Recovery of tetrodotoxin from pufferfish viscera extract by amine-functionalized magnetic nanocomposites

Abstract

Tetrodotoxin (TTX) has been widely used in pharmacology, food poisoning analysis, therapeutic use, and neurobiology. In the last decades, the isolation and purification of TTX from natural sources (e.g., pufferfish) were mostly based on column chromatography. Recently, functional magnetic nanomaterials have been recognized as promising solid phases for the isolation and purification of bioactive compounds from aqueous matrices due to their effective adsorptive properties. Thus far, no studies have been reported on the utilization of magnetic nanomaterials for the purification of TTX from biological matrices. In this work, an effort has been made to synthesize Fe₃O₄@SiO₂ and Fe₃O₄@SiO₂-NH₂ nanocomposites for the adsorption and recovery of TTX derivatives from a crude pufferfish viscera extract. The experimental data showed that Fe₃O₄@SiO₂-NH₂ displayed a higher affinity toward TTX derivatives than Fe₃O₄@SiO₂, achieving maximal adsorption yields for 4epi-TTX, TTX, and Anh-TTX of 97.9, 99.6, and 93.8%, respectively, under the optimal conditions of contact time of 50 min, pH of 2, adsorbent dosage of 4 g L^-1, initial adsorbate concentration of 1.92 mg L^-1 4epi-TTX, 3.36 mg L^-1 TTX and 1.44 mg L^-1 Anh-TTX and temperature of 40 °C. Interestingly, desorption of 4epi-TTX, TTX, and Anh-TTX from Fe₃O₄@SiO₂-NH₂-TTX investigated at 50 °C was recorded to achieve the highest recovery yields of 96.5, 98.2, and 92.7% using 1% AA/ACN for 30 min reaction, respectively. Remarkably, Fe₃O₄@SiO₂-NH₂ can be regenerated up to three cycles with adsorptive performance remaining at nearly 90%, demonstrating a promising adsorbent for purifying TTX derivatives from pufferfish viscera extract and a potential replacement for resins used in column chromatography-based techniques.

Fig. 1. Characteristics of Fe₃O₄, Fe₃O₄@SiO₂, Fe₃O₄@SiO₂–NH₂, Fe₃O₄@SiO₂-TTX, Fe₃O₄@SiO₂–NH₂-TTX. XRD (A), VSM (B), TGA (C), and FT-IR (D).

Fig. 2. EDS spectra of Fe₃O₄ (A), Fe₃O₄@SiO₂ (B), Fe₃O₄@SiO₂–NH₂ (C), Fe₃O₄@SiO₂-TTX (D), Fe₃O₄@SiO₂–NH₂-TTX (E) and SEM image of Fe₃O₄ (F), Fe₃O₄@SiO₂ (G), Fe₃O₄@SiO₂–NH₂ (H), Fe₃O₄@SiO₂-TTX (I), Fe₃O₄@SiO₂–NH₂-TTX (J).

Fig. 3. HPLC spectra of TTXs standards (A) and pufferfish viscera extract (B).

Fig. 4. Variation of 4epi-TTX, TTX, and Anh-TTX concentrations over adsorption time by Fe₃O₄@SiO₂ (A) and Fe₃O₄@SiO₂–NH₂ (B), adsorption yield (C), adsorption capacity (D) and adsorption rate of Fe₃O₄@SiO₂ and Fe₃O₄@SiO₂–NH₂ (E). Adsorption conditions: temperature, 25 °C; pH, 7.0; stirring rate, 150 rpm; adsorbent dosage, 4 g L⁻¹; initial adsorbate concentrations, 1.28 mg L⁻¹ 4epi-TTX, 2.24 mg L⁻¹ TTX, and 0.96 mg L⁻¹ Anh-TTX.

Fig. 5. Adsorption yield of Fe₃O₄@SiO₂ (A) and Fe₃O₄@SiO₂–NH₂ (B) for 4epi-TTX, TTX, and Anh-TTX under different pH. Adsorption conditions: temperature, 25 °C; stirring rate, 150 rpm; adsorbent dosage, 4 g L⁻¹; initial adsorbate concentrations, 1.28 mg L⁻¹ 4epi-TTX, 2.24 mg L⁻¹ TTX and 0.96 mg L⁻¹ Anh-TTX; contact time, 50 min.

Fig. 6. Adsorption yield of Fe₃O₄@SiO₂ (A) and Fe₃O₄@SiO₂–NH₂ (B) for 4epi-TTX, TTX and Anh-TTX under different Fe₃O₄@SiO₂ and Fe₃O₄@SiO₂–NH₂ dosages. Adsorption conditions: temperature, 25 °C; pH, 2.0; stirring rate, 150 rpm; initial adsorbate concentrations, 1.28 mg L⁻¹ 4epi-TTX, 2.24 mg L⁻¹ TTX and 0.96 mg L⁻¹ Anh-TTX; contact time, 50 min.

Fig. 7. Adsorption capacity of Fe₃O₄@SiO₂ and Fe₃O₄@SiO₂–NH₂ for 4epi-TTX (A), TTX (B) and Anh-TTX (C) under different initial concentrations of 4epi-TTX, TTX, and Anh-TTX. Adsorption conditions: temperature, 25 °C; pH, 2.0; stirring rate, 150 rpm; adsorbent dosage, 4 g L⁻¹; contact time, 50 min.

Fig. 8. Adsorption yield of Fe₃O₄@SiO₂ (A) and Fe₃O₄@SiO₂–NH₂ (B) for 4epi-TTX, TTX, and Anh-TTX at different temperatures. Adsorption conditions: pH, 2.0; stirring rate, 150 rpm; adsorbent dosage, 4 g L⁻¹; initial adsorbate concentrations, 1.92 mg L⁻¹ 4epi-TTX, 3.36 mg L⁻¹ TTX, and 1.44 mg L⁻¹ Anh-TTX; contact time, 50 min.

Fig. 9. Recovery yield of 4epi-TTX, TTX, and Anh-TTX from Fe₃O₄@SiO₂-TTX (A) and Fe₃O₄@SiO₂–NH₂-TTX (B) using different solvents. Adsorption conditions: temperature, 40 °C; pH, 2.0; stirring rate, 150 rpm; adsorbent dosage, 4 g L⁻¹; initial adsorbate concentrations, 1.92 mg L⁻¹ 4epi-TTX, 3.36 mg L⁻¹ TTX, and 1.44 mg L⁻¹ Anh-TTX; contact time, 50 min. Desorption conditions: temperature: 50 °C, extraction time 30 min.

Fig. 10. HPLC spectra of 4epi-TTX, TTX, and Anh-TTX recovered from Fe₃O₄@SiO₂-TTX (A) and Fe₃O₄@SiO₂–NH₂-TTX (B) using 1% AA/ACN solvent.

Fig. 11. Adsorptive performance of Fe₃O₄@SiO₂ (A) and Fe₃O₄@SiO₂–NH₂ (B) for sequential adsorption and desorption of 4epi-TTX, TTX, and Anh-TTX. Adsorption conditions: temperature, 40 °C; pH, 2.0; stirring rate, 150 rpm; adsorbent dosage, 4 g L⁻¹; initial adsorbate concentrations, 1.92 mg L⁻¹ 4epi-TTX, 3.36 mg L⁻¹ TTX, and 1.44 mg L⁻¹ Anh-TTX; contact time, 50 min. Desorption conditions: solvent, 1% AA/CAN; temperature, 50 °C; extraction time 30 min.