different analysis methods used to
determination amount of histamine in fish product especially tuna fish which is
the most kind of fish consumed in the middle east because it’s the cheapest
type comparing to another types , these analysis methods used to determine
histamine are HPLC ,GC, TLC and CE.
the comparison between these method
done according to different parameters : sample pretreatment, derivatization required, column used , staitarinay phase used , mobile
phase used , time analysis, lower detection limit , detector , linearity , and
Reagent used .
Key word: histamine, HPLC,
TLC, CE, GC.
Histamine is a chemical messenger
that mediates several cellular responses: inflammatory reactions, allergic reaction,
gastric and acid secretion, limited neurotransmitter action in the brain.
Histamine and receptors are present
essentially in all tissue types, there are especially high concentration of
histamine receptors on cells found in the lungs , skin, blood vessels, an GI
tract . histamine is stored in granules in mast cells throughout the body ,
histamine can be released in response to
tissue injury resulting from cold , heat, toxins ( microbial organism ) , venom
( snacks , insect ) , trauma and ingested histamine rich food . when histamine
released it can bind to histamine receptors ( H1,H2,H3,H4) the most common
histamine receptors (H1 and H2) , several effect are possible happened
according to tissue type and location
when histamine bind to (H1, H2 ) receptors .( see table one) .
Type of histamine receptors
Peripheral sensory neurons
Itching, sometimes pain.
Intestinal smooth muscle
Contraction, cramps, diarrhea.
Production mucus, rhinorrhea, coughs.
Pulmonary smooth muscle
Decrease lung capacity and asthma.
Hypotension, increase heart beat.
Leakage of fluid and protein into
Stimulate gastric acid secretion
that lead to peptic ulcer.
Table 1: effects that present from
binding histamine to histamine receptors.
Diamine oxidase enzyme ( DAO) located in the intestinal mucosa mainly become
active during the digestion of histamine rich food , during metabolism of histamine and if there is not
enough DAO activity( the main cause of enzymatic dysfunction has a genetic
origin ) the imbalance between ingested histamine and the histamine released
from the storage cells leads to histamine accumulation in plasma , that’s
lead to adverse effect on health ( histamine
intolerance , histamine toxication ) , that leads to the following symptoms :
diarrhea , headache , asthma , flushing , irregular heartbeat, hypotension ,
and red welts on the skin , to reduce these problem avoid histamine rich food
that’s shown in( table 2 ).
Citrus food, papaya,
strawberries, pineapple, nuts, peanuts, tomatoes, spinach, chocolate.
Fish , shrimp , egg white
Table 2: histamine rich food.
histamine produce in food by
decarboxylation of histidine amino acid to histamine through microbial ( morganella
morganii ) or enzymatic) proteolytic
enzyme) processes under temperature ubused conditions. the aims of my term
paper to determination of histamine in fish especially tuna fish ( highest type
consumed ) in several advanced techniques ( High pressure liquid chromatography
HPLC , thin layer chromatography TLC , gas chromatography GC , capillary
electrophoresis CE ) and compartion these techniques with each other.
Standard amine solution prepared by
used stock solution containing different amino acid (1000mgl) diluted in HCL (0.1M)
or TCA (5% trichloroacetic acid), added to it 1.7- diaminoheptane as internal
standard to know the concentration of analyte in the standard.
Sample pretreatment in capillary
electrophoresis done by extraction and purification that’s done by homogenized
tuna fish sample with extraction solution either in HCL or in TCA then stored
the sample (at 4c for 7 days) to crystallize most of the fat, centrifugation
and filtration and purification by LLE (liquid- liquid extraction) by
N-butanol then the sample ready to analysis.
the same thing done in HPLC
but after filtration a neceeary step
done which is neutralization ( ph 6-7) to keep native form for amino acid then
purification done by both ( LLE , SFE solid face extraction ) SFE done by
activation ( methanol + distilled water + HCL+ sample extract ) then washing by
potassium citrate buffer and finally elution with potassium citrate buffer –
isopropanol . before the sample injection the derivatization of pre-column by OPA ( o-phathaldialdehyde ) which
is the most common one of derivatization because it react with histamine in 15
second that is very necessary to facilate detection and reduce time of analysis
separation of analyte in CE done by
used capillary electrophoresis column that contain fused silica , the capillary
conducted with sodium hydroxide , water and buffer ( to make histamine more
stable under acidic condition ph = 2.44) the flushed of buffer done in sequence
interval , the capillary inlet must be closed after each flushed interval done
to reduce the time of exposed on the capillary , injection of the sample done
under pressure , temperature constant 25c , and the voltage was gradually
ramped up in different amount to minimize the possible sample loss inside the
Detection of analyte in CE happened
by diode array detector (DAD) 2mg/kg while detection in HPLC happened by both
fluorescence detector 0.5mg/kg and DAD 1mg/kg. the time used for separation of analyte in CE 9 min
while in HPLC 20 min. the retention time used for elution histamine in CE 4min
while in HPLC 8 min. the linearity that done by calibration curve for CE is up
to 1000 mg/kg while in HPLC up to 100
mg/kg . the correlation between HPLC and CE is very good ( the correlation of
histamine = 0.994). See (table 3).
Extraction + purification
Extraction + neutralization +
Correlation with HPLC
0.994 for histamine
Analysis time ( including sample
15min ( 35min)
30min (90min )
Lower detection limit
2-6 mg /kg
1-8.5 mg /kg
Up to 1000
Up to 100
Histamine , tyramine
Histamine, tyramine, cadaverine, putrescine,
Table 3: comparation HPLC versus CE.
Preparation of standard amine done
by stock solution prepared by diluted about (0.2-0.25g) of each amino acid in
10 ml of (TCA) trichloroacetic acid.
derivatizataion of standard amine
done by take 1ml of standard amine in 5ml screw cap tube added to it 1ml of
phosphate buffer (ph=9) , drop of NaOH and 2 ml of dansly reagent ( 50mg of
dansyl chloride and 10 ml acetone ).then cover test tube with aluminum ,
incubated it at 55c for 1hour , to done perfect
densalyation that necessary to reduced total run time .sample
pretreatment in thin layer chromatography (TLC) done by extraction and
derivatization , extraction happened by homogenized 10g of tuna fish sample with 30 ml of 5% hot TCA (80-90c)
for 2 Min then centrifugation ,filtration
the supernatant solution, 1ml of filtrate derivatization similar to
standard amine derivatization.
separation of analyte (mixture of amino acid ) in TLC done by take
10 microlitiers of it and applied on precoated silica gel plate (stationary phase
) then added to it mobile phase
(Chloroform: triethylamine (100:25)) after
that the plate sprayed with isopropanol: triethylamine (8:2) to enhance the
flouoresence. While in HPLC the separation of analyte done by injection
(5-8microlitter) of it in c18 column and added mobile phase (methanol: water
(70:30)) after that to make good separation of all amine by increasing methanol
concentration gradually (75%, 80%, 100%).
Detection in TLC done by UV at 365nm, while in
HPLC done at 245. the lower detection limit in HPLC (5-10 ng)while in TLC (15-20 ng ) , the analysis time used for one
sample in HPLC 30 min while in TLC 10 min , the number of sample that can be
analysis at same time in TLC is 10-12 comparing to HPLC one sample can analysis
, so the cost in TLC lower than HPLC because the amount of reagent used lower
than HPLC ( 1ml for one sample in TLC
comparing to HPLC 30 ml for one sample ) , also the cost of HPLC higher it need
expensive solvents, high operational
skill and careful maintenance. the linearity done by HPLC up to 100ng while in
TLC up to 300ng , the repapilty for HPLC lower than 3% while in TLC lower than
8% ,the senseviety of HPLC 9.35 while TLC 0.84 , the correlation for HPLC ( 0.998-0.999) while TLC (0.996-0.999) that’s
mean there are marginal differences between two method. (See table 4) .
Lee than 3%
less than 8%
time used for analysis
number of sample that can
analysis at same time
10-12 in one plate
lower detection limit
amount of reagent used for
analysis one sample
(Table 4: compartion HPLC versus TLC).
Preparation of standard solution in
gas chromatography done by dissolving 50mg of internal standard (1, 9 nanodiol)
and histamine in 100 ml methanol. compared these with CE they used
different internal standard which is
(1.7-diaminoheptane ) while in GC ( 1,9 nanodiol ) that is necessary for both
techniques to known concentration of analyte in the standard , while in HPLC
standard solution can prepared with and without internal standard according
to the aims of the analysis , while in
thin layer chromatography they don’t used internal standard because the system
of it is very simply compared to other techniques it does not contain column
only stationary phase formed as plate .
sample pretreatment in GC done by extraction
and derivatization that’s done by dissolving sample of tuna fish in alkaline
methanol after that the extractor sample derivatization with pentafluoropionic
anhydride to make it volatile , compared to HPLC derivatization of pre-column
necessary to rapid analysis procedure , also in TLC derivatization is very
necessary to reduce time of analysis while in CE derivatization is not
necessary and not applied because during analysis buffer flushed to make
histamine more stable and to reduce amount of lost histamine .
Injection of sample done by used
three different polarity columns (Cp-sill 5CB, Cp-sill 8CB, and CP-sill 9CB)
and nitrogen gas used to make separation and to make reactant between
stationary phase and analyte more active under oven condition (160-280c) and
the separation done according to polarity.
Detection done by flame ionization detector
(FID) the lower detection limit (5microgram /g). the analysis time used to
analysis less than 20 min compared to
another technique ( HPLC 30min ,TLC
10min, CE 15 min ) which is the third
one arrangement according to consuming time , the retention time used to
detect histamine differ according to column used ( 4.66min, 4.77min,5.24min ) as
the above arrangement used , according
to that CP-sil5cB the more efficient column used to detection histamine comparing
to another columns , recovery for it ( 2.7-7.8% ) . (See table 5).
Extraction , purification
Extraction, neutralization, purification,
Can do with it and with out of it
(50 mg of dansyl chloride and 10
(Cp-sill 5CB, Cp-sill 8CB, CP-sill
Liquid (buffer , NaOH , water )
Liquid ( water + methanol )
Liquid (chloroform: triethylamine)
Inert gas (nitrogen gas )
Lower detection limit
2-6 mg /kg
5 microgram /g
diode array detector ( DAD)
Fluorescence detector and diode
flame ionization detector(FID)
Up to 1000 ng
Up to 100 ng
Up to 300ng
Table 5: CE, TLC, HPLC, GC
the most problem that’s happened in
HPLC method is derivatization step that’s need more time to done which is
required controlled reaction conditions , also can effect peak brooding and sample dilution , so analysis without
derivatization is not improved good detection . HPLC method need high technical
skill, time and cost, it’s more applicable in determine information about the
composition of biogenic amine in food sample, that’s mean it’s suitable for
food quality control.
TLC method that’s features in rapid
method which required 10 min , and less expensive so it’s sutabile for analysis
different sample at the same time , according to that it’s more applicable in
fish industry to detect biogenic amine especially toxic histamine.
GC method that’s accurate method, quicker than
HPLC about 20 min need to complete analysis, it’s more applicable in determine
histamine and another biogenic amine in fish and other food.
CE has higher theoretical
resolution than other analysis methods , no organic solvent used so the cost of
reagent and capplieries is reduced , quicker than HPLC and GC , more accurate
method no interference without result , not required derivatization, its applicable to determine histamine
concentration in marine product .
According to previous, histamine
produced in food by decarboxylation of histidine amino acid to histamine
through microbial or enzymatic process under temperature ubused conditions.
histamine can determine by several
methods (HPLC, CE, TLC, GC) according to the comparison between these method
the best one is CE that’s the highest theoretical resolution than other method,
no organic solvent used so coast reduced, quicker than HPLC and GC time consumed
15min, more accurate method no interference between the result, not required
derivatization step that’s consumed more time.