Determination of Dyes in Fish Tissue by HPLC/UV

March 2, 2012
Brian Kinsella

Craig A. Perman

The Application Notebook

Volume 0, Issue 0

UCT Application Note

Daniel A. Fonseca, Brian Kinsella, Thomas August and Craig A. Perman, UCT, LLC, Bristol, USA.

Various triarylmethane and fluorescent dyes are used in aquaculture as antibacterial, antifungal and anthelmintic agents for the treatment of fish disease. The US Foods and Drug Administration (FDA) has banned the importation of certain fish from several countries due to possible contamination of these food products. Most published methods only detect malachite green, crystal violet and their metabolites. UCT has developed a procedure for the extraction and analysis of nine dyes used in aquaculture using a carboxylic acid ion-exchange SPE cartridge.

1) Sample Preparation

a. Prepare tilapia fish tissue (purchased at a local grocery store) by homogenization with dry ice.

b. Add 100 µL of each dye standard (50 µg/mL) to one gram of fish tissue.

c. Vortex for 30 s.

d. Add 10 mL of acetonitrile/100 mM ammonium acetate buffer at pH 3 (90:10, v/v).

e. Vortex for 30 s.

f. Centrifuge at 4500 rpm for 5 min and use the supernatant for analysis.

g. Add 10 mL of 100 mM phosphate buffer (pH 7.0) to the supernatant (1:1 dilution).

h. Prepare the vacuum manifold or automated SPE system using EUCCX156 cartridge(s).

Dyes included in the study

2) Condition Cartridges (no vacuum used)

a. Add 3 mL of methanol to each cartridge.

b. Add 3 mL of reagent grade water to each cartridge.

c. Add 1 mL of 100 mM phosphate buffer (pH 7.0).

3) Extract Sample (no vacuum used)

a. Extract sample using 1–2 mL/min flow-rate.

4) Wash Cartridge

a. Add 3 mL of 100 mM phosphate buffer solution to cartridge.

b. Slowly draw through.

c. Dry cartridge for 10–15 min at full vacuum.


5) Elute Dye Analytes

a. Add 3 mL of (95:5) methanol/acetic acid solution (adjusted to pH 2.5).

b. Collect eluate using 1–2 mL/min flow rate.

c. Evaporate eluate to dryness under N2 at < 40 °C.

d. Redissolve residue using 500 µL of methanol:H2O (50:50, v/v).

LC/UV Analysis


Waters 1525 Binary HPLC Pump, 717plus autosampler and 487 UV Detector (other instrumentation may be used)

LC/UV Parameters

Column: Selectra C18, 150 × 2.1 mm, 3 µm particle size (or equivalent)

Injection: 20 µL

Oven temp: 20 °C

Data mode: Dual absorbance: 265 and 600 nm

Pump mode: Gradient

Mobile Phase:

(A) Deionized (DI) H20 with 10 mM ammonium acetate and 5 mM octanesulfonic acid

(B) Methanol with 10 mM ammonium acetate and 5 mM octanesulfonic acid


Water soluble dyes used in fish aquaculture can be readily analysed with good recovery using UCT's EUCCX156 carboxylic acid cation exchange SPE cartridge. No oxidation to the leuco metabolites is required prior to LC/UV detection. An LC/MS/MS method is being developed to detect the dyes at lower concentrations according to FDA and EU criteria and will be published shortly.


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