35.1.

32

paper and wash again with H2O. Prepare resin fresh weekly and
store under H2O. Place glass wool plug in base of tube, B(a), and
slurry in enough resin to form 8 cm bed. Maintain H2O level above
top of resin bed at all times. Do not regenerate resin in packed
column; rather, use batch regeneration in beaker when necessary.
Wash column with ca 10 mL H2O before applying each extract.

AOAC Official Method 977.13

Histamine in Seafood
Fluorometric Method
First Action 1977
Final Action 1987

(b) Phosphoric acid.To prepare 1.19M phosphoric acid, dilute

79.33 mL 85% (15M H3PO4) to 1 L. For other concentrations of
H3PO4, the volume required for 1 L 1.19M H3PO4 = 17 493/(density
H3PO4 % H3PO4). Standardize 5.00 mL by titration with 1.00M
NaOH to phenolphthalein end point and adjust concentration if
necessary.

Codex-AdoptedAOAC Method*

Caution: See Appendix B, Laboratory Safety for Safe Handling

of Alkaliessodium hydroxide; Safe Handling of
Acidsphosphoric and hydrochloric acids; and Safe
Handling of Special Chemical Hazardsmethanol.
Dispose of waste solvents in an appropriate manner
compatible with applicable environmental rules and
regulations.

(c) o-Phthaldialdehyde (OPT) solution.0.1% (w/v). Dissolve

100 mg OPT in 100 mL distilled-in-glass methanol. Store in amber
bottle in refrigerator. Prepare fresh weekly.

See Tables 977.13A and B for the results of the interlaboratory

study supporting acceptance of the method, and Table 977.13C for
recovery data.

(d) His ta mine stan dard so lu tions.Store in re frig er a tor.

(1) Stock solution.1 mg/mL as free base. Accurately weigh ca
169.1 mg histamine2HCl (98%) into 100 mL volumetric flask, and
dissolve and dilute to volume with 0.1M HCl. Prepare fresh weekly.
(2) Intermediate solution.10 mg/mL. Pipet 1 mL stock solution into
100 mL volumetric flask, and dilute to volume with 0.1M HCl. Prepare
fresh weekly. (3) Working solutions.0.5, 1.0, and 1.5 mg/5 mL. Pipet
1, 2, and 3 mL intermediate solution into separate 100 mL volumetric
flasks, and dilute each to volume with 0.1M HCl. Prepare fresh daily.

A. Principle

Product is extracted with 75% (v/v) methanol. Extract is passed

through ion exchange column. o-Phthaldialdehyde solution is added
to eluate to form fluorescent histamine derivatives. Fluorescent
intensity of derivatives is measured using fluorometer and histamine
is quantified using external standards.

(e) Methanol.75% (v/v). Place 75 mL MeOH (distilled in

glass) into 100 mL volumetric flask or stoppered graduated cylinder.
Dilute to volume with H2O. Swirl flask while adding H2O.

B. Apparatus

Rinse all plastic and glass containers with HCl (1 + 3) and H2O
before use.
(a) Chromatographic tube.200 7 id mm polypropylene tube
fitted with small plastic stopcocks and ca 45 cm Teflon tubing.
Control flow rate at >3 mL/min by adjusting height of column relative
to tubing outlet. Alternatively, use 2-way valve in place of tubing.
(b) Photofluorometer.With medium pressure Hg lamp with
excitation at 350 nm and measuring emission at 444 nm.
(c) Repipets.1 and 5 mL.

D. Preparation of Standard Curve

Pipet duplicate 5 mL aliquots of each working standard solution into

separate 50 mL glass or polypropylene Erlenmeyers. Pipet in 10 mL
0.1M HCl to each flask and mix. Pipet in 3 mL 1M NaOH and mix.
Within 5 min, pipet in 1 mL OPT solution and mix immediately. After
exactly 4 min, pipet in 3 mL 1.19M H3PO4 and mix immediately. It is
important to mix thoroughly after each addition and at least once during
OPT reaction. (Run 610 OPT reactions simultaneously by adding
reagents to Erlenmeyers in set order.) Prepare blank by substituting
5 mL 0.1M HCl for histamine solution. Within 1.5 h, record
fluorescence intensity (I) of working standard solutions with H2O in
reference cell, using excitation wavelength of 350 nm and emission
wavelength of 444 nm. Plot I (corrected for blank) against mg
histamine/5 mL aliquot.

C. Reagents

(a) Ion-exchange resin.Bio-Rad AG 1-X8, 50100 mesh

(Bio-Rad Laboratories, 1000 Alfred Nobel Dr, Hercules, CA 94547,
USA; www.biorad.com) or Dowex 1-X8, 50100 mesh. Convert to
-OH form by adding ca 15 mL 2M NaOH/g resin to beaker. Swirl
mixture and let stand <30min. Decant liquid and repeat with
additional base. Thoroughly wash resin with H2O, slurry into fluted

Table 977.13A. Interlaboratory study results for determination of histamine in tuna by fluorometric method (based on
collaborative study results of the original method)
Test sample

Acceptable skipjack packed in water

Histamine,
mg/100 g

Avg. histamine
found, mg/100 g

sr

RSDr, %

sR

RSDR,
%
HorRat

1.4

0.643

46.9

1.009

73.6

Skipjack with 25 mg histamine added/100 test sample

26

25.8

0.950

3.7

1.383

5.4

0.54

Decomposed albacore packed in water

30

31.6

2.053

6.5

3.473

11.0

0.97

Decomposed yellowfin packed in water

20

19.8

0.941

4.7

1.729

8.7

0.65

Decomposed skipjack packed in oil

120

125.6

6.432

5.1

8.807

7.0

0.94

Decomposed yellowfin packed in oil

200

198.8

4.801

2.4

10.6555

5.4

0.47

a
Blind duplicates.
b

Approximate composition.

2012 AOAC INTERNATIONAL

Table 977.13B. Interlaboratory study results for determination of histamine in canned tuna and frozen mahimahi by fluorometric
a
method (generated for modified method using 75% (v/v) methanol)
Test sample

Mean, mg/g

sr, mg/g

sR, mg/g

9.896

0.884

0.915

1.710

1.710

1
2

13.0

RSDr, %

RSDR, %

HorRat

9.01

9.32

2.475

2.562

0.83

13.17

13.17

4.788

4.788

1.22

5.627

1.205

1.295

21.42

23.03

3.374

3.626

1.88

7.831

1.084

1.084

13.84

13.84

3.035

3.035

1.19

20.28

1.140

2.077

5.62

10.24

3.192

5.816

1.01

58.02

2.111

5.466

3.64

9.42

5.911

15.305

1.09

6.575

14.050

4.15

8.87

18.410

39.340

1.19

158.4

a
Based on results received from 16 laboratories.
b
r = 2.8 sr.
c
R = 2.8 sR.

E. Determination

Extract prepared 10 g test portion with 75% (v/v) methanol as in

957.07C (see 35.1.31), paragraph 1. Pass 45 mL H2O through
column, B(a), and discard eluate. Pipet 1 mL extract onto column
and add 45 mL H2O. Immediately initiate column flow into 50 mL
volumetric flask containing 5.00 mL 1.00M HCl. When liquid level
is ca 2 mm above resin, add ca 5 mL H2O and let elute. Follow with

Table 977.13C. Recovery of histamine added to canned tuna

a
(generated for modified method using 75% methanol)
Background,
mg/g

Found,
mg/g

Recovered,
mg/g

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

Lab

Avg. rec., %

a
Added 50.15 mg histamine/g.
b

Data from Laboratories I, L, and M were excluded because results for the
background or the spike were outliers.

2012 AOAC INTERNATIONAL

H2O in larger portions until ca 35 mL has eluted. Stop column flow,

dilute to volume with H2O, stopper, and mix. Refrigerate eluate.
Pipet 5 mL eluate into 50 mL Erlenmeyer, and pipet in 10 mL 0.1M
HCl. Proceed as in D, beginning Pipet in 3 mL 1M NaOH . . ..
If test sample contains >15 mg histamine/100 g product, pipet
1 mL test solutionOPT mixture into 10 mL beaker containing
exactly 2 mL blankOPT mixture, and mix thoroughly. Read
fluorescence of new solution. Dilute and mix aliquots with
blankOPT mixture as needed to obtain measurable reading. This
approximation indicates proper dilution of eluate required prior to
second OPT reaction needed for reliable quantitation of test
solution. Alternatively, use sensitivity range control of fluorometer
(if in stru ment has one) to es ti mate di lu tion. Use these
approximations to prepare appropriate dilution of aliquot of eluate
with 0.1M HCl, and proceed as in D, beginning Pipet in 3 mL 1M
NaOH . . ..
F. Calculations

Plot of I (measured by meter deflection or recorder response and

corrected for blank) against mg histamine/5 mL test solution should be
straight line passing through origin with slope = m = [(Ia /1.5) + Ib + 2Ic ]/3.
mg Histamine/100 g fish = (10)(F)(1/m)(Is)
mg Histamine/g fish = 10 (mg histamine/100 g fish)
where Is, Ia, Ib, and Ic = fluorescence from test solution, 1.5, 1.0, and
0.5 mg histamine standards, respectively; and F = dilution factor =
(mL eluate + mL 0.1M HCl)/mL eluate. F = 1 for undiluted eluate.
If calibration plot is not linear, use standard curve directly for
quantitation. Each subdivision on abscissa should be 0.1 mg
histamine/5 mL test solution. Read all values from curve to nearest
0.05 mg histamine/5 mL test solution.
mg Histamine/100 g test portion = (10)(F)(W)
mg Histamine/g test portion
= 10 (mg histamine/100 g test portion)
where W = mg histamine/5 mL test solution as determined from
standard curve.
Reference: JAOAC 60, 1125, 1131(1977).
CAS-51-45-6 (histamine)
Revised: June 2003, September 2012, November 2012

* Codex Stan 36-1981, Rev. 1-1995, Codex Standard for Quick Frozen
Finfish, Uneviscerated and Eviscerated. Codex Stan 70-1981, Rev.
1-1995, Codex Standard for Canned Tuna and Bonita. Codex Stan
94-1981, Rev. 1-1995, Codex Standard for Canned Sardines and
Sardine-Type Products. Codex Stan 119-1981, Rev. 1-1995, Codex
Standard for Canned Finfish. Codex Stan 165-1989, Rev. 1-1995,
Codex Standard for Quick Frozen Blocks of Fish Fillet, Minced Fish
Flesh, and Mixtures of Fillets and Minced Fish Flesh. Codex Stan
166-1989, Rev. 1-1995, Codex Standard for Quick Frozen Fish Sticks
(Fish Fingers), Fish Portions, and Fish FilletsBreaded or in Batter.
Codex Stan 190-1995, Codex General Standard for Quick Frozen Fish
Fillets.

2012 AOAC INTERNATIONAL