1146 DEVRIES ET AL: JOURNAL OF AOAC INTERNATIONAL VOL. 82,
5,1999
FOOD COMPOSITION AND ADDITIVES
Studies in Improvement of Official Method 996.06
JONATHAN W. DEVRIES and LORI KJOS
Medallion Laboratories, 9000 Plymouth Ave N, Minneapolis, MN 55427
LINDA GROFF and BOB MARTIN
Hershey Foods Corporation, Technical Center, 1025 Reese Ave, Box 805, Hershey, PA 17033
KRISTI CERNOHOUS and HASMUKH PATEL
Medallion Laboratories, 9000 Plymouth Ave N, Minneapolis, MN 55427
MARK PAYNE
Hershey Foods Corporation, Technical Center, 1025 Reese Ave, Box 805, Hershey, PA 17033
HARRY LEICHTWEIS and MIKE SHAY
Medallion Laboratories, 9000 Plymouth Ave N, Minneapolis, MN 55427
LEO NEWCOMER
Hershey Foods Corporation, Technical Center, 1025 Reese Ave, Box 805, Hershey, PA 17033
Quantitation of fat in foods has been performed
successfully with AOAC Official Method 996.06. A
number of situations have been encountered that
render the method note, "Note: For any unknown
or uncalibrated peaks, use the nearest calibrated
fatty acid response factors and conversion fac-
tors," inaccurate. Identification of extraneous com-
pounds and availability of additional standard fatty
acid methyl esters combined with mass spectral
data lead to the recommendation of modifications
in Official Method 996.06. The stepwise perfor-
mance of the method remains unchanged.
nalysis of fat in foods by AOAC Official
A Method 996.06 involves the summation of the quanti-
ties of fatty acids present in the sample, expressed as
triglyceride equivalents. AOAC Official Method 996.06 also
quantitates fractions of the fat (saturates, monounsaturates,
etc.) by summing up the respective component fatty acids of
the fraction. At the time Method 996.06 was adopted, calibra-
tion standards were not available for a number of the fatty ac-
ids present in low quantities in fats in foods. Therefore, the
method specifies: "Note: For any unknown or uncalibrated
peaks, use the nearest calibrated fatty acid response factors
and conversion factors."
Following adoption as an Official Method of Analysis,
Method 996.06 has now been used extensively on a wide vari-
ety of matrixes with good success. Commensurate with this
use, 3 things have occurred leading to a study of method im-
provements and a subsequent recommendation for minor
modifications to Official Method 996.06: (1) Additional fatty
acid standards have become available since adoption of
Method 996.06. (2) A number of nonfatty-acid peaks (albeit
Received December 30, 1998. Accepted by JL April 20, 1999.
No.
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minor peaks) in the chromatographic traces of derivatized food
sample extracts have been identified. (3) Additional fatty acid
peaks in the chromatograms have been separated by better col-
umns and identified by mass spectrometry (MS). The first event
indicates that supplier references and calculation factors of the
method should be updated. The second indicates that the word-
ing of the method relating to peaks of unknown identity should
be changed, and the third indicates a need for updating the chro-
matographic column used for identification and for including a
table of peak relative retention times to assist analysts in identi-
fying additional minor peaks in their chromatograms.
Analysis and Discussion
All analyses were performed as specified in
Method 996.06 in laboratory 2. Likewise, laboratory 1
performed Method 996.06 as written, but used an SP2560
100 m x 0.25 mm with 0.20 pun film capillary column
(Supelco, Inc., Bellefonte, PA) in place of the SP2340 60 m
x 0.25 mm with 0.20 ^.m film capillary column. Data gener-
ated from the SP2560 column is placed in brackets [ ], when-
ever data were generated from both columns. Data generated
using the SP2340 column is not bracketed. Operating parame-
ters for the SP2560 column were column length, 100 m; carrier,
He at 20 cm/s; split ratio, 139:1; injector temperature, 225 °C,
detector temperature, 285 °C; initial temperature, 100°C, hold
for 4 min, ramp at 3°C/min to 240°C, hold for 15 min. Oper-
ating parameters for the SP2340 column were column length,
60 m; carrier, He at 18 cm/s; split ratio, 200:1; injector tempera-
ture, 225 °C, detector temperature, 285 °C; initial temperature,
100°C, hold for 4 min, ramp at 3°C/min to 240°C, hold for
17 min. Both columns have the same functionality—a
poly(biscyanopropylsiloxane) film coated on inert fused sil-
ica—but the SP2560 column gives slightly better resolution of
certain combinations of fatty acid methyl ester (FAME) peaks.
 DEVRIES ET AL: JOURNAL OF
During analysis of samples for major chocolate syrup
manufacturers for fat content by Method 996.06, a peak of
retention time 28.6 [26.94] min positioned between C12:0 at
27.9 [25.58] min and C14:0 [C13:0] at 31.0 [28.15] min for
the SP2340 [SP2560] column (Figure 1) was observed in
some of the syrup samples. Analysis of the ingredients used
in the formulation of the chocolate syrups showed the same
peak to be present and in proportionally higher quantities in
some of the high-fructose corn syrups used for manufactur-
ing chocolate syrup. Minor peaks like these can have a major
impact on fat quantitation for products containing less than
0.5 g fat per serving.
MS analysis the peak showed the following characteristic
mass peaks (m/z): 130,115,99,88, and 55. The compound rep-
resented by the peak was tentatively identified as levulenic acid,
an apparent by-product of production of high-fructose corn
syrup. An authentic sample of levulenic acid (Acros, Inc., Pitts-
burgh, PA) was obtained and compared chromatographically
and by MS (Figure 2). There is excellent agreement between
the authentic sample of levulenic acid and the peaks found in
the chocolate and high-fructose corn syrups.
Sorbic Acid in Chocolate Syrup sample
o c . Syrup 111
* Sorbic Acid
fYA-A-
I ' I ' r
11 12 13 14 15 16 24
E l u t i o n Time
Figure 1. Identification of levulenic and sorbic acid in chocolate syrup sample.
AOAC INTERNATIONAL VOL. 82, No. 5,1999 1147
Eight samples of chocolate syrups from 4 manufacturers
and 2 samples of high-fructose corn syrup from 2 suppliers
were split into subsamples and analyzed in the 2 laboratories
for total fat. Excellent agreement in data was obtained be-
tween the laboratories (Table 1).
A second unknown peak was also observed in the choco-
late syrup samples. This peak occurs at 23.0 [18.93] min,
while C8:0 peak occurs at 20.4 [15.69] min and C10:0 at 24.5
[20.39] min on SP2340 [SP2560]. On the basis of retention
time and makeup of the sample, this peak was tentatively
identified as sorbic acid. Chromatographic comparisons and
MS of this peak confirmed it to be sorbic acid, the potassium
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salt of which is used to inhibit mold growth in high-moisture
products (Figures 1 and 3).
Another common preservative for foods is the sodium salt
of benzoic acid. A sample of benzoic acid was obtained and
analyzed by Method 996.06. A peak of retention time
[24.56] min, compared with CI 1:0 at [22.99] min and C12:0
at [25.58] min, on [SP2560] was found. Analysis of an orange
soda beverage, a product listing potassium benzoate as an ad-
ditive, also showed a peak between CI 1:0 and C12:0 (Fig-
12i22:50 31Augl998
* Levulenic Acid
O -H
3 a
rP* n IV ftnH A - A - ft ft A
I ' I ' I '
25 26 33 34 35 36 37 38 39
Minutes
1148 DEVRIES ET AL: JOURNAL OF AOAC INTERNATIONAL VOL. 82, No. 5,1999

ures 4 and 5). Figure 6 shows an example of a chromatogram
showing all 3 compounds present with peaks eluting during a
fatty acid analysis. Figure 6 is a chromatogram of raspberry
angel food cake in which sorbic acid, benzoic acid, and
levulenic acid were found.
In AOAC Official Method 996.06, Tables 996.06D and E
list the conversion factors for all the fatty acids that occur in
major quantity in foods. Foods contain a number of additional
fatty acids present in small quantity (usually less than 0.1%).
GC/MS was used to establish the identity and retention times
for a number of these fatty acids present in minor amounts. In
the course of this identification effort, it was discovered that
60 m SP2340 column, slightly improved resolution of fatty
acid peaks could be obtained. In particular, the diffi-
cult-to-separate pair of peaks for the FAMES of CI8:3 and
C20:1 and the trio of peaks for the FAMES of C22:1, C20:3,
and C20:4 are resolved. The SP2560 column has the same
chemical makeup as the SP2340 column but has improved
resolution, perhaps in part due to its extended length. In addi-
tion to the GC/MS identifications, a number of fatty acid stan-
dards have become available and were purchased (Sigma,
Inc., St. Louis, MO). These were used to establish the reten-
tion times for myristelaidic acid (14: It), palmitelaidic (16: It),
conjugated linoleic (18:2 conj), docosatrienoic acid (22:3),

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by using a 100 m SP2560 column in place of the traditional and docosatetraenoic acid (22:4). The retention times are as

Abundance Scan 1
Abundance Scan 16' 99
900000-
1400000-
850000-
115
1300000 800000
115 55
55
1200000 750000

700000-1
1100000
650000
1000000
600000

900000 550000-1
B
500000
800000-
450000
700000
400000 88
600000 88
350000

500000 A 300000 -

250000 :
400000
200000 :
300000-
150000

200000- 100000
130
50000H
100000- 130

0-^ 172 m/z-->

0
50
illv100 L40 171
150 20<
m/z--> 50 100 150 200
Figure 2. (A) Mass spectrum—extraneous peak in fat profile of chocolate syrup at 26.94 min. (B) Mass
spectrum—levulenic acid.
 DEVRIES ET AL: JOURNAL OF AOAC INTERNATIONAL VOL. 82, No. 5,1999 1149

Table 1. Comparison of fat results by 2 laboratories

Labi Lab2 a Lab 2

Sample total fat, % total fat, % total fat, %

Chocolate syrup #1 1.15 1.29 1.2

Chocolate syrup #2 1.05 1.22 1.13
Chocolate syrup #3 1.07 1.12 1.01
Chocolate syrup #4 1.09 1.23 1.14
Chocolate syrup #5 1.12 1.25 1.13
Chocolate sundae syrup 1.15 1.2 1.11
Chocolate syrup #6 1.08 1.15 1.09

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High-corn fructose #1 0 0.13 0
Chocolate syrup #7 0.64 0.68 0.63
High-corn fructose #2 0 0.09 0
Average 0.835 0.936 0.844

Lab 2, first column, includes sorbic acid and levulenic acid in the calculation of total fat, while the second column does not.

Abundance Scan
Abundance Scan
b7
340000-
5000000
320000- i:.1

300000- 4500000

280000 -
111
4000000
260000-

240000-
3500000
220000- B
200000-
95
3000000

180000-

160000 - 2500000

140000 -
6 s 2000000-
120000 -

100000-
1500000

80000-

60000- 1000000

40000-
1
500000
20000-
II 1 >7

ril Ji
l.ull 1 167
• Vi | i '. . i
m/z--> 5C ) 3.00 ISO 2
m/z-->

Figure 3. (A) Mass spectrum—extraneous peak in fat profile of chocolate syrup at 18.93 min. (B) Mass
spectrum—sorbic acid.
1150 DEVRIES ET AL: JOURNAL OF AOAC INTERNATIONAL VOL.
follows: 14:lt is 32.01, 16:lt is 36.20, 18:2 conj ranges from
45.05 to 45.40, 22:3 is 51.98, and 22:4 is 52.28. The conju-
gated fatty acid, linoleic acid (18:2 conj), occurs and is sold as
a mixture of isomers having double bonds primarily at the
9,11 and 10, 12 positions. The areas of the peaks occurring
between 45.05 and 45.40 min in the standards and samples are
summed for quantitation purposes.
Table 2 lists the retention times and relative retention times
of the FAMEs of the fatty acids for which GC/MS identifica-
tion or identification by comparison to known standards has
been completed in our laboratories. GC/MS was performed
with the analytical conditions of Method 996.06 and the
SP2560 column (chromatographic parameters listed earlier in
this paper), electron impact MS, and the Wiley/NIST MS li-
brary. Table 3 lists the respective conversion factors for all the
identified FAMEs to their corresponding fatty acids and their
triglyceride equivalents.
In addition to the fatty acids studied as part of this work,
AOAC Official Method 994.15 provides detail on identifica-
tion of certain cis and trans isomers of octadecanoic,
octadecenoic, octadecadienoic, and octadecatrienoic acids.
Unfortunately, Figure 994.15 is not accompanied by a table to
assist the analyst in identifying the peaks. Therefore, we have
SODA
\fi —
83
I ' I ' " I""
11 12 13 19 20 21
E l u t i o n Time
Figure 4. Identification of benzoic acid in orange soda beverage.
82, No. 5,1999
constructed such a table (Table 4) based on the references in
Method 994.15 to aid in peak identification.
Recommendations
On the basis of our results and analysis, we recommend
that the current tables—996.06D and E—be deleted, that an
updated table of relative retention times (shown as Table 2 in
this paper) be inserted as table 996.06D in the method, and
that an updated table of conversion factors (shown as Table 3
in this paper) be inserted as 996.06E.
We further recommend that the column requirements for
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996.068(b) be changed to a performance-based specification
as follows: Capillary column capable of separating the FAME
pair of adjacent peaks of C18:3 and C20:1 and the FAME trio
of adjacent peaks of C22:l, C20:3, and C20:4 with a resolu-
tion of 1.0 or greater. SP2560 100 m x 0.25 mm with 0.20 \xm
film is suitable.
We further recommend that the wording in Method 996.06,
section G, regarding the handling of peaks of unknown iden-
tity be changed from "For any unknown or uncalibrated peaks,
use the nearest calibrated fatty acid response factors and con-
version factors" to "Note: Peaks of known identity with
38 39
Minutes
 DEVRIES ET AL: JOURNAL OF AOAC INTERNATIONAL VOL. 82, No. 5,1999 1151

known relative retention times are listed in Table 996.06D.
When peaks of unknown identity are observed during the
chromatographic run, attempts should be made to accurately
identify such peaks by mass spectrometry, Fourier-transform
infrared spectroscopy, etc. Peaks of unknown identity should
not be included in the summation when quantifying fat in the
test sample."
Because virtually all the fatty acids in foods have now been
identified and are quantifiable by this method, Method 996.06
should be considered as a substitute or replacement for
Method 979.19 for the identification and quantification of
polyunsaturated fatty acids. In addition, trans-fatty acids
should be identified and quantified using Method 996.06.
We further recommend that the following be added to
Method 996.06 section G, just prior to the final Note: Calcu-
late the amount of polyunsaturates in the test sample (w/w; ex-
pressed as polyunsaturated fats; sum of C16:2, C18:2, C18:3,
C20:4> C22:4» C22:6>etc'^as ^ ° ^ o w s - I nc l u <i e only the all-ds iso-
meric fatty acids for nutrition labeling purposes:
Polyunsaturated fat, % =
^polyunsaturated W/W sample ) x 100

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Abundance Scan Abundance Scan
320000- 105 105
320000 -
300000
300000
280000-
280000
260000 - 77
260000
240000-
77 240000
220000
220000

200000 :
200000
B
180000-
180000

160000 :
160000

140000 - 140000 51
136
120000 : 120000
136
100000 51
100000H

80000- 80000

60000- 60000

40000 40000-

20000 20000-

m/z-->
p. ifh^Mt
50
,11, jt.ll, i| -,. I, | ,
100
100 150
150 m/z-->
r-f^
50
1 100 150

Figure 5. (A) Mass spectrum—extraneous peak in fat profile of orange soda at 24.56 min. (B) Mass spectrum—sorbic acid.
1152 DEVRIES ET AL: JOURNAL OF AOAC INTERNATIONAL VOL. 82, No. 5,1999

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11 12 13 14 15 16 17 IB 19 20 21 22 23 24 25 26 27 26 29 30 31 32 33 34 35 36 37 38 39
Blution Time Minute8

Figure 6. Identification of sorbic, benzoic, and levulenic acid in raspberry angel food cake.

Table 2. Retention and relative retention times of fatty acids on SP2560 column

Relative retention time

Description Retention time, min (relative to 11:0 as internal standard)

4:0 Butyric 10.49 0.46

6:0 Caproic 12.36 0.54
8:0 Caprylic 15.69 0.68
10:0Capric 20.39 0.89
11:0 Undecanoic 22.99 1.00
12:0 Laurie 25.58 1.11
13:0Tridecanoic 28.15 1.22
14:0 Myristic 30.65 1.33
14:1 Myristoleic 32.63 1.42
14:1 frans-Myristelaidic 32.01 1.39
15:0 Pentadecanoic 33.04 1.44
15:1 Pentadecenoic 34.98 1.52
16:0 Palmitic 35.41 1.54
16:1 frans-Palmitelaidic 36.39 1.58
16:1 Palmitoleic 36.88 1.60
17:0Margaric 37.54 1.63
17:1 Margaroleic 38.92 1.69
18:0 Stearic8 39.78 1.73
18:1 trans 6-Petroselenic 40.50 1.76
 DEVRIES ET AL: JOURNAL OF AOAC INTERNATIONAL VOL. 82, No. 5,1999 1153

Table 2. (continued)

Relative retention time

Description Retention time, min (relative to 11:0 as internal standard)

18:1 frans-Elaidic 40.61 1.77

18:1 trans 11 -Vaccenic 40.72 1.77
18:1 Petroselenic 40.90 1.78
3
18:1 Oleic 40.99 1.78
18:1 Vaccenic 41.18 1.79
18:1 Octadecenoic 41.54 1.81
18:2 frans-Linolelaidic 41.69 1.81

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18:2 trans 9-Linolelaidic 42.11 1.83
18:2 trans 12-Linolelaidic 42.53 1.85
18:2 Linoleic 42.87 1.86
20:0 Arachidic 43.75 1.90
18:3g-Linolenic 44.25 1.92
20:1 Eicosenic cis 5 44.42 1.93
20:1 Eicosenic trans 11 44.45 1.93
20:1 Eicosenic cis8 44.67 1.94
20:1 Eicosenic cis 11 44.82 1.95
20:1 Eicosenic cis 13 44.99 1.96
a
18:3Linolenic 45.02 1.96
18:2 Linoleic conjugated 45.35 1.97
18:2 Linoleic conjugated 45.40 1.97
21:0 Heneicosanoic 45.69 1.99
18:2 Linoleic conjugated 46.18 2.01
18:4 Octadectetraenoic 46.39 2.02
20:2 Eicosadienoic 46.65 2.03
22:0 Behenic 47.46 2.06
20:3 g-Eicosatrienoic 47.94 2.09
22:1 Cetoleic 48.27 2.10
22:1 Erucic 48.50 2.11
20:3 Eicosatrienoic 48.68 2.12
20:4 Arachidonic 48.94 2.13
23:0 Tricosanoic 49.22 2.14
22:2 Docosadienoic 50.17 2.18
24.0 Lignoceric 50.79 2.21
20:5 Eicosapentaenoic 50.96 2.22
24:1 Nervonic 51.92 2.26
22:3 Docosatrienoic 51.98 2.26
22:4 Docosatetraenoic 52.28 2.27
22:5 Docosapentaenoic 54.75 2.38
22:6 Docosahexaenoic 55.82 2.43

For additional detail on the elution pattern of the 18 carbon fatty acids, see AOAC Official Method 994.15.
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CO o 0. o_ CL 0- ^ ^ C73 u u < LU LU < LU X CO LU a Q U Q O i- <L
cu o o
o
o
O T- o o
T- T- O t - CM CO
•* oo CM CO • * in o o CM CO i
i -
^- in CO o o
,
r~

m m CO co i^ 1^ 00 00 00 CO 00 O o O o CM CM CM CM CM CM CM CO
Q • * CO 00
CM
CM CM CM ,—
CM CM CM CM CM CM CM CM CM CM ^- CM
CM •*
 DEVRffiS ET AL: JOURNAL OF AOAC INTERNATIONAL VOL. 82, No. 5,1999 1155

Table 4. Identification of peaks in Method 994.15, Figure 994.15f

Peak ID Peak ID Peak ID Peak ID

1 18:0 11 18:1*13c 21 18:2ctb 31 18:3*9c,12c,15t

2 18:1*6-8t 12 18:2tt 22 18:2A9c,12t 32 18:3*9t,12c,15c
3 18:1 *9t 13 18:1*14 23 Unidentified 33 18:3*9c,12t,15c
4 18:1 *10t 14 18:2tt 24 18:2*9t,12c 34 18:3»9c,12c,15c
5 18:1*11t 15 18:1A15C 25 Unidentified 35 20:1
6 18:1*12t 16 18:2tt 26 18:2*9c,12c 36 18:2 Conjugated
unidentified isomer

Downloaded from https://academic.oup.com/jaoac/article/82/5/1146/5683723 by guest on 01 February 2021

7 18:1 *9c+18:1 *13t 17 18:2tc 27 18:2»9c,15c 37 18:2 Conjugated
+18:1*14t unidentified isomer
8 18:1*10c 18 18:2ct 28 Unidentified 38 18:2 Conjugated
unidentified isomer
9 18:1*11c 19 18:2*9t,12t 29 20:0 39 18:2 Conjugated
unidentified isomer
10 18:1*12c+18:1*15t 20 18:2tcb 30 18:3 Unidentified 40 18:2 Conjugated
isomer unidentified isomer

fl
Ratnayake, W.M.N., Hollywood, R., O'Grady, E., and Beare-Rogers, J.L. (1990) Determination of cis and frans-Cctadecenoic Acids in
Margarines by Gas Liquid Chromatography/lnfrared Spectrophotometry, J. Am. Oil Chem. Soc. 67, 804-810.
* Nonmethylene interrupted.

We further recommend that the title of Method 996.06 be References

changed from "Fat (Total, Saturated, and Monounsaturated)
in Foods" to "Fat (Total, Saturated, and Unsaturated) in
Foods" and that a table be set up in Method 994.15 based on
Table 4 of this paper to facilitate analyst identification of chro-
matographic peaks.
(1) Official Methods ofAnalysis (1995) Method 996.06, Fat (To-
tal, Saturated, and Monounsaturated) in Foods, AOAC
INTERNATIONAL, Gaithersburg, MD
(2) House, S.D., Larson, P.A., Johnson, R.R., DeVries, J.W., &
Martin, D.L. (1994) J. AOAC Int. 77,960-965
(3) House, S.D. (1997) J. AOAC Int. 80, 555-563