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ses (2). One problem with fat analysis is the wide variety of
available methods (36), each with advantages and deficiencies
with regard to quantitation of fat. In general, all ether-extractable material that is unbound from the food matrix is
gravimetrically determined to be fat. Often, exhaustive extraction using hydrolysis followed by ether phase partition yields
high results because extraneous ether-soluble materials also are
measured. In some cases, simple refluxing in ether is insufficient
to extract all fat from the matrix, resulting in low recoveries.
The task force recommended that FDA adopts a chemical
definition of fat, to dissuade use of methods that might yield a
biased result. Therefore, the comments prefacing the regulations state: the agency has decided to define total fat as total
lipid fatty acids, that is, the sum of fatty acids from mono-, di-,
and triglycerides, free fatty acids, phospholipid fatty acids, and
sterol fatty acids, and the agency has decided to require
that the declaration of total fat be expressed as the amount of
triglyceride that would provide the analytically measured
amount of total lipid fatty acids in the food (1). These statements had several consequences on the state-of-the-art analysis
of fat.
The definition of total fat specifically requires measurement
of fatty acids that originate from all lipidic sources, but it also
limits what can be measured and reported as fat. Ether-soluble
materials that are not fatty acids are not to be included in the
total fat content. The further stipulation that total fat be expressed as the equivalent triglyceride amount of analytically
measured fatty acids means each fatty acid measured must be
converted to its triglyceride equivalent. Then these triglyceride
equivalents are summed to give total fat. The chemical and
mathematical nuances of this requirement are described in the
paper by House et al. (7). Furthermore, non-fatty-acid material
that might be extracted into the organic phase cannot be
counted as fat. This material includes glycerol, low molecular
weight carbohydrates and their polymerization products...
amino acids, and urea salts (2). It was clear that conventional
gravimetric analysis was not sufficient to meet the new NLEA
requirements.
A. Principle
Fat and fatty acids are extracted from food samples by hydrolytic methods (acidic hydrolysis for most samples, alkaline
hydrolysis for dairy products, and combination for cheese samples). Pyrogallic acid is added to minimize oxidative degradation of fatty acids during analysis. A triglyceride, triundecanoin
(C11:0), is added as internal standard. Fat is extracted into ether
and then methylated to fatty acid methyl esters (FAMEs) using
BF3 in methanol. FAMEs are quantitatively measured by capillary gas chromatography (GC) against C11:0 internal standard.
Total fat is calculated as the sum of individual fatty acids expressed as triglyceride equivalents. Saturated and monounsaturated fats are calculated as the sum of respective fatty acids.
Monounsaturated fat includes only the cis form.
B. Apparatus
(a) Gas chromatograph.Equipped with hydrogen flame
ionization detector, capillary column, split mode injector, oven
temperature programming sufficient to implement a holdramp-hold sequence. Operating conditions: temperature (C):
injector, 225; detector, 285; initial tempperature, 100 (hold
4 min); ramp, 3C/min; final temperature, 240; hold 15 min;
carrier gas, helium; flow rate, 0.75 mL/min; linear velocity,
18 cm/s; split ratio, 200:1.
(b) Capillary column.60 m 0.25 mm id, 0.20 m film,
fused silica capillary, SP-2340 phase. (Note: FAMEs of C18:3
and C20:1 may coelute after several hundred injections.)
(c) Analytical balance.Capable of weighing to
0.0001 g.
(d) Mojonnier flasks.
(e) StoppersSynthetic rubber or cork.
(f) Mojonnier centrifuge basket.
C. Reagents
(a) Pyrogallic acid.Analytical reagent grade.
(b) Hydrochloric acid (HCl).12N (concentrated) and
8.3N. To make 8.3N HCl, add 250 mL 12N HCl to 110 mL
H2O. Mix well. Store at room temperature.
(c) Ammonium hydroxide (NH4OH).58% (w/w), analytical reagent grade.
(d) Diethyl ether.Purity appropriate for fat extraction.
(e) Petroleum ether.Anhydrous; analytical reagent grade.
(f) Ethanol.95%; analytical reagent grade.
(g) Toluene.Nanograde.
(h) Chloroform (CHCl3).Analytical reagent grade.
(i) Sodium sulfate(Na2SO4).Anhydrous.
(j) Boron trifluoride (BF3) reagent.7% BF3 (w/w) in
methanol, made from commercially available 14% BF3 solution. Prepare in the hood.
(k) Diethyl etherpetroleum ether mixture.1 + 1, v/v.
(l) Triglyceride internal standard solution.C11:0 - triundecanoin; 5.00 mg/mL in CHCl3. Accurately weigh 2.50 g
C11:0 - triundecanoin into 500 mL volumetric flask. Add ca
400 mL CHCl3 and mix until C11:0 is dissolved. Dilute to volume with CHCl3. Invert flask at least 10 additional times.
Triglyceride internal standard solution is stable up to 1 month
when stored in refrigerator.
(m) Fatty acid methyl esters (FAMEs) standard solutions.(1) Mixed FAMEs standard solution.Reference
mixture containing series of FAMEs, including C18:1 cis and
trans (available as GLC-85 from Nu Chek Prep, Elysian, MN,
or equivalent). To prepare mixed FAMEs standard solution,
break top of glass vial, open, and carefully transfer contents to
3 dram glass vial. Wash original vial with hexane to ensure
complete transfer and add washes to 3 dram glass vial. Dilute
to ca 3 mL with hexane. (2) C11:0 FAME standard solution.
C11:0 - undecanoic methyl ester in hexane. Use only in preparation of individual FAME standard solutions, (3). To prepare
C11:0 FAME standard solution, break top of glass vial open and
carefully transfer contents to 50 mL volumetric flask. Wash
D. Extraction of Fat
Finely grind and homogenize test samples prior to extraction of fat.
(Note: When running matrixes of unknown composition, it
may be necessary to analyze sample without addition of internal standard to ensure against interferences. Should interfering
peak be found, the area of C11:0 internal standard peak must be
corrected before performing calculations. Instead, use 2.0 mL
chloroform.)
(a) Foodstuffs excluding dairy products and cheese.Accurately weigh ground and homogenized sample (containing ca
100200 mg fat) into labeled Mojonnier flask. Force sample
into flask as far as possible. Add ca 100 mg pyrogallic acid,
C(a), and 2.00 mL triglyceride internal standard solution, C(l).
Add a few boiling granules to flask. Add 2.0 mL ethanol and
mix well until entire sample is in solution. Add 10.0 mL 8.3N
HCl and mix well. Place flask into basket in shaking water bath
at 7080C and set at moderate agitation speed. Maintain
40 min. Mix contents of flask on Vortex mixer every 10 min to
incorporate into solution particulates adhering to sides of flask.
After digestion, remove flask from bath and allow to cool to
room temperature. Add enough ethanol to fill bottom reservoir
of flask and mix gently.
(b) Dairy products.Accurately weigh ground and homogenized sample (containing ca 100200 mg fat) into labeled
Mojonnier flask. Force sample into flask as far as possible. Add
ca 100 mg pyrogallic acid, C(a), and 2.00 mL triglyceride internal standard solution, C(l). Add a few boiling granules to
flask. Add 2.0 mL ethanol and mix well until entire sample is
in solution. Add 4.0 mL H2O and mix well. Add 2.0 mL 58%
NH4OH, C(c), and mix well. Place flask into basket in shaking
water bath at 7080C set at moderate agitation speed. Maintain 10 min. Mix contents of flask on Vortex mixer every 5 min
to incorporate into solution particulates adhering to sides of
flask. After digestion, remove flask from water bath and add a
few drops of phenolphthalein. Keep solution basic (pink) with
addition of ammonium hydroxide. Add enough ethanol to fill
bottom reservoir of flask and mix gently.
(c) Cheese.Accurately weigh ground and homogenized
sample (containing ca 100200 mg fat) into labeled Mojonnier
flask. Force sample into flask as far as possible. Add ca 100 mg
pyrogallic acid, C(a), and 2.00 mL triglyceride internal standard solution, C(l). Add a few boiling granules to flask. Add
2.0 mL ethanol and mix well until entire sample is in solution.
Add 4.0 mL H2O and mix well. Add 2.0 mL 58% NH4OH,
C(c), and mix well. Place flask into basket in shaking water
bath at 7080C set at moderate agitation speed. Maintain
20 min. Mix contents of flask on Vortex mixer every 10 min to
incorporate into solution particulates adhering to sides of flask.
Add 10.0 mL 12N HCl and place flask into boiling steam bath
and maintain 20 min. Mix flask contents every 10 min on Vortex mixer. Remove flask from steam bath and allow to cool to
room temperature. Add enough ethanol to flask to fill bottom
reservoir and mix gently.
Add 25 mL diethyl ether to Mojonnier flask from (a), (b),
or (c). Stopper flask and place in centrifuge basket. Place basket
in wrist action shaker, securing flask in shaker with rubber tubing. Shake flask 5 min. Rinse stopper into flask with diethyl
etherpetroleum ether mixture, C(k). Add 25 mL petroleum
ether, stopper flask, and shake 5 min. Centrifuge flask (in basket) 5 min at 600 rpm. (Note: If centrifuge is not available, allow contents to set at least 1 h until upper layer is clear.) Rinse
stopper into flask with diethyl etherpetroleum ether mixture.
Decant ether (top) layer into 150 mL beaker and carefully rinse
lip of flask into beaker with diethyl etherpetroleum ether mixture. Slowly evaporate ether on steam bath, using nitrogen
stream to aid evaporation. Residue remaining in beaker contains extracted fat.
E. Methylation
Dissolve extracted fat residue in 23 mL chloroform and 2
3 mL diethyl ether. Transfer mixture to 3 dram glass vial and
then evaporate to dryness in 40C water bath under nitrogen
stream. Add 2.0 mL 7% BF3 reagent, C(j), and 1.0 mL toluene,
C(g). Seal vial with screw cap top containing Teflon/silicone
septum. Heat vial in oven 45 min at 100C. Gently shake vial
ca every 10 min. (Note: Evaporation of liquid from vials indicates inadequate seals; if this occurs, discard sample and repeat
the entire procedure.) Allow vial to cool to room temperature.
Add 5.0 mL H2O, 1.0 mL hexane, and ca 1.0 g Na2SO4, C(i).
Cap vial and shake 1 min. Allow layers to separate and then
carefully transfer top layer to another vial containing ca 1.0 g
Na2SO4. (Note: Top layer contains FAMEs including FAME of
triglyceride internal standard solution.)
Inject FAMEs onto GC column or transfer to autosampler
vial for GC analysis.
F. GC Determination
Relative retention times (vs FAME of triglyceride internal
standard solution) and response factors of individual FAMEs
can be obtained by GC analysis of individual FAME standard
solutions and mixed FAME standard solution.
Inject ca 2 L each of individual FAME standard solutions
and 2 L of mixed FAMEs standard solution. Use mixed
FAMEs standard solution to optimize chromatographic response before injecting any test samples.
After all chromatographic conditions have been optimized,
inject test samples from E.
G. Calculations
Total fat is the sum of fatty acids from all sources, expressed
as triglycerides. Expressing measured fatty acids as
triglycerides requires the mathematical equivalent of condensing each fatty acid with glycerol. For every 3 fatty acid molecules, 1 glycerol (HOCH2CHOHCH2OH) is required. Essentially, 2 methylene groups and 1 methine group are added to
every 3 fatty acids.
Calculate retention times for each FAME in individual
FAMEs standard solutions, C(m)(3), by subtracting retention
time of C11:0 peak from retention time of fatty acid peak. Use
these retention times to identify FAMEs in mixed FAMEs
standard solution. Use additional FAME solutions (from the
same supplier) when necessary for complete verification of
FAME identity.
Calculate response factor (Ri) for each fatty acid i as follows:
Ri =
Psi
WC11:0
PsC11:0
Wi
WFAMEi =
WTGi
Wsample
100
Saturated Wi
100
Wsample
Monounsaturated Wi
100
Wsample
(Note: Samples containing hydrogenated fat will yield complicated chromatograms due to large numbers of isomers
formed during hydrogenation process. One general indication
of hydrogenation is presence of C18:1 trans peak(s). For chromatograms of hydrogenated fats, use the following guidelines
to calculate FAME peak areas: Trans peaks elute prior to cis,
therefore, include all peaks between C18:1 cis and C18:2 cis,cis
in calculation of C18:2 peak area. Often C18:1 trans peak consists of broad series of peaks [due to positional isomers from
hydrogenation]; include all of these in C18:1 trans peak area.)
Ref.: J. AOAC Int. 80, 555563(1997)
Results and Discussion
Each laboratory received 3 replicates of an oat-based cereal
(fat content about 7%) to serve as a precollaborative test sample
(Table 2). Results were analyzed for accuracy (versus the
authors laboratory) and precision. Once a laboratory submitted
satisfactory results for total fat, it was sent the collaborative
study samples. No laboratory was required to repeat analysis of
pretest samples.
Samples consisted of 8 pairs of blind duplicates, representing a wide variety of matrixes (Table 1). Also listed in Table 1 is the approximate sector of the food triangle, recommended for use by the AOAC Task Force on Methods for
Nutrition Labeling Analyses (8), into which each sample belongs.
Each laboratory was instructed to analyze each sample
(116) only one time. The exception would be obvious mistakes that would necessitate reruns. Sample weights for each
Collaborators Comments
On the basis of comments from collaborators, the following
additions, corrections, and other recommended changes to the
method are reflected in the method described:
(1) Additional instructions on how to interpret complicated
chromatograms, usually from samples containing hydrogenated fat, are now included.
(2) Conversion factors to transform from FAME weights to
fatty acid weights in the report sheet have been changed to exhibit 4 significant figures rather than 2.
(3) There were a few comments regarding the use of hazardous chemicals. However, we cannot at this time eliminate
liquid organic solvents.
Recommendation
On the basis of the results of this study it is recommended
that the hydrolytic extractionGC method for determining fat
(total, saturated, and monounsaturated) in foods be adopted
first action.
Acknowledgments
I thank each of the collaborators and their associates for their
contribution, patience, and persistence in completing this study:
References
(1) Fed. Reg. (1990) 58, 6312964
(2) Carpenter, D.E., Ngeh-Ngwainbi, J., & Lee, S. (1993) in
Methods of Analysis for Nutrition Labeling, Chapter 5, D.M.
Sullivan & D.E. Carpenter (Eds), AOAC INTERNATIONAL, Arlington, VA
(3) Official Methods of Analysis (1990) 15th Ed., AOAC, Arlington, VA, sec. 920.39C
(4) Daugherty, C., & Lento, H. (1983) J. Assoc. Off. Anal. Chem.
66, 927932
(5) Sheppard, A.J., Hubbard, D.W., Prosser, A.R., Shen, J.C.-S.,
ODell, R.G., & Jones, S.T. (1989) Lipid Manual, U.S. Food
and Drug Administration, Washington, DC
(6) Ngeh-Ngwainbi, J. (1992) Analysis of Fat in Low Fat Cereal
Products, presented at the 106th Annual AOAC INTERNATIONAL Conference, Cincinnati, OH
(7) House, S.D., Larson, P.A., Johnson, R.R., DeVries, J.W., & Martin, D.L. (1994) J. AOAC Int. 77, 960965
(8) Wolf, W.R. (1993) in Methods of Analysis for Nutrition Labeling, Chapter 7, D.M. Sullivan & D.E. Carpenter (Eds),
AOAC INTERNATIONAL, Arlington, VA
Table 996.06A. Method performance for determination of total fat in foodstuffs by hydrolytic extractiongas
chromatography
Samplea
_
x, %
sr
sR
rb
Rc
RSDr, %
RSDR, %
No. of
laboratories
excludedd
Wheat-based cereal
Peanut butter
Fish sticks
Parmesan cheese
Chocolate cake (baked)
Fruit snack
Ground beef
Yogurt
1.96
46.3
11.2
26.5
13.3
3.92
21.91
1.46
0.208
0.86
0.354
0.540
0.929
0.087
1.11
0.131
0.260
2.37
0.541
4.17
1.95
0.146
1.82
0.222
0.582
2.41
0.991
1.51
2.60
0.244
3.11
0.367
0.728
6.64
1.51
11.7
5.46
0.409
5.10
0.622
10.6 1
1.85
3.14
2.04
7.00
2.22
5.06
8.98
13.3
15.12
14.80
15.8
14.7
13.74
18.32
15.2
2/10
2/10
1/10
1/10
a
b
c
d
Blind duplicates.
r = 2.8 sr.
R = 2.8 sR.
Outliers by either Cochran or Grubbs tests.
Table 996.06B. Method performance for determination of saturated fat in foodstuffs by hydrolytic extractiongas
chromatography
Samplea
Wheat-based cereal
Peanut butter
Fish sticks
Parmesan cheese
Chocolate cake (baked)
Fruit snack
Ground beef
Yogurt
a
b
c
d
_
x, %
sr
sR
rb
Rc
RSDr, %
RSDR, %
No. of
laboratories
excludedd
0.493
8.72
3.00
17.4 1
3.56
1.27
9.98
0.986
10.0391
0.257
0.223
0.311
0.171
0.0242
0.636
0.119
0.0522
1.81
0.572
2.46
0.304
0.0362
1.39
0.170
0.109
0.720
0.624
0.871
0.479
0.0678
1.78
0.333
0.146
5.07
1.60
6.89
0.851
0.101
3.89
0.476
7.92
2.95
7.44
1.79
4.81
1.90
6.38
12.1
10.6
20.7
19.1
14.1
8.55
2.83
13.9
17.2
1/10
1/10
1/10
2/10
1/10
Blind duplicates.
r = 2.8 sr.
R = 2.8 sR.
Outliers by either Cochran or Grubbs tests.
Table 996.06C. Method performance for determination of monounsaturated fat in foodstuffs by hydrolytic
extractiongas chromatography
Samplea
Wheat-based cereal
Peanut butter
Fish sticks
Parmesan cheese
Chocolate cake (baked)
Fruit snack
Ground beef
Yogurt
a
b
c
d
_
x, %
sr
sR
rb
Rc
RSDr, %
RSDR, %
No. of
laboratories
excludedd
0.280
22.3
1.83
6.43
3.79
1.08
8.88
0.345
0.0320
0.411
0.165
0.271
0.413
0.0453
0.930
0.0222
0.0560
1.11
0.313
1.09
1.27
0.734
1.96
0.0542
0.0896
1.15
0.462
0.759
1.16
0.127
2.60
0.0621
0.157
3.11
0.876
3.05
3.56
2.06
5.49
0.152
11.4
1.84
9.02
4.20
10.9
4.17
10.5
6.42
20.0
4.96
17.1
17.0
33.5
67.7
22.0
15.1
2/10
1/10
2/10
1/10
Blind duplicates.
r = 2.8 sr.
R = 2.8 sR.
Outliers by either Cochran or Grubbs tests.
fi
0.8627
0.8923
0.9114
0.9247
0.9300
0.9346
0.9386
0.9421
0.9417
0.9453
0.9449
0.9481
0.9477
0.9507
0.9503
0.9530
0.9527
0.9524
0.9520
0.9517
0.9570
0.9568
0.9557
0.9554
0.9604
0.9602
0.9593
0.9590
0.9963
fTGi
0.9868
0.9897
0.9915
0.9928
0.9933
0.9937
0.9945
0.9944
0.9948
0.9947
0.9950
0.9950
0.9953
0.9952
0.9955
0.9955
0.9954
0.9954
0.9954
0.9959
0.9959
0.9958
0.9957
0.9962
0.9962
0.9961
0.9961
0.9965
ID No.
1, 15
6, 91
2, 16
7, 10
3, 14
8, 11
4, 13
5, 12
5
5
3
4
8
6
4
6
Statistics
Test 1
Test 2
Test 3
Mean
CV, %
1
2
3
4
5
6
7
8
b
9
10
6.32
7.32
NAa
7.45
7.42
7.43
7.06
7.50
7.15
7.29
6.71
7.28
NA
7.52
7.41
7.59
7.05
7.40
7.19
6.59
6.70
7.23
NA
7.52
7.34
7.54
7.07
7.50
7.16
6.97
6.58
7.28
7.50
7.49
7.52
7.06
7.47
7.17
6.95
3.34
0.62
0.53
0.77
1.09
0.14
0.77
0.29
5.04
Average
SD
CV, %
7.21
0.39
5.41
7.27
0.29
3.93
7.26
0.29
3.93
Laboratory ID
a
b
15
16
1.83
2.15
2.21
2.01
1.58
2.49
2.10
2.10
1.96
2.02
1.66
2.14
1.9
1.93
1.66
2.07
1.91
2.06
2.06
1.31
44.10
48.60
47.92
45.62
44.15
47.74
a
51.41a
46.40
49.54
b
30.97c
42.20
48.96
46.23
43.82
43.59
47.27
a
45.96a
45.22
49.40
b
30.18c
14
11.10 10.80
11.34
11.15
12.02 11.45
10.59 11.15
10.58 10.76
11.09
11.40
a
12.84a a10.54a
11.22
11.25
11.63 12.68
1b7.22c 1b7.37c
13
12
10
11
28.60
27.89
32.66
18.94
25.16
28.05
25.64
26.84
30.81
20.07
28.90
27.22
32.3
20.67
25.14
26.98
26.28
26.78
30.92
19.30
12.80
14.22
14.97
12.06
12.91
13.63
16.20
14.63
14.66
17.87
11.00
14.28
13.96
12.93
12.82
13.88
13.45
14.65
14.76
10.01
4.20
4.00
3.92
3.73
3.85
3.66
3.99
NAb
4.04
a
2.44a
4.00
3.87
3.91
3.97
3.92
3.65
3.98
NA
4.01
a
8.04a
20.70
24.79
21.46
20.87
19.86
23.80
22.51
20.74
23.97
a
5.33a
20.30
22.14
23.63
19.02
18.82
22.69
22.29
22.86
24.02
a
13.93a
1.63
1.46
1.50
1.32
1.31
1.38
1.25
1.19
1.67
1.33
1.69
1.46
1.95
1.38
1.33
1.67
1.25
1.26
1.88
1.30
15
0.49
0.54
0.57
b
0.79b
0.42
0.51
0.54
0.53
0.50
0.48
0.43
0.54
0.47
b
0.76b
0.44
0.51
0.51
0.52
0.52
0.37
16
8.31
7.97
9.82
9.82
8.88
8.63
b
11.40b1 b11.30b1
8.23
8.12
a
6.64a a8.91a
9.70
8.92
8.63
8.39
9.75
9.71
5.03
4.46
3
a
11.10a1
2.97
3.61
4.06
2.74
2.70
3.40
2.91
3.02
1.89
14
13
12
10
11
2.87a
2.92
2.97
3.99
2.82
2.99
2.84
2.91
3.29
1.95
19.20
17.70
20.50
13.50
16.30
18.20
17.30
17.90
20.20
13.10
19.40
17.30
20.3
14.60
16.30
17.80
17.70
17.90
20.30
12.70
3.39
3.56
3.64
3.98
3.10
3.54
4.04
3.53
3.58
2.29
3.17
3.56
3.43
4.22
3.08
3.58
3.43
3.56
3.60
1.27
1.26
1.31
1.25
b
1.68b
1.23
1.34
1.31
1.27
1.31
a
0.92a
1.20
1.28
1.27
b
1.78b
1.25
1.28
1.31
1.29
1.30
a
3.68a
9.88
7.87
9.91
11.001
9.05
11.301
11.101
9.79
10.901
a
1.87a
9.68
6.14
10.8 1
9.95
8.58
10.601
10.801
11.001
11.201
a
6.45a
1.11
0.94
0.95
1.04
0.85
0.90
0.85
0.82
1.09
0.80
1.12
0.94
1.37
1.27
0.86
1.09
0.85
0.87
1.21
0.80
Table 5. Collaborative study results for monounsaturated fat, by laboratory ID and sample number
Sample
Laboratory ID
11
12
13
14
15
16
17
18
19
10
a
b
c
15
16
14
0.23
0.32
0.39
0.36
0.21
0.28
0.29
0.27
0.27
a
0.29a
0.21
0.32
0.32
0.35
0.22
0.27
0.28
0.26
0.29
a
0.17a
20.70
23.10
22.20
23.80
21.30
22.90
a
24.10a
22.30
23.70
b
12.80c
20.40
23.30
21.8
22.40
21.10
22.70
a
21.20a
21.70
23.60
b
12.90c
1.78
1.92
2.43
2.16
1.66
1.79
2.13
1.79
1.94
1.12
1.74
1.89
1.97
1.94
1.69
1.91
1.65
1.79
2.13
1.14
4
a
2.11a
7.50
8.39
5.40
5.68
6.88
6.18
6.58
7.10
4.65
13
12
10
11
6.99a
6.78
8.21
6.00
5.65
6.32
6.36
6.55
7.14
4.41
1.02
2.76
4.65
5.14
3.47
4.39
5.79
2.53
4.63
2.93
2.32
2.80
4.34
5.43
3.44
4.50
4.80
2.47
4.65
3.67
0.57
1.09
0.50
2.25
0.24
0.87
2.04
NAb
1.04
1.27
0.53
1.03
0.55
2.38
0.23
0.96
2.03
NA
1.04
3.51
9.31
7.90
9.25
9.96
8.72
10.20
9.87
9.62
10.40
a
2.74a
9.13
6.11
10.4
9.29
8.32
9.96
9.90
10.401
10.101
a
6.11a
0.37
0.39
0.37
a
0.46a
0.29
0.34
0.30
0.26
0.40
0.31
0.39
0.38
0.38
a
0.25a
0.30
0.41
0.30
0.29
0.44
0.29