Method Validation for Determination of Methyltestosterone in Nile Tilapia by Supercritical Fluid with Carbon Dioxide as a Green Extraction Technique for HPLC Analysis
Methyltestosterone (MT) is a synthetic anabolic steroid hormone. It is worldwide known for its use in fisheries and aquaculture to induce a mono-sex population, especially in Nile tilapia (Oreochromis niloticus) production. In this study, the quantitative analysis for residual methyltestosterone in Nile tilapia tissue was conducted using HPLC (high-performance liquid chromatography). A green extraction technique was carried out. Supercritical fluid with carbon dioxide was conducted at 31 ⁰C, 75 bars, and 10 minutes. The analysis was performed on RP-C18 (4.6 mm × 250 mm, 5 µm) using the mobile phase water-acetonitrile (30:700, v/v), 1.0 ml min-1 of flow rate, temperature at 25 ⁰C, and wavelength of 245 nm. A good linearity was obtained for methyltestosterone detection between concentrations of 25.0 – 600.0 µg L-1, R2 = 0.9991 and LOD = 8.0 µg L-1. Samples were spiked with methyltestosterone at levels of 10.0, 20.0, 30.0, and 40.0 ng g-1 and used to validate the method. The mean recovery value greater than 95% was achieved and RSD was less than 5%. These established results demonstrate the applicability of supercritical fluid carbon dioxide as a green extraction technique for residual methyltestosterone extraction in fish tissue.
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