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A collaborative study was conducted to test a modification to the AOAC fluorometric method for histamine (AOAC Official Method 977.13) that substitutes 75% methanol as the extracting solvent. All
other steps remain unchanged. The extracts prepared with 75% methanol were also used to collaboratively test a gas chromatographic (GC) method
for determination of putrescine and cadaverine in
seafood. In the GC method, the extracted diamines
are converted to fluorinated derivatives, the reaction mixtures are passed through solid-phase extraction columns, and the derivatives are quantitated by electron capture GC after separation on an
OV-225 column. Fourteen laboratories using the
GC method for putrescine and cadaverine and
16 laboratories using the fluorometric method for
histamine analyzed 14 canned tuna and raw mahimahi (including blind duplicates and a spike) containing 0.22.6 ppm putrescine, 0.69.1 ppm
cadaverine, and 0.6154 ppm histamine. At the
5 ppm level, recoveries ranged from 71 to 102% for
putrescine and 77 to 112% for cadaverine; the respective repeatability relative standard deviations
(RSDr) were 5.2 and 15%, and the respective reproducibility relative standard deviations (RSDR)
were 8.8 and 18%. At the 50 ppm level, histamine recoveries ranged from 84 to 125%, RSDr was 3.6%,
and RSDR was 9.4%. The GC method for determination of putrescine in canned tuna and cadaverine in
canned tuna and mahimahi has been adopted first
Submitted for publication February 16, 1996.
The recommendation was approved by the Methods Committee on
Commodity Foods and Commodity Products, and was adopted by the
Official Methods Board of the Association. See Official Methods Board
Actions (1996) J. AOAC Int. 79, 76A, and Official Methods Board
Actions (1996) The Referee, June issue.
or many years considerable interest has focused on finding reliable chemical indicators of decomposition to
characterize spoilage in seafood. There are several potential areas for biochemical evaluation to assess quality. For example, metabolic products from actions of enzymes that remain
active in fish after death have been investigated as indicators of
decomposition. These metabolites, which include amines, lactic acid, and hypoxanthine, support growth of bacteria that have
invaded muscle tissue from the gills, intestines, and surface of
the fish.
Metabolites from this microbial action also are candidate
indicators of decomposition. Microbial spoilage produces enzymes called decarboxylases. Decarboxylation of the amino
acids ornithine and lysine produces the diamines putrescine and
cadaverine, respectively. Quantitative analyses by gas chromatography (GC) of a variety of seafood (cod, sole, mahimahi,
and tuna) have shown low levels of putrescine and cadaverine
in fish of acceptable quality and increasing concentrations as
spoilage proceeds. Histamine is formed from decarboxylation
of free histidine. Because there is essentially no free histamine
in fresh tuna, the presence of histamine indicates decomposition and, at sufficiently high levels, has the potential for scombroid poisoning. The U.S. Food and Drug Administration
(FDA) has applied a defect action level (DAL) for histamine as
an index of decomposition and scombroid poisoning since 1978.
Formation of specific decomposition products depends on
the microbial flora present and the marine species. The products formed are a result of storage time and temperature. In
many cases, either cadaverine or putrescine is the major product.
The relation between changes in amines and decomposition
of marine products has been studied by many investigators.
Cadaverine, putrescine, tryptamine, and agmatine have been
Study Design
This study involved determination of putrescine, cadaverine, and histamine in canned tuna and raw frozen mahimahi.
The test samples contained putrescine levels ranging from 0.1
to 10 ppm, cadaverine levels ranging from 0.1 to 15 ppm, and
histamine levels ranging from 5.0 to 100 ppm (ppm = g/g =
10 mg/100 g). There were 7 blind duplicates (including a
spike), 1 blank, and 1 practice test sample, for a total of 16 determinations. Except for the spike, analytes were naturally incurred. Laboratories were asked to analyze all test samples in
duplicate and report all data to 0.01 ppm. They were asked to
send their results, including all calculations and chromatograms, with their comments to the study organizer.
Statistical Calculations
The conventional AOAC (IUPAC-87) Components of
Variance with standard outlier tests were used for analyses of
the data (15, 16).
996.07 Putrescine in Canned Tuna, and Cadaverine
in Canned Tuna and Mahimahi,
Gas Chromatographic Method
First Action 1996
(Applicable to determination of 0.3 g putrescine/g in canned
tuna and 0.5 g cadaverine/g in canned tuna and mahimahi.)
Caution: See Appendix B, safety notes on evaporators,
blenders, flammable solvents, toxic solvents, ethyl acetate, hydrochloric acid, hexane, methanol, and petroleum ether. Pen-
tafluoropropionic anhydride causes severe burns. Avoid contact with skin and eyes. Wear gloves and use effective fume
removal device. Dispose of waste solvents in an appropriate
manner compatible with applicable environmental rules and
regulations.
Method Performance:
See Tables 996.07A and B for method performance data and
Tables 996.07C and D for recovery data.
A. Principle
Putrescine and cadaverine are extracted from test sample
with 75% methanol and then converted to fluorinated derivatives. Reaction mixture is passed through solid-phase extraction column, and derivatives are quantitated by electron capture
gas chromatography (GC) after separation on an OV-225 column.
B. Apparatus
(a) Gas chromatographic (GC) sysem.Equipped with
Ni pulsed electron capture detector. Operating conditions: injection port, 210C; detector, 320C; GC column, 170180C;
nitrogen flow, 25 mL/min; electrometer range, 1010 (full
scale). Detector makeup purge gas as needed (e.g.,
40 mL/min).
(b) GC column.1800 2 mm id, glass column, packed
with 3% OV-225 (50% cyanopropylphenyl and 50% methyl)
on 100120 mesh Gas-Chrom Q, or equivalent capillary column. Condition 2 h with gas flow at room temperature, increase temperature gradually (ca 6C/min) to 240C, and hold
16 h. Retention times of pentafluoropropionic derivatives of
putrescine, cadaverine, and hexanediamine should be 7, 9, and
12 min, respectively.
(c) Solid-phase extraction (SPE) tubes.3 mL, SUPELCLEAN LC-Alumina-N SPE tubes (available from Supelco
Inc., Bellefonte, PA 16823-0048, USA, or equivalent). Check
activity of each lot of SPE tubes as follows: Pass 2 mL petroleum ethertoluene solvent, C(k), through SPE tube and then
add to tube 50 L Sudan I dye solution, C(l). Add a few drops
of petroleum ethertoluene solvent, C(k), to remove dye from
top frit. Pass 3 mL solvent through column. If band of dye stays
in the top 4 mm of SPE tube, the batch is satisfactory for purification of calibration standard and samplePFP reaction mixtures.
(d) Analytical balance.
(e) Rotary evaporator.Capable of maintaining 50
5C.
(f) Water bath.Capable of maintaining 50 5C and
60 5C.
(g) Glassware.Volumetric flasks, 100 mL (glass-stoppered) and 1 L; round or flat bottom 24/40 flasks, 100 mL;
graduated cylinders, 100 and 500 mL (glass-stoppered).
(h) Blender.For test sample preparation.
(i) Pipets.Capable of accurately delivering 1, 2, 3, 5, 10,
and 25 mL.
(j) Micropipets.Capable of accurately delivering 50,
150, and 500 L.
(k) Folded filter paper.
(l) Glass syringe.1.0 mL, calibrated in 0.01 mL.
63
C. Reagents
(a) Pentafluoropropionic (PFP) anhydride.Refrigerate
and protect from moisture (available from Pierce Chemical
Co., Rockford, IL 61105, USA).
(b) Ethyl acetate.Distilled-in-glass. Suitable for GC.
(c) Toluene.Distilled-in-glass. Suitable for GC.
(d) Hexane.Suitable for GC.
(e) Hydrochloric acid (HCl).1 and 0.1N.
(f) Ethyl acetatetoluene solvent.30% Ethyl acetate in
toluene. Transfer 150 mL ethyl acetate to stoppered graduated
cylinder, add 350 mL toluene, and mix.
(g) Methanol.75%, v/v. Place 750 mL MeOH (distilledin-glass) into 1 L volumetric flask. Dilute to volume with H2O.
Swirl flask while adding H2O.
(h) Hexanediamine internal standard solutions.(1)
Hexanediamine standard stock solution.Dissolve 163.0 mg
hexanediamine dihydrochloride in 0.1N HCl in 100 mL volumetric flask and dilute to volume with 0.1N HCl. (2)
20 g/mL.Pipet 2 mL hexanediamine standard stock solution, (1), into 100 mL volumetric flask and dilute to volume
with 0.1N HCl. (3) Hexanediamine internal standard working
solution.5 g/mL. Pipet 25 mL 20 g/mL hexanediamine
standard solution into 100 mL volumetric flask and dilute to
volume with 0.1N HCl.
(i) Putrescinecadaverine standard stock solution.Dissolve 91.4 mg putrescine dihydrochloride and 171.3 mg
cadaverine dihydrochloride in 0.1N HCl in 100 mL volumetric
flask and dilute to volume with 0.1N HCl.
(j) Putrescinecadaverine standard working solutions.
(1) Solution A.10 g cadaverine/mL and 5 g putrescine/mL as free base. Pipet 1 mL putrescinecadaverine standard stock solution, (i), into 100 mL volumetric flask and dilute
to volume with 0.1N HCl. (2) Solution B.1 g cadaverine/mL and 0.5 g putrescine/mL as free base. Pipet 10 mL solution A into 100 mL volumetric flask and dilute to volume
with 0.1N HCl.
(k) Petroleum ethertoluene solvent.4 + 1, v/v. For
checking activity of SPE tubes.
(l) Sudan I dye solution.For checking activity of SPE
tubes. Dissolve 0.01 g Sudan I dye (97%) in 8 mL petroleum
ethertoluene solvent, (k).
per, mix, and heat 30 min at 50C in water bath. (Note: Because
of pressure increase as PFP reaction is heated, stoppers should
be secured with restraining clip.)
Swirl solution at least once during reaction. After reaction
of PFP anhydride with residue of putrescinecadaverine calibration standard solution, the resulting putrescinecadaverine
calibration standardPFP reaction mixture should remain clear.
Within 2 h after removal from water bath, proceed to next step
to purify putrescinecadaverine calibration standardPFP reaction mixture using SPE tube.
Add 2 mL toluene, C(c), to putrescinecadaverine calibration standardPFP reaction mixture. Add 2 mL hexane, C(d),
to each SPE tube and let flow through by gravity. Discard hexane. Add 150 L putrescinecadaverine calibration standard
PFPtoluene reaction mixture to top of SPE tube. Start collecting effluent when mixture is added. Add 3 or 4 drops 30% ethyl
acetatetoluene solvent, C(f), to tube. After mixture passes into
frit, add 2 mL 30% ethyl acetatetoluene solvent. Add additional 6 mL 30% ethyl acetatetoluene solvent (total of 8 mL)
and collect entire effluent. Effluent is stable at least 1 week
when stored in refrigerator in the dark.
F. GC Determination
Adjust GC system to give full-scale recorder response for injection of 1 L derivatized putrescinecadaverine calibration
standard solution IV from D. For products containing lower levels
of analytes (e.g., 05 g cadaverine/g), set derivatized putrescine
cadaverine calibration standard solution III (5 g/g) full scale.
Correlation coefficient of standard curve should be 0.9.
Inject 12 L effluent of sample from E onto GC system.
(Note: Full calibration of GC system with duplicate injections of each of putrescinecadaverine calibration standard solutions is required only at the beginning of analysis, unless instrument is shut down between analyses. When analyzing test
portions on succeeding days, rerun derivatized putrescine
cadaverine calibration standard solutions II and III through GC
system to ensure that calibration of instrument is stable.)
G. Calculations
Perform following calculations:
(1) Calculate response ratio of cadaverine (RSc) in each putrescinecadaverine calibration standard solution as follows:
RSc =
PSc
PSh
PTc
PTh
Wc F 10
Wt
sults and the megabore column results for both putrescine and
cadaverine in all test samples. Retention times obtained with
the megabore column (7, 10, and 12 min; 180C; 25 mL
N2/min) were comparable with those obtained with the packed
column, and the peaks were well separated. The method will
work equally well with either column.
Recommendation
The data indicate good within-laboratory and among-laboratories agreement for the test samples, despite instrument
problems in one laboratory and possible analyst error in 2 laboratories. On the basis of the results of this study, we recommend
that the GC method for determining putrescine and cadaverine
in tuna and mahimahi be adopted first action. We also recommend that the fluorometric Method 977.13 be modified in section D, Determination to read, Extract prepared sample with 75%
MeOH as in 957.07C, paragraph 1, and that the calculation be
changed to report histamine in ppm = g/g = 10 mg/100 g.
Acknowledgments
We thank the following collaborators for their participation:
Roberta J. Berg, U.S. Food and Drug Administration,
Atlanta, GA
Gary Yuen, U.S. Food and Drug Administration, Brooklyn, NY
Richard T. Newton and Julie A. Rorberg, U.S. Food and
Drug Administration, Seattle, WA
Roberta Wagner and Christine Whitby, U.S. Food and Drug
Administration, Baltimore, MD
William Hummer, Terry S. Thomas, and Jennifer C. Personeau, U.S. Food and Drug Administration, Denver, CO
Lydell B. Hansen, U.S. Food and Drug Administration,
Dallas, TX
Don Mowdy and Ed Jester, U.S. Food and Drug
Administration, Dauphin Island, AL
Beth Atienza, Starkist, Terminal Island, CA
Judy Krzynowek, National Marine Fisheries, Gloucester, MA
Mike Jahncke, National Marine Fisheries, Pascagoula, MS
Dave McLachlan and Mike Gilgan, Department of Fisheries
and Oceans, Halifax, Nova Scotia, Canada
Henry Chin, National Food Processors Association, Dublin, CA
Karen Kennedy, Department of Fisheries and Oceans, St.
Johns, Newfoundland, Canada
Claude DesJardins, Department of Fisheries and Oceans,
Quebec, Canada
Gerald Shum, Department of Fisheries and Oceans,
Toronto, Ontario, Canada
Abe Adams, Bumble Bee Seafood, Santa Fe Springs, CA
The statistical analysis was provided by Richard H. Albert
and Richard F. Newell, Division of Mathematics, U.S. Food
and Drug Administration, Washington, DC.
References
(1) Meitz, J.L., & Karmus, E.J. (1978) J. Assoc. Off. Anal.
Chem. 61, 139145
(9) Nagayama, T., Tamura, Y., Maki, T., Kan, K., Naoi, Y., &
Nishima, T. (1985) J. Hyg. Chem. 31, 362370
(10) Yamanaka, H. (1990) Food Rev. Int. 6, 591602
(11) Morrow, J.D., Margolies, G.R., Rowland, J., & Roberts, J.L.,
II (1991) N. Engl. J. Med. 324, 716720
(12) Taylor, S.L., Hui, J.Y., & Lyons, D.E. (1984) in Seafood Toxins, E.P. Ragelis (Ed.), ACS Symposium Series 262,
American Chemical Society, Washington, DC, pp. 417430
(13) Baranowski, J.D., Frank, H.A., Brust, P.A., & Premaratne,
R.J. (1990) J. Food Prot. 53, 217222
(14) Karolus, J., Leblanc, D., March, A.S., Mshan, R., & Furgalack, T.H. (1985) J. Food Prot. 48, 166168
(15) Horwitz, W. (1988) J. Assoc. Off. Anal. Chem. 71, 160172
(16) Horwitz, W. (1988) Pure Appl. Chem. 60, 855864
(17) Horwitz, W., Kamps, L.R., & Boyer, K.W. (1980) J. Assoc.
Off. Anal. Chem. 63, 13441354
(18) Horwitz, W. (1982) Anal. Chem. 54, 67A76A
Table 996.07A. Method performance for determination of putrescine in canned tuna by gas chromatographya
Sample
1
2
3
4
5
6
a
b
c
Mean, g/g
sr, g/g
sR, g/g
RSDr, %
RSDR, %
rb
Rc
0.323
0.184
0.524
0.778
2.606
4.545
0.053
0.028
0.089
0.058
0.196
0.234
0.064
0.080
0.141
0.072
0.209
0.399
16.49
15.57
17.09
7.51
7.51
5.17
19.75
43.6
26.86
9.23
8.03
8.77
0.148
0.078
0.249
0.162
0.549
0.655
0.179
0.224
0.395
0.202
0.585
1.117
Table 996.07B. Method performance for determination of cadaverine in canned tuna and mahimahi by gas
chromatographya
Sample
Canned tuna
Canned tuna
Canned tuna
Canned tuna
Canned tuna
Canned tuna, spiked
Mahimahi
a
b
c
Mean, g/g
sr, g/g
sR, g/g
RSDr, %
RSDR, %
rb
Rc
0.564
1.064
1.118
2.573
9.120
5.171
8.390
0.056
0.082
0.099
0.076
0.515
0.779
0.418
0.104
0.104
0.117
0.111
0.648
0.914
1.239
9.91
7.71
8.88
2.98
5.65
15.06
4.45
18.40
9.80
10.49
4.55
7.1
17.68
14.77
0.157
0.230
0.277
0.213
1.442
2.181
1.170
0.291
0.291
0.328
0.311
1.814
2.559
3.469
Background, g/g
Found, g/g
Recovered, g/g
Recovery, %
0.35
0.31
0.35
0.32
0.33
0.42
0.24
0.31
0.26
0.40
4.60
4.80
5.35
4.82
4.78
4.65
4.88
4.83
4.83
4.51
4.00
4.15
4.66
4.74
4.71
4.59
4.57
3.74
4.61
4.42
4.25
4.45
5.04
4.51
4.43
4.30
4.56
4.51
4.50
4.18
3.58
3.73
4.42
4.50
4.40
4.28
4.31
3.48
4.21
4.02
86.38
90.45
102.44
91.67
90.04
87.40
92.68
91.67
91.46
84.96
72.76
75.81
89.84
91.46
89.43
86.99
87.60
70.73
85.57
81.71
87.05
Average recovery, %
a
b
Background, g/g
Found, g/g
Recovered, g/g
Recovery, %
0.53
0.54
0.46
0.57
0.50
0.58
0.59
0.72
0.60
0.71
5.10
5.40
5.33
5.48
5.35
5.33
5.40
5.30
5.33
4.58
5.38
5.32
5.43
5.50
5.38
5.66
5.52
4.41
6.13
6.27
4.57
4.87
4.79
4.94
4.89
4.87
4.83
4.73
4.83
4.08
4.80
4.74
4.84
4.91
4.66
4.94
4.92
3.81
5.42
5.56
91.95
97.99
96.38
99.40
98.39
97.99
97.18
95.17
97.18
82.09
96.58
95.37
97.38
98.79
93.76
99.40
98.99
76.66
109.05
111.87
96.58
Average recovery, %
a
b
Putrescinecadaverine
standard working solution
Volumea, mL
Equivalent cadaverine
level in test sample, g/g
B
B
B
A
0.5
1.0
5.0
1.0
0.5
1
5
10
0.25
0.5
2.5
5
Description
Canned tuna
0.31 0.04
0.5 0.10
8.96 0.76
Canned tuna
0.2 0.05
1.04 0.05
12.08 0.35
Canned tuna
0.51 0.07
1.03 0.08
4.78 0.39
Canned tuna
0.71 0.08
2.47 0.09
7.41 0.29
Canned tuna
2.57 0.16
9.41 0.38
20.35 1.54
4.67 0.34
5.03 0.49
56.98 2.50
Mahimahi
1.54 0.13
8.14 0.30
153.43 10.67
Each value is the mean standard deviation from analyses of 6 test portions of each collaborative test sample to determine homogeneity.
Table 2. Statistical analysis of collaborative results for fluorometric determination of histamine (ppm) in canned
tuna and raw frozen mahimahi
Test sample
Parameter
No. of laboratories
No. of results
No. of accepted results
Mean, ppm
sr, ppm
RSDr, %
sR, ppm
RSDR , %
Horwitz ratio
16
32
28
9.896
0.884
9.01
0.915
9.32
0.82
16
32
28
13.0
1.71
13.17
1.71
13.17
1.21
3
16
32
26
5.627
1.205
21.42
1.295
23.03
1.87
16
32
26
7.831
1.084
13.84
1.084
13.84
1.18
16
32
28
20.28
1.140
5.62
2.077
10.24
1.01
16
32
28
58.02
2.111
3.64
5.466
9.42
1.08
7
16
32
30
158.4
6.575
4.15
14.05
8.87
1.19
Average
a
b
c
d
0.40
0.30
0.31
0.31
0.33
0.37
0.31
0.32
0.33
0.32
0.44
0.39
0.20
0.27
0.33
0.29
0.23
0.28
b,d
0.94b,d
b,d
0.54b,d
0.50
0.30
0.28
0.35
d
0.70d
d
0.59d
0.30
0.30
0.20
0.20
0.11
0.08
0.19
0.21
0.24
0.20
0.20
0.17
0.11
0.16
0.18
0.23
0.12
0.13
a
NDa
0.08
0.35
0.29
0.17
0.25
0.17
0.14
0.33
0.37
0.10
0.10
0.50
0.60
0.45
0.48
0.54
0.54
0.45
0.45
0.57
0.55
0.54
0.46
0.62
0.40
0.48
0.57
0.46
0.49
0.76
0.88
0.60
0.465
0.50
0.29
0.85
0.58
0.30
0.30
0.75
0.80
0.69
0.77
0.81
0.77
0.64
0.65
0.81
0.77
0.88
0.79
0.73
0.79
0.72
0.76
0.88
0.72
d
1.25d
d
1.12d
0.84
0.67
0.79
0.85
0.86
0.89
d
0.40d
d
0.35d
2.60
2.60
2.68
2.46
2.58
2.60
2.44
2.48
2.72
2.59
2.66
2.71
2.21
2.77
2.94
2.94
2.55
2.47
c,d
4.02c,d
c,d
3.66c,d
3.14
2.56
2.66
2.34
2.57
2.27
b
3.15b
b
1.45b
4.60
4.80
5.35
4.82
4.78
4.65
4.88
4.83
4.83
4.51
4.00
4.15
4.66
4.74
4.71
4.59
4.57
3.74
b
7.60b
b
10.80b
4.61
4.42
b,d
1.93b,d
b,d
5.09b,d
3.91
3.84
c,d
2.10c,d
c,,d
1.55c,d
0.80
0.90
1.90
1.84
3.01
2.96
1.41
1.46
1.70
1.60
1.60
1.63
2.55
2.50
1.65
1.40
1.04
1.15
c,d
6.39c,d
c,d
7.66c,d
3.23
3.03
1.51
1.50
1.27
1.27
0.70
0.70
0.32
0.18
0.52
0.78
2.61
4.55
1.70
ND = not determined.
Removed as outlier by Cochrans test.
Removed as outlier by Grubbs test.
Removed as outlier by paired Grubbs test.
0.55
0.50
0.55
0.52
0.47
0.45
0.55
0.58
0.48
0.52
0.61
0.54
0.67
0.51
0.77
0.66
0.52
0.67
0.72
0.69
a, b
1.16a, b
a,b
0.73a,b
0.62
0.63
C
1.33c
C
0.82c
0.35
0.40
0.56
1.00
1.10
1.10
1.00
1.03
1.02
1.08
1.05
1.03
1.00
1.05
1.10
1.20
0.96
1.11
1.14
1.10
1.07
1.25
1.25
a, b
0.99a, b
a,b
2.67a,b
1.20
1.11
0.77
1.04
1.00
0.90
1.06
1.10
1.20
1.09
1.05
0.98
0.99
1.07
1.15
0.94
1.04
1.06
0.93
1.09
1.12
1.16
1.30
1.20
1.22
1.17
1.35
1.34
0.97
1.14
1.18
b
1.33b
b
1.65b
a
4.20a
a
1.10a
1.12
2.60
2.60
2.61
2.55
2.56
2.42
2.50
2.53
2.53
2.47
2.74
2.66
2.53
2.59
2.77
2.77
2.63
2.53
2.47
2.55
a,b
5.72a,b
a,b
3.28a,b
2.56
2.73
2.30
2.54
b
1.30b
b
1.65b
2.57
9.30
9.20
9.56
9.64
8.99
8.91
9.40
9.27
8.89
9.30
9.45
9.54
7.74
9.01
9.58
9.80
9.51
9.25
9.71
9.59
a,b
11.60a,b
a,b
3.02a,b
8.73
8.37
8.82
7.05
9.90
8.60
9.12
5.10
5.40
5.33
5.48
5.35
5.33
5.40
5.30
5.33
4.58
5.38
5.32
5.43
5.50
5.38
5.66
5.52
4.41
6.13
6.27
5.02
7.58
2.50
5.40
4.79
4.71
3.60
3.60
5.17
7.05
7.40
8.66
8.56
9.01
8.67
8.32
8.32
8.20
7.70
8.43
8.95
7.61
8.31
11.7
10.89
5.88
6.37
9.49
10.66
8.64
8.10
8.10
8.38
7.74
7.79
8.00
8.00
8.39
1
10.0
10.0
9.5
10.2
10.1
9.2
9.2
8.7
9.8
9.2
9.5
9.8
10.3
12.8
9.8
8.6
a,c
9.6a,c
a,c
16.6a,c
9.4
9.3
9.0
11.5
a,b,c
52.2a,b,c
a,b,c
107.7a,b,c
7.7b,c
b,c
5.6b,c
10.4
9.7
9.0
11.0
10.9
10.4
9.9
15.0
13.0
13.0
13.9
11.4
14.9
12.1
11.9
11.7
12.2
10.9
13.5
11.3
16.9
12.1
13.6
16.3
11.5
11.9
12.0
b,c
9.0b,c
b,c
7.0b,c
a,b,c
75.5a,b,c
a,b
196.9a,b,c
12.0
12.5
12.5
12.4
14.0
15.0
13.3
13.2
13.0
5.0
8.0
4.9
4.6
6.2
6.8
4.7
4.4
5.1
4.9
a,b,c
5.2a,b,c
a,b,c
15.6a,b,,c
5.4
6.1
7.3
4.8
b,c
19.9b,c
b,c
6.9b,c
5.1
4.8
6.5
4.5
a ,b,c
11.8a,b,c
a ,b,c
84.0a,b,c
6.7
4.3
5.2
6.0
6.5
9.5
3.8
5.2
5.6
8.0
8.0
8.4
7.4
5.2
9.4
7.3
7.6
7.1
7.8
9.2
7.8
8.0
7.8
7.1
9.1
9.6
8.6
7.5
7.8
b,c
4.5b,c
b,c
5.5b,c
a,b,c
51.7a,b,c
a,b,c
98.0a,b,c
8.6
6.8
7.9
6.9
c
12.5c
c
8.0c
7.4
7.3
7.8
23.0
23.0
22.0
20.5
24.4
22.4
21.8
21.9
18.7
19.5
19.8
20.0
20.6
20.0
17.9
17.2
a,c
21.0a,c
a,c
9.1a,c
20.0
19.5
20.0
21.0
b,c
80.4b,c
b,c
73.1b,c
18.2
16.8
17.2
20.3
21.0
24.0
17.3
19.9
20.3
69.0
66.0
63.3
62.2
67.0
72.4
55.0
58.1
58.4
55.1
58.1
51.8
61.4
57.8
54.1
54.1
58.3
45.3
55.6
56.1
53.0
53.0
b,c
111.0b,c
b,c
104.2b,c
53.8
52.8
54.3
57.6
59.0
59.0
53.3
53.3
58.0
151.0
156.0
159.8
161.1
153.8
157.3
141.9
146.8
163.0
162.0
149.6
146.3
157.5
148.0
167.6
154.1
131.6
143.1
173.6
168.9
144.0
144.0
b,c
278.8b,c
b,c
296.7b,c
175.3
194.5
184.1
172.5
178.0
161.0
152.5
154.3
158.4
Table 6.
Laboratoryb
Background, ppm
Found, ppm
Recovered, ppm
Recovery, %
10.00
9.85
9.65
8.95
9.50
9.65
11.55
9.20
9.30
10.25
10.10
10.00
10.70
69.00
66.00
63.30
62.20
67.00
72.40
55.00
58.10
58.40
55.10
58.10
51.80
61.40
57.80
54.10
54.10
55.60
56.10
53.00
53.00
54.30
54.30
59.00
59.00
53.30
53.30
59.00
56.00
53.45
52.35
57.35
62.75
46.05
49.15
48.90
45.60
48.45
42.15
49.85
46.25
44.90
44.90
46.30
46.80
42.75
42.75
44.20
44.20
49.00
49.00
42.60
42.60
117.65
111.67
106.58
104.39
114.36
125.12
91.82
98.01
97.51
90.93
96.61
84.05
99.40
92.22
89.53
89.53
92.32
93.32
85.24
85.24
88.14
88.14
97.71
97.71
84.95
98.89
96.41
Average recovery, %
a
b
Mean, ppm
b
sr , ppm
b
RSDr , %
b
sR , ppm
b
RSDR , %
c
Mean, ppm
c
sr , ppm
c
RSDr , %
c
sR , ppm
c
RSDR , %
a
b
c
0.28
0.31
0.29
0.29
0.28
0.30
0.29
0.01
5.05
0.01
5.05
0.32
0.05
16.49
0.06
19.75
0.17
0.14
0.13
0.12
0.13
0.25
0.16
0.05
32.3
0.05
32.3
0.18
0.03
15.57
0.08
43.6
0.57
0.38
0.46
0.52
0.54
0.56
0.51
0.08
16.2
0.08
16.2
0.52
0.09
17.09
0.14
26.85
0.73
0.82
0.75
0.88
1.14
0.74
0.84
0.18
20.8
0.18
20.8
0.78
0.06
7.51
0.07
9.23
2.90
2.73
2.50
2.61
2.80
2.80
2.72
0.08
3.04
0.16
5.77
2.61
0.20
7.51
0.21
8.03
5.21
4.90
4.59
4.44
4.92
4.06
4.69
0.38
8.07
0.42
8.89
4.55
0.23
5.17
0.40
8.77
1.35
1.86
1.52
1.44
1.47
1.57
1.54
0.21
13.99
0.21
13.99
1.70
0.08
4.41
0.76
44.37
Mean, ppm
b
sr , ppm
b
RSDr , %
b
sR , ppm
b
RSDR , %
c
Mean, ppm
c
sr , ppm
c
RSDr , %
c
sR , ppm
c
RSDR , %
a
b
c
0.46
0.49
0.56
0.49
0.47
0.64
0.52
0.08
14.7
0.08
14.7
0.63
0.05
9.91
0.10
18.40
1.21
1.10
1.06
1.24
1.25
1.06
1.15
0.12
10.1
0.12
10.1
1.06
0.08
7.71
0.10
9.80
1.25
1.38
1.13
1.47
1.29
1.23
1.29
0.15
11.7
0.15
11.7
1.12
0.09
8.88
0.12
10.49
2.53
2.44
2.67
3.00
2.70
2.59
2.66
0.15
5.52
0.20
7.67
2.57
0.08
2.98
0.11
4.33
9.74
9.53
9.57
9.48
9.24
9.28
9.47
0.09
1.00
0.20
2.15
9.12
0.52
5.65
0.65
7.10
5.68
5.23
5.64
5.35
5.64
4.31
5.31
0.59
11.0
0.59
11.0
5.17
0.78
15.06
0.91
17.68
17.22
19.31
18.40
18.10
18.10
18.64
18.30
10.89
10.7
10.89
10.7
18.39
10.42
14.45
11.24
14.77