Eicosapentaenoic acid enrichment from sardine oil by argentation chromatography

Article abstract of DOI:10.1021/jf071407r

Eicosapentaenoic acid (EPA) derived from chemically hydrolyzed sardine oil was concentrated by urea fractionation using methanol at different temperatures (2, 4, and 6°C) and urea/fatty acid ratios (2:1, 3:1, and 4:1 w/w) and purified by argentation neutral alumina column chromatography. The individual fatty acids were determined as fatty acid methyl esters (FAME) by gas-liquid chromatography and gas chromatography-mass spectroscopy as FAME and N-acyl pyrrolidides. In the mass fragmentation pattern of FAME, the base peak was assigned to be the 1-methoxyethenol moiety (m/z = 74) obtained by McLafferty rearrangement. Formation of the cyclic tropylium ion (m/z = 91) in fatty acids with four or more double bonds was apparent in FAME-PUFAs. The base peak of N-acyl pyrrolidides was the McLafferty rearrangement ion, 1-(pyrrolidin-1-yl) ethenol (m/z = 113). The highest concentration of EPA (47.78%) was obtained at the crystallization temperature of 4°C with a urea/fatty acid ratio of 4:1 (w/w) with 93.74% yield. After complexation of saturated and less unsaturated fatty acids by urea complexation, argentation chromatography resulted in an EPA of high purity (99.6%) with an overall recovery of 54.09% using 50% diethyl ether/n-hexane as eluting solvent. The peroxide (POV) and thiobarbituric acid (TBS) values were found to be highest (4.0 mequiv of O₂/kg and 5.2 mg of malondialdehyde/kg, respectively) during urea fractionation at the higher crystallization temperature (6°C) and higher urea/fatty acid ratio (4:1).

Full text of DOI:10.1021/jf071407r

7586 J. Agric. Food Chem. 2007, 55, 7586−7595
Eicosapentaenoic Acid Enrichment from Sardine Oil by
Argentation Chromatography
KAJAL CHAKRABORTY* AND R. PAUL RAJ
Physiology Nutrition and Pathology Division, Central Marine Fisheries Research Institute, Ernakulam
North P.O., P.B. No. 1603, Cochin-682018, Kerala, India
Eicosapentaenoic acid (EPA) derived from chemically hydrolyzed sardine oil was concentrated by
urea fractionation using methanol at different temperatures (2, 4, and 6 °C) and urea/fatty acid ratios
(2:1, 3:1, and 4:1 w/w) and purified by argentation neutral alumina column chromatography. The
individual fatty acids were determined as fatty acid methyl esters (FAME) by gas-liquid chromatog-
raphy and gas chromatography-mass spectroscopy as FAME and N-acyl pyrrolidides. In the mass
fragmentation pattern of FAME, the base peak was assigned to be the 1-methoxyethenol moiety
(m/z ) 74) obtained by McLafferty rearrangement. Formation of the cyclic tropylium ion (m/z ) 91)
in fatty acids with four or more double bonds was apparent in FAME-PUFAs. The base peak of
N-acyl pyrrolidides was the McLafferty rearrangement ion, 1-(pyrrolidin-1-yl)ethenol (m/z ) 113). The
highest concentration of EPA (47.78%) was obtained at the crystallization temperature of 4 °C with
a urea/fatty acid ratio of 4:1 (w/w) with 93.74% yield. After complexation of saturated and less
unsaturated fatty acids by urea complexation, argentation chromatography resulted in an EPA of
high purity (99.6%) with an overall recovery of 54.09% using 50% diethyl ether/n-hexane as eluting
solvent. The peroxide (POV) and thiobarbituric acid (TBS) values were found to be highest (4.0 mequiv
of O2/kg and 5.2 mg of malondialdehyde/kg, respectively) during urea fractionation at the higher
crystallization temperature (6 °C) and higher urea/fatty acid ratio (4:1).
KEYWORDS: Sardine oil; eicosapentaenoic acid (EPA); fatty acid methyl esters (FAME); urea fractionation;
argentation column chromatography
INTRODUCTION
prepare n3 concentrates as FFA after chemical hydrolysis of
marine oils. The technologies available for purifying individual
PUFAs and PUFA concentrates from fish oil are based on
differences in physicochemical properties associated with the
number of double bonds in the molecule or the chain length
like urea complexation method (7). Recently, argentation silica
gel column chromatography has been employed to obtain high-
purity EPA methyl ester from hydrolysates of fish oil that
contained other polyunsaturated fatty acid esters including
linolenic acid (18:3n3) and oleic acid (18:1n9) (8, 9). Partial
success in purification of linolenic and oleic acid has been
achieved by using a mixture of acetonitrile and propionitrile
(1:2) at -60 °C (10). Argentation silica gel chromatography of
urea inclusion adducts from cod liver oil yielded highly pure
DHA in the process (100% purity, 64% yield), while for EPA,
the recovery in the combined process was 29.6%, and the final
purity was 90.6%. The recovery in the urea inclusion method
was strongly enhanced by application of orbital agitation during
the crystallization process, in which EPA yield increased from
60-70% without agitation to 90-97% at 800 rpm (11). EPA
and DHA have been concentrated from shark liver (Isurus
oxyrinchus) in one single step, in which fish liver oil was
simultaneously extracted, saponified, and concentrated. Ad-
ditionally, the PUFA concentrate was winterized to crystallize
The n3 and n6 long-chain polyunsaturated fatty acids (LC-
PUFAs), viz., eicosapentaenoic acid (EPA, 20:5n3), docosa-
hexaenoic acid (DHA, 22:6n3), and arachidonic acid (AA, 20:
4n6), are essential fatty acids in the diet of a majority of marine
finfish and crustaceans, especially for the larvae and broodstock
because they cannot synthesize it de novo from precursor
molecules (1, 2). Diets deficient in these PUFAs particularly
EPA have been found to have a negative effect on ovarian
development, fecundity, and egg quality (3). The important
natural sources of n3 LC-PUFAs are marine fish oils such as
sardine, mackerel, cod, shark, and menhaden, which contain
PUFA levels of about 30%. For this reason, marine fish oils
are preferentially used as raw material to prepare n3 PUFA
concentrates (4). However, they contain relatively high levels
of triacylglycerols and sterols, which are too heterogeneous,
rendering it difficult to isolate individual fatty acids efficiently
with a single separation method. Additionally, it was reported
that n3 PUFAs were moderately absorbed by the intestine as
triglycerides and most promptly absorbed when free fatty acids
(FFA) were given orally (5, 6). Therefore, it is convenient to
* To whom correspondence should be addressed. Tel: +91-484-2394867,
+91-484-4069936. Fax: 0091-0484-2394909. E-mail: kajal_iari@yahoo.com.
10.1021/jf071407r CCC: $37.00
© 2007 American Chemical Society
Published on Web 07/25/2007

Eicosapentaenoic Acid Enrichment from Sardine Oil
J. Agric. Food Chem., Vol. 55, No. 18, 2007 7587
the lower hydroalcoholic phase containing the saponified fatty acids
was adjusted to 1.0 with HCl-H2O (1:1 v/v, 15 mL), and the resultant
free fatty acids were recovered by extraction with n-hexane (6 × 75
mL), washed with water to neutral pH, and dried over anhydrous
MgSO4. The residual solids were removed with a Buchner funnel under
suction, and the solvent was evaporated to recover the free fatty acids,
dried by passing N2, weighed (34 g), and converted to methyl esters
for further fractionation and gas-liquid chromatographic (GLC) and
gas chromatographic-mass spectroscopic (GC/MS) analysis.
the remaining saturated fatty acids, resulting in a further increase
in the concentration of DHA and EPA (12). The polyunsaturated
fatty acids EPA and AA have been purified from the red
microalga Porphyridium cruentum by the urea inclusion method
followed by silica gel column chromatography of the urea
concentrate. Total AA and EPA recoveries reached 39.5% and
50.8%, respectively, for purity and approximately 97% for both
fatty acids (13). Solvent winterization of seed oil and free fatty
acids (FFAs) was employed to obtain γ-linolenic acid (GLA)
concentrates from seed oils of two Boraginaceae species, Echium
fastuosum and Borago officinalis. Different solutions of seed
oils and FFA from these two oils at 10%, 20%, and 40% (w/w)
were crystallized at 4, -24, and -70 °C, respectively, using
n-hexane, acetone, diethyl ether, isobutyl alcohol, and ethanol
as solvents. Best results were obtained for B. officinalis FFAs
in n-hexane, reaching a maximum GLA concentration of 58.8%
(14). Also, there are reports for the production of EPA or DHA
concentrates by a combination of techniques such as molecular
distillation, fractional distillation, liquid chromatography, and
supercritical fluid extraction (8).
Formation of the Fatty Acid-Urea Inclusion Complex. The urea-
fatty acid complexation was accomplished following the procedures
(7, 9) with modification. In the first step of crystallization, urea was
added to methanol in the ratio of 1:3 (w/v). The mixture was heated at
elevated temperature (65-75 °C) with constant stirring until a clear,
homogeneous solution was formed, and the fatty acids derived from
saponification were applied in incremental portions into the resulting
urea solution, the urea/fatty acid ratios being 2:1, 3:1, and 4:1 (w/w).
The solution was cooled to room temperature at a cooling rate of 0.5
°C/min and subsequently crystallized at three different sets of temper-
atures (2, 4, and 6 °C) for 24 h. The crystals were thereafter kept at
-20 °C for 24 h. After thawing, the urea crystals (urea complex
fraction) were separated from the mother liquor (non-urea complex
fraction) by vacuum filtration. The filtrate containing the unsaturated
fatty acids was evaporated using a rotary vacuum evaporator to remove
the residual methanol. The solid residue thus obtained was diluted with
water (200 mL) and acidified with dilute HCl (6 M, 30 mL) to pH
4-5 under stirring to remove traces of residual urea and methanol.
The liberated fatty acids after acidification with dilute HCl were
extracted with different solvents (n-hexane, chloroform, dichlo-
romethane), and hexane was found as the best with higher recovery
percentage of fatty acids. The fatty acids were extracted with n-hexane
three times (3 × 100 mL) in a separatory funnel to cause the phase
separation of urea and concentrated PUFAs. The upper layer of
n-hexane containing the PUFAs was separated and dried over anhydrous
MgSO4. The resulting PUFA concentrate was dissolved in methanol,
added with TBHQ (0.01% w/v) as an antioxidant to increase the stability
of the fatty acids, and kept under a blanket of N2 at -20 °C for 48 h.
Acid-Catalyzed Transesterification of Fatty Acid to Methyl
Esters (FAMEs). Lipid from the crude sardine oil was extracted by
using CHCl3-CH3OH-H2O (2:4:1 v/v/v) (17). The lipid extract thus
obtained was saponified with 0.5 N KOH in CH3OH. After removal of
the nonsaponifiable material with n-hexane and acidification with 1 N
HCl, the saponifiable materials were extracted with petroleum ether-
diethyl ether (1:1 v/v) and transesterified to furnish fatty acid methyl
esters (FAME) by reaction (30 min under reflux) with a methylating
mixture (14% BF3/MeOH, 5 mL) in a boiling water bath under an inert
atmosphere of N2 (18). The FAME thus obtained was cooled to ambient
temperature, and distilled water (20 mL) was added. The solution was
extracted with n-hexane (10 mL × 6), and the upper n-hexane layer
was removed and concentrated under an inert atmosphere of N2. The
resulting FAME concentrate was reconstituted in petroleum ether,
flushed with N2 in glass vials, and stored in deep freeze (-20 °C) until
required for GC/GC-MS analyses. Analysis was performed in triplicate.
Similarly, the fatty acids obtained by urea-aided crystallization at various
combinations of temperature (2-6 °C) and urea/fatty acid ratios (2:1,
3:1, and 4:1) were transesterified for analysis by GC/GC-MS. The
PUFA concentrates obtained during urea fractionation were directly
methylated to furnish the FAMEs, which were extracted with n-hexane,
and concentrated under inert atmosphere of N2 to yield a residue. The
residue was immediately dissolved in n-hexane (30 mL) to be applied
on a chromatography column.
PUFAs are widely available in a large variety of marine
organisms such as microalgae, polychaetes (15, 16), finfish, and
shellfish, but sardine oil is easily available and inexpensive and
contains a considerable amount of PUFAs (33.26%), particularly
EPA (>15%), an essential fatty acid for larval and broodstock
nutrition in marine aquaculture. The present paper highlights a
method for purification of sardine fatty acids with the goal to
get a PUFA concentrate with high EPA. The objective of this
study is to purify EPA from sardine oil in three main steps:
(1) saponification of fish oil to derive free fatty acids, (2)
enrichment of PUFA content from the mixed fatty acid
concentrate by urea fractionation at three different temperatures
(2, 4, and 6 °C) and urea fatty acid ratios (2:1, 3:1, and 4:1
w/w), and (3) separation of the individual n3 fatty acids,
particularly high-purity EPA, by normal pressure argentation
alumina column chromatography. The oxidative stability of fatty
acid concentrates at different stages of purification was also
studied.
MATERIALS AND METHODS
Chemicals and Reagents. All solvents used for sample preparation
were of analytical grade, and the solvents used for MS analyses were
of LC grade (E-Merck, Darmstadt, Germany). Analytical grade solvents
were redistilled in an all-glass system. The solvents were nitrogen
degassed prior to use. Doubly distilled water was used throughout this
work, while all reagents used were of analytical grade and purchased
from E-Merck. Standards of fatty acid methyl ester (Supelco TM 37
component FAME mix, catalog no. 47885-U) and boron trifluoride/
methanol (14% BF3 in methanol, w/v) were procured from Sigma-
Aldrich Chemical Co. Inc. (St. Louis, MO). Crude sardine oil
(Sardinella longipes) was obtained from a local fishmeal plant located
in Cochin, India, bleached with 4% activated charcoal, and stabilized
with butylated hydroxyquinone (TBHQ, 0.01% w/v). The oil was stored
under nitrogen at -20 °C, in a 2 L sealed dark amber glass container,
until used. All glassware was rinsed with CHCl3-CH3OH (2: l v/v)
and dried under N2.
Argentation Column Chromatographic Fractionation of FAMEs.
The methyl ester of EPA was fractionated from other PUFAs by means
of normal pressure liquid column chromatography with AgNO3-
impregnated neutral alumina as the stationary phase. Silver nitrate
powder (AgNO3, 5 g) was added to ethanol (80% v/v, 30 mL) and
dissolved by stirring for 10 min. About 50 g of neutral alumina (0.06-
0.2 mm, MW 101.96), column chromatography grade, was slurried in
ethanol (96% v/v, 100 mL) and AgNO3 solution (10 mL) under stirring
for 2 h. Ethanol was evaporated under vacuum at 60 °C, and the silver-
impregnated alumina was activated by overnight heating (110-120 °C)
Saponification and Extraction of Fatty Acid from Sardine Oil.
Sardine oil (50 g) was saponified by refluxing with 100 mL of alkaline
solution [prepared by dissolving 250 g of NaOH and 2.5 g of Na2-
EDTA in a 1.5 L solution of water/methanol (1:1) at 60 °C] under
continuous stirring for 30-45 min under an inert atmosphere of N2
following the procedure adopted by Haagsma with modification (9).
The hydrolyzed mixture was diluted with water (50 mL), and the
unsaponifiable matter was extracted successively with diethyl ether (50
mL × 3) and n-hexane (5 × 75 mL) and discarded. The pH value of

7588 J. Agric. Food Chem., Vol. 55, No. 18, 2007
Chakraborty and Raj
in the oven to prepare Ag-alumina powder. This material was cooled
and kept in the dark in a desiccator until use. The slurry of Ag-alumina
gel (500 mg) in n-hexane (5 mL) was poured into a water-jacketed
column (45 cm × 50 mm i.d.) previously half-filled with n-hexane.
The packed height of 0.5 cm diameter glass chromatography columns
was maintained at 9 cm. The methylated extract (24 mg) obtained in
the transesterification step (by using a U/FA ratio of 4:1 at a temperature
of 4 °C) was dissolved in n-hexane (5 mL) and applied on the
chromatography column. The column was eluted with a sequence of
solvents (5-50% diethyl ether/n-hexane) passed successively through
the column with a flow rate of 1.5 mL/min. The eluates were collected
as fractions corresponding to the applied solvents. The EPA was purified
by passing through the column along with 50% diethyl ether/n-hexane.
n-Hexane was removed from the fractionated liquid layer to obtain
highly purified EPA (∼2.11 g).
Methyl Linoleate. EI-MS m/z (relative intensity, %): 294 (M+,
52.46), 263 (24.59), 220 (8.20), 178 (13.11), 164 (19.67), 150 (21.31),
136 (18.03), 123 (18.85), 109 (37.70), 95 (70.49), 81 (100), 67 (91.80),
55 (50.82).
Methyl Linolenate. EI-MS m/z (relative intensity, %): 292 (M+,
16.67), 261 (5.00), 236 (6.67), 173 (6.67), 163 (6.67), 149 (20.00),
135 (20.00), 121 (25.00), 108 (56.67), 95 (58.33), 79 (100), 67 (56.67),
55 (35.00).
Methyl Arachidonate. EI-MS m/z (relative intensity, %): 318 (M+,
1.82), 290 (1.82), 264 (1.82), 175 (5.45), 150 (7.27), 133 (7.27), 105
(30.91), 91 (70.91), 79 (100), 67 (80.00), 55 (49.09).
Methyl Eicosapentaenoate. EI-MS m/z (relative intensity, %): 315
(M+, 1.67), 175 (6.67), 161 (8.33), 145 (11.67), 131 (18.33), 119
(31.67), 108 (31.67), 91 (70.00), 79 (100), 67 (68.33), 55 (48.33).
Methyl Docosahexaenoate. EI-MS m/z (relative intensity, %): 342
(M+, 0.60), 145 (4.20), 131 (6.60), 119 (10.80), 108 (11.40), 91 (28.20),
79 (100), 67 (20.40).
Mass Spectroscopic Analyses of N-Acyl Pyrrolidide Derivatives.
The following are the mass spectrometric data of N-acyl pyrrolidide
derivatives.
1-(Pyrrolidin-1-yl)hexadecan-1-one/Palmitoylpyrrolidine. EI-MS m/z
(relative intensity, %): 309 (M+, 16.00), 294 (2.00), 168 (8.00), 140
(10.00), 126 (16.00), 113 (100), 98 (8.00), 70 (12.00), 55 (14.00).
1-(Pyrrolidin-1-yl)octadec-9-en-1-one. EI-MS m/z (relative intensity,
%): 335 (M+, 27.56), 250 (8.62), 236 (10.34), 208 (6.90), 196 (5.17),
182 (12.07), 126 (53.45), 113 (100), 98 (18.97), 85 (8.62), 72 (20.69),
55 (27.59).
Silver Ion Thin-Layer Chromatography (AgNO3 TLC). The
recovered fatty acid methyl esters from urea fractionation and column
chromatography were resolved by TLC (5 cm × 20 cm), precoated
with silica gel, and impregnated with AgNO3. Silica gel G (15.0 g)
was mixed with a 10% (w/v) solution of AgNO3 (40 mL) in methanol/
water (9:1 v/v) and spread in a uniform thickness (0.25 mm). Plates
were drained, air-dried, activated at 110-120 °C in dim light for 30
min, and stored in a light-tight desiccator container. The methyl esters
were applied to the plate as a narrow band. The plates were developed
twice in n-hexane/diethyl ether/acetic acid (94:5:1 v/v/v) to separate
individual bands. The bands were stained with 2,7-dichlorofluorescein
in alcohol (0.1% w/v) and examined under UV light.
Gas-Liquid Chromatography and Gas Chromatography-Mass
Spectrometry (GC/MS) Analysis of FAMEs. FAMEs were analyzed
by an Perkin-Elmer AutoSystem XL gas chromatograph (Perkin-Elmer)
equipped with an Elite-5 (cross-bond 5% diphenyl polsiloxane-95%
dimethyl polsiloxane) capillary column (30 m × 0.53 mm i.d., 0.50
µm film thickness; Supelco, Bellfonte, PA) using a flame ionization
detector (FID). The oven temperature ramp was 110 °C for 1.0 min,
followed by an increase of 45 °C/min to 250 °C, where it was held for
1.0 min, followed by an increase of 30 °C/min to 250 °C, where it was
held for 1.0 min, followed by an increase of 25 °C/min to 285 °C,
where it was held for 2.0 min, until all peaks had appeared. Ultrahigh
purity He (>99% purity) was used as the carrier gas at a flow rate of
1 mL/min. The injector temperature was maintained at 285 °C. The
injection volume was 1 µL. The detector temperature was 290 °C.
FAMEs were identified by comparison of retention times with the
known standards (37-component FAME mix; Supelco).
The GC-MS analyses were performed using the electronic impact
(EI) ionization mode in a Varian GC (CP-3800) interfaced with a Varian
instrument 1200L single quadruple mass spectrometer for confirmation
of fatty acid identification. FAMEs were derivatized to N-acyl pyrro-
lidides by condensation of fatty acid methyl ester with a mixture of
pyrrolidine (1 mL) and acetic acid (0.1 mL) at 100 °C under reflux (2
h) for GC-MS analyses (19). The GC apparatus was equipped with
WCOT fused silica capillary column of high polarity (DB-5; 30 m ×
0.25 mm i.d., 0.39 mm o.d., and 0.25 µm film thickness; Varian). The
polymeric stationary phase was nonpolar (VF-5MS, 5% phenyl-
substituted methylsiloxane). The carrier gas was ultrahigh purity He
(99.99% purity) with a constant flow rate of 1 mL/min. The injector
and detector temperature were maintained isothermal at 300 °C. The
injection volume was 1 µL. Samples were injected in split (1:15) mode
at 300 °C into the capillary column similar to that used for the GC
analyses, and the oven was identically programmed. The ion source
and transfer line were kept at 300 °C. Mass spectra were analyzed using
Varian Workstation (version 6.2) software.
1-(Pyrrolidin-1-yl)octadeca-9,12-dien-1-one. EI-MS m/z (relative
intensity, %): 333 (M+, 77.97), 290 (10.17), 236 (15.25), 222 (20.34),
182 (16.95), 168 (15.25), 140 (22.03), 126 (44.07), 113 (100), 98
(25.42), 70 (42.37), 55 (49.15).
1-(Pyrrolidin-1-yl)octadeca-9,12,15-trien-1-one. EI-MS m/z (relative
intensity, %): 331 (M+, 44.00), 182 (22.00), 168 (24.00), 140 (26.00),
126 (60.00), 113 (100), 98 (30.00), 72 (64.00), 55 (42.00).
1-(Pyrrolidin-1-yl)icosa-5,8,11,14-tetraen-1-one. EI-MS m/z (relative
intensity, %): 357 (M+, 18.97), 232 (10.34), 180 (10.34), 126 (13.79),
113 (100), 85 (17.24), 70 (22.41), 55 (27.59).
1-(Pyrrolidin-1-yl)icosa-5,8,11,14,17-pentaen-1-one. EI-MS m/z (rela-
tive intensity, %): 355 (M+, 3.85), 286 (7.69), 232 (7.69), 126 (13.46),
113 (100), 85 (17.31), 72 (26.92), 55 (21.15).
1-(Pyrrolidin-1-yl)octadeca-9,12-dien-1-one. EI-MS m/z (relative
intensity, %): 381 (M+, 3.91), 312 (7.05), 272 (7.29), 232 (16.22),
218 (15.76), 192 (8.24), 166 (23.67), 153 (22.85), 113 (100), 98 (46.62),
72 (21.98).
Purification Index. The purification indices of the fatty acids were
calculated following an earlier procedure (8). The yield obtained is
defined as the ratio of the weight of the particular component in the
product before purification and that of the component after purification.
For a process starting from the substance “A” to the end product “B”,
the yield of the component “x” in the process A-B may be defined as
Yx
) (Xb/Xa) × 100 ) (Px /Px )(Wx /Wx ) × 100, where Xa and Xb
(a-b)
b
a
b
a
represent the concentrations of the compound “x” (e.g., EPA) in the
products A and B. Wx and Wx are the weights of the component “x”
a
b
in the product “b” and reactant “a” before and after purification,
respectively.
Peroxide Value (POV), Thiobarbituric Acid Reactive Substances
(TBS), and Conjugated Diene (CD) Values. The formation of primary
products of lipid oxidation (peroxides) at various stages of purification
was evaluated by peroxide value (20). POV was calculated as the
reactive oxygen content and expressed as millimoles of free iodine per
kilogram of lipid. To supplement POV values, the level of lipid
peroxidation was measured colorimetrically to indicate the presence
of malondialdehyde (MDA, CHOCH2CHO) in thiobarbituric acid
reactive substance (TBS) assay (21, 22). TBS was calculated and
expressed as milligrams of MDA per kilogram of sample. Conjugated
diene (CD), another specific parameter to determine the formation of
oxidation products, was used to measure the contents of the conjugated
diene and conjugated triene of fatty acid concentrates at 233 and 265
nm, respectively (23).
Mass Spectroscopic Analyses of FAME Derivatives. The following
are the mass spectrometric data of FAME derivatives.
Methyl Palmitate. EI-MS m/z (relative intensity, %): 270 (M+,
61.11), 239 (15.74), 227 (31.48), 213 (7.41), 199 (14.81), 185 (12.96),
171 (12.96), 157 (7.41), 143 (31.48), 129 (11.11), 87 (74.07), 74 (100),
55 (18.52).
Methyl Oleate. EI-MS m/z (relative intensity, %): 296 (M+, 20.00),
111 (76.67), 264 (33.33), 222 (26.67), 180 (18.33), 166 (23.33), 152
(23.33), 123 (23.33), 110 (38.33), 97 (75.00), 83 (70.00), 74 (66.67),
69 (78.33), 55 (100).
Statistical Analyses. Percentage composition individual fatty acid
methyl esters were subjected to a one-way analysis of variance

Eicosapentaenoic Acid Enrichment from Sardine Oil
J. Agric. Food Chem., Vol. 55, No. 18, 2007 7589
Figure 1. Tentative mass fragmentation pattern of FAME and N-acyl pyrrolidides.
(CH2)nCH3]+ in the homologous series. The McLafferty ion was
found to be less conspicuous in EI-MS of LC-PUFAs having
more double bonds (n > 4), which undergoes complex rear-
rangement under high-energy EI conditions. The abundance of
these fragment ions in SFA and MUFA-FAME was found to
be higher than in the EI-MS spectra of LC-PUFAs. Formation
of the cyclic tropylium ion (m/z ) 91) in fatty acids with four
or more double bonds was apparent in PUFAs. Cleavage of the
tail from the n6 terminal moiety in the methylene-interrupted
bond to yield a fragment ion at m/z ) 150 was found to be
characteristic of n6 PUFA methyl esters, while for n3 PUFA
methyl esters the characteristic fragment ion was apparent at
m/z ) 108 following cleavage of the tail from the n6 terminal
moiety. The EI-MS spectrum of methyl linoleate has an
abundant molecular ion (m/z ) 294) and base peak as the
McLafferty ion (m/z ) 74). In methyl linolenate, a fragment
ion at m/z ) 150 was obvious, which is characteristic for fatty
acids with an n6 terminal moiety, while one at m/z ) 108 defines
an n3 terminal group as in methyl eicosapentaenoate and methyl
docosahexaenoate. In the mass fragmentation pattern of methyl
arachidonate the molecular ion is barely distinguishable, but
the characteristic ion for the n3 moiety, at m/z ) 108, is
apparent. In the mass fragmentation pattern of LC-PUFAs with
more double bonds (n g 4), the tropylium ion (m/z ) 91) does
stand out as the diagnostic fragment ion. However, in FAME
EI-MS spectra it is difficult to determine the location of the
double bond from the spectra. The reason for this is the
migration of the double bond along the long aliphatic chain due
to destabilization of the positive charge by electron ionization
in the mass spectrometer. This drawback is encountered by the
EI-MS of fatty acid pyrrolidine derivatives (N-acyl pyrrolidides),
which minimize the migration, as they are able to stabilize the
(ANOVA) using SPSS (version 10.0) software, and significant means
were compared by Tukey’s multiple range tests. A significance level
of 95% (p ) 0.05) was used throughout. Arc sin transformation was
used prior to statistical analyses of FAME data expressed in percentages.
All measurements were performed at least in triplicate (n ) 3), and
values were averaged. On the basis of the significance of treatments
LSD at the 5% level of significance (p ) 0.05) was computed.
RESULTS AND DISCUSSION
Gas Chromatographic-Mass Spectroscopic Analyses of
Fatty Acid Methyl Esters and N-Acyl Pyrrolidides. GC-MS
of FAME was used to determine primarily the chain length and
the degree of unsaturation with the molecular ion (M+). Mass
spectrometry of FAME with EI ionization provides meager
information only related to the structure of fatty acids, particu-
larly for LC-PUFAs. The molecular ions for SFA and MUFA-
FAME were conspicuous as obvious from the mass spectra, and
hence the molecular weight is discernible from the EI-MS
spectra of FAME. An ion at (M - 31)+ represents the loss of
the -OCH3 group, thus confirming the molecular structure as
methyl ester. In the mass fragmentation pattern of FAME, the
base peak was assigned to be the 1-methoxyethenol moiety (m/z
) 74) obtained by McLafferty rearrangement per se with the
loss of the McLafferty ion (m/z ) 222) (Figure 1). The
molecular ion undergoes further rearrangement to furnish
1-methoxyprop-2-en-1-ol (m/z ) 88). The characteristic ion for
SFA is formation of the McLafferty ion (m/z ) 74) by
McLafferty rearrangement, while MUFA methyl esters exhibited
the formation of a stable and abundant ion at M - 74 after the
loss of the McLafferty ion. The spectra further contained lower
m/z fragment ions at a difference of m/z ) 14, definitely
supporting the general structure of FAME, viz., [-CH3OCO-

7590 J. Agric. Food Chem., Vol. 55, No. 18, 2007
Chakraborty and Raj
charge on the heterocyclic pyrrolidine moiety. In the mass
fragmentation pattern of N-acyl pyrrolidides, the base peak was
assigned to be the McLafferty rearrangement ion, 1-(pyrrolidin-
1-yl)ethenol (m/z ) 113), with the concurrent loss of the M -
113 fragment ion. The molecular ion undergoes further rear-
rangement to furnish 1-(pyrrolidin-1-yl)prop-2-en-1-ol (m/z )
127). A uniform distribution of fragment peaks is apparent at
every m/z ) 14 units, except in the vicinity of the double bond,
where the interval is m/z ) 12 units. For example, in the mass
spectrum of 1-(pyrrolidin-1-yl)octadeca-9,12,15-trien-1-one, the
double bonds in positions 9 and 12 are located by the gaps of
m/z ) 12 units between ions at m/z ) 196, 208 and 236, 248,
respectively.
acid concentrate with comparatively lower PUFA recovery
(51.57%). Consequently, the yield of EPA (YEPA(b-c)) was
recorded to be higher at 4 °C and U/FA ratio of 4:1 (93.74%)
as compared to that at 6 °C (84.37%) (Table 2). Linolenic acid
(18:3n3) was found to be concentrated at 4 °C and U/FA ratio
of 4:1 with a recovery of 53.16%. On the basis of these results,
PUFA concentrate obtained from sardine oil at 4 °C temperature
of urea crystallization by using a U/FA ratio of 4:1 was selected
for subsequent purification by argentation column chromatog-
raphy.
The purity of 16:1n7, the predominant MUFA, was reduced
by 62.4% at the U/FA ratio of 4:1 and a temperature of 4 °C.
The yield of total MUFA (YMUFA(b-c)) was found to be 19.04%
at the U/FA of 3:1, and at U/FA of 2:1, the recovery was
recorded as 29.88%, which were comparatively higher than that
Saponification and Extraction of Fatty Acids. Among
saturated fatty acids (SFAs), 14:0 was found to be predominant
(7.04% TFA), while 16:1n7 contributed the major share (>31%
TFA) among all individual fatty acids in the crude sardine oil
(Table 1). EPA and DHA were found to be the major n3 PUFAs
contributing to 17.8% and 7.67% of TFA, respectively. The n6
fatty acids have a minor share in the total fatty acid content of
sardine oil. Solvent extraction resulted in marginal increase of
unsaturation (0.85%) in the fatty acid profile, the PUFA
exhibited an increase of 6.49%, while MUFA and SFA reduced
by 2.96% and 6.91%, respectively (Table 1). The n3 fatty acids
exhibited an increase of 5.73% in the solvent extract of fatty
acids.
obtained at U/FA ratio of 4:1 at 4 °C (YMUFA
) 9.70%)
(b-c)
(Table 2). At higher temperature (6 °C), the urea fractionation
could not resolve the SFAs and MUFAs effectively, resulting
in lower concentration of PUFAs in the urea concentrate
(YPUFA
) 80.81%) (Table 2). It is likely that, at lower
(b-c)
temperature (2 °C), the reaction kinetics to form the urea
inclusion complex with SFAs and MUFAs was relatively
inefficient, resulting in higher MUFA in the extract. On the
contrary, at higher temperature (>2 °C), the urea-fatty acid
complex dissociates, resulting in a relatively higher amount of
SFA and MUFA in the fatty acid concentrates. The urea
complexation reaction raised the EPA content by 2.6-fold, thus
allowing further purification by column chromatography.
LC-PUFA Enrichment by Urea Fractionation. The free
fatty acids (34 g) derived from sardine oil were subjected to
urea crystallization using methanol as solvent to obtain PUFAs
of high purity. Urea complexation of fatty acids is used to
concentrate PUFAs from SFAs and MUFAs, where urea
occludes straight-chain compounds such as long-chain saturated
and monounsaturated fatty acids in a hexagonal crystalline
structure, excluding methylene-interrupted polyunsaturated fatty
acids (having the -CdC-C-CdC- moiety) due to the
irregularities in their molecules caused by the bends at each
double bond (7). The interfering SFAs and most of the MUFAs
(long- and straight-chain molecules) were removed in the form
of urea inclusion compound, while the PUFAs remain in
solution. Further, as oxidized products do not form urea adducts,
the peroxidation of n3 PUFAs could be avoided during the
extraction of free acids from fish oil triglycerides. The urea
inclusion method allows handling of large quantities of material
in a more efficient way than fractional crystallization or selective
solvent extraction. One of the major parameters quantifying the
tendency of fatty acids and esters to combine with urea is the
urea adductability values (UAV). The UAV is equal to the
carbon number of SFAs and decreases with the unsaturation
number. The LC-PUFAs, such as EPA and DHA, have UAV
below 4 and, therefore, a very low tendency to form inclusion
compounds (7).
Separation of LC-PUFAs by Argentation Column Chro-
matography. Purification of fatty acid methyl esters was
performed as these compounds exhibit more stability and better
chromatographic behavior than free fatty acids. Normal pressure
column chromatography on neutral alumina impregnated with
AgNO3 was used to separate different fatty acids according to
the differences in their degree of unsaturation. The resolving
power of Ag-alumina is largely attributed to a reversible
charge-transfer complexation of Ag+ ion with -CdC- double
bonds of unsaturated compounds. The stability of the complex
increases with an increasing number of double bonds but
decreases with the increasing chain length. The extent and the
strength of complexation control the mobility of a solute (fatty
acids), as does the polarity of the mobile phase. Under identical
conditions, alumina with a greater surface density of silver is
better at resolving unsaturated fatty esters than silica gel with
a lower Ag+ loading. The separation achieved by argentation
column chromatography was continuously monitored by AgNO3
TLC and GC/GC-MS.
The fatty acid profiles of the various solvent fractions obtained
during the course of argentation chromatography are shown in
Table 3. The fractions containing 5% diethyl ether/n-hexane
eluted a substantial amount of MUFA esters 16:1n7, 18:1n9,
and 17:1 (75.96%), which associates weakly to the stationary
phase. The fraction from 25% diethyl ether/n-hexane eluted EPA
Different classes of fatty acid composition in the crude sardine
oil, solvent-extracted sardine oil, urea concentrate at three
different temperatures (2, 4, and 6 °C), and urea/fatty acid ratio
(U/FA ) 2:1, 3:1, and 4:1 w/w) vis-a`-vis sardine oil are depicted
in Table 1. Among the three different temperatures of crystal-
lization and U/FA ratio, higher U/FA ratio (4:1), irrespective
of temperature of crystallization, furnished higher PUFA
concentrate with higher recovery. At 6 °C, the total PUFA was
found to be 53.59%, with a recovery of 80.81% at the U/FA
ratio of 4:1, while at 4 °C the corresponding values were
ester of higher purity (PEPAd ) 59.36%) and recovery (YEPA
(c-d)
) 30.33%). The process was continued by using 50% diethyl
ether/n-hexane, eluting primarily 20:5n3, and halted the elution
of all MUFAs and diunsaturated and triunsaturated methyl esters.
Combining the solvent fractions of 5%, 20%, and 50% diethyl
ether/n-hexane, the final purity of EPA obtained in this step
was 99.56%, and the total recovery was calculated as 54.09%
(Table 3). It is apparent that the preceding urea process removes
nearly all SFAs, most of the MUFAs, and also significantly
reduces the linoleic (18:2n6) and linolenic (18:3n3) acid levels.
The DHA ester having a larger number of double bonds was
considerably higher (PPUFA ) 78.35%, YPUFA
) 80.28%)
c
(b-c)
(Tables 1 and 2). The reduction in yield at 2 °C may be due to
poor complexation of SFAs and MUFAs, thus furnishing a fatty

Eicosapentaenoic Acid Enrichment from Sardine Oil
J. Agric. Food Chem., Vol. 55, No. 18, 2007 7591
P
P
P
P
p
P
P
P
P
b
b
P
a
p
a

7592 J. Agric. Food Chem., Vol. 55, No. 18, 2007
Chakraborty and Raj
Y
Y
Y
Y
p
Y
Y
Y
a
Y
p
a

Eicosapentaenoic Acid Enrichment from Sardine Oil
J. Agric. Food Chem., Vol. 55, No. 18, 2007 7593
Table 3. Percent Composition (Pxd) and Recovery (Yx d)) of Fatty Acids in Three Different Eluates Using Three Different Solvent Systems (5%,
(c
-
25%, and 50% Diethyl Ether in n-Hexane) by Argentation Chromatographya
diethyl ether (%)/n-hexane (v/v)
25%
5%
50%
fatty acid
Px
Yx
Px
Yx
Px
Yx
(c-
d
(c-
d)
d
(c-
d)
d
d)
SFAs
12:0
ND
ND
ND
ND
ND
0.00
0.00
0.00
0.00
0.00
±
±
±
±
0.00
0.00
0.00
0.00
ND
0.00
0.00
0.00
0.00
0.00
±
0.00
0.00
0.00
0.00
ND
ND
ND
ND
ND
0.00
0.00
0.00
0.00
0.00
±
0.00
0.00
0.00
0.00
14:0
16:0
17:0
ND
ND
ND
ND
±
±
±
±
±
±
∑
SFA
MUFAs
16:1n7
18:1n9
17:1
43.15
25.84
±
±
0.51
1.29
2.45
97.42
±
0.07
14.74
ND
ND
ND
14.74
±
1.38
31.71
0.00
0.00
0.00
25.81
±
±
±
±
2.38
0.00
0.00
0.00
ND
ND
ND
ND
ND
0.00
0.00
0.00
0.00
0.00
±
±
±
±
0.00
0.00
0.00
0.00
100
100
±
±
0.00
0.00
6.98
ND
±
20:1n11
0.00
98.52
±
0.00
∑
MUFA
75.96
PUFAs
18:2n6
18:3n3
18:4n3
20:4n6
20:5n3
22:5n3
22:6n3
1.51
14.12
ND
±
±
0.26
1.17
17.35
43.44
0.00
0.00
1.14
0.00
2.14
15.36
0.93
±
±
±
±
±
±
±
1.15
3.04
0.00
0.00
0.12
0.00
0.26
2.13
ND
ND
ND
59.36
3.47
5.41
±
0.16
23.32
0.00
0.00
±
±
±
±
±
±
±
3.49
0.00
0.00
0.00
2.25
2.09
0.56
ND
ND
ND
ND
99.56
ND
0.36
99.92
0.52
0.00
0.00
0.00
0.00
54.09
0.00
0.55
79.72
0.46
±
±
±
±
±
±
±
0.00
0.00
0.00
0.00
0.15
0.00
0.12
ND
0.00
2.12
ND
±
0.19
0.12
±
±
±
2.24
0.18
0.57
30.33
29.22
7.72
68.51
1.20
±
0.08
1.43
±
±
0.03
∑
PUFA
19.18
1.32
70.37
1.58
LSD (p
)
0.05)
a Px
)
percentage composition of individual fatty acids in the column chromatographic eluates using urea
all other notations are as indicated under Table 1. On the basis of the significance of treatments LSD at the 5% level of significance (p
presented as mean values of three samples (mean standard deviation). ND fatty acids identified on the GC trace but not integrated by the instrument.
−
fatty acid concentrate obtained at 4
°C using U/FA ) 4:1;
d
)
0.05) was computed. Data
±
)
found to coelute with EPA in this final fraction containing 50%
diethyl ether/n-hexane.
Table 4. Changes in Various Lipid Oxidation Parameters [Peroxide
Value (POV) and Thiobarbituric Acid Reactive Substances (TBS)]
during Downstream Processing of EPA
Silver Ion Thin-Layer Chromatography (AgNO3 TLC).
TLC was used as a first-hand tool to know about the progress
of purification of PUFAs from other monoenoic and saturated
fatty acids during argentation column chromatography. The
results were confirmed by using GC/GC-MS to know about the
individual fatty acids. The first eluent from column chroma-
tography using 5% diethyl ether/n-hexane reveals the separation
of FAME up to trienes (18:3n3), the more unsaturated such as
18:2n6 and 18:3n3 were found to be staying at the base of TLC
chromatogram (using n-hexane/diethyl ether/acetic acid, 94:5:1
v/v/v, as developing solvent). The uppermost band (Rf: 0.86)
of saturated fatty acids and the second band of monoenoic fatty
acids (Rf: 0.49) were apparent. Tetraene, pentaene, and hexaene
methyl esters appeared to be concentrated to >90% in the lower
band of the TLC plates (Rf: 0.18-0.33). The second eluent
(25% diethyl ether/n-hexane) enabled the complete separation
of all FAMEs except the saturated and monoenes which
comigrate near the solvent front. The eluates from the chro-
matographic column using 50% diethyl ether/n-hexane reveal
the separation of EPA from other fatty acids as evident from
the TLC chromatogram (Rf: 0.23).
POV
(mequiv of
O2/kg)
TBS
(mg of malon-
dialdehyde/kg)
solvent extraction
solvent extract of fatty acids
4.1
±
0.28
6.8
±
0.83
urea crystallizationa
crystallization temp 2
U/FA, 2:1
U/FA, 3:1
U/FA, 4:1
crystallization temp 4
U/FA, 2:1
U/FA, 3:1
U/FA, 4:1
crystallization temp 6
U/FA, 2:1
U/FA, 3:1
°
°
°
C
C
C
2.6
2.8
4.2
±
±
±
0.09
0.16
0.37
4.1
3.8
4.3
±
±
±
0.38
1.07
0.95
2.3
3.5
3.9
±
±
±
0.04
0.10
0.27
3.2
3.6
3.6
±
±
±
0.18
0.29
0.62
4.0
5.8
6.3
±
±
±
0.88
0.53
1.32
5.2
6.9
9.4
±
±
±
0.09
1.25
1.37
U/FA, 4:1
column chromatographyb
5% diethyl ether/hexane
20% diethyl ether/hexane
50% diethyl ether/hexane
2.1
2.9
2.6
±
±
±
0.12
0.37
0.08
3.0
2.8
2.1
±
±
±
0.22
0.17
0.44
LSD (p
)
0.05)
0.88
1.03
Lipid Oxidation. Lipid oxidation is a complex process in
which unsaturated fatty acids react with molecular oxygen,
usually via a free radical mechanism, to form hydroperoxides,
the primary oxidation products (24, 25). Peroxide, conjugated
diene, and TBS values were used as indices to assess the level
of lipid oxidation in sardine oil. Changes in peroxide and TBS
values of sardine oil during various stages of purification are
shown in Table 4. The peroxide value measures the formation
of peroxide or hydroperoxide groups that are initial products
of lipid oxidation. Hydroperoxides decompose to create an
assortment of nonvolatile and volatile compounds, such as
polymeric triacylglycerides and aldehydes, respectively (26).
a POV and TBS values as a factor of temperature of crystallization (2, 4, and
°C) at three different urea/fatty acid ratios (2:1, 3:1, and 4:1) vis-a`-vis sardine oil
6
and solvent extract of fatty acids. b POV and TBS values of the eluates from
argentation column chromatography (5%, 25%, and 50% diethyl ether in n-hexane)
as compared to the fatty acid concentrate obtained in urea fractionation at 4
using a urea/fatty acid ratio of 4:1. Data presented as mean values of three samples
(mean standard deviation).
°C
±
Peroxide values were found to be very marginal in fatty acid
concentrates during solvent extraction (4.1 mequiv of O2/kg).
This may be attributed to the antioxidant activity of TBHQ used

7594 J. Agric. Food Chem., Vol. 55, No. 18, 2007
Chakraborty and Raj
in the oil and lower atmospheric exposure. The POV of fatty
acid concentrates rose to 6.3 mequiv of O2/kg at higher
crystallization temperature (6 °C) and urea/fatty acid ratio (4:
1) and declined during subsequent steps of purification. No
significant changes in POV were apparent during urea frac-
tionation at lower temperatures (2 and 4 °C) (Table 4). The
peroxidation at higher crystallization temperature (6 °C) for urea
fractionation was probably due to the release of prooxidants
from the oil (9). A decrease in POV (2.53 ( 0.19 mequiv of
O2/kg) was noticeable during column chromatographic purifica-
tion of fatty acids.
preparation and purification of EPA would contribute to the
industrial application to obtain concentrated EPA.
ACKNOWLEDGMENT
The authors are thankful to Prof. (Dr.) Mohan Joseph Modayil,
Director, CMFRI, Cochin, for providing necessary facilities to
carry out the work. Thanks are also due to Dr. Shibu M. Eappen,
Scientist B, Sophisticated Test and Instrumentation Centre,
Cochin University of Science and Technology, Kerala, for
recording the mass spectral data.
Thiobarbituric acid (TBS) values have been used to measure
the concentration of relatively polar secondary reaction products,
especially aldehydes. The TBS value, which measures the
malondialdehyde concentration (end product of lipid oxidation)
followed a similar pattern. The initial value of TBS was found
to be 6.8 mg of malondialdehyde/kg, suggesting that marginal
lipid oxidation occurred during solvent extraction (Table 4).
Higher TBS values were apparent at the higher temperature of
crystallization (6 °C) during the course of urea-fatty acid
complexation, thus indicating the formation of secondary lipid
oxidation products at a higher temperature. Gray (27) reported
that the TBS value correlate well with the peroxide value only
in oils containing fatty acids with g3 double bonds such as
sardine oil fatty acid concentrate.
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In conclusion, the present study enabled the development of
a process of two-step purification for achieving concentrated
EPA from sardine oil using urea complexation followed by
argentation chromatography on neutral alumina. Among three
different temperatures of crystallization and U/FA, higher U/FA
ratio (4:1 w/w), irrespective of temperature of crystallization,
furnished higher recovery of PUFA as well as EPA. The highest
concentration of EPA (47.78%) was obtained at the crystalliza-
tion temperature of 4 °C with a U/FA ratio of 4:1 (w/w) with
a recovery of 93.74%. After complexation of saturated and less
unsaturated free fatty acids by urea complexation, argentation
neutral alumina column chromatography resulted in EPA of high
purity (99.6%) with an overall recovery of 54.09% using 50%
diethyl ether/n-hexane as eluting solvent. The oxidative stability
of fatty acid concentrates at different stages of purification
revealed that no significant changes in POV, TBS, and CD
values were found during urea fractionation at lower temper-
atures (2 and 4 °C). This established procedure including

Eicosapentaenoic Acid Enrichment from Sardine Oil
J. Agric. Food Chem., Vol. 55, No. 18, 2007 7595
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Received for review May 13, 2007. Accepted June 7, 2007.
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