Return to study ST003017 main page

MB Sample ID: SA327634

Local Sample ID:Sp03
Subject ID:SU003131
Subject Type:Mammal
Subject Species:Florida manatee (Trichechus manatus latirostris)
Age Or Age Range:Adult

Select appropriate tab below to view additional metadata details:

Sample Preparation:

Sampleprep ID:SP003137
Sampleprep Summary:Lithium heparinized manatee plasma samples, quality control samples, extraction and solvent blanks, and pooled group samples were extracted via the Folch extraction method (2:1 chloroform: methanol) (Folch et al., 1957). The plasma samples were thawed on ice and briefly homogenized. Next, 50 µL of each sample was added to a glass test tube. Three quality control samples were prepared the same manner using 50 µL of SRM 1950. All samples, except the solvent blanks, were spiked with 50 µL of deuterium-labeled internal standards from 10 different lipid classes at known concentrations (diluted 1:10) (Splash Lipidomix, Avanti Polar Lipids, Alabaster, AL). Biphasic extraction was conducted by adding 3 mL of 2:1 chloroform:methanol solution to each test tube including the quality control samples and extraction blanks. The samples were then vortexed for 10 s, 500 µL of Optima-grade water was added, and the tubes were inverted 5 times. All samples were centrifuged at 2500 g for 5 min at 4 °C. The resulting bottom lipid containing layer was then removed and transferred to a new glass test tube using glass Pasteur pipettes. Samples were divided into sub-groups determined by study site, sex, and reproductive status. Extract pools were created by equal contributions from each sample within a group. Finally, the samples, controls, and pools were evaporated under a steady stream of nitrogen at 25 °C for about 25 min. The samples were then reconstituted with 100 µL of isopropanol and vortexed for 10 s. The reconstituted solutions were then transferred to autosampler vial inserts inside glass autosampler vials for UHPLC-MS/MS analysis.
Sampleprep Protocol Filename:Sample Prep.txt
  logo