Page 55 - Application Handbook - Liquid Chromatography

LAAN-J-LC-E108

LC
Liquid Chromatograph
High-Speed Analysis of Amino Acids and

35 Histamine in Fish Sauce via Automated
OPA Pre-column Derivatization

Processed foods and fish containing a significant amount of histamine have resulted in several cases of allergic food
poisoning. In Japan, no standard values have been set for histamine concentrations in foods. However, the FDA has
specified levels of 50 mg/kg max. for foods in general, the EU has specified 100 mg/kg max. for marine products, and the
Codex Alimentarius standard specifies 400 mg/kg max. for fish sauce.

Simultaneous Analysis of Amino Acids and Histamine in Fish Sauce
Amino acids and histamine were derivatized via o-phthalaldehyde (OPA) and chloroformic acid 9-fluorenylmethyl
(FMOC) using the automatic pretreatment function of the Nexera X2 Ultra High Performance Liquid Chromatograph
(UHPLC) system's SIL-30AC autosampler. A simultaneous analysis of primary amines (OPA derivatives) and
secondary amines (FMOC derivatives), which differ in detection wavelength, was enabled by the wavelength switching
function of the RF-20Axs fluorescence detector.
mV

2750 2 10 100 mV
2500 1 15
75
2250
50
2000
25 14
1750 12
0
1500 7.5 8.0 8.5 min
1250 8 19
4 6 18
1000
7 17 20
750
500 13 16
5
250 9 11 15
3
0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 min
Pretreatment Procedure
1. Mix a 0.5 mol/L aqueous trichloroacetic acid solution with the sample in a 2:1 ratio (v/v ratio), and then centrifuge the
mixture (10,000 rpm for 5 minutes).
2. Mix a 0.3 mol/L aqueous sodium hydroxide solution with the supernatant in a 1:3 ratio (v/v ratio).
3. Dilute the mixture 100 fold with a 0.1 mol/L boric acid buffer solution and then filter it through a 0.2 µm rated
membrane filter before proceeding with the analysis.
Column: YMC Triart C18 1.9 µm Peaks:
(75 mmL. × 3.0 mmI.D., 1.9 µm) 1. Aspartic Acid 7. Threonine 14. Histamine
Mobile phase: A) 20 mmol/L (Potassium) Phosphate buffer (pH 6.5) 2. Glutamic Acid 8. Citrulline 15.Tryptophan
B) Acetonitrile/Methanol/Water = 45/40/15 (v/v/v) 3. Asparagine 9. Arginine 16. Phenylalanine
Gradient Elution 4. Serine 10. Alanine 17. Isoleucine
Flow rate: 0.8 mL/min 5. Histidine 11. Tyrosine 18. Leucine
Column temp.: 35 °C 6. Glycine 12. Valine 19. Lysine
Injection volume: 1 µL 13. Methionine 20. Proline

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First Edition: May, 2013
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Shimadzu Corporation
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© Shimadzu Corporation, 2013