Tetracycline antibiotics (TCAs) are used globally in aquaculture to control disease and promote growth. The tetracyclines used in veterinary medicine in Japan account for more than half the total of antibiotics used.¹ The overuse of TCAs can result in their presence in seafood for human consumption.

This is a serious problem in Japan where seafood is consumed in copious amounts and prawns are the Japanese consumer's favourite choice. A report that 2.3 ppm of oxytetracycline (OTC) was detected in imported Japanese Tiger Prawn (Marsupenaeus japonicus) magnified these concerns. This level is greater than the minimal risk level (MRL) of 0.1 µg/g in fish — singly or in combination — the EU has set for OTC, chlortetracycline (CTC) and tetracycline (TC).² Correspondingly, the Codex MRL for OTC is 0.2 µg/g for Black Tiger Prawn (Penaeus monodon),³ which is consistent with the value set for fish by the Japan Ministry of Health, Labor, and Welfare.⁴

The EU, Japan and the US import more than 70% of the world seafood market⁵ and it is essential to monitor the safety of seafood for consumers in relation to the presence of tetracyclines.

The ideal method must be simple, quick, cost-effective and cause negligible harm to the environment and the analyst. High performance liquid chromatography (HPLC) is now the preferred method for analysis of many substances, including TCAs of biological interest.⁶ HPLC interfaced with ultraviolet (UV), fluorescence (F),^8,9 photo-diode array (PDA),^10–12 and mass spectrometery (MS) detection,^7,13 have been reported. MS is very expensive and is not available in a lot of labs for routine analysis, particularly in developing countries.

Environmental Impact

The methods mentioned in references 8–13 share crucial disadvantages. They all consume organic solvents in the HPLC mobile phases and in the solutions used to extract and deproteinize the sample during the sample preparation stage.The disposal of organic solvents is a global environmental problem in terms of risk to humans and environmental impact.^4–17

Incineration of waste organic solvents has steadily increased over the past 10 years but is very expensive.^18,19 Reducing or, if possible, eliminating the use of organic solvents is, therefore, an important goal in terms of environmental conservation, human health and the economy.

All these techniques also involve at least one extracting/purifying step using large amounts of organic solvent, which consumes time and money if a large number of samples need to be analysed. Additionally, most of these methods did not detect OTC, CTC and TC simultaneously. This article describes an inexpensive technique that does not use organic solvents to determine OTC, CTC, TC in prawns. This involves a fast sample preparation technique followed by RP-HPLC interfaced with a PDA detector using 100% aqueous conditions for the mobile phase and sample preparation stage.

Experimental

Procedure: Black Tiger Prawn tissues were minced fully and used as the blank samples. An accurately weighed 0.1 g sample was taken into a 1.5 mL microcentrifuge tube and homogenized with a hand-held ultrasonic-homogenizer (model HOM-100, 2 mm i.d. probe, Iwaki Glass Co. Ltd, Funabashi, Japan) for 30 s with 0.6 mL of a 20% (w/v) trichloroacetic acid (TCA) solution. After being homogenized, the capped tube was centrifuged at 12000 g for 5 min. The supernatant liquid was filtrated through the 0.45 µm filter unit and the filtrate was then injected into an HPLC system.

HPLC: The HPLC system included a model PU-980 pump and DG-980-50 degasser (Jasco Corp., Tokyo, Japan) equipped with a model CO-8010 column oven (Tosoh Corp., Tokyo, Japan), as well as a model SPD-M10A VP PDA detector (Shimadzu Scientific Instruments, Kyoto, Japan).

Operating conditions: The analytical column was a Capcell Pak 70 C1 UG120 (S-5) packed with C1 methyl-silica, 5 µm d_p, 35 mm × 4.6 mm i.d. (Shiseido Co. Inc., Tokyo, Japan) equipped with a guard column (5 mm × 4.6 mm i.d.) containing the same packing material; the isocratic mobile phase was 0.1 mol/L citric acid; the flow-rate was 1.0 mL/min; column temperature was 45 °C; injection volume was 20 µL; and the analytical time was less than 4 min. The PDA detector was operated at 200–380 nm. Monitoring the wavelength was adjusted to 363 nm, which represents an average maximum wavelength for all the target compounds.