. As indica ed
abo e, one o he main di e ences be ween HPLC and UHPLC columns is he pa icle
size o he s a iona y phase. Smalle pa icles make he column mo e e icien and hey
end o allow solu es o ans e in o and ou o he pa icle mo e quickly because hei
di usion pa h leng hs a e sho e . Thus, he solu e is elu ed as a na ow peak because i
spends less ime in he s a iona y and s agnan mobile phase whe e band b oadening
occu s. A highe esolu ion be ween analy es can be expec ed. This echnique can also
g ea ly educe he un ime, he eby a oiding he isk o deg ada ion, a p ocess caused
by he high sensi i i y o hese compounds o physical and chemical ac o s.
Sánches-Sil a e al. (2012) de eloped and op imized a me hod o de e mine AX
by UHPLC wi h a DAD me hod in sh imp cons i uen s (exoskele on, head and mea ) o
bo h aw and cooked sh imp and in e men ed sh imp was e. Mo eo e , he me hod was
also used o e alua e he mig a ion o as axan hin om ac i e packaging in o ood
simulan s. Sepa a ion and quan i ica ion we e pe o med wi h a UPLCW BEH
analy ical column o 5 cm. Mobile phases we e (A) ACN–me hanol (con aining 0.05 M
ammonium ace a e)–DCM (75:20:5, / / ); (B) ul apu e wa e . AX de ec ion was
moni o ed a 480 nm. The analy ical me hod was alida ed acco ding o US Food and
D ug Adminis a ion (Food and D ug Adminis a ion and Cen e o D ug E alua ion
and Resea ch, 1994) guidelines. The me hod p esen ed good in a-assay p ecision (RSD
= 3.6%) and i also p esen ed good in e -assay p ecision (RSD = 4.7%), e alua ed by
analysis o h ee independen samples in h ee consecu i e days. The limi o de ec ion
was 0.054 µg/mL and he limi o quan i ica ion was 0.16 µg/mL. The limi o de ec ion
was lowe han he one ound by López-Ce an es e al. (2006), which used he same
mobile phase sol en s. Reco e y was de e mined using he s anda d addi ion p ocedu e
and he esul was sa is ac o y (90.5%) o 8 mg o added as axan hin.
3.3 MS o iden i ica ion and quan i ica ion.
In HPLC, UV–VIS ins umen s a e he mos common de ec o s used o iden i y
AX. Howe e , gi en ha he UV– is spec a o many ca o enoids a e simila and a

Más e Uni e si a io en Oceanog a ía Tania Da ias He nández
31
numbe o s uc u ally ela ed molecules coelu e, many esea che s ha e complemen ed
he iden i ica ion o ca o enoids using o he de ec ion me hods like NMR and IR,
be ween hem. Among hose, mass de ec o s ha e shown g ea ad an ages o he
analysis o hese subs ances, including he elucida ion o hei s uc u e on he basis o
he molecula mass and hei agmen a ion pa e n. These p ope ies acili a e he
quan i ica ion o indi idual ca o enoids ha coelu e.
The agmen pa e n obse ed in he ca o enoid mass spec a depends on he
ioniza ion echnique, including elec on impac (EI), as a om bomba dmen (FAB),
ma ix-assis ed lase deso p ion/ioniza ion (MALDI), elec osp ay (ESI), a mosphe ic
p essu e chemical ioniza ion (APCI) and mo e ecen ly, a mosphe ic p essu e
pho oioniza ion (APPI) and a mosphe ic p essu e solids analysis p obe (ASAP) and he
composi ion o he mobile phase used.
APCI has been used o success ully ionize no only ca o enes bu also ca o enoid
es e s , he eby demons a ing he sui abili y o his app oach o ionize ca o enoids wi h
di e en pola i ies. A highly p omising echnique o ionize nonpola compounds, such
as ca o enoids, is APPI. This me hod has ecen ly been in oduced as a new ioniza ion
me hod o LC–MS and can be conside ed complemen a y o he o he wo a mosphe ic
p essu e ioniza ion (API) echniques, namely ESI and APCI al hough esea ch is
equi ed o es he e ec i eness o his echnique o ionize di e se ca o enoids.
Thus, al hough some ca o enoids show he same o a e y simila agmen a ion
pa e n (meaning ha hei s uc u es a e simila and he e o e hey migh coelu e),
di e ences be ween he in ensi ies o hei agmen s ha e been epo ed. These
di e ences can be used o dis inguish he molecules. Mo eo e , hese di e ences can
p o ide an insigh in o he p edominan ca o enoid when coelu ion occu s. In addi ion,
LC–MS has been used no only o cha ac e ize ca o enoids bu also o quan i y hem.
The la e is possible because o he low de ec ion limi s and wide linea dynamic ange
alues exhibi ed by he mass de ec o s.
In his sense, Vilasoa-Ma ínez e al. (2008) con i med AX o ms in c abs wi h a
LC–MS sys em which ha e a ODS2 column and mobile phases: A (me hanol–
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ace oni ile, 14:86 / ) and B (hexane–dichlo ome hane, 50:50 / ) wi h a g adien
elu ion and di e en low a es De ec o ope a ed unde he ollowing condi ions:
a mosphe ic p essu e chemical ioniza ion posi i e (APCI+); p obe empe a u e 450ºC,
cone ol age (+) 20V, d ying gas ni ogen a 425 L/h, APCI gas ni ogen a 175 L/lh.
In he same manne , Hol in e al. (2009) de eloped a g adien me hod, desc ibed
p e iosuly, o sepa a e and de e mine he composi ion o he complex as axan hin
ex ac by HPLC-MS using an APCI in e ace and an ion ap. The de ec ion was
pe o med using APCI in he posi i e ioniza ion mode. The ol age o he co ona needle
was se o 4 kV. Ni ogen was used as he d ying gas as well as he ca ie gas a a low
a e o 5 L/min, wi h a nebulize p essu e o 65 psi. The ioniza ion chambe empe a u e
was se o 450°C and he d y gas empe a u e was held a 350°C. The compound
s abili y was se o 80% and he ap d i e le el o 70%. In his s udy, mono acid es e s
o AX a e he mos common ca o enoids ound in he mic oalgae Haema ococcus
plu ialis. He e, in addi ion o mino amoun s o palmi inic acid (C16:0), single and
polyunsa u a ed a y acids o he C18 amily (C18:1, C18:2 and C18:3) we e ound o
be he p incipally occu ing a s. In addi ion o he all- ans compound, he 9- and 13-cis
isome s we e ound o be he mos common con igu a ions in he algal ex ac .
Cas o-Puyana e al. (2013) cha ac e ized he ex ac s ob ained om Neochlo is
oleoabundans wi h a LC equipped wi h a diode-a ay de ec o as discussed abo e and
di ec ly coupled o an ion ap mass spec ome e ia an APCI in e ace, in posi i e
ioniza ion mode using he ollowing pa ame e s: capilla y ol age, −3.5 kV; d ying
empe a u e, 350 °C; apo ize empe a u e, 400 °C; d ying gas low a e, 5 L/min;
co ona cu en (which se s he discha ge ampe age o he APCI sou ce), 4,000 nA;
nebulize gas p essu e, 60 psi. A ange om m/z 150 o m/z 1,300 was acqui ed.
Combining he da a ob ained om he analysis o he ex ac s by LC-DAD and LC-MS,
au ho s we e able o conduc , o he i s ime, a en a i e iden i ica ion o di e en
ca o enoids, including di e en AX o ms (monoes e s and dies e s), p esen in N.
oleoabundans ex ac s unde ce ain g ow h condi ions.
Tandem mass spec ome y (MS/MS) p o ides many ad an ages o he analysis
o ca o enoids. LC–MS/MS o e s added selec i i y and speci ici y o he simple LC–
Más e Uni e si a io en Oceanog a ía Tania Da ias He nández
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MS sys ems. This mo e selec i e de ec ion me hod educes in e e ence by impu i ies in
he ex ac and allows he ollowing: (a) a minimal sample clean-up (leading o a high
sample h oughpu ); (b) dis inguishing be ween ca o enoids ha coelu e; (c) in o ma ion
abou s uc u al isome s; and (d) a dec ease in o e all analysis ime. Thus, using LC–
MS/MS, i is possible o dis inguish be ween s uc u al isome s. In his sense, Ri e a e
al. (2011) ha e e i ied he use o ansi ions o imp o ing he selec i i y o ca o enoid
analysis. They obse ed ha an he axan hin and as axan hin coelu e unde he
ch oma og aphic condi ions used in he UHPLC analysis. Howe e , hese ca o enoids
we e dis inguished using he speci ic ansi ions ound o each ca o enoid using APCI.
An he axan hin was iden i ied using he MS/MS ansi ions 585.3 > 93.1 and 585.3 >
105.2, while as axan hin p esen ed he ansi ions 597.6 > 147.1 and 597.6 > 579.4.
Nei he compound showed he co esponding ansi ions o i s coun e pa species.
Thus, he MS/MS ansi ions allow he indi idual quan i ica ion o hese subs ances in
spi e o he ac ha hey show he same ch oma og aphic e en ion ime. MS/MS
spec a ha e also p o en especially aluable o con i ming he p esence o speci ic
componen s.
Howe e , ca o enoid analysis is some imes di icul wi h so ioniza ion
echniques such as ESI because hese molecules ail o ionize e icien ly wi h hese
sys ems. Consequen ly, he mass spec a p esen poo s uc u al in o ma ion wi h a lack
o molecula ions. Mo eo e , he mul iple agmen s obse ed o en do no p o ide any
aluable in o ma ion abou he s uc u al cha ac e is ic o he compound. Howe e ,
ca o enoid ioniza ion can be imp o ed by adding chemical compounds ha acili a e
ioniza ion like ammonium ace a e, ace ic acid and halogen-con aining eluen s.
Quali a i e analysis o ca o enoids can be used o many pu poses, among hese
o (a) ob ain a apid o e iew o he ca o enoids p esen in a sample; (b) s udy
ca o enoid composi ions in hei na u al en i onmen ; (c) s udy he con o ma ional
changes o ca o enoids; and (d) classi y samples.
Tania Da ias He nández Más e Uni e si a io en Oceanog a ía
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4. Conclusions and u u e ends.
Mic oalgae, sh imp and c ab species can be conside ed as a no el po en ial
sou ce o na u al ca o enoids, including AX, which can be used in aquacul u e eeds. A
end o inc easing na u al ca o enoid sou ces, mainly om ma ine o igin, in
aquacul u e die s may o ce he indus y o be e de e mine AX in he na u al sou ces
and i s ole in ish physiology, in o de o g ow h owa ds sus ainabili y o he esou ces
and he indus y. Resea ch is being conduc ed o s udy he mo phological and
physiological changes associa ed wi h accumula ion o ca o enoids and he e ec o he
g ow h condi ions on he o e p oduc ion o hem and how ac in aquacul u e ishes.
Fo ha , AX de e mina ion in ma ine o ganisms is e y impo an . In ac , one
o he mos c i ical s eps in he de e mina ion o AX is he sample p epa a ion due o
hei physical-chemical cha ac e is ics. S udy o new ex ac ion and clean-up p ocedu es
a e necessa y. In his sense, u u e ends in his ield ha e o be o ien ed owa d he
de elopmen o new ex ac ion p o ocols o clean-up o ma ine o ganism samples.
Rega ding he cu en ins umen a ion, se e al echniques can be used o
imp o e he sepa a ion and de ec ion o di e en AX isome s and hei de i a i es.
HPLC and mo e ecen ly, UHPLC a e used o hei sepa a ion. Bo h ch oma og aphic
sys ems a e usually linked o DAD de ec o s and o MS and MS/MS de ec o s. The
la e p o ides mo e con i ma i e in o ma ion, he eby allowing he analysis o
coelu ing compounds. Quali a i e o semi-quan i a i e analysis can be ca ied ou using
a la ge numbe o spec oscopic and mass spec ome ic me hods (e.g. MS-TOF). This
a ie y o de ec ion echniques will con ibu e o ex ending in o ma ion abou
ca o enoids, including AX o ms, such as hei dis ibu ion in hei na u al en i onmen
and he ype o chemical changes hey unde go in ma ine o ganisms.
5. Acknowledgmen s.
I would like o exp ess hanks o Depa men o Chemis y o he Uni e si y o
Las Palmas de G an Cana ia and o my di ec o s Mª Es he To es Pad ón, Zo aida Sosa
Fe e a and Lidia Robaina Robaina.
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Alumna: Tania Da ias He nández
Pa a la elabo ación de es e abajo he ealizado las siguien es ac i idades:
a) Es udio bibliog á icos sob e el conocimien o, la de e minación, la impo ancia,
las aplicaciones de as axan ina en di e en es ma ices biológicas,
b) Pa e expe imen al, ealizando algunas ex acciones con encionales e
in es igando la p epa ación del pa ón de as axan ina. Además, he abajado con
un sis ema UHPLC.
Du an e el pe iodo en el que he ealizado las ac i idades mencionadas an e io men e
he enido una elación luida con mis compañe os de labo a o io y con mis di ec o as,
sin iéndome pa e in eg ada del g upo de in es igación de Análisis Químico
Medioambien al (AQMA).
Algunos aspec os posi i os del T abajo Fin de Más e (TFM) son el habe ap endido
cuál es el compo amien o de la as axan ina, pa a qué si e y la impo ancia que iene,
abaja en el labo a o io ap endiendo cómo hace las ex acciones de as axan ina de
algunos peces y maneja el UHPLC. Además, me he sen ido implicada en el abajo
ealizado y engo una buena elación con el g upo AQMA.
Algunos aspec os nega i os del mismo han sido la demo a en la llegada del pa ón
de as axan ina, las complicaciones en de e mina dicho compues o y no ob ene los
esul ados espe ados, inicialmen e, pa a ealiza el abajo expe imen al que habíamos
p og amado en un p ime momen o.
Realiza es e abajo me ha supues o ene una isión global de lo qué es el abajo
de in es igación, apo ándome un conocimien o impo an e sob e el ema elegido pa a
mi TFM. El inal de es e abajo me ha causado mucho in e és sob e las u u as
endencias que puede ene es e campo, quedándome con ganas de con inua abajando
y ampliando mis conocimien os.