92 ROZEMA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO.
1, 2008
FOOD CHEMICAL CONTAMINANTS
Proposed Modifications to AOAC 996.06, Optimizing the
Determination of Trans Fatty Acids: Presentation of Data
BRENT ROZEMA, BARBARA MITCHELL, DOUG WINTERS, ANDREW KOHN, DARRYL SULLIVAN, and ERIN MEINHOLZ
Covance Laboratories Inc., 3301 Kinsman Blvd, Madison, WI 53704
The increased focus on the accuracy of trans fatty
acid data generated using current methodologies
has resulted in research initiatives to optimize the
quality of these assays. In this study, scientists
combined the established methodology from
AOAC 996.06 and the American Oil Chemists
Society method Ce 1h-05, as well as other
independent research. As a result, method
modifications are proposed that could allow for a
more accurate determination of trans fat than the
current AOAC 996.06 method. Validation data from
this study are presented. The authors encourage
peer review and offer to facilitate a collaborative
validation to update AOAC 996.06.
O
n January 1, 2006, legislation in the United States went
into effect requiring the labeling of trans fat in food and
dietary supplements. As a result, nutrition labels are
required to include trans fat and disclose amounts >0.5 g.
Foods that contain less than this can claim “0 g of trans fat per
serving” (Canadian regulations allow this statement for levels
<0.2 g). The methods of analysis for trans fat in these products
have subsequently become increasingly important, and have
also come under intensive scrutiny by manufacturers and
consumers alike. These methods need to evolve in order to
accurately analyze foods against these regulatory requirements
while maintaining the highest degree of scientific accuracy (1).
AOAC Method 996.06 (1) for the analysis of trans fat is
specified as one of the preferred methods by the U.S. Food and
Drug Administration (FDA; 2). It is specifically the method of
preference for foods that contain low levels of trans fat, as
opposed to ingredients, such as frying oils, that contain high
levels. This method, originally developed in 1996, involves the
extraction of fat and fatty acids from food by acidic or alkaline
hydrolysis. After hydrolysis, the fats are extracted into mixed
ethers and converted into fatty acid methyl esters (FAMEs).
The FAMEs are a mixture of all of the cis and trans isomers
derived from the food product. A profile of the isomers is
obtained using gas chromatography (GC). When available,
individual standards are employed to identify and quantitate the
individual isomers in the sample. When standards are not
Received May 14, 2007. Accepted by AP October 24, 2007.
Corresponding author's e-mail: brent.rozema@covance.com
available, the quantitation is performed using the response
factors of isomers of similar chemical structure. Identification is
Downloaded from https://academic.oup.com/jaoac/article/91/1/92/5656206 by guest on 01 February 2021
then established using peak retention times and comparison of
the fatty acid profile to the peak assignments in the method.
Quantitation of each analyte is then performed relative to a
C11:0 (undecanoic acid) internal standard. Using this FAME
profile, trans fats are identified and summed to generate a total
trans fat value.
After the development and validation of Method 996.06,
the American Oil Chemists Society (AOCS) performed
additional research to optimize trans fatty acid isomer
separation and identification. This was done using a round
robin, multilaboratory validation process and culminated in
the release of method No. Ce 1h-05 in 2005 (3). As a result,
additional fatty acid isomers are more clearly separated, and at
least 15 trans isomers can now be specifically identified and
quantitated using the AOCS conditions. Because analytical
standard materials for direct calibration are not available for
all isomers separated, some isomers were identified using
techniques such as Fourier transform infrared spectrometry
and silver-ion chromatography.
Due to the fact that the AOCS method was written
specifically for the analysis of pure oils and fats, it does not
include the sample preparation procedures documented in the
AOAC Method (i.e., the separation of the fats from food
matrixes). To compile the data presented, we have continued
to follow the sample extraction as written in AOAC
Method 996.06, but have employed the improved analytical
separation of the GC conditions as documented in the
AOCS method.
It should be noted FDA district laboratories currently use
both AOAC and AOCS methods. In addition, the AOAC
guidelines allow the use of alternate steps within the scope of a
method, provided that all potential parameters related to the
accuracy and reliability of the data are validated. Different GC
conditions in this analysis are inherent to the proposed
modifications, as they affect the determination of trans fat.
The GC column temperature program, in particular, is critical
in analysis of trans fats, as minimal changes have a great
effect on the separation of the trans isomers (4). The other
component in the modifications is in the interpretation and
quantitation of the chromatography. Measures to standardize
the procedure have been established in an attempt to quantify
trans fat accurately and consistently.
 ROZEMA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 1, 2008 93

Table 1. GC conditions of AOAC 996.06 versus the modified method

AOAC 996.06 Modified method

GC detector Hydrogen flame ionization Hydrogen flame ionization

Capillary column packing 100% Cyanopropanol 100% Cyanopropanol
Capillary column dimensions 100 m ´ 0.25 mm, 0.20 mm film thickness 100 m ´ 0.25 mm, 0.20 mm film thickness
Suggested capillary column SP 2560 SP 2560, HP-88, CP-Sil 88
models
Injector temp., °C 225 250
Detector temp., °C 285 300
Split ratio 200:1 100:1
Injection volume, mL 2 0.2

Downloaded from https://academic.oup.com/jaoac/article/91/1/92/5656206 by guest on 01 February 2021

Carrier gas Helium Helium (HE) or hydrogen (H)
Flow rate, mL/min 0.75 1.0 (He), 1.2 (H)
Linear velocity, cm/s 18 18 (He), 30 (H)
Hold-ramp-hold sequence Initial temp. 100°C; hold 4 min; Helium: initial temp. 170°C; hold 17.5 min;
ramp 3°C/min; final temp. 240°C; ramp 5°C/min; final temp. 200°C; hold 2 min; ramp 5°C/min;
hold 15 min final temp. 210°C; hold 22.5 min
Hydrogen: initial temp. 170°C; hold 11.2 min;
ramp 7.9°C/min; final temp. 200°C; hold 1.3 min; ramp 7.9°C/min;
final temp. 210°C; hold 16.0 min

Figure 1. 18:1 and 18:2 cis and trans isomers in USDA Grade AA butter, identified individually but integrated for
quantitation using the proposed method.
94 ROZEMA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 1, 2008

Table 2. Typical levels of trans fat in a variety of foods Table 3. Precision data for trans fat in 4 different
using the modified method matrixes

Food product Total trans fat, % Shortening

Cheese
Potato chips 11.0 Oil Cereal cracker 18:1t 18:2t
Butter 4.04
French fries 2.76 Mean trans fat, 5.54 0.77 1.14 12.7 2.84
%, as acids
Fried corn tortilla chips 2.59
a
SD, % 0.154 0.014 0.023 0.222 0.146
Sweetened condensed milk 1.80
b
RSD, % 2.77 1.83 2.02 1.75 1.62
Beef/hamburger 0.86
Bread 0.25 a
SD = Standard deviation.
b
Corn cereal 0.14 RSD = Relative standard deviation.

Downloaded from https://academic.oup.com/jaoac/article/91/1/92/5656206 by guest on 01 February 2021

Peanut butter 0.06
Granola bars 0.02

to the profile of the identified isomers in the AOCS method.

Over the last 2 years, the revisions and recommendations set
forth in this paper have been used to analyze hundreds of food
and supplement matrixes. As performance checks are imperative
with any method, every analysis we perform includes the testing
of a control material. The control material used for fatty acid
profiles including trans fat is a cheese cracker matrix. Statistics
on this material are the foundation of our study, although we also
present the chromatography of U.S. Department of Agriculture
(USDA) Grade AA butter and analytical data on cereal,
shortening, and a proprietary oil blend.
METHOD
The method of analysis for trans fat used in this evaluation
combines aspects of both AOAC 996.06 and AOCS Ce 1h-05.
Proposed revisions to AOAC 996.06 occur in the
GC conditions and the identification and quantitation of trans
fat in subsequent chromatography. Suggested modifications
to the GC parameters in AOAC 996.06 are presented in
Table 1 as a side-by-side comparison. Calculation of data
using the modified method is explained below.
The quantitation of major peaks in the profile is performed
using external standards relative to a C11:0 internal standard.
However, standard materials for many of the minor
components in the profile are not available. As a result,
identification of some important isomers is done by comparison
Total trans fat in this method is defined as the trans isomers of
the 18:1 and 18:2 fatty acids, which are quantitated and
summed to yield total trans fat (see the note below concerning
other trans fatty acids). The peaks 9 trans 18:1 (elaidate) and 9
trans, 12 trans, 18:2 (linolelaidate) are identified and
quantitated using external standards. Additional 18:1 and 18:2
trans fat isomers are quantitated using guidelines from the
AOCS method, in which the regions of 18:1 and 18:2 trans
isomers are quantified as grouped peaks, identification being
confirmed by retention times of other known peaks.
Quantitation of these peaks is performed using the response
factors of elaidate and linolelaidate, respectively. Trans 18:1
isomers are considered to be the peaks immediately near 9 trans
18:1 elaidate (typically 4 trans–14 trans 18:1) but prior to 9 cis
18:1, as well as 16 trans 18:1 (which elutes after 9-cis 18:1).
Trans 18:2 isomers are considered to be the peaks between
15 cis 18:1 and 9 cis, 13 cis 18:2. Figure 1 is a chromatogram of
18:1 and 18:2 cis and trans isomers in butter. Isomers are
identified individually, but integrated by region to demonstrate
the quantitation of 18:1 and 18:2 trans by the method described.
These peak regions are subsequently calculated against the
most similar external standard (elaidate for 18:1 trans, and
linolelaidate for 18:2 trans). One exception to the grouped
quantitation is the inclusion of 16 trans 18:1, which elutes
between known 18:1 cis peaks. Total 18:1 trans, total
18:2 trans, and total trans fatty acids can be reported using this

Table 4. Precision statistics for total fatty acids in the cheese cracker matrix control (n = 47)

As acids

Total quantitated fat,

Total Saturated fat Monounsaturated fat Polyunsaturated fat Trans fat as triglycerides

Mean, % 7.04 6.41 10.15 1.14 25.97

SD, % 0.134 0.117 0.183 0.023 0.461
RSD, % 1.91 1.83 1.80 2.02 1.78
 ROZEMA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 1, 2008 95

Table 5. Trans fatty acid statistics, modified method versus AOAC 996.06

USDA Grade AA butter Cheese cracker matrix

Modified methoda AOAC 996.06a Modified methodb AOAC 996.06a

18:1 trans 18:2 trans 18:1 trans 18:2 trans 18:1 trans 18.2 trans 18:1 trans 18:2 trans

Mean, % 3.37 0.68 2.07 1.10 0.82 0.32 0.71 0.38

SD, % 0.178 0.058 0.073 0.051 0.015 0.009 0.024 0.012
RSD, % 5.28 8.53 3.53 4.64 1.81 2.68 3.38 3.20

a
n = 16.
b
n = 47.

Downloaded from https://academic.oup.com/jaoac/article/91/1/92/5656206 by guest on 01 February 2021

methodology. This method of identification is supported by
research published in Food Chemistry (5).
Note: Some coelution of cis and trans isomers occurs in the
current methodology and in this method. For example, 6 cis
18:1 coelutes with 13–14 trans 18:1, 14 cis 18:1 with 16 trans
18:1, and 15 trans 18:1 with 9 cis 18:1. However, because
individual peak identification cannot be confirmed, we have
followed the AOCS method of identification that takes a
conservative approach and quantitates all peaks in the 18:1
and 18:2 trans regions as trans fat. This is a result of the fact
that the relative amounts of cis versus trans in these minor
peaks cannot be determined. Although some trans may be
overestimated by quantitating entire regions of peaks, the
research reported in Food Chemistry (5) shows that other
trans are underestimated because the small isomers are hidden
under the much larger cis peaks. The net result is, therefore,
considered to be negligible (5). This also ensures, in the
interest of human health, that significant trans fatty acids are
quantitated. Regions of other trans fatty acids, such as 14:1,
16:1, 18:3, and 20:1, are not addressed, as they are typically
not found in significant levels. Also, conjugated 18:2 linoleic
and conjugated 18:3 linolenic acids (CLA) can be determined
independently. Because these conjugated isomers are not
classified or quantified as part of trans fat, they are out of the
scope of the proposed method changes.
Another item to consider in regards to using external
standards in any analysis of fatty acids is that commercially
available fatty acid standards are not 100.0% pure (i.e., 99.5 to

Figure 2. Cis and trans isomers in USDA Grade AA butter using the GC conditions and integration guidelines in
the proposed method.
96 ROZEMA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 1, 2008
Figure 3. Cis and trans isomers in USDA Grade AA butter using AOAC 996.06 GC conditions and integration
guidelines.
99.8%); impurities typically are other fatty acids. When a
standard solution is prepared with multiple analytes, trace
contaminants in each reference standard could increase the
actual concentration of another fatty acid in the mixture. There
is not really a clean way to eliminate such interference, and it
has generally been accepted that in the absence of absolutely
pure reference materials, it is a negligible source of error to
the assay.
Method Evaluation
This method has been evaluated on a variety of matrixes.
Table 2 shows trans fat levels in different food products, while
Table 3 contains precision data for different types of
matrixes (6). Tables 4 and 5 contain expanded precision data
and specific statistics on 18:1 and 18:2 trans fat in butter and
the cheese cracker matrix.
Possibly the best evaluation of the method is to show
chromatograms and results from the same food product
analyzed by both methods. Figures 2 and 3 show the
difference between the modified method and AOAC 996.06 at
the 18:1 and 18:2 trans region in USDA Grade AA butter. Key
differences in the chromatography are the amount of sample
injected on the column, the carrier gas (hydrogen is used with
the modified method), and the temperature program. See
Table 1 for a complete listing of method differences.
Downloaded from https://academic.oup.com/jaoac/article/91/1/92/5656206 by guest on 01 February 2021
In the chromatograms, note that the modified method
produces better separation of 18:1 trans peaks. The 4–11t
18:1 peaks, while still not baseline resolved, can be seen
using modified conditions. Also, the 13–14t 18:1 peak is
very well resolved when compared to the AOAC 996.06 in
which it is completely hidden under the large 18:1 cis (oleic
acid) peak. Another difference is that the 16t 18:1 peak is
slightly better defined in the modified method, and the
modified approach identifies it and subsequent 14–15 cis
18:1 peaks. This, in turn, lowers the total 18:2 trans when
compared to the approach in AOAC 996.06 (that method
quantitates all of these peaks as 18:2 trans). A final point of
interest with the modified method is that by using hydrogen
as the carrier gas, the retention time of the trans region is
better defined at 17–19 min than the AOAC Method is at
44–47 min.
This method has demonstrated a high degree of precision at
low levels. The limit of quantitation (LOQ) typically
employed by Covance is 0.01%, based upon the successful
analysis of the low standard in a 5 point standard curve. The
limit of detection (LOD) has not been established for the
assay, but as Tables 2–5 demonstrate, foods with very low
levels of trans fat can be analyzed successfully. Table 5 shows
18:1 and 18:2 trans in butter and the cheese cracker matrixes.
The components have relative standard deviation (RSD)
values <10%, with mean levels of 0.32–3.37%.
 ROZEMA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 1, 2008 97
Table 5 shows a comparison of the statistics for the total
18:1 and 18:2 trans in butter and the cheese cracker using the
2 methods. The modified method displays less precision for
butter than the AOAC Method, but better precision with the
cheese cracker [although the number of trials (n) of 47 versus
16 must be considered with regard to the cheese cracker]. The
modified method quantitates more 18:1 trans. This can be
attributed to the separation of 13t, 14t 18:1, and less 18:2
trans; as 16t 18:1 and 14c, 15c 18:1 are (correctly) not
included. The net result in the case of butter and this cheese
cracker is that the modified method quantitates more trans fat
than AOAC 996.06. The conclusion, however, is that the
modified method is more accurate and precise for cis and
trans determination.
Conclusions
A more accurate determination of trans fat in all food
samples may be possible using these modifications. The
authors will solicit comments prior to conducting a single
laboratory validation (SLV) on a cheese cracker matrix. Upon
successful completion of the SLV, a collaborative effort to
revise AOAC 996.06 would be proposed.
Acknowledgments
We wish to acknowledge the editorial contributions of
Richard Crowley (Covance Laboratories) in the preparation
of this manuscript.
References
(1) AOAC Official Method 996.06 (2005) Official Methods of
Analysis of AOAC INTERNATIONAL, 18th Ed., AOAC
INTERNATIONAL, Gaithersburg, MD
(2) Federal Register (July 11, 2003) U.S. Food and Drug
Administration, Washington, DC
(3) AOCS Official Method Ce 1h-05 (2005) Official Methods
Downloaded from https://academic.oup.com/jaoac/article/91/1/92/5656206 by guest on 01 February 2021
and Recommended Practices of the AOCS, The American
Oil Chemists Society, Champaign, IL
(4) Ratnayake, W.M.N. (2004) J. AOAC Int. 87, 523–539
(5) Golay, P.-A., Dionisi, F., Hug, B., Giuffrida, F., & Destaillats,
F. (2006) Food Chem. 101, 1115–1120
(6) Kohn, A., & Mitchell, B. (2006) Lipid Technol. 18, 279–282