1. Separation and Determination of Biogenic Amines by CE Using Salicylaldehyde-5-sulfonate as Derivatizing Reagent

2. CE determination of BA
Direct UV detection: histamine, tyramine, tryptamine, phenylalanine
Advantage (o: simply and clear conditions
Disadvantage /o: only four from eight important BA
low responses of His and Pea
Derivatization – UV or fluorescent detection
Advantage (o: all eight BA
Disadvantage /o: due to loss of the charges of BA‘s amino group is necessary use MECK

3. (!) NEW (!) derivatization reagent salicylaldehyde-5-sulfonate (SAS)
1. R. Driouich, T. Takayanagi, M. Oshima, S. Motomizu, J. of Chromatogr., 2001, 943, 95.
2. R. Driouich, T. Takayanagi, S. Motomizu, Analytical Sciences, 2001, 17, a343.
3. R. Driouich, T. Takayanagi, M. Oshima, S. Motomizu, J. of Pharm. Bioch. Anal., 2003, 20, 1523.
histamine1,2, tyramine2
linear n-aklylamines1,2 and n-alkyldiamines3
lysine3 and diaminopimelic acid3
isoniazid3

4. Salicylaldehyd-5-sulfonic
Formation BA-SAS +
Salicylaldehyd-5-sulfonic
acid
oxim
Conditions of derivatization reaction:
low basic pH (8.0)
20 times excess of concentration of derivatization solution to the amine
presence of ethanol (10–60 %)

5. Formation BA-SAS Cu chelate complex→ +
Cu 2+
chelate
oxim

6. RESULTS and DISCUSSION
Derivatization process:
250 μl sample
250 μl SAS 20 mg/ml 20 mM borate buffer (pH 8.0)
500 μl EtOH
AND/NOT ▪ 200 μl 20 mM CuCl2 (NiCl2, CoCl2)
Dividing of BA into groups:
only BA-SAS:
BA-SAS-Cu:
without any reaction:
Cadaverine, Tyramine, Tryptamine, Phe
Histamine, Putrescine, Spermidine
Spermine

7. CE conditions Instrument: HP CE 3D, HP ChemStation®
Capillary: 40/46.5 cm, 50 μm I.D.
Applied voltage: 25 kV (ramp time 1.25 min)
Capillary temperature: 25 °C
Detection wavelengths: 200, 220, 235, 320 nm
Injection: 25 mbar 10 s
Migrating solution (BGE): 30 mM phosphate buffer (pH 7.81) + 40% (v/v) EtOH

8. Electrophoregram for biogenic amines after derivatization by SAS
Electrophoregram for biogenic amines after derivatization by SAS. Sample solution: 500 μM biogenic amines. Signals: 1, EOF; 2, cadaverine; 3, putrescine; 4, spermidine; 5, tyramine; 6, tryptamine; 7, histamine; 8, 2-phenylalanine; 9, impurity.

9. Electrophoregram for biogenic amines after derivatization by SAS as Cu chelate complex. Sample solution: 200 μM biogenic amines. Signals: 1, EOF; 2, putrescine; 3, histamine; 4, impurity; 5, spermidine; 6, putrescine ??2x putrescine??.

10. Table: Detection limit of the propose method for the determination of the biogenic amines before and after derivatization.
before
mg.l-1
BA-SAS
BA-SAS-Cu
2-phenylalanine
1.9
4.0 -
Histamine
7.6 / 4.5*
9.5
1.2
Tyramine
1.4
3.9
Tryptamine
1.3 / 2.4*
2.9
Cadaverine
Putrescine
10.6
Spermidine
15.7
* 235 nm

11. Conclusion
Complete derivatization and CZE separation of seven biogenic amines have been developed.
Use salicylaldehyde-5-sulfonate as a derivatization reagent has provided simple and sensitive analysis of the biogenic amines.
Detection limit of BA is 10 μmol/l as a BA-SAS-Cu chelate complex and μmol/l as a BA-SAS.
Determination of all biogenic amines into single analysis isn‘t possible due to substantially different structure of these amines => specific reaction.
Cu cation is best for the formation of chelate (water soluble) complex. Ni or Co anions reacts more specific.