Astaxanthin determination in marine biological samples : an overview

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 Más e Uni e si a io en Oceanog a ía Tania Da ias He nández 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) Tania Da ias He nández Más e Uni e si a io en Oceanog a ía 14 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 16 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 Más e Uni e si a io en Oceanog a ía Tania Da ias He nández 17 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 Tania Da ias He nández Más e Uni e si a io en Oceanog a ía 18 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. Más e Uni e si a io en Oceanog a ía Tania Da ias He nández 19 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 Tania Da ias He nández Más e Uni e si a io en Oceanog a ía 20 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 21 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 Tania Da ias He nández Más e Uni e si a io en Oceanog a ía 22 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 Más e Uni e si a io en Oceanog a ía Tania Da ias He nández 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 30 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– Tania Da ias He nández Más e Uni e si a io en Oceanog a ía 32 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 33 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 34 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. Más e Uni e si a io en Oceanog a ía Tania Da ias He nández 35 6. Re e ences. Al ami ano, R. C., D dák, M., Simon, P., Smelík, A., & Simko, P. (1992). S abili y o ed bee pigmen concen a e in maize s a ch. Jou nal o he Science o Food and Ag icul u e, 58(4), 595–596. Ama , E. C., Ki on, V., Sa oh, S., & Wa anabe, T. (2001). In luence o a ious die a y syn he ic ca o enoids on bio-de ence mechanisms in ainbow ou , Onco hynchus mykiss (Walbaum). Aquacul u e Resea ch, 32, 162–173. Anan o anich, S., Gulick, P., & Ib ahim, R. K. (1995). Fla onol sul o ans e ase-like cDNA clone om Fla e ia biden is. Plan physiology, 107(3), 1019–20. Bakke , J., & Timbe lake, C. F. (1997). Isola ion, Iden i ica ion, and Cha ac e iza ion o New Colo -S able An hocyanins Occu ing in Some Red Wines. Jou nal o Ag icul u al and Food Chemis y, 45(1), 35–43. Ba ua, A. B. (2001). Imp o ed no mal-phase and e e sed-phase g adien high- pe o mance liquid ch oma og aphy p ocedu es o he analysis o e inoids and ca o enoids in human se um, plan and animal issues. Jou nal o Ch oma og aphy A, 936(1), 71–82. Be anco , M. B., No d um, S., A alah, E., Caballe o, M.J., Bení ez-San ana, T. Robaina, L. & IZQUIERDO, M.S. (2011). Po en ial o h ee new k ill meal p oduc s o seab eam la al p oduc ion. Aquacul u e Resea ch 19, 395-406. Bi keland, S., & Bje keng, B. (2004). Ex ac abili ies o as axan hin and p o ein om muscle issue o A lan ic salmon (Salmo sala ) as a ec ed by b ine concen a ion and pH. Food Chemis y, 85(4), 559–568. Bje keng, B., Følling, M., Lagocki, S., S o ebakken, T., Olli, J. J., & Als ed, N. (1997). Bioa ailabili y o all-E-as axan hin and Z-isome s o as axan hin in ainbow ou (Onco hynchus mykiss). Aquacul u e, 157(1), 63–82. Tania Da ias He nández Más e Uni e si a io en Oceanog a ía 36 Bje keng, B. (2000). Ca o enoid pigmen a ion o salmonid ishes- ecen p og ess. A ances en nu ición acuícola V. Memo ias del V simposium in e nacional de nu ición acuícola, 19-22. Bligh, E. G., & Dye , W. J. (1959). A apid me hod o o al lipid ex ac ion and pu i ica ion. Canadian Jou nal o Biochemis y and Physiology, 37(8), 911–917. Bowen, J., Sou a , C., Se wa a, R. D., La Gocki, S., Whi e, D. A., Da ies, S. J., & Young, A. J. (2002). U iliza ion o (3S,3’S)-as axan hin acyl es e s in pigmen a ion o ainbow ou (Onco hynchus mykiss). Aquacul u e Nu i ion, 8(1), 59–68. Bu le, L., C amp on, V., & Williams, P. (2001). The e ec o eed pigmen ype on lesh pigmen deposi ion and colou in a med A lan ic salmon, Salmo sala L. Aquacul u e Resea ch, 32(2), 103–111. . Caballo, C., Cos i, E. M., Sicilia, M. D., & Rubio, S. (2012). De e mina ion o supplemen al eeding needs o as axan hin and can haxan hin in salmonids by sup amolecula sol en -based mic oex ac ion and liquid ch oma og aphy–UV/VIS spec oscopy. Food chemis y, 134(2), 1244-1249. Cas o-Puyana, M., He e o, M., U e a, I., Mendiola, J. A., Ci uen es, A., Ibáñez, E., & Suá ez-Al a ez, S. (2013). Op imiza ion o clean ex ac ion me hods o isola e ca o enoids om he mic oalga Neochlo is oleoabundans and subsequen chemical cha ac e iza ion using liquid ch oma og aphy andem mass spec ome y. Analy ical and bioanaly ical chemis y, 405(13), 4607–16. Ce on, M. C., Campos, I., Sanchez, J. F., Acién, F. G., Molina, E., & Fe nandez-Se illa, J. M. (2008). Reco e y o lu ein om mic oalgae biomass: de elopmen o a p ocess o Scenedesmus alme iensis biomass. Jou nal o ag icul u al and ood chemis y, 56(24), 11761-11766. Choube , G., & Hein ich, O. (1993). Ca o enoid pigmen s o he g een alga Haema ococcus plu ialis: assay on ainbow ou , Onco hynchus mykiss, pigmen a ion in compa ison wi h syn he ic as axan hin and can haxan hin. Aquacul u e,112(2),217–226. Más e Uni e si a io en Oceanog a ía Tania Da ias He nández 37 Co al, H.G., Hube man, A., De la Lanza, G., and Mon oy-Ruiz, J.( 1997). Pigmen a ion o he ainbow ou (Onco hynchus mykiss) wi h oil-ex ac ed as axan hin om he langos illa (Pleu oncodes planipes). A chi os La inoame icanos de Nu ición., 47:237–241. Co al, G., Hube man, A., de la Lanza, G., & Mon oy-Ruiz, J. (1998). Muscle Pigmen a ion o Rainbow T ou (Onco hynchus mykiss) Fed on Oil-Ex ac ed Pigmen om Langos illa (Pleu oncodes planipes) Compa ed wi h Two Comme cial Sou ces o As axan hin. Jou nal o Aqua ic Food P oduc Technology, 7(2), 31–45. Co al-Hinos oza, G. N., & Bje keng, B. (2002). As axan hin om he ed c ab langos illa (Pleu oncodes planipes): op ical R/S isome s and a y acid moie ies o as axan hin es e s. Compa a i e Biochemis y and Physiology Pa B: Biochemis y and Molecula Biology, 133(3), 437–444. Cyano ech, 2000. www.cyano ech.com/h ml/as a/na u/na u ose.h ml. Da achai, J., Piya i a i i o akul, S., and Menas e a, P. 1999. E ec o as axan hin on g ow h and su i al o Penaeus monodon la ae., In: P oceedings o he 37 h Kase sa Uni e si y Annual Con e ence. pp. 36–41. De la Fuen e, J.C., Oya zun, B., Quezada, N., del Valle, J. M., Solubili y o ca o enoid pigmen s (lycopene and as axan hin) in supe c i ical ca bon dioxide. Fluid Phase Equilib. 2006, 247, 90–95. Delgado-Va gas, F., Jiménez, A. R., & Pa edes-López, O. (2000). Na u al pigmen s: ca o enoids, an hocyanins, and be alains--cha ac e is ics, biosyn hesis, p ocessing, and s abili y. C i ical e iews in ood science and nu i ion, 40(3), 173–289. Dissing, B. S., Nielsen, M. E., E sbøll, B. K., & F osch, S. (2011). Mul ispec al imaging o de e mina ion o as axan hin concen a ion in salmonids. PloS one, 6(5), 19032. Tania Da ias He nández Más e Uni e si a io en Oceanog a ía 38 Félix-Valenzuela,L.,Higue a-Ciapa a, I, Goycoolea-Valencia F., & A guemes-Monal W. (2001).Supe c i ical CO2/ e anol ex ac ion o as axan hin om blue c ab (Callinic es Sapidus) shell was e. Jou nal o Food P ocess Enginee ing, 24(2), 101–112. Fel l, L., Pacako a, V., S ulik, K., & Volka, K. (2005). Reliabili y o Ca o enoid Analyses: A Re iew. Cu en Analy ical Chemis y, 1(1), 93–102. Flo es-Co e a, L. B., & Sánchez, S. (2001). Coppe bu no i on limi a ion inc eases as axan hin p oduc ion by Pha ia hodozyma in a chemically de ined medium. Bio echnology Le e s, 23(10), 793–797. Fu , H. C. (2004). Analysis o e inoids and ca o enoids: p oblems esol ed and unsol ed. The Jou nal o nu i ion, 134(1), 281S–285S. Ga cía-Fonseca, S., Balles e os-Gómez, A., Rubio, S., & Pé ez-Bendi o, D. (2008). Coace a i e ex ac ion o Och a oxin A in wines p io o liquid ch oma og aphy/ luo escence de e mina ion. Analy ica chimica ac a, 617(1), 3-10. Ga cía Rome o, J., Kalinowski, T., Izquie do, M.S. & Robaina, L. (2010). Ma ine and eshwa e c ac meals in die s o ed po gy (Pag us pag us): e ec on g ow h, ish composi ion and skin colou . Aquacul u e Resea ch, 41,1748-1758. Ga cía Rome o, J., Ginés, R., Izquie do, M.S. & Robaina, L. (2013) . E ec o die a y subs i u ion o ish meal o ma ine c ab and echinode m meals on g ow h pe o mance, ammonia exc e ion, skin colou , and lesh quali y and oxida ion o ed po gy (Pag us pag us). Aquacul u e. In p ess. AQUA-D-13-00950 Goodwin, T. (1986). Me abolism, nu i ion, and unc ion o ca o enoids. Annual e iew o nu i ion, 6, 273–293. G anado-Lo encio, F., He e o-Ba budo, C., Blanco-Na a o, I., & Pé ez-Sac is án, B. (2010). Sui abili y o ul a-high pe o mance liquid ch oma og aphy o he de e mina ion o a -soluble nu i ional s a us ( i amins A, E, D, and indi idual ca o enoids). Analy ical and bioanaly ical chemis y, 397(3), 1389–93. Más e Uni e si a io en Oceanog a ía Tania Da ias He nández 39 Hanne H. F Re sgaa d., B ockho , P. B., & Jensen, B. (1998). Biological Va ia ion o Lipid Cons i uen s and Dis ibu ion o Tocophe ols and As axan hin in Fa med A lan ic Salmon ( Salmo sala ). Jou nal o Ag icul u al and Food Chemis y, 46(3), 808–812. He e o M, Mendiola JA, Cas o-Puyana M, Ibáñez E (2012) In: Hayes M (ed) Ma ine bioac i e compounds: Sou ces, cha ac e iza ion and applica ions. Sp inge , New Yo k, pp 55–98 Higue a-Ciapa a, I., Félix-Valenzuela, L., & Goycoolea, F. M. (2006). As axan hin: a e iew o i s chemis y and applica ions. C i ical e iews in ood science and nu i ion, 46(2), 185–96. Hol in, K., Kuehnle, M., Rehbein, J., Schule , P., Nicholson, G., & Albe , K. (2009). De e mina ion o as axan hin and as axan hin es e s in he mic oalgae Haema ococcus plu ialis by LC-(APCI)MS and cha ac e iza ion o p edominan ca o enoid isome s by NMR spec oscopy. Analy ical and bioanaly ical chemis y, 395(6), 1613–22. Hou, X., Li, Y., Wu, G., Wang, L., Hong, M., & Wu, Y. (2010). De e mina ion o Pa a Red, Sudan Dyes, Can haxan hin, and As axan hin in Animal Feeds Using UPLC. Jou nal o Ch oma og aphic Science, 48(1), 22–25. Huang, M., & Liu, D. (2013). High-pe o mance hin laye ch oma og aphy de e mina ion o as axan hin in Euphausia supe ba oil. Chemis y o Na u al Compounds, 49(1), 145–147. Igene, 2003. www.igene.com/s udy1.h ml. Jaime, L., Rod íguez-Meizoso, I., Ci uen es, A., San oyo, S., Sua ez, S., Ibáñez, E., & Seño ans, F. J. (2010). P essu ized liquids as an al e na i e p ocess o an ioxidan ca o enoids' ex ac ion om Haema ococcus plu ialis mic oalgae. LWT-Food Science and Technology, 43(1), 105-112. 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.