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MB Sample ID: SA245787

Local Sample ID:bivalves_21C_C125_02
Subject ID:SU002545
Subject Type:Invertebrate
Subject Species:Ruditapes philippinarum
Taxonomy ID:129788
Height Or Height Range:length: 3.81 ± 0.42 cm; and width: 3.06 ± 0.51 cm
Gender:Male and female

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Sample Preparation:

Sampleprep ID:SP002551
Sampleprep Summary:Metabolite extraction was performed using a water/methanol/chloroform method, as described in (Hines, Oladiran, Bignell et al., 2007). Briefly, the clams´ soft tissue (0.15 g per sample) was ground with a pestle and mortar, in liquid nitrogen, and then transferred to a microtube, followed by the addition of cold methanol (600 µL), ultrapure water (128 µL) and chloroform (300 µL). The mixture was vortexed, left in ice for 10 min and centrifuged (2,500 g, 4 °C, 10 min). The top layer was transferred into a microtube to which chloroform (300 µL) and water (300 µL) were added. The mixture was vortexed and centrifuged (2,500 g, 4 °C, 10 min). The upper layer (aqueous) was transferred into vials, dried in a centrifugal vacuum concentrator (UNIVAP 100H) and stored at −80 °C until NMR analysis.
Sampleprep Protocol Filename:Bivalves_Experimental_Procedure.docx
Processing Storage Conditions:-80℃
Extraction Method:Water/methanol/chloroform method, as described in (Hines, Oladiran, Bignell et al., 2007)
Extract Storage:-80℃
Sample Resuspension:The dried polar extracts of clam samples were resuspended in 600 μL of sodium phosphate buffer (0.1 M in D2O, 99.96% D, pH 7.4, containing 0.5 mM sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, TSP-d4, chemical shift referencing). The mixture was vortexed and centrifuged (16,000 g, 10 min, room temperature) and 550 μL were transferred into 5 mm NMR tubes.
Sample Spiking:0.5 mM sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP-d4), as a chemical shift reference.
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