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AGRICULTURAL MATERIALS
Development of Optimized Extraction Methodology for
Cyanogenic Glycosides from Flaxseed (Linum usitatissimum)
VÉRONIQUE J. BARTHET and RAY BACALA
Canadian Grain Commission, Grain Research Laboratory, 1404-303 Main St, Winnipeg, MB, Canada, R3C 3G8
A reference method (higher accuracy) and a
routine method (higher throughput) were
developed for the extraction of cyanogenic
glycosides from flaxseed. Conditions of
(essentially) complete extraction were identified by
comparing grinding methods and extraction
solvent composition, and optimizing
solvent-to-meal ratio, extraction time, and repeat
extraction. The reference extraction method
consists of sample grinding using a high-speed
impact plus sieving mill at 18 000 rpm with a
1.0 mm sieve coupled with triple-pooled extraction
in a sonicating water bath (40°C, 30 min) using 75%
methanol. The routine method differs by the use of
a coffee mill to grind samples and a single
extraction. The 70 and 80% methanol solutions
were equal and superior to other combinations
from 50 to 100% aqueous ethanol or methanol. The
extraction efficiencies of the routine method
(relative to the reference method) was 87.9 ± 2.0%
SD (linustatin) and 87.6 ± 1.9% SD (neolinustatin)
using four composite samples that were generated
from seeds of multiple cultivars over two crop
years and locations across Western Canada.
Ground flaxseed was stable after storage at room
temperature, refrigeration, or freezing for up to
7 days, and frozen for at least 2 weeks but less
than 2 months. Extracts were stable for up to
1 week at room temperature and at least 2 weeks
when refrigerated or frozen.
Flaxseed (Linum usitatissimum L.) contains the
cyanogenic glycosides linustatin (2-[6-b-D-glucosyl-b-
D-glucopyranosyloxy]-2-methylpropionitrile) and
neolinustatin ((R)-2-[6-b-D-glucosyl-b-D-gluco- pyranosyloxy]
-2-methylbutyronitrile) in significant amounts (1). The
corresponding monoglycosides linamarin (2-b-D-glucopyrano-
syloxy-2-methylpropionitrile) and lotaustralin ((R)-2-b-D-
glucopyranosyloxy-2-methylbutyronitrile) are present in
immature seed (2, 3), but diminish to trace levels in mature
seed (3, 4). Cyanogenic glycosides are retained in meal after
oil extraction and readily liberate cyanide upon acid
hydrolysis and by endogenous seed enzymes. As a result, the
usefulness of flaxseed and flaxseed meal in livestock and
poultry feed products is limited.
Extraction and analytical methods for cyanogenic
glycosides from flax have been recently reviewed (5).
Analytical assays fall into two broad categories: those that
hydrolyze the glycosides and measure liberated cyanide and
those that extract the intact glycosides and analyze them
chromatographically. Examples of the former category
include acid hydrolyzed or enzyme-linked colorimetric
tests (3, 6, 7), and autohydrolysis followed by quantitation of
liberated cyanide by LC (8). Examples of the latter category
include TLC (3, 9–12), RPLC (2, 4, 6, 13–19), and
GC (2, 20, 21). Various extraction methodologies are also
found in the literature. The most widely used extraction
method involves extraction of ground seed with 70%
methanol in a 30°C sonicating water bath for 1 h (13, 17).
Variations on this method involve shortening the extraction
time to 30 min (4) and using 70% ethanol (6) or 80%
methanol (14, 15) instead of 70% methanol. Other methods
include overnight room temperature extraction of unground
seed in methanol on a shaker (2), grinding in liquid nitrogen
followed by extraction with boiling 80% ethanol (3), grinding
to pass a 1 mm sieve and shaking in 0.1 M orthophosphoric
acid for 1 h (22), and electric homogenization in water
followed by autohydrolysis (8). Another method involves
extraction of glycosides with 70% ethanol (7°C, 1 h),
evaporation to dryness, resuspension in methanol and then
chloroform (1:2 ratio), clarification by centrifugation,
evaporation of the supernatnant, and redissolution of the dried
material in 15% aqueous methanol (19).
Only two comparisons between analytical methods have been
published. Haque and Bradbury (7) compared an acid hydrolysis
colorimetric technique to an enzyme-linked colorimetric
technique and demonstrated that the acid hydrolysis method was
the less accurate and less reproducible of the two. Kobaisy et
al. (6) demonstrated that two enzyme-linked colorimetric tests
were equally accurate to an RPLC method. Although this
establishes the relative performance of these assays, there is a
lack of published data demonstrating the repeatability, precision,
and accuracy of any one method.
Recently, we developed and validated a GC method
for measurement of linustatin and neolinustatin in
flaxseed (20). During development, preliminary
478 BARTHET & BACALA: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 2, 2010
Received March 27, 2009. Accepted by EB August 14, 2009.
Corresponding author’s e-mail: veronique.barthet@grainscanada.gc.ca
investigations demonstrated that certain factors have a great
effect on extraction efficiency. The choice of grinder affected
the extraction efficiency by up to 12% and the use of three
different extraction methodologies affected extraction
efficiency by up to 18%. Given the variability of other
published extraction methodologies, it is likely that the
extraction efficiency would vary even more across all
published methods. This is a major shortcoming, as the
results for any one analytical method, no matter how
well-characterized it is, cannot be any better than the
extraction method. This study reports the development of
exhaustive and routine extraction methodology for
cyanogenic glycosides from flaxseed.
Materials and Methods
Materials and Reagents
Linustatin and neolinustatin were purchased from
Chromadex Inc. (Santa Ana, CA). Methyl-a-D-glucopyranoside,
phenyl-b-D-glucopyranoside, and 1-methylimidazole were
purchased from Sigma-Aldrich (St. Louis, MO).
Bistrimethylsilylacetamide and trimethylsilylchlorosilane
(TMCS) were purchased from Regis Technologies Inc.
(Morton Grove, IL). All other solvents were of ACS grade or
better, and purchased from Fisher Scientific (Whitby, ON) or
VWR International (Mississauga, ON), with the exception of
anhydrous ethanol, which was purchased from Commercial
Alcohols Inc. (Toronto, ON).
Equipment
The GC apparatus consisted of an Agilent 6890 gas
chromatograph equipped with an Agilent 7683 series
autoinjector (10 mL syringe) and a flame ionization detector.
Data collection and analysis were carried out using Agilent
Chemstation software, Version A.09.03, Build 1417. The
column was a Supelco (St. Louis, MO) SPB-17 column
(30 m ´ 0.32 mm, 0.25 mm film thickness). Hydrogen carrier
gas was produced using a Parker (Haverhill, MA) hydrogen
generator, Model 75-34. Sample grinders included an
in-house constructed high-speed impact grinder, a Black and
Decker Smartgrind coffee mill, and a Retsch Model ZM200
mill with a 1.0 mm sieve (Haan, Germany). Extraction
equipment included a Tissue Tearor Model 985-370
high-speed electric homogenizer (Biospec Products Inc.,
Bartlesville, OK), a Crest Tru-Sweep Model 575HT
ultrasonic water bath (Trenton, NJ) and a vortex mixer
(various models). Centrifugation was performed on a
Beckman GS-6R centrifuge (Fullerton, CA) equipped with a
GH 3.8 rotor.
Flaxseed Samples
Samples were derived from No. 1 Canada Western
flaxseed collected from the Canadian Grain Commission’s
2005 and 2006 harvest surveys. The FX2005 and FX2006
composites were composites from 2005 and 2006,
respectively, consisting of a variety of cultivars (food and
industrial use) and growing locations across Western Canada.
FX2006 MB Bethune consisted of seed of the Bethune
BARTHET & BACALA: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 2, 2010 479
Figure 1. Comparison of extraction solvents for extraction of linustatin and neolinustatin from flaxseed. Bars
annotated with the same letter and case are not significantly different at the 95% confidence interval (n = 6).
cultivar from 72 growing locations in Manitoba during the
2006 crop year. FX2006 SK Vimy was a composite of
seed of the Vimy cultivar from 57 growing locations across
Saskatchewan.
Analytical Method
Preparation of trimethylsilyl ester (TMS) derivatives and
analysis by GC were carried out as previously described (20).
Positive displacement pipets were used to measure extracts at
all points of sample preparation.
General Extraction Method
Samples were ground using either a coffee grinder or a
Retsch mill (1.0 mm sieve, 18 000 rpm). Ground meal
samples were blended manually for 2 min, and replicate
samples (0.51 ± 0.1 g) were weighed into 20 mL glass vials.
Extraction solvent (5 mL) was added to each vial, and vials
were capped. All samples were briefly agitated immediately
before extraction in an ultrasonic water bath set at 40°C for
30 min. The water bath temperature typically increased to
47–49°C during this interval. The extraction solvent was
turbid and milky in appearance after extraction. After cooling
for 15 min, samples were centrifuged at 1500 ´ g for 10 min.
Extracts were analyzed within 24 h of preparation and stored
at –20°C unless otherwise indicated.
Comparison of Extraction Solvents
A single batch of FX2005 composite seed was ground in
the Retsch mill and extracted (n = 6 for each treatment) using
combinations of aqueous ethanol or methanol (50, 60, 70, 80,
90, or 100% alcohol), as described.
Effect of Solvent-Meal Ratio on Extraction
A single bulk sample of FX2006 composite was ground in
the coffee mill. Sextuplicate extractions were performed using
the general method with 75% methanol at each of the
480 BARTHET & BACALA: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 2, 2010
Table 1. Effect of varying solvent-to-ground seed ratio on extraction of cyanogenic glycosides from flaxseed
Ratio,
mL solvent:g seed
Linustatin,
mg/100 g seed SD
Neolinustatin,
mg/100 g seed SD SNK groupa
20:1 290 3277 2A
16:1 290 2277 1A
10:1 289 5277 5A
6.7:1 281 4267 5A, B
5:1 279 6265 6B
2.5:1 250 12 239 12 C
aSNK = Student-Newman-Keuls.
Figure 2. Comparison of time for the extraction of linustatin and neolinustatin from flaxseed. Bars annotated with
the same letter and case are not significantly different at the 95% confidence interval (n = 6).
following solvent-to-meal ratios: 20:1 (0.25 g ground seed,
5 mL extraction solvent); 16:1 (0.25 g, 4 mL); 10:1 (0.5 g,
5 mL); 6.67:1 (0.75 g, 5 mL); 5:1 (1 g, 5 mL); and 2.5:1 (2.5 g,
5 mL). The volumes of extract used to prepare GC samples
were varied as follows to ensure that peak areas were within
the calibration curve: 20:1 (200 mL); 16:1 (150 mL); 10:1
(100 mL); 6.67:1 (75 mL); 5:1 (50 mL); and 2.5:1 (25 mL).
Multiple Extraction of Flaxseed Meal
Single, double, triple, quadruple, and quintuple extractions
were compared using FX2005 composite seed. A single batch
of seed was ground in the Retsch mill and extracted (n = 6 for
each treatment) with 75% methanol in water using the general
method (10:1 solvent-to-meal ratio). Extracts were pooled for
each sample and filtered through by syringe filter (0.45 mm,
47 mm diameter, nylon) before analysis.
Effect of Extraction Time on Extraction Efficiency
A single bulk sample of ground meal was prepared using
the Retsch mill. Extraction samples were weighed using the
general method (n = 6 for each treatment) and were
triple-extracted. The duration of extraction was set at 5, 15,
30, or 60 min per extraction round. Extracts were pooled for
each sample and filtered before analysis as described in the
multiple extraction experiment.
Determination of Extraction Efficiencies of Routine
Extraction Methods
Two routine methods were compared to the established
reference method in order to calculate the extraction
efficiencies of each method. The reference method used seed
ground in the Retsch mill and triple extraction with 75%
methanol. The routine methods used the coffee mill to grind
samples and a single extraction with either 80% ethanol or
75% methanol. All three methods used the sonicating water
bath, as described in the general extraction method. Four seed
samples were used for comparison: FX2005 composite;
FX2006 composite; FX2006 MB Bethune; and FX2006 SK
Vimy. Six extracts were prepared from each meal sample
from each grinder.
Evaluation of Meal and Extract Stability
The stability of ground meal and extracts was assessed
using FX2005 composite seed. Extracts (n = 6 per treatment)
were stored at room temperature, refrigerated temperature
(3°C), and freezer temperature (–18°C) after 1 and 7 days of
storage. Meal stability was assessed at room temperature (2, 4,
6, and 24 h) and at refrigerator and freezer temperatures (1, 7,
14, and 28 days). Meal samples (n = 6 per treatment) were
pre-weighed into extraction vials with tightly fitting caps and
PTFA-lined septa prior to storage. All meal samples were
extracted and analyzed immediately after removal from
storage on the same day along with stored extract samples. All
treatment groups were compared to freshly ground and
extracted seed.
Statistical Analysis
All statistical analyses were performed using SAS 9.1 with
Enterprise Guide 4.1. Significant differences between
treatment groups were tested by analysis of variance; when
detected, treatment groups were grouped using the
Student-Newman-Keuls (SNK) multiple-range test. Grubbs’
test for outliers was used for treatments when the RSD
exceeded 7.5%. All tests were performed at the 95%
confidence interval.
Results and Discussion
Development of the extraction methodology was
conducted by first selecting an extraction solvent.
Combinations of aqueous ethanol or methanol (50–100%,
v/v) were evaluated. Water was also evaluated; however,
extracted mucilage was not soluble in the derivatization
cocktail and rendered the samples non-analyzable.
Methanol-containing solvent systems extracted more
linustatin than neolinustatin in the corresponding amounts of
ethanol (Figure 1). The difference between 90 and 100%
BARTHET & BACALA: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 2, 2010 481
Table 2. Comparison of grinding methods, extraction method, and multiple extraction on yield of cyanogenic
glycosides from flaxseed
Extraction
solvent Grinder Extraction method
Sequential
extraction
Linustatin,
mg/100 g seed SD
Neolinustatin,
mg/100 g seed SD
SNK
groupa
75% Methanol Retsch mill Sonicating bath Single 199 8169 7C
Retsch mill Sonicating bath Double 219 3186 3A
Retsch mill Sonicating bath Triple 222 3188 3A
Retsch mill Sonicating bath Quadruple 221 4186 3A
Retsch mill Sonicating bath Quintuple 218 2185 2A
Retsch mill Polytron and sonicating bath Single 203 12 174 11 B, C
Coffee mill Sonicating bath Single 195 2164 2B
80% Ethanol Coffee mill Sonicating bath Single 169 3144 2D
aSNK = Student-Newman-Keuls.
ethanol was striking, and demonstrated the importance of the
presence of water in the extraction solvent. The difference
between 90 and 100% methanol showed the same trend. The
largest amounts of both linustatin and neolinustatin were
extracted with 70 and 80% methanol. Although the means for
80% methanol were numerically lower for both species, the
difference was not statistically significant. The 75% methanol
was selected as the extraction solvent for further work to
provide a buffer region to control for variability of seed
moisture and extraction solvent batches.
The ratio of solvent volume to mass of ground seed was set
at 10:1 (5 mL to 0.5 g) early in development. To evaluate the
sensitivity of the extraction to this parameter, the ratio was
varied as widely as could reasonably be accommodated by the
constraints of the equipment in use: 20:1; 16:1; 10:1; 6.67:1;
5:1; and 2.5:1 (Table 1). Two outliers were identified using
the Grubbs test, one in each of the 20:1 and 6.67:1 treatment
groups. Upon elimination of the outliers, the 20:1, 16:1, 10:1,
and 6.67:1 groups were statistically equivalent and showed
superior extraction to the 5:1 (which was the same as 6.67:1)
and 2.5:1 groups. When the data were tested without removal
of outliers, all ratios were equal except the 2.5:1 group, which
was significantly lower. The solvent-to-ground seed ratio of
10:1 was maintained for both the routine and reference
extraction method.
Extraction efficiency was surprisingly insensitive to
extraction time (Figure 2). There was no difference between 5,
15, 30, or 60 min for linustatin, whereas 5, 30, and 60 min
were significantly higher than 15 min for neolinustatin. This
difference (15 min being inferior to 5 and 30 min) is likely
anomalous and not an actual relationship, and was likely
observed due to the small variability between replicate
extractions within the treatment group. A 30 min extraction
time was selected to provide control for any foreseeable
differences in sonicating water baths between laboratories.
The effect of multiple extraction was evaluated by
subjecting meal samples to up to five replicate extractions
(Table 2). Double, triple, quadruple, and quintuple extraction
were statistically equal in efficiency and better than single
extraction. The effect of further particle size reduction
(beyond that of the Retsch mill) was investigated by
performing a single extraction with 75% methanol and
electric homogenizer before sonication. Although the yields
for linustatin and neolinustatin were numerically marginally
higher than single extraction without the electric
homogenizer, the difference was not significant. Importantly,
the SD value increased dramatically with the incorporation of
the extra step. The use of addition particle reducing steps was
not considered any further. Two candidate routine methods
using a coffee mill rather than the Retsch and either 75%
methanol (routine methanol method) or 80% ethanol (rapid
ethanol method) were also investigated. The extraction
efficiency was expectedly lower for both methods. When the
coffee mill is compared to the Retsch (single extraction, 75%
methanol), the results from the coffee mill are significantly
lower than those from the Retsch mill. This may be attributed
directly to the superior ability of the Retsch (impact plus
sieving) mill over the coffee mill (impact only) at particle size
reduction. This difference also illustrates the importance of
adequate and consistent grinding in any extraction method.
The extraction efficiency of the routine ethanol and
methanol methods was determined using four flax composites
(Table 3). The 2005 and 2006 composites represented an
average sample matrix that would be expected from a
Canadian sample, whereas the MB Bethune and SK Vimy
represented the two most popular cultivars grown in Canada.
Typically, Bethune comprises over 55% and Vimy 10–15% of
total Canadian flax (Canadian Grain Commission). The mean
extraction efficiencies were 87.9 ± 2.0% SD (linustatin) and
87.6 ± 1.9% SD (neolinustatin) for the routine methanol
extraction, and 80.2 ± 1.9% SD (linustatin) and 80.1 ± 1.9%
SD (neolinustatin) for the routine ethanol extraction. Both
methods showed low variability in extraction efficiency, but
the routine methanol method was the better choice because of
482 BARTHET & BACALA: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 2, 2010
Table 3. Extraction efficiency of routine methods relative to reference extraction methoda
Extraction method Seed sample Linustatin, % of reference SD Neolinustatin, % of reference SD
Single extraction 2005 composite 85.0 1.9 85.3 1.9
75% Methanol 2006 composite 90.0 2.5 89.6 2.4
Sonicating bath, 40°C2006 MB Bethune 88.5 1.8 88.9 1.7
2006 SK Vimy 88.0 1.5 86.7 1.3
Average 87.9 2.0 87.6 1.9
Single extraction 2005 composite 78..1 0.9 78.9 0.9
80% Ethanol 2006 composite 83.4 2.2 83.7 2.0
Sonicating bath, 40°C2006 MB Bethune 81.0 2.0 81.6 2.1
2006 SK Vimy 78.5 2.3 77.3 2.5
Average 80.2 1.9 80.1 1.9
aTreatments annotated with the same letter and case are not statistically different at the 95% confidence interval (n = 6).
bSNK = Student-Newman-Keuls.
the higher recovery. The use of a routine extraction method
and extraction efficiency correction increased the overall
measurement uncertainty, compared to using the reference
method for every sample. The choice between routine and
reference method should be made by balancing the need for
high throughput versus best possible accuracy.
The establishment of sample stability is critical to the
development of a method, as it defines sample storage
conditions, stability windows and stability of ground meal
samples; extracts were assessed by storage of samples and
comparison to freshly ground, freshly extracted samples
(Table 4). Stability of ground meal was assessed at room
temperature over 2, 4, and 6 h periods to simulate laboratory
conditions where seed may be ground several hours before
extraction. Meal and extract storage were also assessed at
room temperature, refrigeration, and freezer conditions over
1 and 7 days. Finally, meal was assessed at 2 weeks and at
2 months in the freezer. The freshly ground and extracted
sample yielded the highest assay results for both analytes and
were not statistically different from any storage conditions
other than meal at 1 day under refrigeration (linustatin),
extract after 7 days at room temperature (linustatin), and meal
after 2 months in the freezer (both analytes). The difference
observed in the 1 day meal treatment was likely an anomaly
arising from the low SD values observed, as the meal was
statistically equivalent to the fresh sample after 1 and 7 days at
room temperature (a harsher condition), as well as 7 days
under refrigeration. The statistically lower results for the
extract after 7 days at room temperature and meal after
2 months in the freezer must be accepted, however, as no
longer-term samples were analyzed. When the statistics were
repeated at the 99% confidence interval (data not shown), all
storage conditions were statistically inseparable from the
freshly ground and extracted sample, with the exception of the
2 month meal sample. It was concluded that ground meal
could be stored at room temperature for at least 24 h and up to
a week without analyte degradation, and extracts may be
stored at room temperature for up to 1 day, but should be
refrigerated or frozen thereafter.
In conclusion, the reference extraction method for
linustatin and neolinustatin from flax consisted of sample
grinding using a Retsch ZM200 mill (or equivalent) at
18 000 rpm with a 1.0 mm sieve coupled with triple-pooled
extraction in a sonicating water bath (40°C, 30 min) using
75% methanol. The routine method differed by the use of a
coffee mill to grind samples and a single extraction. The
BARTHET & BACALA: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 2, 2010 483
Table 4. Stability of ground flaxseed meala
Time
Linustatin,
mg/100 g seed SD SNK groupbNeolinustatin,
mg/100 g seed SD SNK groupb
Fresh Temperature 209 3A176 2a
Meal samples
2 h R.T.c207 2A, B 175 2a, b
4 h R.T. 206 1A, B 173 1a, b
6 h R.T. 207 1A, B 174 1a, b
24 h R.T. 205 3A, B 175 2a, b
7 days R.T. 207 3A, B 176 3a
24 h 3°C202 3B172 3a, b
7 days 3°C208 2A, B 176 2a
24 h –18°C203 2A, B 174 2a, b
7 days –18°C207 2A, B 176 1a
2 weeks –18°C208 1A, B 174 2a, b
2 months –18°C197 7C170 6b
Extracts
24 h R.T. 205 2A, B 175 2a, b
24 h 3°C205 3A, B 175 2a, b
24 h –18°C205 2A, B 175 2a, b
7 days R.T. 202 2B172 2a, b
7 days 3°C204 5A, B 173 4a, b
7 days –18°C205 3A, B 174 3a, b
aTreatments annotated with the same letter and case are not statistically different at the 95% confidence interval (n = 6).
bSNK = Student-Newman-Keuls.
cR.T. = Room temperature.
largest barrier to method transferability is the lack of a
standardized or certified reference material. This means that,
due to differences in grinding equipment and with no
reference material for comparison, conditions of (essentially)
complete extraction must be re-established in every laboratory
for the reference extraction method.
Acknowledgments
This is Canadian Grain Commission paper No. 1026.
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