MA 102 / ANALYSIS OF BIOGENIC AMINES
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, the only sample preparations necessary are centrifugation/filtration.
Extraction Procedure:
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, 80 % HPLC Methanol and 20 % 0.1 N HCl, 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
Analytical Conditions
Column: ALKION™ cation-exchange, K+ form, 4 x 150 mm,
Catalog No. 9410917 ALKION™ Guard column, 3 x 20 mm, Catalog No. 9493020
Temperature: 45 °C
Flow Rate: 0.8 mL/min
Mobile Phase: K600, K563, K130
Post-column Conditions
Post-Column System: Pinnacle PCX
Reactor Volume: 0.15 mL
Temperature: 45 °C
Reagent: 300 mg of OPA, 2 g of Thiofluor™, 3 mL of 30 % Brij® 35 in 950 mL of OD104
Flow Rate: 0.3 mL/min
Detection: Fluorometer λex: 330 nm, λem: 465 nm
|
Time (Min) |
K600 % |
K563 % |
K130 % |
|---|---|---|---|
|
0 |
100 |
0 |
0 |
|
6 |
100 |
0 |
0 |
|
15 |
0 |
100 |
0 |
|
21 |
0 |
100 |
0 |
|
21.1 |
0 |
0 |
100 |
|
23 |
0 |
0 |
100 |
|
23.1 |
100 |
0 |
0 |
|
29 |
100 |
0 |
0 |
