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Method Abstract
for Post-column Liquid Chromatography 102
Here you can download this material in PDF format*
Histamine and other polyamines such as Putrescine,
Cadaverine, Spermidine and Spermine are products
of decomposition. The level of histamine, in
particular, has been used as a regulatory
guideline for degree of decomposition in fish.
Also, Histamine and Tyramine levels are of
interest to producers of wine and cheese. The
polyamines, Spermidine, Spermine and Putrescine
are elevated in neoplastic and damaged, e.g.,
burned, tissue. In fact, the level of circulating
and urinary polyamines is an indicator of total
body damage. The levels can be monitored for the
effect of diet and other modalities on recovery.
This method employs ion-exchange chromatography
so, after extraction, centrifugation/filtration
are the only sample preparations necessary.
Weigh 10 g of sample into a
small glass blender cup (any fortifications should
be added at this time). Add 50 mL of extraction
solution, Catalog No. 1700-1119, and homogenize
for two minutes. Centrifuge for five minutes. Mix
equal portions of the supernatant with the mobile
phase (K600). Allow to coagulate at -4 °C for 15
minutes, then centrifuge for five minutes. The
clear supernatant is filtered (0.45 μm Nylon) and
placed in an autosampler vial.
METHOD
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Analytical Conditions |
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COLUMN: |
ALKION™ cation-exchange,
K+ form, 4 x 150 mm,
Catalog No. 9410917
ALKION™ Guard
column, 3 x 20 mm,
Catalog No. 9493020 |
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TEMPERATURE: |
40 °C |
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FLOW RATE: |
0.8 mL/min |
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MOBILE PHASE: |
K600, K563, K130 |
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Post-Column Conditions |
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POST-COLUMN SYSTEM: |
Pinnacle PCX |
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REACTOR VOLUME: |
0.15 mL |
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TEMPERATURE: |
45 °C |
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REAGENT: |
300 mg of OPA, 2 g of Thiofluor™,
3 mL of 30% Brij® 35 in 950 mL of OD104 |
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FLOW RATE: |
0.3 mL/min |
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DETECTION: |
Fluorometer
 ex:
330 nm
em:
465 nm |
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0 |
100
|
0
|
|
12 |
100
|
0
|
|
12.1 |
0
|
100
|
|
15 |
0
|
100
|
|
15.1 |
100
|
0
|
|
20 |
100
|
0
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| 1 |
Histamine |
| 2 |
Putrescine |
| 3 |
Cadaverine |
|
|
|
|
 |
|
0 |
100
|
0
|
0
|
|
6 |
100
|
0
|
0
|
|
15 |
0
|
100
|
0
|
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21 |
0
|
100
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0
|
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21.1 |
0
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0
|
100
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23 |
0
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0
|
100
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23.1 |
100
|
0
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0
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29 |
100
|
0
|
0
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| 1 |
Histamine |
| 2 |
Putrescine |
| 3 |
Cadaverine |
| 4 |
Spermidine |
| 5 |
Spermine |
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| |
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