epa and dha extraction

Separations

ZINC ION DOPED SOLID-PHASE EXTRACTION OFEICOSAPENTAENOIC ACID AND DOCOSAHEXAENOICACID FROM ANTARCTIC KRILL

Baokun Tang, Minglei Tian, and Kyung Ho RowDepartment of Chemical Engineering, Inha University, Incheon, Korea

Antarctic krill crude extracts contain high levels of eicosapentaenoic acid (EPA) and

docosahexaenoic acid (DHA). Accordingly, the solid phase extraction of EPA and DHA

from Antarctic krill crude extracts has attracted signicant research interest. This study

compared the extraction of EPA and DHA from Antarctic krill crude extracts using an

aminopropyl, zinc ion-doped silica, and C18 and zinc ion-doped C18 solid-phase column.

The best extraction effect was obtained using the zinc ion-doped C18 SPE with water con-

taining methanol as the eluant. The efciency increased gradually with increasing methanol

concentration from 12.5 to 25% in the washing stage, and when pure methanol (5.0mL) or

acetonitrile (3.0mL) was used as the eluant. To detect EPA and DHA, the acids were rst

converted to their methyl esters and detected by gas chromatography with ame ionization

detection (GCFID). In the zinc ion-doped C18 elution fractions, EPA and DHA were

isolated from the crude extracts in high yield (8591% (r2 4.86.3%)).

Keywords: Antarctic Krill; Aminopropyl; Docosahexaenoic acid; Eicosapentaenoic acid; Zinc ion doped

C18; Zinc ion doped silica

INTRODUCTION

Antarctic krill is a species of krill found in the Antarctic waters of the SouthernOcean. Beginning with the discovery of Antarctic Krill, many researchers have paidincreasing attention to this potential source of food (Hamner et al. 1983; Ross andQuetin 1986; Hempel and Hempel 1986). Estimates suggest that although the stockof Antarctic krill in the Southern Ocean is difcult to determine accurately, the abun-dance of biomass is likely to be the highest of any multi-cellular animal species onEarth (Nicol, James, and Pitcher 1987; Priddle et al. 1998). Reports in the 1980s sug-gested that this abundant source may allow yearly catches in the range of 50100 mil-lion tons, which was in the same order as the total catch of sh in the world at that

Received 27 April 2012; accepted 4 June 2012.

This study was a part of the project titled Korea Sea Grant Program (Gyeong-gi Sea Grant)

funded by the Ministry of Land, Transport, and Maritime Affairs, Korea.

Address correspondence to Kyung Ho Row, Department of Chemical Engineering, Inha Univer-

sity, Incheon 402-751, Korea. E-mail: [email protected]

Analytical Letters, 45: 26752686, 2012

Copyright # Taylor & Francis Group, LLCISSN: 0003-2719 print=1532-236X online

DOI: 10.1080/00032719.2012.702179

2675

time (Ellingsen and Mohr 1979). In recent years, the annual sustainable capture fromAntarctic krill has increased to 70200 million tons (Suzuki and Shibata 1990). Bycomparison, the total annual capture from all sheries has been approximately130 million tons since 2000 (Gigliotti et al. 2011). This suggests that Antarctic krillmay be comparable to the biomass of all other aquatic species currently harvested.

Eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA) are twoomega-3 polyunsaturated fatty acids (PUFA) that are essential to normal growthand health. An adequate dietary intake of EPA and DHA is considered benecialfor alleviating a range of chronic diseases, such as cardiovascular, hypertensive,inammatory and autoimmune disorders, depression and certain disrupted neuro-logical functions (Simopoulos 1991, 1997; Dyerberg 1986; Mehta et al. 1988; Kin-sella 1986; Shahidi and Wanasundara 1998; Mansour 2005). Fricke et al. (1984)reported that free fatty acids (FFA) (8-16%) in the main lipid cases of Antarctic krill,and EPA and DHA were identied as the major components of FFA (Fricke et al.1984; Kolakowska, Kolakowski, and Szczygielski 1994).

The general chemical and biochemical composition of Antarctic krill has beenthe subject of many studies (Ellingsen and Mohr 1979; Fricke et al. 1984; Kola-kowska et al. 1994; Kolakowski 1986). A small number of comprehensive studiesexamined PUFA but most used crude preparations and detection methods (Wilsonet al. 1993; Belarbi, Molina, and Chisti 2000; Fountoulaki et al. 2003; Giogios et al.2009). Few studies of EPA and DHA in Antarctic krill have been reported. This maybe because Antarctic krill contains large quantities of saturated and unsaturated fattyacids as well as other compounds, such as lipids, sterols, amino acids, alcohols, andso forth (Fricke et al. 1984). Isolating EPA and DHA from these complicated com-positions in a sample is quite difcult. Moreover, it is difcult to detect EPA andDHA correctly and accurately among the many compounds in the samples. Someresearchers attempted a range of techniques to isolate and prepare PUFA from shell-sh, sh oil and edible oils etc. samples. These include solid phase extraction,reversed-phase high-performance liquid chromatography, silver-ion high-perfor-mance liquid chromatography, argentated silica gel chromatography, aminopropylsolid phase extraction, and so forth (Mansour 2005; Wilson et al. 1993; Lacazeet al. 2007; Adlof and List 2004; Guil-Guerrero, Campra-Madrid, andNavarro-Juarez 2003). On the other hand, these processes involved the use of toomany processing operations that reduce the target recovery and magnication time.Furthermore, HPLC may be better used to separate and detect the targets of PUFA(Mehta et al. 1988; Adlof and List 2004) but it is an expensive technique that is noteasily scalable to obtain large quantities of puried targets. Therefore, there is nosimple and agreed method for further purifying PUFA from different samples.

More recently, solid-phase extraction (SPE) was applied to the isolation of lipidclasses by step-wise sequential elution. SPE with commercially available and auton-omous sorbent cartridges was applied recently and offers a viable alternative to con-ventional sample preparation methods (Gelencser et al. 1995). Speed, reliability, lowcost, and the use of minimal amounts of solvent are the main advantages of SPE overthin layer chromatography (TLC) and column chromatography (Giacometti, Milose-vic, and Milin 2002). Different types of SPE packing, such as silica, C18, and bondedaminopropyl-silica have been used to separate targets into classes (Giacometti et al.2002; Poerschmann et al. 2006; Kaluzny et al. 1985). The SPE isolation of the PUFA

2676 B. TANG ET AL.

fraction from lipid samples can be achieved using small volumes of solvent and with ahigh degree of purity and high target recovery (Lacaze et al. 2007).

Normally, the PUFA in samples is rst transformed to methyl esters, and thendetected by capillary column gas chromatography with ame ionization detection(GCFID) (Lacaze et al. 2007; Brondz 2002). The esterication of PUFA is essentialfor accurate detection. The reaction conditions were optimized in another study(Tang et al. 2012).

This report describes the use of different packingmaterials for isolating EPA andDHA fromAntarctic krill by solid phase extraction, as well as an efcient SPEmethodto characterize the EPA and DHA composition in different Antarctic krill samples.

EXPERIMENTAL

Chemicals

Methanol, dichloromethane, hexane, acetonitrile (HPLC grade), and 95% sulfu-ric acid (extra pure)were supplied byDuksanPureChemical Co., Ltd. (Ansan,Korea).Double distilled water was ltered through a vacuum pump (Waters, Milford, MA,USA) and a lter (HA-0.45, Waters, Milford, MA, USA) prior to use in SPE washingor elution. Methanol and acetonitrile were used in the SPE washing or elution stage,and dichloromethane was utilized as the extractant in Soxhlet extraction. Methanoland sulfuric acid were employed as the esterifying agent and catalyst, respectively,for EPA and DHA esterication. There is a key sulfuric acid to methanol ratio (v=v)in esterication (as described in a following section). Hexane was used to extract theEPA and DHA methyl esters in the mixture solution after esterication. The C18(15 mm, E. Merck, Germany), aminopropylsilyl (50 mm, Alltech, US), silica (25 mm,YMC, Japan), and zinc sulfate (extra pure, Duksan pure chemical CO. LTD, Korea)were used as the SPE solid phase in the packing cartridge.

The EPA (C20:5, analytical standard), DHA (C22:6, purity98%) EPA methylester (analytical standard), DHA methyl ester (analytical standard), and 3,5-di-tert-butyl-4-hydroxytoluene (BHT) (analytical standard, as anti-oxidant) were acquiredfromSigmaAldrich, Co. andused to optimize the esterication process andquantitat-ive analysis. The BHT (0.01%, based on PUFA or PUFA esters solution) was added toall solutions, which included EPA, DHA, EPAmethyl esters, and DHAmethyl esters,to prevent oxidation. Both EPA andDHAmethyl ester standardswere initially dilutedin acetonitrile to give a calibration stockmixture solution (both 5.0mg=mL) dependingon the composition of the EPA and DHA methyl esters. The stock mixture solutionsand acetonitrile were combined to produce seven calibration solutions (ranging from0.0001 to 1.0mg=mL depending on the EPA and DHA methyl esters composition)for quantitative analysis of the EPA and DHA methyl esters in each sample solution.Standard EPA and DHA mixture solutions (both 1.0mg=mL) based on acetonitrilewere used to optimize the esterication process.

Antarctic Krill Preparation

Whole fresh and dried Antarctic krill samples were supplied by Insung Foodand Biological Products Co., Ltd. (Seoul, Korea). The Antarctic krill was collected

SOLID-PHASE EXTRACTION FROM ANTARCTIC KRILL 2677

from the Southern Ocean in July 2011 and processed by Insung Food and BiologicalProducts Co., Ltd. The fresh Antarctic krill measured 45 cm from head to tail andthe dried product measured 23 cm. The fresh and dried krill blocks were trans-ported to the laboratory in heavily insulated industrial strength boxes lled withdry ice. Upon arrival, the samples were removed from the boxes and stored immedi-ately in a refrigerator until needed.

PUFA Extraction

The structure of one whole Antarctic krill includes mainly head and pleonparts. In this study, the two parts of fresh or dried Antarctic krill were separatedusing stainless tweezers and examined. The fresh or dried head and pleon sampleswere extracted with a dichloromethane solvent using a Soxhlet extraction method(AOAC 1998). After extraction, 0.01% BHT (v=v, based on the extraction solution)for anti-oxidation of EPA and DHA were added to the solution. All the sample solu-tions were stored in a refrigerator.

Solid-Phase Extraction

The SPE was used to separate PUFA from a complicated mixture of com-pounds. The separation of EPA and DHA from the Antarctic krill crude extractswas achieved using different stationary phases, such as C18, aminopropyl-silica, zincsulfate-silica, and zinc sulfate-C18. Each type of SPE cartridge was prepared using asimilar process as follows: 200mg of adsorbent (20mg zinc sulfate if needed) for col-umn chromatography was added to methanol (20mL). The mixture was agitated for20min and the slurry mixture was added to the cartridge (9 cm 0.9 cm i.d.). Meth-anol was pumped using a vacuum pump. The packed SPE cartridges were activatedin a dryer heated to 50C, at which point they were removed from the dryer andcooled to room temperature.

The SPE cartridges were conditioned with water (1.0mL) and methanol(1.0mL), and each Antarctic krill crude extract (1.0mL) was loaded onto a cartridge.During SPE, all the crude extracts were the same as the Soxhlet extracts of fresh Ant-arctic krill pleon. Each SPE process is reported in the following section. The inter-ferents were washed with watermethanol, and the PUFAs were eluted withmethanol or acetonitrile into a 5mL glass tube. The contents of the tubes were ester-ied and detected as follows.

GC-FID Analysis

The optimization conditions for the esterication of EPA and DHA arereported elsewhere. Therefore, the EPA and DHA from the SPE fractions were ester-ied directly under the optimal conditions. The EPA and DHA methyl esters stan-dards as well as the fractions of the EPA and DHA esters obtained from the SPEfractions were analyzed by GC=FID on a Yong Lin Instrument (Korea) GC-6100with a DB-1701 capillary column (30m 0.320mm 1.00 mm) (Agilent Technolo-gies) and detected using a FID detector. Ultra-high purity hydrogen (purity99.999%) was used as the carrier gas with a ow rate of 1.80mL=min. The oven

2678 B. TANG ET AL.

temperature was programmed as follows: initial temperature of 140C increasing to220C at 5C=min, followed by a further increase to 280C at 10C =min, and held atthat temperature for 4min. The total time for a single GC run was 26min. The FIDand injector temperature was 300C and 280C, respectively. The injection was per-formed in split mode at a rate of 42:1.

RESULTS AND DISCUSSION

SPE of EPA and DHA From Antarctic Krill Extracts

SPE is adsorption chromatography using columns of different materials and isuseful for isolating target molecules from complicated mixtures, making them easy todetect. The methods used to obtain fractions rich in PUFA are commonly based onthe differences in polarity and spatial conguration of the PUFA present in the crudeextracts. These differences are associated mainly with the number of double bonds inthe carbon chain. Therefore, PUFA can be extracted and separated according totheir degree of unsaturation. The results of the extraction of PUFA using differentsolid phase columns were compared.

The crude extracts and fractions obtained by SPE were analyzed by gas chroma-tography to establish the recovery. The initial amount of EPAorDHA in theAntarctickrill crude extract loading on SPE was detected and recorded. After the SPE process,the amount of PUFA in the different fractions was also detected and recorded. Therecovery of EPA or DHA in SPE was calculated using the following equation:

Recovery % Amount of target in SPE fractionAmount of target in loading crude extract

100% 1

In this paper, the capacity factor (k) of the target on each SPE fraction was calculatedusing the equation described by Gelencser et al. (1995):

Capacity factor k Amount of target adsorbed on the stationary phaseAmount of target in SPE fraction

2

In the aforementioned equation, the corresponding capacity factor of a targetwas calculated when each of the SPE fractions was nished. Capacity factor meanschanges in the target between the solid and mobile phases.

Aminopropyl Bonded Silica Column

The SPE isolation of the fatty acids fraction from the other lipid classes wasachieved using aminopropyl-silica cartridges (Wilson et al. 1993; Lacaze et al.2007; Giacometti et al. 2002; Kaluzny et al. 1985). Therefore, the separation ofEPA and DHA from krill crude extracts was attempted to achieve bonded phaseaminopropyl-silica cartridges.

The crude extracts were applied to silica-aminopropyl columns, as listed inTable 1. The recovery and capacity factor of EPA and of DHA in each SPE stepis also shown. In the loading step fraction, the amounts of EPA and DHA were


121.0 and 110.3 mg, respectively, corresponding to 17.5% and 16.1% recovery in thecrude sample. The two targets in the crude extracts were not absorbed completely bythe silica-aminopropyl solid phase, and the amount ofEPAabsorbedwas less than thatof DHA. After loading, 5.3% and 3.7% recovery of EPA and DHA, respectively, werealso washed with 1.0mL of a methanol to water mixture (2.5%, v:v) was rst used towash the referents in the crude extracts. Although some referents were washed in thisfraction, a certain amount of the targetswas alsowashed away. Therefore, the referentsand targets could not be separatedwell. Increasing themethanol content in thewashingsolution to 5.0% resulted in more EPA and DHA being washed with the referents, aslisted in Table 1. One reason may be the bonding force between the column azyl andlow concentration of carboxyl groups. Accordingly, the targets combined with theinterferents were washed easily in the same washing fraction. In addition, the capacityfactors in the washing steps were small, meaning the targets interacted weakly with thesolid phase. Despite more referents being washed in this fraction, the targets were alsolost from the solid phase. Consequently, in the next elution fraction, only 66.2% and74.8% of EPA and DHA, respectively, were obtained in the elution fraction. On theother hand, the total recovery of EPA and DHAwere both approximately 100%. Thiscompared favorably with the SPEmethod detailed by Kaluzny et al. (1985), where therecovery of one C18:1 unsaturated fatty acid contained in a lipid calibration standardwas 101%. This was better than a similar SPEmethod reported by Lacaze et al. (2007))where the recovery ofEPAandDHAfrom shellsh extractswas 8490%.Although thetotal recovery of EPA or DHA was high, the recovery in the elution fraction was notideal and the targetswere not separated completely from the referents. Possibly, the dis-advantage of the silica-aminopropyl SPE of EPA andDHA can be overcome by select-ing an appropriate washing and elution solution system. The solution system mightrequire further research. This study attempted to change the solid phase to achievethe research objective.

Zinc Ion Doped Silica Column

The use of Ag doped silica solid phase column extraction of PUFA had beendescribed (Belarbi et al. 2000; Guil-Guerrero et al. 2003; Ryu et al. 1997). The Ag

Table 1. Amounts, recovery, and capacity factor of EPA and DHA from the Antarctic krill extracts using

silica-aminopropyl SPE

Solvent EPA DHA

Methanol

content (%, v:v)

Volume

(mL)

Amount

(mg)Recovery

(%)

Capacity

factor (k)

Amount

(mg)Recovery

(%)

Capacity

factor (k)

Loading

step

0 1.0 121.0 17.5 4.8 110.3 16.1 5.2

Washing

step

2.5 1.0 37.1 5.3 14.6 25.4 3.7 21.7

5 1.0 73.3 10.4 6.4 34.1 5.1 14.7

Elution

step

100 2.0 462 66.2 514 74.8

Total 693 99.4 683 99.7

Note: means that the value is not in the range of determination.

2680 B. TANG ET AL.

doped silica gel fractionation of PUFA is based on the bonding force between theAg blank outermost orbit and the olenic p electrons of PUFA. Considering thatZn2 has a similar electron conguration to Ag, the Zn2 doped silica solid phasewas selected to isolate EPA and DHA from the krill crude extracts. The retention ofEPA and DHA by zinc ion chromatography, which was attributed primarily to theinteraction of Zn2 ions and the olenic p electrons of the sample molecule(s), can becorrelated to some extent with these double bonds in the targets. The highly unsatu-rated PUFA might be retained more strongly at the Zn2 doped silica column.

The Zn2 doped silica solid phase was applied to the extraction of EPA andDHA from krill crude extracts. Table 2 lists these SPE fractions and target recovery,capacity factor. The recovery of EPA and DHA in the loading fraction was 19.5 and16.6%, respectively, which suggests that the absorbing force of Zn2 doped silica wasinsufcient to retain the two targets in 1.0mL of the krill crude extracts. A washingfraction containing 2.5% methanol resulted in 11.8% and 10.5% recovery of EPA andDHA, respectively. Increasing the methanol fraction to 5% resulted in EPA andDHA recovery of 24.7 and 23.6%, respectively, based on the crude extract. Thecapacity of the two targets in the loading and washing fractions were small, so mostof the EPA and DHA had been obtained in the two fractions. The interferents obvi-ously decreased and there was greater EPA and DHA purity (41.9 and 42.9% recov-ery, respectively) in the elution fraction. The total recovery of EPA or DHA (97.9and 93.6% recovery, respectively) was similar to that reported by Guil-Guerreroet al. (2003) in that several unsaturated fatty esters were obtained by Ag doped sil-ica extraction. The objective of isolating the targets from the crude extracts waseffective using this method but it was not enough to completely separate the targetsfrom the crude mixtures. Therefore, a search was made to identify a perfect solidphase to achieve this research aim.

C18 Column

After previously employing aminopropyl-silica and ion-doped silica to purifyPUFA, a solid phase of C18 was assessed for purifying PUFA with a range of chain

Table 2. Amounts, recovery, and capacity factor of EPA and DHA from Antarctic krill extracts using

silica-zinc ion SPE

Solvent EPA DHA

Methanol

content (%, v:v)

Volume

(mL)

Amount

(mg)Recovery

(%)

Capacity

factor (k)

Amount

(mg)Recovery

(%)

Capacity

factor (k)

Loading

step

0 1.0 131.4 19.5 4.1 112.3 16.6 5.0

Washing

step

2.5 1.0 83.2 11.8 5.8 71.3 10.5 6.9

5 1.0 170.6 24.7 1.8 153.7 23.6 2.1

Elution

step

100 2.0 292.4 41.9 292.6 42.9

Total 677.6 97.9 629.9 93.6



lengths and unsaturation (Lacaze et al. 2007; Giacometti et al. 2002; Kaluzny et al.1985; Ryu et al. 1997; Ulberth and Achs 1990; Wilson and Sargent 1992). Althoughone paper (Mansour 2005) reported that RP-HPLC (C18 packed) had been opti-mized for the isolation and detection of PUFA, there is no published method usingC18 as a solid phase in the cartridge to extract and purify PUFA. Therefore, C18 as asolid phase in the cartridge was developed as follows.

Table 3 lists the sequence of operations in the isolation of EPA and DHAfrom an Antarctic krill extract by C18 SPE. The table also shows the EPA andDHA recovery at various steps, such as loading, washing, and elution. NeitherEPA nor DHA were detected in the loading fraction or 5% methanol washing frac-tion. There might be a relationship between the length of the compounds and C18,which shows that longer chain compounds are eluted later on a C18 solid phasethan shorter chain analogs (Mansour 2005). Therefore, when a polar solvent isused for washing, the longer chains of EPA and DHA are retained on the column,and the shorter chain interferents are washed away. Hence, the capacity factors ofthe two targets in the washing step were obviously higher than the previous twomethods. When the methanol content in the washing solution was increased, someless polar interferents were washed and only a small amount of the targets washedaway (recovery

C18 Column and Zinc Sulfate

To better isolate EPA and DHA completely from the crude extracts, themethod of C18 SPE was improved by doping Zn

2 into C18 as a solid phase. The rea-son for combining C18 and zinc ions was that while high recovery of the targets in theelution fraction was maintained, the targets were isolated completely from the crudeextracts. In C18 SPE, although a high elution recovery was achieved, interferentswith a similar length chain to the targets were also combined with the targets inthe washing and elution fraction. This method employed C18 with a long chain targetand Zn2 with double bonds of the target to isolate it from the interferents. Thiscombined the advantages of the ion force and C18 alkyl chain force. The recoveryof EPA and DHA in the elution fraction was 96.8 and 95.2%, respectively, as shownin Table 4. Although the capacity factor of the target on Zn2-C18 phase was smallerthan the C18 phase in the washing and elution stage, the targets were apparentlyseparated from the interferents on the Zn2-C18 phase, as shown in Figure 1. Theunsaturated targets were adsorbed strongly on the Zn2-C18 stationary phase dueto the formation of very stable coordination complexes, and require pure methanolor acetonitrile to elute them.

Based on the aforementioned discussion, apparently different effects on differ-ent SPE were observed. The C18 and zinc-doped C18 SPE were better methods forenriching the EPA and DHA targets. Moreover, the latter was the best methodfor enriching the targets. Highly unsaturated EPA and DHA are strongly adsorbedon the zinc ion-doped C18 stationary phase due to the formation of stable forces.Therefore, the targets can remain in the column during the washing stage and canbe eluted with high recovery in the elution fractions.

Applications

Table 5 lists the results of the zinc ion-doped C18 SPE to isolate EPA and DHAfrom 12 Antarctic krill samples. The targets were then transformed to methyl esters

Table 4. Amounts, recovery, and capacity factor of EPA and DHA from Antarctic krill extracts using zinc

ion-doped C18 SPE

Solvent EPA DHA

Methanol

content (%, v:v)

Volume

(mL)

Amount

(mg)Recovery

(%)

Capacity

factor (k)

Amount

(mg)Recovery

(%)

Capacity

factor (k)

Loading

step

0 1.0

Washing

step

12.5 5.0

15 3.0 12.5 1.8 54.6

20 3.0 22.3 3.1 30.7 15.9 2.4 40.7

25 3.0 45.1 6.4 13.9 29.1 4.4 21.2

Elution

step

100 2.0 604.5 85.5 584.4 88.4

Total 673.2 96.8 629.3 95.2



and detected by GC=FID. Higher recovery of EPA and DHA was noted in eachsample. The application of the developed zinc ion-doped C18 SPE method to the12 samples showed similar EPA and DHA recovery with some differences betweenspecies; the recovery of EPA and of DHA ranged from 85 to 91%. In fresh samples,80% water was detected in the fresh sample. Overall, the amount of target in thefresh samples was lower than in the dried sample.

CONCLUSIONS

A zinc ion doped C18 SPE method was developed to isolate EPA and DHAfrom complex Antarctic krill crude extract mixtures. This method was more effective

Table 5. Amounts of EPA and DHA from different Antarctic krill crude extracts, and recovery of EPA

and DHA from the different Antarctic krill extracts using zinc ion doped C18 SPE

Sample Target Amount mg g1 Recovery (%)

Fresh

Head EPA 4.87 0.23 86.7 5.3DHA 4.26 0.22 89.4 6.1

Pleon EPA 2.79 0.13 85.5 5.6DHA 2.62 0.14 88.4 5.9

Dried

Head EPA 27.80 1.08 87.2 4.8DHA 24.68 1.01 90.1 5.1

Pleon EPA 19.89 0.78 91.1 6.3DHA 18.46 0.68 90.4 5.7

Note: Amount is expressed as the amount of EPA or DHA in sample and represents means standarddeviation of 3 separate experiments.

Figure 1. GC chromatograms of different fractions of zinc ion-doped C18 SPE of Antarctic krill extract.

2684 B. TANG ET AL.

in separating the targets than the other SPE techniques. A gradient increase in themethanol to water ratio in the extraction procedure was used to extract the two tar-gets from different types of samples. This method, which showed good selectivityand high recovery, was applied successfully to extract the EPA and DHA presentin different species. After separation, the targets were esteried and analyzed byGC=FID. This method was applied to the extraction and detection of PUFA from12 Antarctic krill samples. The amounts of EPA and DHA in the fresh Antarctic krilldetected were respectively, 4.87 and 4.26mg=g in the head, and 2.79 and 2.62mg=g inthe pleon. In the dried Antarctic krill, the amount of EPA and DHA was, respect-ively, 27.80 and 24.68mg=g in the head, and 19.89 and 18.46mg=g in the pleon.

REFERENCES

Adlof, R., and G. List. 2004. Analysis of triglyceride isomers by silver-ion high-performanceliquid chromatography Effect of column temperature on retention times. J. Chromatogr. A1046: 109113.

AOAC. 1998. Ofcial methods of analysis (16th ed., CD-ROM 3rd rev.). Washington, DC:AOAC International.

Belarbi, E. H., E. Molina, and Y. Chisti. 2000. A process for high yield and scaleable recoveryof high purity eicosapentaenoic acid esters from microalgae and sh oil. Enzyme Microb.Technol. 26: 516529.

Brondz, I. 2002. Development of fatty acid analysis by high-performance liquid chromato-graphy, gas chromatography, and related techniques. Anal. Chim. Acta. 465: 137.

Dyerberg, J. 1986. Linolenate derived polyunsaturated fatty acids and prevention of athero-sclerosis. Nutr. Rev. 44: 125134.

Ellingsen, T., and V. Mohr. 1979. A new process for the utilization of Antarctic krill. ProcessBiochem. 14: 1419.

Fountoulaki, E., M. N. Alexis, I. Nengas, and B. Venou. 2003. Effects of dietaryarachidonicacid (20:4n-6) on growth, body composition, and tissue fatty acid prole of gilthead breamngerlings (Sparus aurata L.). Aquaculture 225: 309323.

Fricke, H., G. Gercken, W. Schreiber, and J. Oehlenschlager. 1984. Lipid, sterol and fatty acidcomposition of antarctic krill (euphausia superba dana). Lipids 19: 821827.

Gelencser, A., G. Kiss, Z. Krivacsy, Z. Varga-Puchony, and J. Hlavay. 1995. Method for thedetermination of capacity factor on solid phase extraction cartridges: Part I. J. Chromatogr.A 693: 217225.

Giacometti, J., A. Milosevic, and C. Milin. 2002. Gas chromatographic determination of fattyacids contained in different lipid classes after their separation by solid-phase extraction. J.Chromatogr. A 976: 4754.

Gigliotti, J. C.,M. P. Davenport, S. K. Beamer, J. C. Tou, and J. Jaczynski. 2011. Extraction andcharacterisationof lipids fromAntarctic krill (Euphausia superba).FoodChem.125: 10281036.

Giogios, I., K. Grigorakis, I. Nengas, S. Papasolomontos, N. Papaioannou, and M. N. Alexis.2009. Fatty acid composition and volatile compounds of selected marine oils and meals. J.Sci. Food Agric. 89: 88100.

Guil-Guerrero, J. L., P. Campra-Madrid, and R. Navarro-Juarez. 2003. Isolation of somePUFA from edible oils by argentated silica gel chromatography. Grasas Aceites 54: 116121.

Hamner,W.M., P. P.Hamner, S.W. Strand, andR.W.Gilmer. 1983. Behavior of antarctic krill,euphausia superba: chemoreception, feeding, schooling, and molting. Sci. 220: 433435.

Hempel, I., and G. Hempel. 1986. Field observations on the developmental ascent of larvaleuphausia superba (Crustacea). Polar Biol. 6: 121126.


Kaluzny, M. A., L. A. Duncan, M. V. Merritt, and D. E. Epps. 1985. Rapid separation oflipid classes in high yield and purity using bonded phase columns. J. Lipid Res. 26: 135140.

Kinsella, L. E. 1986. Food components with potential therapeutic benets: The n-3 polyunsa-turated fatty acids of sh oils. Food Technol. 40: 8997.

Kolakowska, A., E. Kolakowski, and M. Szczygielski. 1994. Winter season krill (Euphausiasuperba Dana) as a source of n-3 polyunsaturated fatty acids. Food = Nahrung. 38: 128134.

Kolakowski, E. 1986. Changes of non-protein nitrogens fractions in Antarctic krill (Euphausiasuperba Dana) during storage at 3C and 20C. Z. Lebensm. Unters. Forsch. 183: 421425.

Lacaze, J. C. L., L. A. Stobo, E. A. Turrell, and M. A. Quilliam. 2007. Solid-phase extractionand liquid chromatographymass spectrometry for the determination of free fatty acids inshellsh. J. Chromatogr. A 1145: 5157.

Mansour, M. P. 2005. Reversed-phase high-performance liquid chromatography puricationof methyl esters of C16C28 polyunsaturated fatty acids in microalgae, including octaco-saoctaenoic acid [28:8(n-3)]. J. Chromatogr. A 1097: 5458.

Mehta, L., L. M. Lopez, D. Lowton, and T. Wargovich. 1988. Dietary supplementation withomega-3 polyunsaturated fatty acids in patients with stable coronary disease: effects on indi-ces of platelet and neutrophil function and exercise performance. Am. J. Med. 84: 4552.

Nicol, S., A. James, and G. Pitcher. 1987. A rst record of daytime surface swarming byeuphausia lucens in the southern benguela region. Mar. Biol. 94: 710.

Poerschmann, J., U. Trommler, W. Biedermann, U. Truyen, and E. Lucker. 2006. Sequentialpressurized liquid extraction to determine brain-originating fatty acids in meat products asmarkers in bovine spongiform encephalopathy risk assessment studies. J. Chromatogr. A1127: 2633.

Priddle, J., I. L. Boyd, M. J. Whitehouse, E. J. Murphy, and J. P. Croxall. 1998. Estimates ofsouthern ocean primary production constraints from predator demand and nutrient draw-down. J. Mar. Sys. 17: 275288.

Ross, R. M., and L. B. Quetin. 1986. How productive are Antarctic Krill. BioSci. 36: 264269.Ryu, S., J. Lee, and H. Hur. 1997. Method for separating and purifying -linolenic acid fromperilla oil. US Patent 5.672.726, Assigned to the Republic of Korea as represented by theRural Development Administration (30.09.1997).

Shahidi, F., and U. N. Wanasundara. 1998. Omega-3 fatty acid concentrates: nutritionalaspects and production technologies. Trends Food Sci. Technol. 9: 230240.

Simopoulos, A. P. 1991. Omega-3 fatty acids in health and disease and in growth and devel-opment. Am. J. Clin. Nutr. 54: 438463.

Simopoulos, A. P. 1997. Essential fatty acids in health and chronic disease. Am. J. Clin. Nutr.70: 560S569S.

Suzuki, T., and N. Shibata. 1990. The utilization of Antarctic Krill for human food. Food Rev.Int. 6: 119147.

Tang, B., M. Tian, Y. R. Lee, and K. H. Row. 2012. Optimized analytical conditions foreicosapentaenoic and docosahexaenoic acids in Antarctic krill using gas chromatography.Anal.Lett. 45:18851893.

Ulberth, F., and E. Achs. 1990. Argentation chromatography of fatty acid methyl esters usingsilver-loaded solid-phase extraction columns. J. Chromatogr. A 504: 202206.

Wilson, R., R. J. Henderson, I. C. Burkow, and J. R. Sargent. 1993. The enrichment of n-3polyunsaturated fatty acids using aminopropyl solid phase extraction columns. Lipids 28:5154.

Wilson, R., and J. R. Sargent. 1992. High-resolution separation of polyunsaturated fatty acidsby argentation thin-layer chromatography. J. Chromatogr. A 623: 403407.

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epa and dha extraction

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