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Abstract
The study evaluated β-ecdysone concentration (C27H44O7), which a major molting hormone, in crab hemocyte plasma of commercial softshell crab. Quantification of β-ecdysone level by High Performance Liquid Chromatography (HPLC) will supported for utilization of suitability quality diet and regime feeding for molting crab stage. Samples were collected in Can Gio farm, salinity at 15 ‰, the body weight of 5 crab samples ranges from 130 to 150g. Blood was drawn from the crab via cheliped by using a syringe contained 1-ml heparinized (citrate/EDTA anticoagulant).
Samples were centrifuged at 14000 rpm in 15 minutes; the plasmas in supernatant were collected and stored at -30ºC. β-ecdysone solutions were enriched through AccuBond SPE ODS-C18 columns and eluted with 5% methanol solvent. The extracts then were vacuum dried and diluted with 60 μl methanol. Before injecting (20 μl) into the HPLC, samples were filtered through a 0.22 μm membrane. For analysis conditions, Ecosil C18 column was used for separating, mobile phase MeOH:water (v:v) in 60:40 ratio, flow rate 0.5 ml/min, wavelength 244 nm and oven at 40ºC.
The retention time of β-ecdysone standard was in range of 7.7-8.9 min, peak reach at 8.376 min. In the chromatogram of crab blood plasma samples, peaks appear in range of 7.7-8.9 min, peak at 8.156 min, with 89.43% recovery efficiency. At the pre-molting period, the highest β-ecdysone content in hemocyte plasma was detected at 45ng/ml the crab and the lowest at 20ng/ml. The determination of β-ecdysone concentration in hemocyte plasma can help to select appropriate diets for effectively molting process and optimization feeding time.
Key words: High Performance Liquid Chromatography (HPLC), Scylla paramamosain, β-ecdysone