Recommended Protocol for Histamine Quantification
SCOPE AND APPLICATION
Principle: Histamine is extracted with methanol
and derivatized with o-phthalaldehyde (OPT) to generate
the fluorescent product (see figure below). This method
is used to determine the histamine content in raw,
precooked, and canned tuna.1-4
1.2 Interference: All methods for histamine
determination are overwhelmed with interfering substances
which have to be removed in order to accurately measure
the histamine present. The two naturally-occurring
substances that cause the most interference are histidine
and spermidine since they also react with OPT to form
fluorescent products.5 However, spermidine,
the major contaminant in extracts, can be separated
from histamine on cellulose phosphate cation-exchange
columns.6 There is also variability due
to the pH and temperature sensitivity of the
Because of the ubiquity of interfering fluorophors,
all reagents used must be of the highest obtainable
purity. Exposure of any of the materials involved
to rubber or silicones may produce erratic results.8
It is recommended that polyethylene labware be used
in place of glass, due to an observed loss of fluorescence.9
All labware should be acid washed and rinsed in distilled
water. New solution must be prepared after four to
seven days, due to an observed increase in blank readings.8
SUMMARY OF METHOD
The histamine-containing materials are homogenized
and extracted with methanol. The extract can then
be passed through an anion exchange column to remove
any remaining interfering substances. The elutant
is reacted with the OPT reagent and allowed to stand
for 4 minutes. The mixture is acidified with H3PO4
and the corresponding fluorescence is read on a calibrated
è è è è
3. APPARATUS AND EQUIPMENT
Assorted Class A calibrated pipettes.
BioSystems TD-700 Laboratory Fluorometer with standard
photomultiplier tube (PMT) (P/N 7000-009)
Near UV mercury-vapor lamp (P/N 10-049)
300 nm - 400 nm Excitation Filter (P/N 10-069R)
410 nm - 500 nm Emission Filters (P/N 10-059R and
10x10 mm Square Methacrylate Cuvettes (3.5 ml) (P/N
3.1 Labware. All reusable labware (glass,
polyethylene, Teflon, etc.) should be cleaned by soaking
in laboratory grade detergent and water for 4 hours,
rinsed with tap water, deionized water, and methanol.
It is recommended that polyethylene ware be used due
to absorbance observed when using glass.
3.1.2 Graduated cylinder, 100-mL.
3.1.3 Assorted volumetric flasks for preparing
Chromatographic columns (Kontes #K-422250).
REAGENTS AND STANDARDS
Ion exchange resin: Sigma 1X8-200, chloride form 100-200
mesh; or BioRad AG1-X8, 50-100 mesh, chloride form,
Cat. No. 140-1431, or equivalent.
1.0N Sodium hydroxide: Dissolve 40g NaOH in 1
liter of distilled water.
2.0N Sodium hydroxide: Dissolve 80g NaOH in 1
liter of distilled water.
Histamine dihydrochloride: MCB #HX0440 or J.T.
1.0N Hydrochloric acid: Add 83 ml concentrated
HCl to about 500 ml distilled water. Cool and bring
to 1 liter volume with distilled water.
0.1N Hydrochloric acid: Add 100ml 1N HCl to about
500 ml distilled water. Cool and bring to 1 liter
volume with distilled water.
Methanol, reagent grade.
0.1% o-Phthalaldehyde(OPT reagent): Phthalic dicarboxaldehyde
(Aldrich, Milwaukee, WI), or o-Phthaldialdehyde (Sigma,
St. Louis, MO), C6H4(CHO)I2,
F.W. 134.13. Dissolve 0.10 g OPT in 100 ml methanol.
Store in an amber bottle and refrigerate when not
in use. Prepare fresh weekly.
3.57N Phosphoric acid: Add 121.8 ml of 85% H3PO4
to about 500 ml distilled water. Bring to 1 liter
volume with distilled water.
Histamine Standard Solution A, 1mg Hm/ml: Weigh
0.1656 g of histamine dihydrochloride into 100 ml
volumetric flask. Dissolve in, and dilute to volume
with 0.1N HCl.
Histamine Standard Solution B, 10 ug Hm/ml: Dilute
1.0 ml Solution A to 100 ml with 0.1N HCl.
Histamine Standard Solution A1(this is our control
solution): Dilute 1.0 ml Solution A to 100 ml with
Histamine Standard Solution C, 0.1 m g Hm/ml:
Dilute 1.0 ml Solution B to 100 ml with 0.1N HCl.
Histamine Standard Solution D, 0.2 m g Hm/ml:
Dilute 2.0 ml Solution B to 100 ml with 0.1N HCl.
Histamine Standard Solution E, 0.3 m g Hm/ml:
Dilute 3.0 ml Solution B to 100 ml with 0.1N HCl.
Prepare Solutions A and B monthly and Solutions C,
D, E, and A1 weekly. Refrigerate solutions when not
Place 20 g of ion exchange resin in a beaker.
5.1.2 Add 2N sodium hydroxide to the resin
in a ratio of 15 ml per gram of resin.
5.1.3 Mix well and allow the resin to settle
for a minimum of 15 minutes, but no more than 30 minutes.
Decant liquid and repeat with additional 2N sodium
5.1.4 Wash resin thoroughly with distilled
water to remove traces of the sodium hydroxide until
pH is less than or equal to 8.5.
5.1.5 Slurry resin with distilled water and
transfer to a funnel containing a fluted filter paper.
Thoroughly wash with distilled water.
5.1.6 Transfer resin to a suitable container
and make sure the distilled water level is above the
resin level at all times.
5.2 Column Preparation:
Slurry sufficient prepared resin into each column
to form a bed 8 cm in height. Maintain a liquid
level above the top of the resin at all times.
5.2.2 Refill columns with fresh resin at
least twice per week.
Install the excitation and emission filters in the
filter cylinder. Then, insert the filter cylinder
into the sample chamber. For additional assistance,
refer to Section III of your TD-700 Operating Manual.
Insert the lamp. Turn on the instrument and allow
to warm-up for 10 minutes. For additional assistance,
refer to Section IV of your TD-700 Operating Manual.
Calibrate instrument in the direct concentration mode
with the prepared histamine standard solutions C,
D, and E. Blank with a reagent blank.
Blend fish in a Waring blender with an equal weight
of deionized water to produce a 1:1 slurry.
7.1.2 Transfer 10.0 g of the slurry to a 150
ml beaker. Add 40.0 ml of methanol and mix thoroughly.
7.1.3 Using Whatman #1 filter paper, or equivalent,
filter the mixture into a suitable container. If the
filtrate is to be saved for later analysis, refrigerate
in a closed container.
NOTE: Evaporation of methanol from the filtrate can
cause erroneous results.
Pass 15-20 ml distilled water through the exchange
column and discard.
7.2.2 Place a 50 ml volumetric flask containing
5 ml 1N HCl at the column outlet.
7.2.3 Pipet 1.0 ml of filtrate (methanol extract)
onto the resin bed with 5-10 ml distilled water.
7.2.4 Immediately initiate column flow. Flow
should be maintained at a rate greater than 3 ml/min.
7.2.5 When liquid level is slightly above the
resin, add about 5 ml distilled water and allow it
to flow through the resin. Repeat with distilled water
in larger increments until total water through column
is about 40 ml.
7.2.6 Discontinue column flow.
7.2.7 Remove volumetric flask and bring to
50 ml volume with distilled water. Store column effluent
in the refrigerator if necessary to postpone determination
for more than 2 hours.
At the beginning of a set of analyses, and again at
the end, pass 1 ml of Solution A1 through one of the
columns and proceed through the procedure as though
it were a fish extract. Fluorescence readings should
be very similar to Solution D reading. If readings
are not within 20% of Solution D, all analyses performed
at the same time are suspect and should be repeated.
Controls and Blanks:
7.3.2 After every 14 samples, 1 ml of methanol
should be put through a column and run through the
procedure as a fish extract. If a reading on one of
these blanks is more than 5 units higher than the
original blank reading, resin contamination is apparent
and corrective action should be taken immediately.
Into separate 25 ml glass stoppered flasks, pipet
5.0 ml of 0.1N HCl (Blank); Solutions C, D, and
E; and each diluted column effluent.
7.4.2 Add 10 ml 0.1N HCl to each flask.
7.4.3 Add 3 ml 1N NaOH. Mix thoroughly.
7.4.4 Within 5 minutes, add 1 ml OPT solution
and mix thoroughly.
7.4.5 After exactly 4 minutes, add
3 ml 3.57N H3PO4 and mix immediately.
7.4.6 Let solutions stand for 15-20 minutes
and then determine the fluorescence intensities
on the TD-700 Laboratory Fluorometer. If a sample
reading is greater than that of Solution E, dilute
25 ml of the column effluent to 100 ml with 0.1N
HCl and proceed from step 7.4.1.
CAUTION: Fish with high salt content may cause problems
with the resin necessitating more frequent changing
7.4.7 If sample dilution was necessary in
step 7.4.6, multiply the obtained result by 4.
Waldron & Bond, FLUOROMETRIC DETERMINATION OF
HISTAMINE IN TUNA: DEVELOPMENT OF METHOD. JAOAC,
Vol. 60, No. 5, 1977.
FLUOROMETRIC DETERMINATION OF HISTAMINE IN TUNA:
COLLABORATIVE STUDY. JAOAC, Vol. 60, No.
FLUOROMETRIC METHOD - OFFICIAL FIRST ACTION. JAOAC,
Vol. 60, No. 2, 1977.
METHODS OF ANALYSIS OF THE AOAC, 13th Edition, 1980.
M.C., S. Rubio, A. Gomez-Hens and M. Valcarcel,
SIMULTANEOUS DETERMINATION OF HISTAMINE AND SPERMIDINE
BY SECOND-DERIVATIVE SYNCHRONOUS FLUORESCENCE SPECTROMETRY,
Anal. Chim. Acta, 193: 349-354 (1987).
Arthur and Petrea Z. Coffman, AN IMPROVED ION-EXCHANGE
PURIFICATION PROCEDURE FROM THE FLUOROMETRIC ASSAY
OF HISTAMINE, Anal. Biochem., 27: 257-261
Rolf and Anna-Lisa Ronnberg, IMPROVED FLUOROMETRIC
ASSAY OF HISTAMINE: CONDENSATION WITH o-PHTHALALDEHYDE
AT -20° C, Anal. Biochem., 60: 560-567 (1974).
Joseph W. and Alta Brand, A FLUOROMETRIC METHOD
TO DETERMINE LEVELS OF HISTAMINE IN HUMAN PLASMA,
J. Allerg., 32: 236-240 (1961).
Dorothy von and David Glick, IMPROVEMENTS IN FLUOROMETRIC
MICRO DETERMINATIONS OF HISTAMINE AND SEROTONIN,
Anal. Biochem., 29: 167-171 (1969).
Extraction: Lorenz, W., L. Benesch, H. Barth,
E. Matejka, R. Meyer, J. Kusche, M. Hutzel and E.
Werle, FLUOROMETRIC ASSAY OF HISTAMINE IN TISSUES
AND BODY FLUIDS: CHOICE OF THE PURIFICATION PROCEDURE
AND IDENTIFICATION IN THE NANOGRAM RANGE, Z.
Anal. Chem., 252: 94-98 (1970).
Blood Extraction: Noah, Joseph W. and Alta
Brand, A FLUOROMETRIC METHOD TO DETERMINE LEVELS
OF HISTAMINE IN HUMAN PLASMA, J. Aller., 32:
236-240 (1961). Noah, Joseph W. and Alta Brand,
SIMPLIFIED MICRO-METHOD FOR MEASURING HISTAMINE
IN HUMAN-PLASMA, J. Lab. Clin. Med., 62: 506-510
Brain: Medina, M. and P.A. Shore, INCREASED
SENSITIVITY IN A SPECIFIC FLUOROMETRIC METHOD FOR
BRAIN HISTAMINE, Biochem. Pharmacol., 15: 1627-
Oates, John A., Edward Marsh and Albert Sjoerdsma,
STUDIES ON HISTAMINE IN HUMAN URINE USING A FLUOROMETRIC
METHODS OF ASSAY, Clin. Chim. Acta, 7: 488-497
Zarower, A., R.H. Dunlop and N.L. Norcross,
FLUOROMETRIC ANALYSIS OF HISTAMINE AND SERATONIN
IN BOVINE MILK, Amer. J. Vet. Res., 26: 1339-1343
Zachariae, H., SKIN HISTAMINE AND DELAYED SKIN
REACTIONS: FLUOROMETRIC DETERMINATIONS ON PATCH
TESTS, Acta. Allerg., 19: 336-350 (1964).
Methods: Michaelson, Arthur, and Petrea Z. Coffman,
AN IMPROVED ION-EXCHANGE PURIFICATION PROCEDURE
FOR THE FLUOROMETRIC ASSAY OF HISTAMINE, Anal.
Biochem., 27: 257-261 (1969).
Hakanson, Rolf and Anna-Lisa Ronnberg, IMPROVED
FLUOROMETRIC ASSAY OF HISTAMINE: CONDENSATION WITH
o-PHTHALALDEHYDE AT -20° C, Anal. Biochem.,
60: 560-567 (1974). Michaelson, I. Arthur and
Petrea Z. Coffman, AN IMPROVED ION-EXCHANGE PURIFICATION
PROCEDURE FOR THE FLUOROMETRIC ASSAY OF HISTAMINE,
Anal. Biochem., 27: 257-261 (1969). Redlich,
Dorothy von and David Glick, IMPROVEMENTS IN FLUOROMETRIC
MICRO DETERMINATIONS OF HISTAMINE AND SERATONINE
, Anal. Biochem., 29: 167-171 (1969). Yusem,
Milton, William E. Delaney, Margaret A. Lindberg
and Edward M. Fashing, AN IMPROVED FLUORIMETRIC
DETERMINATION OF HISTAMINE BY STABILIZING THE o-PHTHALALDEHYDE
REAGENT. Anal. Biochem., 44: 403-409 (1969).
Information: Beaven, Michael A., HISTAMINE,
New Eng. J. Med., 294: 30-36 and 320-325 (1976).
This suggested method for the analysis of histamine
is provided to us from Eddie Robalino, QC Manager
of I.N.E.P.A.C.A (Industria Ecuatoriana Productora
De Alimetos C.A.) in Manta, Ecuador. It has not
been validated by Turner BioSystems.