FOOD COMPOSITION AND ADDITIVES

Determination of trans Unsaturation by Infrared

Spectrophotometry and Determination of Fatty Acid
Composition of Partially Hydrogenated Vegetable Oils and
Animal Fats by Gas Chromatography/Infrared
Spectrophotometry: Collaborative Study
RATNAYAKE: JOURNAL OF AOAC INTERNATIONAL VOL. 78, NO. 3, 1995
WAKISUNDERA M.N. RATNAYAKE
Health Canada, Health Protection Branch, Food Directorate, Nutrition Research Division, Ottawa, ON, K1A 0L2, Canada
Collaborators: J. Alfieri; S. Bacler; B. Ballch; B. Debets; D. Dionne; S. Gould; D.E. Henry; M.R. Lapointe; R. Lahtinen; R.E.
McDonald; G. Pelletier; D. Wolfe

An infrared spectrophotometric (IR) method for the

determination of total trans unsaturated fatty acid
(trans) content and a combined gasliquid chromatographic/infrared spectrophotometric (GC/IR)
method for determination of fatty acid composition
of partially hydrogenated vegetable oils (PHVO)
were studied collaboratively in 12 laboratories using 7 PHVO samples, including 1 pair of blind duplicates. The test samples were methylated and analyzed for total trans content by IR and for fatty acid
composition by GC/IR using a capillary column
coated with SP-2560 or another suitable cyanoalkylsiloxane stationary phase. From the measured IR
absorption, the isolated trans content was calculated using a calibration curve of absorption versus trans content developed with 2-component calibration standard mixtures of methyl elaidate and
oleate. The GC provided the levels of mono-transoctadecadienoates (18:2t), di-trans-octadecadienoates (18:2tt) and mono-trans-octadecatrienoates (18:3t). The trans-octadecenoate (18:1t)
content was calculated with the formula: 18:1t = IR
trans 0.84 (18:2t + 18:3t) 1.74 18:2tt. The cisoctadecenoate (18:1c) content was obtained as the
difference between total octadecenoates (18:1) and
18:1t. Reproducibility relative standard deviations
(RSDR) for 15 to 35% trans content determined by
IR were in the range of 8.811.7%, whereas RSDR
for the test sample with 5% trans content was

34.6%. RSDR values for 18:1t by the GC/IR followed

the same pattern as that of IR trans values: 36.4%
for the test sample with 4.9% 18:1t versus 7.8
12.5% for test samples with 14.9 to 32.6% 18:1t. The
content of 18:1c in the test samples varied
from 24.7 to 34.5% and their RSDR values ranged
from 3.8 to 10.5%. The mean values for 18:1t and
18:1c compared favorably with the absolute levels
determined by a silver nitrate-thin layer chromatography/GC procedure. The IR and GC/IR methods
are recommended for determination of trans content and fatty acid composition, respectively, of partially hydrogenated fats derived from vegetable
oils, terrestrial animal fats or such oils and fats isolated from food products containing >5% trans fatty
acids. For samples containing 5% trans fatty acids, a direct GC method (American Oil Chemists
Society Official Method Ce 1c-89) is available for determination of both trans content and fatty acid
composition, because at lower trans levels, overlap
of 18:1 cis and trans isomers on GC with very polar
capillary columns is negligible. The IR method for
determination of isolated trans unsaturated fatty
acid content in partially hydrogenated fats and the
capillary GC/IR method for determination of total
cis- and trans-octadecenoic isomers and general
fatty acid composition in hydrogenated vegetable
oils and animal fats have been adopted first action
by AOAC INTERNATIONAL.

Submitted for publication March 23, 1994.

The recommendation was approved by the Committee on Food
Nutrition and was adopted by the Official Methods Board of the
Association. See Official Methods Board Actions (1994) J. AOAC Int. 77,
203A, and Official Methods Board Actions (1994) The Referee 18,
October issue.

he widespread use of partially hydrogenated vegetable

oils (PHVO) in many common foods and the widely
publicized adverse health effects of dietary trans fatty
acids (15) create a need for accurate determination of total
trans unsaturation and detailed fatty acid composition, includ-

ing levels of cis- and trans-monounsaturates in PHVO and dietary fats made from PHVO. In Canada (6) and the United States
(7) voluntary labeling regulations of foods require that only
monounsaturates of the cis configuration be declared on the
nutrition label and polyunsaturates are restricted to all cismethylene-interrupted structures. These labeling regulations
also require accurate determination of fatty acid composition in
dietary fats of PHVO origin.
The current American Oil Chemists Society (AOCS) Official Method Ce 1c-89 (revised 1990) has been designed to
evaluate the general fatty acid composition, including the levels
of 18:1c and 18:1t isomers, and the total trans unsaturated fatty
acid (trans) content in hydrogenated and unhydrogenated
vegetable oils, by a direct, one-step capillary gas chromatographic (GC) procedure, using a 60 m 0.25 mm id fused
silica capillary column coated with SP-2340 stationary phase
(8). AOAC INTERNATIONAL recommended the first action
adoption of the same direct GC procedure (9). This direct GC
method was based on the assumption that cis and trans isomers
of 18:1 fatty acid are completely separable on the SP-2340 column. However, because of the complexity of isomers present
in PHVO, a satisfactory separation of 18:1t isomers as a group
from that of cis isomers is not feasible on SP-2340 or any other
currently available GC stationary phases (10, 11). On SP-2340
and other polar columns, 18:1t isomers with values lower
than 11 are well separated from the cis isomers, but the isomers
with high values (18:112 16t) overlap with cis isomers.
Factors such as extent of hydrogenation, level of total trans
content, and amount of sample applied onto GC column could
also influence isomer separation. Because of these overlaps, the
direct GC method gives lower values for 18:1t isomer and consequently higher values for 18:1c (1012).
Ratnayake et al. (11) proposed use of a combined capillary
GC and infrared spectrophotometric (IR) method for accurate
determination of 18:1t and 18:1c isomers in PHVO. Total trans
unsaturation determined by IR was related to the capillary GC
weight percentages of the component trans fatty acid methyl
esters by a mathematical formula:
IR trans = 18:1t + 0.84 18:2t + 1.74
18:2tt + 0.84 18:3t
where 0.84, 1.74, and 0.84 are correction factors relating GC
weight percentages to the IR trans equivalents for 18:2t, 18:2tt,
and 18:3t, respectively. This formula is the basis for determining total 18:1t and 18:1c isomers and hence the general fatty
acid composition of PHVO. In capillary columns coated with
polar cyanoalkylsiloxane stationary phases such as SP-2340
and SP-2560, 18:2t, 18:2tt, and 18:3t are separated as distinct
groups without any serious interferences or overlaps (11, Figure 994.15) and levels of these trans polyunsaturates are obtained directly by GC analysis. IR method provides trans unsaturation and, therefore, total 18:1t is calculated from the
mathematical formula. The content of 18:1c is then calculated
as the difference between total 18:1 fatty acid methyl esters,
which is the sum of all 18:1 isomer peaks in GC and 18:1t.

Because in the above GC/IR method 18:1t and IR trans unsaturation are linearly related, accuracy of 18:1t determination
is dependent solely on the accuracy of total trans unsaturation
determination by IR spectroscopy. During the development of
the GC/IR procedure, Ratnayake et al. (11) used AOCS official
IR method Cd 14-61 (13) for determination of total trans unsaturation of fatty acid methyl esters. However, the AOCS
method, despite using the baseline technique to correct for any
background absorption, suffers from a few drawbacks (14). A
major problem is that samples analyzed as methyl esters produce trans levels which are 1.53% lower for trans values
from 1 to 15% (15). AOAC Official Method 965.34 prescribes
incorporation of correction factors to compensate the lower absorption of methyl esters (16). Another problem is that conjugated trans double bonds absorb very close to the isolated trans
bond and can interfere with the isolated trans measurement (17).
Because of this interference, AOCS official method is applicable
only to samples containing less than 5% conjugated fatty acids.
Madison et al. (18) proposed a 2-component calibration procedure to overcome some of the drawbacks of the AOCS official IR methods. Trans content was calculated using a calibration curve of absorption versus percentage isolated trans
unsaturation developed using a series of carbon disulfide solutions containing different ratios of methyl elaidate and methyl
linoleate. Calibration and test solutions are scanned from 900
to 1500 cm1 against a carbon disulfide blank. A baseline is
drawn between peak minima at about 935 and 1020 cm1, and
the baseline-corrected absorbance of the trans peak (967 cm1)
is obtained. Baseline for the test sample spectrum is drawn exactly as the baseline in the standard spectrum, by overlaying the
2 spectra. This procedure allows to analyze trans contents in
the 0.536% range with increased accuracy. The 2-component
calibration procedure suggested by Madison et al. (18) compensates for the low bias of the AOCS method (13) for methyl
esters, and eliminates the need for calculation of correction factors in AOAC Official Method 965.34 (16).
The present international collaborative study had 2 objectives. One was to evaluate the combined capillary GC/IR procedure of Ratnayake et al. (11) for determination of 18:1t,
18:1c, as well as general fatty acid composition of partially hydrogenated fats. Since the IR procedure proposed by Madison
et al. (18) is an improvement over the current official methods
of AOAC (16) and AOCS (13), the evaluation of an IR procedure similar to that described by Madison et al. was the second
objective of the present collaborative study. Madison et al.
specified a mixture of methyl elaidate and methyl linoleate for
the development of calibration curves, but in the collaborative
study methyl linoleate was replaced by methyl oleate because
of its greater oxidative stability over methyl linoleate and its
availability.
Collaborative Study
Six fat samples (A, B, C1, C2, D, and E), prepared at the
Proctor and Gamble Co., Cincinnati, OH, were used in the collaborative study. Samples C1 and C2 were blind duplicates.
Samples A, B, C1, and C2 were prepared by blending various

ratios of fat extracted from 2 retail samples of margarine (partially hydrogenated soybean, liquid soybean oil, and cottonseed
oil-based margarines) and diluting them with unhydrogenated
corn oil. Samples D and E were prepared by blending the fat
extracted from the 2 retail margarines with partially hydrogenated canola oil (hard stock). All samples were blind coded (each
laboratory had a unique number code) and sent to 19 collaborators. Samples A, B, D, and E were distributed to all participating collaborators. However, due to limited availability, the
blind duplicate samples (C1 and C2) were distributed only to
15 collaborators. Each collaborator was provided with instructions, study protocols, and data report forms. In addition, each
collaborator was provided a reference sample (R, partially hydrogenated soybean oil) with a labeled GC scan of the fatty acid
methyl esters to be analyzed prior to analysis of test samples.
Each collaborator was also provided with authentic standards
of methyl oleate and elaidate for construction of the calibration
curve of IR absorption versus trans content. Collaborators were
instructed to prepare methyl esters from the partially hydrogenated fat samples according to the AOAC Official Method
963.33 that uses boron trifluoride (19). The collaborators were
requested to analyze each sample twice.
Levels of 18:1t and 18:1c isomers in the test samples were
estimated in the authors laboratory by a procedure different
from that of the GC/IR. A known amount (ca 15 mg) of fatty
acid methyl esters of the test samples was fractionated on silver
nitrate-thin layer chromatography (AgNO3-TLC) with development in toluene at 25C (20). The separated 18:1t and 18:1c
bands were extracted quantitatively, then methyl heptadecanoate (internal standard) was added and extracts were analyzed on a Hewlett-Packard 5890 Series II GC system
(Hewlett-Packard Co., Palo Alto, CA) using a SP-2560 flexible
fused silica capillary column (100 m 0.25 mm id, 20 m).
The column oven temperature was programmed at a rate of 1.5C
from 150 to 200C. From GC peak areas, amounts of 18:1t and
18:1c were calculated with respect to the internal standard.
Levels of 18:1c and 18:1t were also determined in authors
laboratory according to the current AOCS official method
Ce 1-89 (8) for determination of fatty acid composition in hydrogenated and unhydrogenated vegetable oils.

Statistical Analyses
Statistical evaluation of collaborative study results was performed with the computer program AOAC BUBR, which was
developed by the AOAC Statistics Committee. The program
calculates the performance parameters according to AOAC
guidelines for collaborative studies (21).
994.14 Isolated trans Unsaturated Fatty Acid
Content, in Partially Hydrogenated Fats
Infrared Spectrophotometric Method
First Action 1994
(Applicable to determination of total isolated [i.e., non-conjugated] trans content in fats and oils containing >5% trans
fatty acids.

Not applicable to samples containing >5% conjugated unsaturation [e.g., tung oil] materials containing functional
groups which modify absorption of C-H deformation around
trans bond [e.g., castor oil containing ricinoleic or ricinelaidic
acids], or any materials where specific groups may absorb close
to 967 cm1 [10.3 m].)
Caution: See Appendix: Laboratory Safety Safe Handling
of Special Chemical Hazardscarbon disulfide. Dispose of
carbon disulfide in an appropriate manner compatible with environmental rules and regulations.
Method Performance:
See Table 994.14 for method performance data.

A. Principle
Isolated trans double bonds (predominant trans configuration in partially hydrogenated fats) show absorption at ca
967 cm1 (10.3 m) deriving from C-H deformation about
trans bond. Isolated trans content is determined by measurement of absorption intensity. Triglycerides or fatty acids are
converted to methyl esters before making IR measurements.
Total isolated trans content is calculated using calibration curve of
absorption versus trans content of calibration solutions.

B. Apparatus
Infrared spectrophotometer (IR).Double-beam IR or
Fourier Transform IR (FTIR); capable of quantitative measurements at 1050900 cm1, with scale readable to 1 cm1; holding fixed thickness cells, 0.11.0 mm, with NaCl or KBr windows. All instruments must be checked for wavelength and
photometric scale accuracy according to manufactures instructions. Chart paper must be linear in either wavelength or wave
number, and calibrated in either transmission, T, or absorbance, A.

C. Reagents
(a) Carbon disulfide (CS2).Dry, ACS grade.
(b) Methyl elaidate stock solution.20 mg/mL. Accurately weigh ca 2000 mg methyl elaidate (purity >99%) to the
nearest 0.1 mg into 100 mL volumetric flask, dilute to volume
with CS2 solution, and mix thoroughly.
(c) Methyl oleate stock solution.20 mg/mL. Prepare as in
(b) using methyl oleate (purity >99%) instead of methyl elaidate.
(d) Calibration solutions.0.8, 1.6, 4, 8, 12, 16, and 20 mg
methyl elaidate/mL in 19.2, 18.4, 16, 12, 8, 4, and 0 mg/mL
methyl oleateCS2 solution, respectively. Accurately add 1, 2,
5, 10, 15, 20, and 25 mL methyl elaidate stock solution, (b),
into separate 25 mL volumetric flasks using pipets. Dilute contents of flasks to volume with methyl oleate stock solution, (d).
Verify weight percentages of methyl elaidate and methyl oleate
by capillary GC analysis as in 994.15. If weight percentages of
methyl elaidate differ from expected values by >5% (for samples containing <10% methyl elaidate) or >2% (for samples
containing >10% methyl elaidate) prepare fresh calibration solutions. Perform step E immediately because of high volatility
of CS2.

D. Preparation of Test Sample

Melt solid fats or free fatty acids on steam bath or in oven at
temperature 10C above melting point. If melted fat is cloudy
filter through filter paper. Using ca 400500 mg sample prepare methyl esters as in 969.33. (Note: Accurate results depend
on purity of fatty acid methyl esters. Before IR analysis remove
excessive levels of impurities [e.g., non-saponifiable matter,
polymers] using suitable cleanup procedure [e.g., saponification followed by extraction of non-saponifiable matter, thinlayer or column chromatography].) Accurately weigh ca 400
500 mg undiluted fatty acid methyl esters (M1) to the nearest
0.1 mg into 25 mL volumetric flask. Dilute to volume with CS2
solution. Perform step E immediately because of high volatility
of CS2.

E. Infrared Determination
Fill cell with CS2 solution, and matching cell with test sample from D or calibration solution from C(d). Use hypodermic
syringe with blunted needle, and cell upright, inject from bottom so bubbles pass up through cell. When using double-beam
IR place cell with CS2 in reference beam. Place cell with test
sample or calibration solution in sample beam. Scan spectrum
(T or A) from 1050 to 900 cm1 at optimal instrument settings.
When using FTIR, initially scan CS2 reference from 1050 to
900 cm1 (background spectrum) and store it in memory of instrument data handling system. Scan test sample or calibration
solution in same range as that for reference. Ratio obtained
spectrum against background spectrum to obtain true T or A.
Measure spectra of calibration solutions in order of increasing
concentrations.

F. Calculations
For each spectrum draw baseline tangent to peak minima
adjacent to 967 cm1 (see Figure 994.14). (Note: It is important
to draw correct baseline because of measurement of baseline
corrected absorption. Absorption minima might vary slightly
between samples. Concentration and amount of trans unsaturation may influence position of absorption minima. Best results are obtained when baseline for sample is drawn exactly as
baseline in spectrum of one of calibration standards having approximately same intensity of absorption at 967 cm1. This can
be obtained by superimposing 2 spectra to draw baseline.)
another straight line parallel to ordinate and passing through
apex of analytical band as in Figure 994.14. (Line meets zero
line of chart at point Z, apex at Y, and baseline tangent at X.)
For transmission spectrum measure distances XZ and YZ.
Calculate absorption of calibration solution, Ai:
Ai = log

XZ
YZ

To calculate absorption of calibration solution, Ai, read AX

at X, and AY at Y:
Ai = AY AX

Plot mg methyl elaidate/mL calibration solution as abscissae versus corresponding Ai values as ordinate. Draw best
straight line through 7 points plotted. For better accuracy determine linear regression equation to fit data. (Note: Once obtained, calibration curve does not have to be repeated as long as
instrument settings, parts, or cells have not been changed.)
Determine absorption of test sample, As, using same procedure as for calibration solutions. Using calibration curve or linear regression equation determine amount of methyl elaidate
(mg M2)/mL test sample solution, D, corresponding to As. Calculate percent trans unsaturated fatty acid content as methyl
elaidate in test sample:
% trans unsaturated fatty acid content (as methyl
M2
elaidate) = 100
M1
where M1 = amount of fatty acid methyl esters in test sample
solution, mg.
Ref.: J. AOAC Int. 78, 783 (1995); J. Chromatogr. Sci. 28,
633 (1990); JAOCS 67, 804 (1990); JAOCS 69, 95 (1992).
994.15 Total cis- and trans-Octadecenoic Isomers
and General Fatty Acid Composition in
Hydrogenated Vegetable Oils and Animal Fats
Capillary Gas Chromatographic/Infrared
Spectrophotometric Method
First Action 1994
(Applicable to partially hydrogenated vegetable oils and terrestrial animal fats containing >5% trans fatty acids. Not applicable to hydrogenated marine oils and partially hydrogenated
fish oils, which contain large levels of cis and trans isomers of
C16, C18, C20, and C22 chain lengths.)
Method Performance:
See Table 994.15 for method performance data.

A. Principle
Total trans isomer content consists of trans fatty acids
(trans-octadecenoate [18:1t]; mono-trans-octadecadienoate
[18:2ct or tc, described as 18:2t]; trans,trans-octadecadienoate
[18:2tt]; and mono-trans-octadecatrienoate [18:3cct, ctc, and
tcc, described as 18:3t]), which occur in hydrogenated vegetable oils and terrestrial animal fats. Total trans content is determined by infrared spectrophotometry (IR) using methyl elaidate
as external standard. Various isomers of 18:2tt, 18:2t, and 18:3t are
resolved; their weight percentages are determined by GC. Based
on IR determination, weight percentage of 18:1t is determined as
described in Calculations. The difference between total methyl
octadecenoate (18:1, as sum of all 18:1 peaks in GC) and calculated 18:1t gives weight percentage of cis-octadecenoate (18:1c).

B. Apparatus
(a) Gas chromatograph (GC).With flame ionization detector, capillary column injection system (split ratio, 1:100).
Operating conditions: injection port, 225C; detector, 250C.

Temperature program: initial, 150C; program rate, 1.0C/min;

final, 200C; final hold, 20 min. (Note: Operator may change
operating conditions to obtain optimum separation of isomeric
fatty acid methyl esters.) Carrier gas, helium or hydrogen
(99.99% purity), with oxygen scrubber in line.
(b) GC column.100 m 0.25 mm fused silica capillary
column coated with SP-2560 (Supelco, Inc., Bellefonte, PA) or
other suitable capillary column coated with cyanoalkylpolysiloxane (e.g., SP-2340, CP SIL-88) that provides same
elution pattern as in Figure 994.15.
(c) GC syringe.Maximum volume 10 L, graduated to 0.1 L.
(d) Infrared spectrophotometer (IR).Double-beam IR or
Fourier Transform IR (FTIR); capable of quantitative measurements at 1050900 cm1, with scale readable to 1 cm1; holding fixed thickness cells, 0.11.0 mm with NaCl or KBr windows. All instruments must be checked for wavelength and
photometric scale accuracy according to manufacturers instructions. Chart paper must be linear in either wavelength or
wave number, and calibrated in either transmission, T, or absorbance, A.

Calculate weight percentage of 18:1t isomers, W18:1t:

% W18:1t = Wtrans (1.74 W18:2tt) 0.84(W18:2t + W18:3t)
where Wtrans = total trans content determined by IR; W18:2tt =
total weight percentage of all 18:2tt isomer peaks in GC; W18:2t
= total weight percentage of all 18:2t isomer peaks in GC; W18:3t
= total weight percentage of all 18:3t isomer peaks in GC; 1.74
and 0.84 = correction factors for trans,trans fatty acids and
mono-trans fatty acids, respectively.
Calculate weight percentage of 18:1c isomer, W18:1c:
%W18:1c = W18:1 W18:1t
where W18:1 = total weight percentage of all the 18:1 isomer
peaks in GC.
Ref: J. AOAC Int. 78, 783 (1995); J. Chromatogr. Sci. 28,
633(1990); JAOCS 67, 804 (1990); JAOCS 69, 95 (1992)

C. Reagents
(a) GC reference standards.Mixture of cis and trans isomers of known composition (Nu Chek Prep, Inc., Elysian, MN,
or Supelco, Inc., Bellefonte, PA).

D. Preparation of Methyl Esters

Melt solid fats or free fatty acids at temperature 10C
above melting point and mix. If cloudy, filter through filter paper. If diluted sample is cloudy due to H2O, add small portion
of anhydrous Na2SO4 to melted sample, mix, and let settle before taking portion for methylation. Using ca 400500 mg fat,
prepare methyl esters as in 969.33.

E. GC Analysis of Fatty Acid Composition

(a) GC performance specifications.Inject 12 L methyl
esters from GC reference standards, C(a), into GC. Select GC
conditions to obtain resolution of methyl esters at least equivalent to that in Figure 994.15.
(b) GC determination.Inject 12 L methyl esters (in
hexane or heptane solution) from test sample into GC. Compare retention times of test sample with those of GC reference
standards (see Figure 994.15).

F. IR Determination of Total trans Content

Perform as in 994.14.

G. Calculations
Calculate weight percentages of fatty acid methyl esters,
WX, assuming unity response factor for each component:
%WX =

PX
100
PT

where PX = GC area counts of specific methyl ester peak; PT =

total area counts of all fatty acid methyl ester peaks in chromatogram.

Results and Discussion

Analytical results were received from 12 of the 19 laboratories. Details of equipment and some of the operating parameters reported by the collaborators are listed in Table 1. Nine of
the 12 collaborators reported using FTIR; 3 collaborators used
conventional dispersive IRs. Nine collaborators reported using
SP-2560 flexible fused silica capillary columns for GC analysis. Others used either SP-2340, SP-2380, or CP-Sil-88. It appears from the data submitted by the collaborators that use of
either FTIR or conventional dispersive IR instrumentation did
not affect the trans unsaturation results. Similarly, the type of
cyanoalkylsiloxane column had no affect on the fatty acid composition data.
The raw data submitted by the collaborators for the 6 test
samples and the reference sample, the statistical calculations of
mean, within laboratory variation, and reproducibility are listed
in Tables 29. Table 10 shows the statistical calculations for the
blind duplicates C1 and C2. A summary of the mean values and
the reproducibility relative standard deviations (RSDR) of the
7 samples is given in Tables 994.14 (% trans data) and 994.15
(fatty acid composition data). Note that the duplicate values
reported in Tables 29 are not blind duplicates, and hence do
not provide a true measure of repeatability. (For the non-blind
duplicates, new terms within laboratory standard deviations
and relative within laboratory standard deviations with corresponding symbols Sw and RSDw were introduced in Tables 29. The normal terms for performance parameters given
in Harmonization Guidelines of AOAC [21] were used only
with the true blind duplicates [Table 10].)
The trans content in the test samples, determined by IR,
ranged from 5.2 to 34.6% and their RSDR values ranged from
8.8 to 34.6% (Table 994.14). Sample A, with the lowest level
of trans unsaturation (5.2%) of all the samples, had the widest
variation in reported IR trans values and the highest RSDR
(34.6%). All other samples tested had greater trans unsatura-

tion, but their RSDR values were much lower, ranging from 8.8
to 11.7%.
The similar RSDR values for 18:1t (Table 994.15) and IR
trans unsaturation reflect the linear relationship between these
2 parameters. Sample A with lowest 18:1t content had the
greatest RSDR, while other samples with higher 18:1t contents
(1533%), had much lower RSDR (7.8 to 12.5%, Table
994.15).
RSDR values for 18:1c ranged from 3.8 to 10.5% and were
slightly lower than those for IR trans and 18:1t. For 16:0, 18:0,
total saturates, and 18:2(n-6), the agreement between the laboratories was excellent, with RSDR values less than 5%. For
18:3(n-3), however, reproducibility was somewhat less satisfactory; the RSDR values ranged from 8.4 to 20.2%. These elevated values may be anticipated for 18:3(n-3), which rarely exceeds 1.0% in partially hydrogenated fats.
A very large variation was observed when analyzing transpolyunsaturated fatty acids. This is to be expected, since the
trans-polyunsaturated fatty acids are a complex mixture of several isomers, most of which constitute less than 0.1% of the
total fatty acids.
The Cochran and Grubbs tests (21) identified a total of
53 outlier values among the 1080 values submitted by the collaborators (Tables 210). The reports of Collaborators 7 and 8
contained 26 and 13 outliers, respectively. No outliers occurred
in the reports of 5 collaborators. In the remaining 4 collaborators reports only 5 or fewer outliers were identified.
Overall, the IR and the GC/IR methods yielded reproducible
results for trans content, saturated fatty acids, 18:1t, 18:1c, and
cis,cis-polyunsaturated fatty acids. The variations obtained for
both major and minor components are reasonable for a study of
this kind. The excellent agreement between the pair of blind
duplicate samples (Table 10) demonstrates that the IR and
GC/IR methods are precise.
Although the Grubbs test did not identify any outliers for
total trans by IR and 18:1t in sample A, the trans values reported by Collaborators 1 and 7 were considerably lower than
those reported by other collaborators, and this large discrepancy could have contributed to the poor agreement between
laboratories for total trans content and 18:1t in sample A. Nevertheless, this suggests that accurate determination of trans
content by IR of samples containing low levels (5%) of trans
unsaturation may be difficult. Direct GC analysis (e.g., AOCS
Official Method Ce 1-89 [8]) is recommended for samples with
trans content 5% when, relative to 18:1c isomers, the proportion of high value 18:1t isomers is low and, consequently, the
overlap of 18:1c and 18:1t isomers in GC cyanosilicone capillary columns is almost negligible.
Table 11 compares the values for 18:1t and 18:1c obtained
from the collaborative study with those determined in the
authors laboratory by 2 other independent methods: AgNO3TLC/GC and AOCS Official Method Ce 1c-89 (8). Mean values obtained in the collaborative study by GC/IR were equivalent to the absolute amounts determined by the tedious,
combined procedure of AgNO3-TLC and GC. This confirms
the accuracy and reliability of the GC/IR method. The AOCS
Official Method (direct GC method) gave substantially lower

values for 18:1t and higher values for 18:1c than those of the
other 2 methods, which is a consequence of ignoring the overlaps of cis and trans isomers (10, 11). The error in determining
the 18:1t and 18:1c by the direct GC method was highest for
samples containing high amounts of trans unsaturation and was
low for sample A, which had the lowest trans content.
Table 12 compares the total trans unsaturation determined
by the IR method described here, with trans unsaturation calculated by summing the 18:1t level determined by AgNO3TLC/GC method and the trans equivalents for 18:2t, 18:1tt,
and 18:3t. The trans equivalents were calculated by multiplying the GC mean weight percent data (Table 994.15) for the
above trans polyunsaturates with the appropriate correction
factors. For mono-trans polyunsaturates, the experimentally
determined correction factor correlating GC weight percent
data to IR trans equivalents is 0.84, whereas for di-trans
polyunsaturates the correction factor is 1.74 (11). Table 12
shows that IR trans values are in close agreement with calculated trans values for all test samples, except for the slightly
higher calculated trans levels for the pair of blind duplicates,
C1 and C2. This discrepancy could be attributed to higher 18:1t
values obtained for C1 and C1 using the AgNO3-TLC/GC
method (Table 11).
Recommendations
The IR method is recommended for the determination of
isolated trans unsaturated fatty acids and the GC/IR method is
recommended for the determination of fatty acid composition,
including the percentages of cis and trans-octadecenoates of
partially hydrogenated fats derived from vegetable oils, terrestrial animal fats or such oils isolated from food products containing >5% trans unsaturation. For samples with 5% trans
unsaturation, AOCS direct GC method Ce 1c-89 is available
(combining the IR and GC data is unnecessary) for determination of 18:1t and 18:1c, as well as total trans content. This is
possible because at lower trans levels, overlap of 18:1t and c
isomers in GC analysis is almost negligible. The GC/IR method
is not applicable to partially hydrogenated fish oils, because
these fats contain a complex mixture of cis and trans isomers
of mono- and polyunsaturated fatty acids with a wider range of
chain lengths.
On the basis of the results of this study it is recommended that
the IR method for determination of isolated trans unsaturated fatty
acid content in partially hydrogenated fats and the capillary GC/IR
method for determination of total cis- and trans-octadecenoic isomers and general fatty acid composition in hydrogenated vegetable oils and animal fats be adopted first action.
Acknowledgments
I thank the following collaborators:
J. Alfieri, Diversified Research Laboratories Ltd., Toronto,
ON, Canada
S. Bacler, Health and Welfare Canada, Winnipeg, MB, Canada
B. Ballch, Kraft General Foods, Inc., Glenview, IL

B. Debets, Union Deutsche Labensmittelwerke GmbH,

Kleve, Germany
D. Dionne, Health and Welfare Canada, Longueuil, PQ,
Canada
S. Gould, Prince Edward Island Food Technology Centre,
Charlottetown, PEI, Canada
D.E. Henry, The Proctor and Gamble Co., Cincinnati, OH
M.R. Lapointe, Health and Welfare Canada, Halifax, NS,
Canada
R. Lahtinen, Raisio Group, Raisio, Finland
R.E. McDonald, U.S. Food and Drug Administration, Summit-Argo, IL
G. Pelletier, Health and Welfare Canada, Ottawa, ON, Canada
D.C. Wolfe, United Catalysts, Inc., Louisville, KY
I extend special thanks to Dan Tallmadge, Proctor and Gamble Co., Cincinnati, OH, for preparation and distribution of the
partially hydrogenated fat samples for this study; David Firestone, U.S. Food and Drug Administration, Washington, DC
and David Berner, AOCS, Champaign, IL, for guidance and
encouragement; Steve Malcolm, Health and Welfare Canada,
Ottawa, for guidance on statistical evaluation; and R. Hollywood, Health and Welfare Canada, Ottawa, for technical assistance with statistical evaluation; R. Peace, Health and Welfare
Canada, Ottawa, for reviewing this paper.
References
(1) Mensink, R.P., & Katan, M.B. (1990) N. Engl. J. Med. 323,
439445
(2) Zock, P.L., & Katan, M.B. (1992) J. Lipid Res. 33, 399410
(3) Mensink, R.P., Zock, P.L., Katan, M.B., & Hornstra, G.
(1992) J. Lipid Res. 33, 14931501
(4) Troisi, R., Willett, W.C., & Weiss, S.T. (1992) Am. J. Clin.
Nutr. 56, 10191024

(5) Willett, W.C., Stampfer, M.J., Manson, J.E., Colditz, G.A.,

Speizer, F.E., Rosner, B.A., Sampson, L.A., & Hennekens,
C.H. (1993) Lancet 341, 581585
(6) Guide for Food Manufactures and Advertisers (1988) Consumer and Products Branch, Bureau of Consumer Affairs,
The Ministry of Consumer and Corporate Affairs, Canada
(7) Fed. Regist. (1993) 58, 21 CFR, Book II, Dept. of Health and
Human Services, U.S. Food and Drug Administration
(8) Official Methods and Recommended Practices of the American Oil Chemists Society (1990) 4th Ed., AOCS,
Champaign, IL, Official Method Ce 1c-89
(9) Firestone, D. (1990) J. Assoc. Off. Anal. Chem. 73, 105
(10) Ratnayake, W.M.N., & Beare-Rogers, J.L. (1990) J. Chromatog. Sci. 28, 633639
(11) Ratnayake, W.M.N., Hollywood, R., OGrady, E., & BeareRogers, J.L. (1990) J. Am. Oil Chem. Soc. 67, 804810
(12) Ratnayake, W.M.N. (1992) J. Am. Oil Chem. Soc. 69, 192
(13) Official Methods and Recommended Practices of the American Oil Chemists Society (1989) 4th Ed., AOCS,
Champaign, IL, Official Method Cd 14-61
(14) Firestone, D., & Sheppard, A. (1992) in Advances in Lipid
Methodology-One, W.W. Christie (Ed.), The Oily Press Ltd.,
Ayr, Scotland
(15) Firestone, D., & LaBouliere, P. (1965) J. Assoc. Off. Anal.
Chem. 48, 437443
(16) Official Methods of Analysis (1990) 15th Ed., AOAC, Arlington, VA, sec. 965.34
(17) OConnor, R.T., (1956) J. Am. Oil Chem. Soc. 33, 115
(18) Madison, B.L., DePalma, R.A., & DAlonzo, R.P. (1982) J.
Am. Oil Chem. Soc. 59, 178181
(19) Official Methods of Analysis (1990) 15th Ed., AOAC, Arlington, VA, sec. 969.33
(20) Ratnayake, W.M.N., & Pelletier, G. (1992) J. Am. Oil Chem.
Soc. 69, 95105
(21) Guidelines for Collaborative Study Procedure to Validate
Characteristics of a Method of Analysis (1989) J. Assoc. Off.
Anal. Chem. 72, 694704

Table 994.14. Method performance for infrared determination of isolated trans unsaturated fatty acids in partially
hydrogenated vegetable oils
Samplea
A
B
C1
C2
b
C1 and C2
D
E
R
a

x, % trans

RSDr, %

RSDR, %

sr

sR

5.2
15.5
18.9
19.1
19.0
30.1
34.6
21.6

4.8
4.2
4.6
3.7
5.8
3.0
1.0
4.1

34.6
11.3
11.7
10.3
10.5
9.0
11.3
8.8

0.3
0.7
0.9
0.7
1.1
0.9
0.3
0.9

1.8
1.8
2.2
2.0
2.0
2.7
3.9
1.9

0.84
1.96
2.52
1.96
3.07
2.52
0.84
2.52

5.04
5.04
7.06
5.49
5.61
7.06
5.04
7.06

AC2 = blends of various ratios of fat extracted from 2 retail samples of margarine (partially hydrogenated soybean, liquid soybean oil, and
cottonseed oil based margarines) diluted with unhydrogenated corn oil; DE = blends of fat extracted from 2 retail margarines with partially
hydrogenated canola oil; R = reference sample (partially hydrogenated soybean soil).
Blind duplicates.

Table 994.15. Method performance for gas chromatographic/infrared determination of fatty acid composition of
partially hydrogenated vegetable oils
Samplea

x, %

RSDr, %

RSDR, %

sr

sR

0.03
0.13
0.11
0.21
0.11
0.10
0.13
0.02

0.25
0.25
0.34
0.39
0.39
0.34
0.36
0.28

0.08
0.36
0.31
0.59
0.31
0.28
0.36
0.06

0.7
0.7
0.95
1.09
0.10
0.95
1.01
0.78

0.03
0.11
0.08
0.08
0.17
0.04
0.20
0.10

0.07
0.23
0.16
0.26
0.32
0.08
0.25
0.25

0.08
0.31
0.22
0.22
0.48
0.11
0.56
0.28

0.20
0.64
0.45
0.73
0.88
0.22
0.7
0.7

0.37
0.27
0.51
0.79
0.40
0.32
0.60
0.58

0.25
0.31
0.98
0.48
0.53
0.42
1.4
0.64

1.04
0.76
1.43
2.21
1.12
0.90
1.68
1.62

1.77
1.41
2.18
1.81
1.90
2.55
2.53
1.87

0.7
0.90
2.55
2.04
3.37
2.69
1.71
2.32

4.96
3.95
6.10
5.07
5.31
7.14
7.08
5.24

0.95
1.75
1.94
2.01
1.84
2.11
3.61
2.10

0.78
1.65
2.46
2.60
3.32
2.86
1.85
1.71

2.66
4.9
5.43
5.63
5.16
5.91
10.11
5.88

16:0
A
B
C1
C2
b
C1 and C2
D
E
R

10.3
9.1
9.7
9.7
9.7
10.7
9.7
10.8

0.3
1.4
1.1
1.2
1.0
1.0
3.7
1.3

2.4
2.8
3.5
4.0
4.0
3.2
3.7
2.6
18:0

A
B
C1
C2
b
C1 and C2
D
E
R

3.8
7.0
6.7
6.7
6.6
7.3
6.9
5.8

0.8
1.6
1.2
1.2
2.6
0.6
2.9
1.8

1.9
3.3
2.4
3.9
4.8
1.2
3.6
4.4

Total saturated fatty acids

A
B
C1
C2
b
C1 and C2
D
E
R

14.9
17.2
17.6
17.3
17.6
19.0
17.5
17.4

0.6
0.7
2.0
1.0
1.1
0.8
2.8
1.4

2.5
1.6
2.9
4.6
2.3
1.7
3.4
3.4

0.09
0.11
0.35
0.17
0.19
0.15
0.50
0.23
18:1t Isomers

A
B
C1
C2
b
C1 and C2
D
E
R

4.9
14.9
17.4
17.5
17.4
26.6
32.6
19.4

5.2
2.1
5.3
4.2
6.9
3.6
1.9
4.3

36.4
9.5
12.5
10.3
10.9
9.6
7.8
9.7

0.25
0.32
0.91
0.73
1.20
0.96
0.61
0.83
18:1c Isomers

A
B
C1
C2
b
C1 and C2
D
E
R

24.9
24.7
28.1
28.2
28.3
34.3
34.3
32.2

1.1
2.4
3.2
3.3
4.2
2.6
1.9
1.9

3.8
7.1
6.9
7.1
6.5
6.1
10.5
6.5

0.28
0.59
0.88
0.93
1.19
1.02
0.66
0.61

Table 994.15. (continued)

Samplea

x, %

RSDr, %

RSDR, %

sr

sR

0.10
0.40
0.22
0.19
0.59
0.16
0.23
0.22

0.53
0.59
0.57
0.38
0.66
0.30
0.26
0.66

0.28
1.12
0.62
0.53
1.66
0.45
0.64
0.62

1.48
1.65
1.60
1.06
1.86
0.84
0.73
1.85

0.07
0.09
0.05
0.04
0.06
0.02
0.05
0.03

0.09
0.50
0.07
0.07
0.16
0.08
0.14
0.08

0.20
0.25
0.14
0.11
0.18
0.06
0.14
0.08

0.25
1.4
0.20
0.20
0.46
0.22
0.39
0.22

NA
0.07
0.15
0.07
0.16
0.26
0.20
0.12

NA
0.08
0.08
0.06
0.34
0.20
0.22
0.25

NA
0.20
0.42
0.20
0.43
0.73
0.56
0.34

0.16
0.39
0.51
0.56
0.51
0.75
0.84
0.48

0.22
0.67
0.7
0.08
0.25
0.48
0.22
0.28

0.45
1.09
1.43
1.57
1.43
2.1
2.35
1.34

18:2(n-6)
A
B
C1
C2
b
C1 and C2
D
E
R

53.0
41.5
33.6
33.5
33.6
13.6
11.2
26.5

0.2
1.0
0.7
0.6
1.8
1.2
2.0
0.8

1.0
1.4
1.7
1.1
2.0
2.2
2.3
2.5
18:3(n-3)

A
B
C1
C2
b
C1 and C2
D
E
R

0.9
0.8
0.8
0.8
0.8
0.9
0.8
1.0

7.8
11.8
5.6
5.2
7.7
2.5
6.8
3.1

9.6
20.2
8.5
8.7
19.9
8.6
17.5
8.4

18:2tt Isomers
A
B
C1
C2
b
C1 and C2
D
E
R

NDc
0.1
0.2
0.01
0.2
0.3
0.03
0.1

NAd
40.2
19.8
25.0
79.3
25.8
28.5
80.0

NA
92.1
96.3
78.9
101.7
100.5
66.9
109.7

NA
0.03
0.03
0.02
0.12
0.07
0.08
0.09

18:2t Isomers
A
B
C1
C2
b
C1 and C2
D
E
R

0.3
0.7
1.5
1.6
1.5
3.3
3.1
2.3

25.8
33.1
17.2
2.1
6.0
5.1
2.6
4.3

49.8
55.0
34.5
35.5
34.2
22.6
27.3
20.8

0.08
0.24
0.25
0.03
0.09
0.17
0.08
0.10

Table 994.15. (continued)

Samplea

x, %

RSDr, %

RSDR, %

sr

sR

NA
0.05
0.04
0.03
0.04
0.12
0.06
0.00

NA
0.06
0.06
0.06
0.06
0.16
0.15
0.08

NA
0.14
0.11
0.08
0.11
0.34
0.17
0.00

NA
0.17
0.17
0.17
0.16
0.45
0.42
0.22

18:3t Isomers
A
B
C1
C2
b
C1 and C2
D
E
R
a

b
c
d

NDc
0.03
0.1
0.04
0.04
0.2
0.2
0.1

NAd
152.3
86.1
75.0
93.81
55.4
31.7
0.0

NA
193.7
136.6
141.6
133.8
76.9
79.7
150.4

AC2 = blends of various ratios of fat extracted from 2 retail samples of margarine (partially hydrogenated soybean, liquid soybean oil, and
cottonseed oil based margarines) diluted with unhydrogenated corn oil; DE = blends of fat extracted from 2 retail margarines with partially
hydrogenated canola oil; R = reference sample (partially hydrogenated soybean soil).
Blind duplicates.
ND = not detected (content of total fatty acids <0.01%).
NA = not applicable.

Table 1. Instrumentation and GC operating parameters reported by collaborating laboratories

Instrumenta

Lab.

IR

Column

GC

Liquid
phase

Dimensions,
m mm

Conditions
Carrier,
gas,
mL/min

Column
temp.,
C

1
2
3
4

Bruker FTIR
Mattson FTIR
PE16PC FTIR
Nicolet FTIR

HP-5890II
PE Sigma 300
HP-5890II
HP-5890A

SP-2560
SP-2560
SP-2560
SP-2340

100 0.25
100 0.25
100 0.25
60 0.25

He, 0.49
He
H2, 0.70
He

150200
150200
150200
150200

5
6

PE-298, D
Pye Unicam, D

HP
HP-5890

CP-SIL-88 50 0.25
SP-2560
100 0.25

He, 0.05
H2, 1.3

150200
125220

7
8
9
10
11
12

PE-597, D
PE-1600 FTIR
Nicolet FTIR
Nicolet FTIR
Nicolet FTIR
PE-1600 FTIR

Varian, 3400
Varian
HP-5890II
HP-5890II
HP-5890
HP-5890II

SP-2380
SP-2560
SP-2560
SP-2560
SP-2560
SP-2560

30 0.25
100 0.25
100 0.25
100 0.25
100 0.25
100 0.25

He, 0.4
H2, 1.8
H2, 0.7
He, 0.33
He, 0.33
H2, 0.7

150200
150200
150220
165200
165200
150200

Program
1C/min to 200C, hold 25 min
hold 10 min, then 2C/min to 200C
1C/min to 200C, hold 10 min
1C/min to 185C, then 10C/min to 225C,
hold 10 min
hold 8 min, then 1C/min to 210C, hold 1 min
hold 1 min, then 1C/min to 175C then,
15C/min to 220C
1C/min
1C/min
1C/min
hold 65 min, then 5C/min to 220C, hold 25 min
hold 75 min, then 7C/min to 220C, hold 40 min
1C/min to 200C, hold 10 min

PE = Perkin Elmer, HP = Hewlett Packard, FTIR = Fourier transform infrared spectrophotometer, D = dispersive spectrophotometer.

Table 2. GC/IR collaborative study% trans by IR

Lab.
Sample
A
B
C1
C2
D
E
R

10

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

1.10
16.00
15.90
20.10
25.30
35.50
20.90

1.10
15.50
15.80
19.10
25.30
35.50
20.90

4.70
13.70
20.60
16.80
26.40
30.50
18.80

4.60
13.30
18.30
17.30
24.50
29.30
21.50

6.20
17.30
20.90
20.90
32.80
37.90
24.40

6.00
17.20
20.90
20.70
32.70
37.70
24.40

5.50
16.50
20.40
20.00
31.60
38.10
23.30

5.60
16.60
20.30
20.60
32.40
38.20
22.70

6.70
17.20
20.70
20.30
31.10
32.70
23.80

7.50
17.40
21.30
21.60
32.50
33.00
24.00

6.90
14.60
18.50
17.40
33.30
39.70
22.00

7.00
12.10
19.90
19.50
30.90
37.40
23.20

2.20
12.10
15.40
16.10
27.30
29.20
17.10

3.00
12.90
17.00
14.70
28.10
34.20
19.80

4.20
14.80
14.70
17.20
27.90
26.10
20.10

4.40
13.30
16.90
17.70
30.30
25.70
19.40

6.40
16.70
19.80
20.60
31.60
36.70
22.40

6.40
16.30
20.30
20.40
32.00
36.70
22.00

X1

11
X2

X1

Sample

Mean

sw

sR

6.10 5.90
16.70 16.70

31.50 30.90
36.40 36.50
22.60 22.30

29.80 30.30
34.60 35.20
19.50 20.60

A
B
C1
C2
D
E
R

5.17
15.54
18.92
19.09
30.06
34.58
21.63

0.25
0.65
0.87
0.71
0.90
1.16
0.88

1.79
1.76
2.21
1.97
2.69
3.91
1.90

Excluding outliers

RSDw RSDR Mean

4.74
4.16
4.62
3.71
2.98
3.36
4.05

34.56
11.31
11.69
10.32
8.94
11.30
8.79

5.17
15.54
18.92
19.09
30.06
34.48
21.63

sw

sR

0.25
0.65
0.87
0.71
0.90
0.33
0.88

1.79
1.76
2.21
1.97
2.69
3.90
1.90

RSDw RSDR
4.74
4.16
4.62
3.71
2.98
0.95
4.05

34.56
11.31
11.69
10.32
8.94
11.31
8.79

Outliers/
No. Outlier
labs lab.*
0/12
0/12
0/10
0/10
0/12
2/12
0/12

6,7

X2

5.90 5.70
16.70 16.30

Statistical evaluation of % trans by IR

Including outliers

12

X1 = Data for experiment 1

X2 = Data for experiment 2
sw = Within laboratory standard deviation
sR = Reproducibility standard deviation
RSDw = Relative within laboratory standard deviation
RSDR = Reproducibility relative standard deviation
*Outlier laboratory, determined by Cochran and/or Grubbs tests

X1

X2

5.40
16.40
20.40
20.50
31.70
36.70
21.60

5.60
16.70
20.30
20.30
31.30
36.50
21.70

Table 3. GC/IR collaborative study results of PHVO sample A

Lab.
FA
16:0
18:0
SAT.
18:1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

X1

X2

X1

X2

X1

X2

X1

X2

10.40
3.80
15.20
0.90
26.20
52.30
1.00
0.00
0.20
0.00
1.10
1.10

10.40
3.80
15.20
0.90
26.20
52.30
1.00
0.00
0.20
0.00
1.10
1.10

10.60
3.60
14.70
4.20
25.80
53.40
0.80
0.00
0.20
0.30
4.70
4.80

10.60
3.60
14.70
4.30
25.90
53.30
1.00
0.00
0.30
0.10
4.60
4.60

10.70
3.70
15.30
5.90
24.80
52.50
0.90
0.00
0.40
0.00
6.20
6.30

10.70
3.70
15.20
5.70
25.10
52.50
0.90
0.00
0.30
0.00
6.00
6.00

10.50
3.80
15.30
5.10
25.50
52.40
0.90
0.00
0.40
0.00
5.50
5.60

10.50
3.80
15.30
5.20
25.50
52.40
0.90
0.00
0.40
0.00
5.60
5.60

X1

6
X2

9.90 9.90
3.80 3.80
14.50 14.40
6.30 7.20
23.90 23.00
53.30 53.50
0.90 0.90
0.00 0.00
0.50 0.40
0.10 0.10
6.70 7.50
6.80 7.60

10

11

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

10.10
3.70
14.50
6.80
23.70
53.40
0.90
0.00
0.10
0.00
6.90
6.90

10.10
3.70
14.40
6.90
23.70
53.40
0.90
0.00
0.20
0.00
7.00
7.10

10.00
2.80
13.00
2.10
23.40
58.90
0.40
0.00
0.10
0.00
2.20
2.20

11.20
2.90
14.30
2.80
24.20
56.80
0.40
0.00
0.20
0.00
3.00
3.00

10.10
3.50
14.30
3.80
26.20
53.90
1.10
0.00
0.50
0.00
4.20
4.30

10.10
3.60
14.10
3.80
25.80
54.10
1.10
0.00
0.80
0.00
4.40
4.60

10.40
3.80
15.10
6.10
24.30
52.60
0.90
0.00
0.30
0.00
6.40
6.40

10.40
3.70
15.00
6.10
24.50
52.70
0.90
0.00
0.40
0.00
6.40
6.50

10.00
3.80
14.80
5.70
24.90
53.00
0.90
0.00
0.20
0.10
5.90
6.00

10.10
3.80
14.90
5.50
25.00
53.10
0.90
0.00
0.20
0.00
5.70
5.70

10.30
3.80
15.00
5.70
25.10
52.80
0.80
0.10
0.30
0.00
6.10
6.10

10.30
3.70
14.70
5.50
25.00
53.10
1.20
0.00
0.40
0.00
5.90
5.90

10.40
3.80
15.00
5.10
25.40
53.00
0.90
0.00
0.30
0.00
5.40
5.40

10.50
3.80
15.10
5.40
25.30
52.80
0.90
0.00
0.20
0.00
5.60
5.60

Statistical evaluation of sample A

Including outliers

FA

Mean

sw

16:0
18:0
SAT.
18:1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

10.34
3.64
14.75
4.88
24.93
53.39
0.90
0.00
0.31
0.00
5.17
5.21

0.25
0.05
0.28
0.25
0.28
0.45
0.07
0.00
0.08
0.00
0.25
0.27

sR

Excluding outliers

RSDw RSDR Mean

0.31 2.38
0.29 1.37
0.53 1.90
1.77 5.20
0.95 1.14
1.52 0.83
0.18 7.86
0.00 0.00
0.16 25.78
0.00 0.00
1.79 4.74
1.80 5.18

2.97
8.02
3.59
36.39
3.79
2.85
19.90
0.00
49.78
0.00
34.56
34.58

10.32
3.75
14.85
4.88
24.93
53.00
0.90
0.00
0.31
0.00
5.17
5.21

sw

sR

0.03
0.03
0.09
0.25
0.28
0.10
0.07
0.00
0.08
0.00
0.25
0.27

0.25
0.07
0.37
1.77
0.95
0.53
0.09
0.00
0.16
0.00
1.79
1.80

RSDw RSDR
0.29
0.84
0.63
5.20
1.14
0.18
7.78
0.00
25.78
0.00
4.74
5.18

2.44
1.88
2.50
36.39
3.79
1.00
9.56
0.00
49.78
0.00
34.56
34.58

12

Outliers/
No. Outlier
labs lab.*
1/12
2/12
1/12
0/12
0/12
1/12
1/12
0/12
0/12
1/12
0/12
0/12

7
7,8
7

7
11

PHVO = Partially hydrogenated vegetable oil

X1 = Data for experiment 1
X2 = Data for experiment 2
sw = Within laboratory standard deviation
sR = Reproducibility standard deviation
RSDw = Relative within laboratory standard deviation
RSDR = Reproducibility relative standard deviation
CAL-trans = Sum of 18:1t, 18:2tt, 18:2t, and 18:3t
*Outlier laboratory, determined by Cochran and/or Grubbs tests
SAT. = Sum of saturated fatty acids
IR-trans = Total trans unsaturation determined by IR
FA = fatty acid

Table 4. GC/IR collaborative study results of PHVO sample B

Lab.
FA
16:0
18:0
SAT.
18:1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

10

11

12

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

9.20
7.20
17.40
15.80
27.90
41.00
0.80
0.00
0.30
0.00
16.00
16.10

9.10
7.10
17.30
15.00
27.50
41.10
0.80
0.00
0.60
0.00
15.50
15.50

9.50
6.90
17.20
12.90
25.90
42.10
0.80
0.10
0.60
0.10
13.70
13.70

9.40
6.70
17.10
12.40
26.00
41.80
1.10
0.10
0.70
0.20
13.30
13.40

9.30
7.10
17.60
16.30
23.20
40.90
0.70
0.20
0.80
0.00
17.30
17.30

9.40
7.10
17.60
16.20
23.20
41.00
0.70
0.20
0.80
0.00
17.20
17.20

9.20
7.10
17.60
15.60
24.00
40.90
0.80
0.10
0.90
0.00
16.50
16.60

9.20
7.10
17.50
15.70
24.00
41.00
0.80
0.10
0.80
0.00
16.60
16.70

8.70
7.10
16.70
15.50
22.80
41.30
0.70
0.00
1.80
0.10
17.20
17.30

8.70
7.20
16.90
16.60
22.00
42.00
0.70
0.00
0.90
0.10
17.40
17.50

9.10
6.80
16.90
14.10
25.40
41.80
0.70
0.10
0.40
0.00
14.60
14.60

8.90
7.00
16.90
11.60
27.70
42.00
0.70
0.10
0.40
0.00
12.10
12.10

8.80
6.70
15.70
12.00
26.50
42.90
0.50
0.00
0.10
0.00
12.10
12.10

9.20
7.00
16.40
12.80
27.40
41.40
0.30
0.00
0.10
0.00
12.90
12.90

8.80
6.70
16.30
13.60
25.00
42.00
1.00
0.00
1.20
0.20
14.80
15.00

8.60
6.40
15.70
13.10
25.50
42.80
0.80
0.00
1.40
0.00
13.30
14.50

9.10
7.10
17.40
15.90
23.70
41.30
0.70
0.10
0.70
0.10
16.70
16.80

9.10
7.10
17.40
15.70
23.50
41.40
0.70
0.00
0.70
0.00
16.30
16.40

8.80
7.10
17.20
15.80
23.70
41.40
0.80
0.10
0.70
0.10
16.70
16.70

8.80
7.10
17.20
15.40
24.00
41.40
0.80
0.10
0.70
0.10
16.30
16.30

9.20
6.90
17.10
15.70
23.00
41.80
0.90
0.10
0.90
0.00
16.70
16.70

9.00
7.00
17.00
15.80
23.50
41.70
0.80
0.20
0.70
0.00
16.70
16.70

9.10
7.40
17.10
15.60
24.20
41.00
0.80
0.10
0.70
0.00
16.40
16.40

9.30
7.40
17.50
15.90
23.70
40.80
0.80
0.10
0.70
0.00
16.70
16.70

Statistical evaluation of sample B

Including outliers

FA

Mean

sw

16:0
18:0
SAT.
18:1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

9.06
7.01
17.04
14.84
24.70
41.53
0.76
0.07
0.73
0.03
15.54
15.63

0.13
0.11
0.17
0.58
0.59
0.40
0.09
0.03
0.24
0.05
0.65
0.58

sR

RSDw RSDR Mean

0.25
1.39
2.80 9.06
0.23
1.63
3.31 7.01
0.63
1.01
3.73 17.23
1.52
3.90 10.28 14.92
1.75
2.39
7.08 24.70
0.59
0.97
1.41 41.53
0.15 11.82 20.21 0.76
0.07 40.20 92.11 0.07
0.39 33.08 54.95 0.73
0.06 152.26 193.65 0.03
1.76
4.16 11.31 15.54
1.72
3.71 10.99 15.84

Excluding outliers

sw

sR

RSDw RSDR

0.13
0.11
0.11
0.32
0.59
0.40
0.09
0.03
0.24
0.05
0.65
0.29

0.25
0.23
0.27
1.41
1.75
0.59
0.50
0.07
0.39
0.06
1.76
1.59

1.39 2.80
1.63 3.31
0.66 1.60
2.12 9.48
2.39 7.08
0.97 1.41
11.82 20.21
40.20 92.11
33.08 54.95
152.26 193.65
4.16 11.31
1.81 10.50

Outliers/
No. Outlier
labs lab.*
0/12
0/12
2/12
1/12
0/12
0/12
0/12
0/12
0/12
0/12
0/12
1/12

7,8
6

PHVO = Partially hydrogenated vegetable oil

X1 = Data for experiment 1
X2 = Data for experiment 2
sw = Within laboratory standard deviation
sR = Reproducibility standard deviation
RSDw = Relative within laboratory standard deviation
RSDR = Reproducibility relative standard deviation
CAL-trans = Sum of 18:1t, 18:2tt, 18:2t, and 18:3t
*Outlier laboratory, determined by Cochran and/or Grubbs tests
SAT. = Sum of saturated fatty acids
IR-trans = Total trans unsaturation determined by IR
FA = fatty acid

Table 5. GC/IR collaborative study results of PHVO sample C1

Lab.
FA
16:0
18:0
SAT.
18:1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

12

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

9.80
6.80
17.80
14.00
31.00
33.20
0.80
0.50
0.10
0.00
15.90
15.60

9.70
7.00
18.00
13.90
31.30
32.50
0.90
0.50
1.10
0.00
15.80
15.50

10.90
6.40
18.10
19.50
26.50
33.60
0.90
0.10
1.00
0.10
20.60
20.70

10.10
6.50
17.40
17.00
29.20
33.50
1.00
0.00
1.30
0.20
18.30
18.50

10.10
6.80
18.00
19.10
26.70
33.20
0.80
0.20
1.70
0.00
21.00
20.90

10.10
6.80
18.00
19.00
26.70
33.20
0.80
0.30
1.70
0.00
20.90
20.90

9.90
6.80
18.00
18.80
27.10
33.30
0.80
0.20
1.60
0.00
20.40
20.50

9.80
6.80
17.80
18.50
27.40
33.20
0.80
0.20
1.70
0.10
20.30
20.50

9.50
7.00
17.40
19.00
25.80
33.20
0.90
0.00
2.00
0.00
20.70
21.00

9.50
6.80
17.00
19.00
25.40
33.20
0.80
0.00
2.70
0.10
21.30
21.70

9.80
6.70
17.40
17.20
28.60
34.00
0.80
0.10
1.20
0.10
18.50
18.60

9.70
6.80
17.40
18.70
27.10
34.00
0.80
0.10
1.20
0.10
19.90
20.10

8.80
5.70
14.70
14.80
29.40
36.10
0.50
0.00
0.70
0.00
15.40
15.50

9.10
5.80
15.00
16.60
28.80
37.80
0.40
0.00
0.50
0.00
17.00
17.10

9.60
6.60
17.00
12.70
32.30
34.60
0.90
0.30
1.74
0.00
14.70
14.70

9.60
6.50
16.80
14.90
30.00
34.80
1.00
0.30
1.70
0.00
16.90
16.90

9.70
6.80
17.60
18.40
27.20
33.50
0.80
0.20
1.20
0.10
19.80
19.90

9.70
6.80
17.60
18.40
27.20
33.50
0.80
0.10
2.00
0.10
20.30
20.60

10.10
6.70
17.70
18.90
27.20
33.20
0.80
0.10
1.60
0.00
20.40
20.60

9.80
6.70
17.40
18.90
27.20
33.20
0.80
0.10
1.50
0.00
20.30
20.50

Statistical evaluation of sample C1

Including outliers

Excluding outliers

FA

Mean

sw

sR

RSDw

RSDR

Mean

sw

sR

16:0
18:0
SAT.
18;1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

9.77
6.65
17.36
17.37
28.11
33.93
0.81
0.16
1.46
0.04
18.92
19.02

0.21
0.08
0.34
0.91
0.88
0.43
0.05
0.03
0.25
0.04
0.87
0.88

0.43
0.35
1.01
2.18
1.94
1.22
0.14
0.15
0.51
0.06
2.21
2.33

2.11
1.21
1.98
5.26
3.14
1.28
6.21
19.76
17.20
86.07
4.62
4.63

4.39
5.26
5.79
12.53
6.89
3.60
17.74
96.26
34.51
136.59
11.69
12.26

9.68
6.74
17.63
17.37
28.11
33.59
0.84
0.16
1.46
0.10
18.92
19.02

0.11
0.08
0.35
0.91
0.88
0.22
0.05
0.03
0.25
0.04
0.87
0.88

0.34
0.16
0.51
2.18
1.94
0.57
0.07
0.15
0.51
0.06
2.21
2.33

RSDw

RSDR

1.12
3.48
1.21
2.40
2.01
2.90
5.26
12.53
3.14
6.89
0.66
1.70
5.58
8.49
19.76
96.26
17.20
34.51
86.07 136.59
4.62
11.69
4.63
12.26

Outliers/
No. Outlier
labs
lab.*
1/10
1/10
1/10
0/10
0.10
1/10
1/10
0/10
0/10
0/10
0/10
0/10

2
7
7

5
7

PHVO = Partially hydrogenated vegetable oil

X1 = Data for experiment 1
X2 = Data for experiment 2
sw = Within laboratory standard deviation
sR = Reproducibility standard deviation
RSDw = Relative within laboratory standard deviation
RSDR = Reproducibility relative standard deviation
CAL-trans = Sum of 18:1t, 18:2tt, 18:2t, and 18:3t
*Outlier laboratory, determined by Cochran and/or Grubbs tests
SAT. = Sum of saturated fatty acids
IR-trans = Total trans unsaturation determined by IR
FA = fatty acid

Table 6. GC/IR collaborative study results of PHVO sample C2

Lab.
FA
16:0
18:0
SAT.
18:1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

12

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

9.90
6.80
17.90
19.00
31.40
33.20
0.90
0.10
1.10
0.00
20.10
20.20

9.90
6.90
18.00
18.00
31.60
32.90
0.90
0.00
1.10
0.00
19.10
19.10

10.20
6.60
17.60
15.50
29.90
34.30
0.90
0.10
1.30
0.10
16.80
17.00

10.40
6.30
17.50
16.20
32.80
37.10
1.00
0.10
0.90
0.20
17.30
17.40

10.00
6.80
18.00
19.10
26.60
33.20
0.80
0.20
1.60
0.10
20.90
21.00

10.00
6.80
17.90
18.90
26.80
33.40
0.80
0.20
1.60
0.10
20.70
20.80

9.90
6.80
17.90
18.30
27.50
33.40
0.80
0.10
1.70
0.00
20.00
20.20

9.90
6.80
17.90
19.00
26.80
33.30
0.80
0.10
1.60
0.00
20.60
20.70

9.20
6.80
17.00
18.10
26.50
33.90
0.80
0.00
2.60
0.00
20.70
20.70

9.10
6.80
16.80
19.40
25.00
33.70
0.80
0.00
2.60
0.00
21.60
22.00

9.70
6.70
17.30
16.00
29.60
34.10
0.80
0.10
1.40
0.10
17.40
17.60

9.60
6.80
17.30
18.20
27.60
34.10
0.80
0.10
1.20
0.10
19.50
19.60

9.00
6.10
15.30
15.60
27.20
38.30
0.50
0.00
0.60
0.00
16.10
16.20

9.20
6.20
15.60
14.20
28.40
37.00
0.40
0.00
0.60
0.00
14.70
14.80

9.30
6.30
16.40
14.60
28.50
33.50
1.25
0.40
1.90
0.50
17.20
17.30

9.70
6.30
16.80
14.90
28.30
34.10
1.10
0.70
1.90
0.20
17.70
17.50

9.80
6.80
17.70
18.90
26.80
33.50
0.70
0.10
1.80
0.00
20.60
20.80

9.80
6.80
17.70
18.60
27.10
33.40
0.80
0.10
1.80
0.10
20.40
20.60

10.00
6.80
17.70
19.00
27.80
33.10
0.90
0.20
1.60
0.00
20.50
20.80

9.90
6.70
17.20
19.00
27.20
33.40
0.80
0.20
1.60
0.00
20.30
20.30

Statistical evaluation of sample C2

Including outliers

Excluding outliers

FA

Mean

sw

sR

RSDw

RSDR

Mean

sw

sR

16:0
18:0
SAT.
18;1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

9.73
6.65
17.26
17.53
28.17
34.16
0.83
0.14
1.53
0.08
19.09
19.26

0.21
0.08
0.17
0.73
0.93
0.71
0.06
0.07
0.09
0.07
0.71
0.69

0.39
0.26
0.79
1.81
2.01
1.53
0.19
0.17
0.55
0.12
1.97
2.04

1.19
1.21
0.98
4.17
3.29
2.08
7.62
50.51
6.20
98.88
3.71
3.56

4.00
3.86
4.58
10.34
7.14
4.47
23.04
119.40
35.71
163.90
10.32
10.60

9.73
6.65
17.26
17.53
28.17
33.53
0.83
0.09
1.59
0.04
19.09
19.26

0.21
0.08
0.17
0.73
0.93
0.19
0.04
0.02
0.03
0.03
0.71
0.69

0.39
0.26
0.79
1.81
2.01
0.38
0.07
0.07
0.56
0.06
1.97
2.04

RSDw

RSDR

1.19
4.00
1.21
3.86
0.98
4.58
4.17
10.34
3.29
7.14
0.57
1.13
5.21
8.66
24.96
78.92
2.10
35.52
75.00 141.56
3.71
10.32
3.56
10.60

Outliers/
No. Outlier
labs
lab.*
0/10
0/10
0/10
0/10
0/10
2/10
2/10
1/10
1/10
1/10
0/10
0/10

2,7
7,8
8
2
8

PHVO = Partially hydrogenated vegetable oil

X1 = Data for experiment 1
X2 = Data for experiment 2
sw = Within laboratory standard deviation
sR = Reproducibility standard deviation
RSDw = Relative within laboratory standard deviation
RSDR = Reproducibility relative standard deviation
CAL-trans = Sum of 18:1t, 18:2tt, 18:2t, and 18:3t
*Outlier laboratory, determined by Cochran and/or Grubbs tests
SAT. = Sum of saturated fatty acids
IR-trans = Total trans unsaturation determined by IR
FA = fatty acid

Table 7. GC/IR collaborative study results of PHVO sample D

Lab.
FA
16:0
18:0
SAT.
18:1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

10

11

12

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

10.60
7.50
19.20
21.70
39.00
13.00
0.80
0.90
2.40
0.10
25.30
25.10

10.60
7.50
19.10
21.40
38.80
13.00
0.90
0.90
2.70
0.10
25.30
24.20

11.00
7.20
19.00
23.80
38.50
13.80
1.00
0.10
2.50
0.30
26.40
26.70

11.40
7.20
19.40
21.60
36.60
13.10
1.00
0.10
2.90
0.30
24.50
25.00

11.00
7.20
19.40
29.00
32.70
13.30
0.80
0.40
3.50
0.20
32.80
33.10

11.00
7.20
19.20
29.10
32.70
13.50
0.80
0.30
3.50
0.20
32.70
33.10

10.80
7.30
19.30
28.00
33.60
13.50
0.90
0.20
3.70
0.20
31.60
32.00

10.70
7.20
19.10
28.80
32.60
13.50
0.90
0.20
3.70
0.20
32.40
32.80

10.20
7.30
18.40
26.90
33.00
13.70
0.90
0.00
5.00
0.10
31.10
31.90

10.10
7.30
18.20
28.60
31.70
13.70
0.90
0.00
4.60
0.10
32.50
33.20

10.60
7.20
18.70
30.00
31.90
13.90
0.90
0.20
3.30
0.20
33.30
33.70

10.60
7.20
18.70
27.60
34.20
13.90
0.90
0.20
3.30
0.20
30.90
31.30

10.30
6.30
16.80
25.90
34.70
14.60
0.40
0.00
1.70
0.00
27.30
27.60

11.00
6.90
18.60
26.40
33.60
18.50
0.50
0.00
2.00
0.00
28.10
28.40

10.30
6.60
17.50
23.30
36.10
15.50
1.30
0.60
3.90
0.30
27.90
28.10

10.21
6.50
17.30
25.80
32.80
14.70
1.50
0.50
3.70
0.70
30.30
30.60

10.90
7.30
19.20
28.10
34.10
13.40
0.80
0.20
3.30
0.20
31.60
31.80

10.70
7.30
19.00
28.10
33.60
13.40
0.80
0.30
3.80
0.20
32.00
32.40

10.30
7.20
18.70
27.60
34.10
13.50
0.90
0.40
3.50
0.30
31.50
31.80

10.40
7.20
18.80
27.00
34.60
13.50
0.90
0.40
3.50
0.30
30.90
31.20

10.90
7.30
19.40
26.70
35.00
13.70
1.00
0.00
3.20
0.50
29.80
30.40

10.90
7.20
18.90
27.20
35.10
13.80
1.00
0.20
3.20
0.10
30.30
30.70

10.70
7.30
18.90
28.80
32.70
13.90
1.00
0.00
3.30
0.20
31.70
32.30

10.90
7.20
18.80
28.00
33.40
13.90
1.00
0.20
3.30
0.20
31.30
31.70

Statistical evaluation of sample D

Including outliers

FA

Mean

sw

16:0
18:0
SAT.
18:1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

10.67
7.15
18.72
26.64
34.38
13.93
0.91
0.26
3.30
0.21
30.06
30.38

0.17
0.13
0.39
0.96
1.02
0.83
0.05
0.07
0.17
0.12
0.90
0.91

sR

RSDw RSDR Mean

0.34 1.63
3.18 10.67
0.29 1.83
4.07 7.26
0.67 2.10
3.60 18.95
2.55 3.59
9.58 26.64
2.11
2.95
6.14 34.38
1.13 5.93
8.13 13.55
0.22 5.50 23.89 0.91
0.26 25.79 100.52 0.26
0.75 5.06 22.58 3.30
0.16 55.43 76.92 0.21
2.69 2.98
8.94 30.06
2.89 2.98
9.50 30.38

Excluding outliers

sw

sR

RSDw RSDR

0.10
0.04
0.15
0.96
1.02
0.16
0.02
0.07
0.17
0.12
0.90
0.91

0.34
0.08
0.32
2.55
2.11
0.30
0.08
0.26
0.75
0.16
2.69
2.89

0.98
3.19
0.62
1.15
0.78
1.69
3.59
9.58
2.59
6.14
1.21
2.19
2.47
8.60
25.79 100.52
5.06 22.58
55.43 76.92
2.98
8.94
2.98
9.50

Outliers/
No. Outlier
labs lab.*
1/12
2/12
2/12
0/12
0/12
2/12
2/12
0/12
0/12
0/12
0/12
0/12

7
7,8
7,8

7,8
7,8

PHVO = Partially hydrogenated vegetable oil

X1 = Data for experiment 1
X2 = Data for experiment 2
sw = Within laboratory standard deviation
sR = Reproducibility standard deviation
RSDw = Relative within laboratory standard deviation
RSDR = Reproducibility relative standard deviation
CAL-trans = Sum of 18:1t, 18:2tt, 18:2t, and 18:3t
*Outlier laboratory, determined by Cochran and/or Grubbs tests
SAT. = Sum of saturated fatty acids
IR-trans = Total trans unsaturation determined by IR
FA = fatty acid

Table 8. GC/IR collaborative study results of PHVO sample E

Lab.
FA
16:0
18:0
SAT.
18:1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

10

11

12

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

9.60
7.20
18.00
32.80
38.60
10.80
0.80
0.40
2.40
0.10
35.50
35.60

9.70
7.30
18.10
33.30
38.70
10.80
0.70
0.10
2.30
0.10
35.50
35.80

10.60
7.10
18.40
28.20
38.20
11.60
0.80
0.10
2.10
0.30
30.50
30.80

9.20
6.70
17.10
27.10
36.50
10.70
0.90
0.10
2.00
0.40
29.30
29.60

10.10
7.00
18.10
34.20
31.50
11.10
0.70
0.50
3.20
0.10
37.90
38.00

10.00
7.00
18.10
34.20
31.70
11.20
0.70
0.40
3.30
0.00
37.70
37.80

9.70
7.00
17.90
34.80
30.40
11.20
0.90
0.20
3.40
0.10
38.10
38.50

9.70
7.00
17.90
34.70
30.80
11.20
0.90
0.30
3.30
0.10
38.20
38.50

9.30
7.00
17.40
28.60
34.80
11.50
0.70
0.00
4.90
0.00
32.70
33.50

9.20
7.00
17.40
28.80
34.40
11.50
0.70
0.10
5.00
0.00
33.00
33.80

9.80
6.70
17.60
36.50
29.60
11.40
0.80
0.30
3.00
0.20
39.70
40.00

9.70
6.80
16.90
34.10
32.20
11.60
0.80
0.30
3.10
0.20
37.40
37.70

9.40
6.30
15.90
27.60
33.20
12.80
0.50
0.00
1.90
0.00
29.20
29.50

10.30
6.90
17.40
32.70
32.60
13.70
0.50
0.00
1.80
0.00
34.20
34.50

9.50
6.80
17.10
22.00
42.60
12.20
1.00
0.60
3.50
0.20
26.10
26.30

9.30
6.20
16.20
21.30
42.70
13.00
1.10
0.60
3.60
0.40
25.70
25.80

9.70
7.00
17.80
32.90
32.50
11.20
0.80
0.40
3.50
0.20
36.70
37.00

9.70
7.00
17.80
33.00
32.40
11.30
0.80
0.50
3.40
0.10
36.70
37.00

9.30
7.00
17.50
32.70
32.90
11.20
0.80
0.50
3.00
0.40
36.40
36.60

9.20
7.00
17.40
32.60
32.90
11.20
0.80
0.50
3.20
0.40
36.50
36.70

10.00
7.10
18.10
31.90
34.10
11.60
0.80
0.10
2.80
0.30
34.60
35.10

9.50
6.90
17.30
31.00
34.00
11.20
1.00
0.30
3.80
0.30
35.20
35.70

9.70
7.00
17.60
33.30
32.60
11.20
0.80
0.40
3.00
0.20
36.70
39.90

9.70
7.10
17.70
33.00
32.70
11.10
0.80
0.40
3.10
0.20
36.50
36.70

Statistical evaluation of sample E

Including outliers

FA

Mean

sw

16:0
18:0
SAT.
18:1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

9.66
6.92
17.53
31.48
34.28
11.51
0.80
0.30
3.11
0.18
34.58
34.89

0.13
0.20
0.50
1.19
0.66
0.32
0.05
0.08
0.22
0.06
1.16
1.16

sR

Excluding outliers

RSDw RSDR Mean

0.36 3.73
0.25 2.87
0.60 2.83
3.99 3.78
3.61 1.92
0.74 2.80
0.14 6.79
0.20 28.45
0.82 7.01
0.15 31.49
3.91 3.36
3.90 3.34

3.73
3.62
3.40
12.66
10.52
6.39
17.50
66.93
26.33
79.33
11.30
11.18

9.66
6.92
17.53
32.60
34.28
11.23
0.80
0.30
3.09
0.18
34.48
34.79

sw

sR

0.13
0.20
0.50
0.61
0.66
0.23
0.05
0.08
0.08
0.06
0.33
0.34

0.36
0.25
0.60
2.53
3.61
0.26
0.14
0.20
0.84
0.15
3.90
3.89

RSDw RSDR
3.73
2.87
2.83
1.88
1.92
2.03
6.79
28.45
2.58
31.49
0.95
0.97

3.73
3.62
3.40
7.78
10.52
2.33
17.50
66.93
27.25
79.73
11.31
11.19

Outliers/
No. Outlier
labs lab.*
0/12
0/12
0/12
2/12
0/12
2/12
0/12
0/12
1/12
0/12
2/12
2/12

7,8

7,8

11

6,7
6,7

PHVO = Partially hydrogenated vegetable oil

X1 = Data for experiment 1
X2 = Data for experiment 2
sw = Within laboratory standard deviation
sR = Reproducibility standard deviation
RSDw = Relative within laboratory standard deviation
RSDR = Reproducibility relative standard deviation
CAL-trans = Sum of 18:1t, 18:2tt, 18:2t, and 18:3t
*Outlier laboratory, determined by Cochran and/or Grubbs tests
SAT. = Sum of saturated fatty acids
IR-trans = Total trans unsaturation determined by IR
FA = fatty acid

Table 9. GC/IR collaborative study results of PHVO sample R

Lab.
FA
16:0
18:0
SAT.
18:1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

10

11

12

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

X1

X2

11.00
5.90
17.90
18.60
35.90
25.80
1.00
0.10
2.10
0.00
20.90
20.80

10.80
5.90
17.80
18.30
35.70
25.70
1.00
0.30
2.40
0.00
20.90
21.10

11.40
5.70
17.70
16.80
33.60
26.80
0.90
0.00
1.80
0.60
18.80
19.20

10.90
5.80
17.10
19.50
31.70
27.40
0.90
0.10
1.80
0.40
21.50
21.80

11.20
5.80
18.00
22.00
29.60
26.30
0.90
0.20
2.50
0.00
24.40
24.70

11.40
5.80
18.10
22.30
29.20
26.30
0.90
0.00
2.50
0.00
24.40
24.80

10.90
5.90
17.90
20.90
30.70
26.20
1.00
0.10
2.60
0.10
23.30
23.60

10.90
5.80
17.80
20.20
31.30
26.20
0.90
0.10
2.60
0.10
22.70
23.00

10.40
6.00
17.30
22.10
28.90
26.90
0.90
0.00
2.10
0.00
23.80
24.20

10.40
6.00
17.20
22.00
28.40
26.40
1.00
0.00
2.40
0.00
24.00
24.40

10.80
5.80
17.40
20.40
31.30
26.80
0.90
0.10
2.30
0.10
22.00
22.90

10.70
5.90
17.40
20.40
31.40
26.80
0.90
0.10
2.30
0.10
23.20
22.90

10.60
5.10
15.90
16.10
31.40
29.70
0.50
0.00
1.20
0.00
17.10
17.30

10.80
5.00
16.00
19.00
32.70
29.20
0.50
0.00
1.00
0.00
19.80
20.00

10.80
5.70
16.90
16.80
33.00
28.40
1.10
0.53
2.90
0.00
20.10
21.20

10.60
5.50
16.50
16.70
34.30
27.90
1.10
0.20
2.80
0.00
19.40
19.70

10.80
5.80
17.60
19.60
31.90
26.20
0.90
0.10
2.90
0.20
22.40
22.80

10.80
5.80
17.60
19.30
32.20
26.30
0.90
0.10
2.80
0.20
22.00
22.40

10.50
5.80
17.40
20.10
31.50
26.20
1.00
0.20
2.40
0.20
22.60
22.90

10.50
5.80
17.40
19.80
31.80
26.20
1.00
0.20
2.40
0.20
22.30
22.60

10.50
5.80
17.10
17.30
35.20
26.10
1.10
0.10
2.50
0.00
19.50
19.90

10.70
6.10
17.60
17.30
34.10
26.20
1.10
0.10
2.50
0.00
20.60
19.90

11.70
5.70
17.70
19.70
32.20
26.60
0.90
0.00
2.40
0.00
21.60
22.10

10.70
5.80
17.00
19.70
33.10
26.20
1.00
0.00
2.40
0.00
21.70
22.10

Statistical evaluation of sample R

Including outliers

FA

Mean

sw

16:0
18:0
SAT.
18:1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

10.83
5.75
17.35
19.37
32.16
26.78
0.93
0.11
2.32
0.09
21.63
21.89

0.25
0.10
0.23
0.83
0.61
0.23
0.03
0.09
0.10
0.04
0.88
0.79

sR

RSDw RSDR Mean

0.33 2.28
3.04 10.79
0.25 1.77
4.40 5.75
0.58 1.35
3.37 17.35
1.87 4.29
9.65 19.37
2.10 1.89
6.53 32.16
1.05 0.87
3.93 26.54
0.15 3.12 16.69 0.96
0.12 79.94 109.72 0.11
0.48 4.32 20.75 2.32
0.15 44.54 167.18 0.05
1.90 4.05
8.79 21.63
1.97 3.62
9.01 21.89

Excluding outliers

sw

sR

RSDw RSDR

0.02
0.10
0.23
0.83
0.61
0.22
0.03
0.09
0.10
0.00
0.88
0.79

0.28
0.25
0.58
1.87
2.10
0.66
0.08
0.12
0.48
0.08
1.90
1.97

1.34
2.64
1.77
4.40
1.35
3.37
4.29
9.65
1.89
6.53
0.82
2.50
3.13
8.37
79.94 109.72
4.32 20.75
0.00 150.37
4.05
8.79
3.62
9.01

Outliers/
No. Outlier
labs lab.*
1/12
0/12
0/12
0/12
0/12
1/12
1/12
0/12
0/12
1/12
0/12
0/12

12

7
7

PHVO = Partially hydrogenated vegetable oil

X1 = Data for experiment 1
X2 = Data for experiment 2
sw = Within laboratory standard deviation
sR = Reproducibility standard deviation
RSDw = Relative within laboratory standard deviation
RSDR = Reproducibility relative standard deviation
CAL-trans = Sum of 18:1t, 18:2tt, 18:2t, and 18:3t
*Outlier laboratory, determined by Cochran and/or Grubbs tests
SAT. = Sum of saturated fatty acids
IR-trans = Total trans unsaturation determined by IR
FA = fatty acid

Table 10. Pair of blind duplicatessamples C1 and C2a

Including outliers

Excluding outliers

FA

Mean

sr

sR

RSDr

RSDR

Mean

sr

sR

RSDr

RSDR

Outliers/No.
labs

Outlier
lab.c

16:0
18:0
SAT.
18:1t
18:1c
18:2(n-6)
18:3(n-3)
18:2tt
18:2t
18:3t
IR-trans
CAL-trans

9.73
6.68
17.30
17.45
28.16
34.05
0.82
0.15
1.49
0.07
19.04
19.10

0.12
0.14
0.16
1.26
1.12
0.57
0.07
0.11
0.08
0.04
1.22
1.17

0.39
0.29
0.86
1.92
1.87
1.31
0.15
0.15
0.51
0.10
2.04
2.13

1.19
2.07
0.95
7.24
3.97
1.66
8.23
73.03
5.57
61.75
6.42
6.12

4.01
4.39
4.97
11.00
6.65
3.85
17.85
97.88
34.29
147.30
10.72
11.14

9.72
6.76
17.54
17.45
28.16
33.69
0.83
0.15
1.49
0.04
19.04
19.10

0.12
0.09
0.10
1.26
1.12
0.59
0.00
0.11
0.08
0.04
1.22
1.17

0.39
0.13
0.43
1.92
1.87
0.68
0.05
0.15
0.51
0.05
2.04
2.13

1.19
1.28
0.57
7.24
3.97
1.75
0.00
73.02
5.57
109.26
6.42
6.12

4.01
1.90
2.43
11.00
6.05
2.01
5.61
97.88
34.29
132.09
10.72
11.14

0/10
1/10
1/10
0/10
0/10
1/10
2/10
0/10
0/10
1/10
0/10
0/10

7
7

7
7,8

a
b

Calculated using average values of samples C1 and C2 for each laboratory.

FA = fatty acid; SAT. = sum of saturated fatty acids; sr = repeatability standard deviation; sR = reproducibility standard deviation; RSDr =
repeatability relative standard deviation; RSDR = reproducibility relative standard deviation; CAL-trans = sum of 18:1t, 18:2tt, 18:2t, and
18:3t; IR-trans = total trans unsaturation determined by IR.
Outlier laboratory, determined by Cochran and/or Grubbs tests.

Table 11. Comparison of 18:1t and 18:1c levels of test samples determined by AgNO3-TLC/GC, GC/IR, and direct GC
18:1t, %
Sample
A
B
C1
C2
D
E
R
a

b
c

18:1c, %
c

AgNo3-TLC/GC

GC/IR

Direct GC

AgNO3-TLC/GC

GC/IR

Direct GC

5.1
15.2
18.9
18.9
26.1
31.9
19.9

4.9
14.9
17.4
17.5
26.6
32.6
19.4

4.4
12.3
14.7
14.7
19.6
23.4
16.8

24.7
24.1
27.2
27.2
35.0
33.0
31.0

24.9
24.7
28.1
28.2
34.4
34.3
32.2

25.9
26.0
30.4
30.4
41.8
41.6
36.0

Values (n = 1) determined in authors laboratory. The 18:1t and 18:1c isomers isolated by AgNO3-TLC were quantitatively analyzed by GC in
the presence of 17:0 internal standard.
Mean values (n = 12) from GC/IR collaborative study.
Values (n = 1) determined in authors laboratory using AOCS Official Method Ce 1c-89.

Table 12. Comparison of total trans unsaturation calculated by combining AgNO3-TLC/GC (18:1t) and GC (18:2t +
18:2tt + 18:3t) data obtained from IR determinations
Trans equivalents, %
Sample
A
B
C1
C2
D
E
R
a

18:1ta

18:2tb

18:ttb

18:3tb

Calculated transc, %

IR trans, %

5.1
15.2
18.9
18.9
26.1
31.9
19.9

0.3
0.6
1.2
1.3
2.8
2.6
1.9

0.0
0.1
0.3
0.2
0.4
0.5
0.2

0.0
0.0
0.0
0.1
0.2
0.1
0.1

5.4
15.9
20.4
20.5
29.5
35.1
22.1

5.2
15.5
18.9
19.1
30.1
34.6
21.6

18:1t values were determined in authors laboratory by AgNO3-TLC/GC method (see Table 11). (Note: Correction factor for converting GC
weight percentage data of 18:1t to IR-trans equivalent is 1.)
Mean values (n = 12) of GC weight percentage data from GC/IR collaborative study (Tables 39) were converted to IR-trans equivalents
using correction factors (0.84 for 18:2t and 18:3t, and 1.74 for 18:2tt).
Sum of trans equivalents for 18:1t, 18:2t, 18:2tt, and 18:3t.

Figure 994.14. IR spectrum of partially hydrogenated

canola oil methyl esters (2% solution in CS2).

Figure 994.15. C18 region of the gas chromatogram of the fatty acid methyl esters from partially hydrogenated soybean oil, using 100 m 0.25 mm fused silica capillary column coated with SP2560.