Electric homogenizer

Metabolites were extracted from whole embryos using the two-step methanol:water:chloroform (2:1.8:2 final solvent ratio) protocol as described previously58 (link). Briefly, pre-weighed frozen samples were homogenized in 8 μL/mg cold methanol and 3.2 μL/mg cold water using an electric homogenizer (Tiangen Biotech, China). All of the remaining solvents (8 μL/mg chloroform and 4 μL/mg water) were added to the homogenates. The samples were vortexed for 1 min, left on ice for 10 min to partition and then centrifuged for 20 min at 12,000 rpm at 4 °C. The upper layers were diluted 10-fold and then transferred into 1.5 mL auto-sampler vials.

During the embryo-larvae exposure test, solutions were collected for determination of PCB atropisomers at the beginning of treatment and 24 h post exposure. Water samples (1 L) were subjected to five liquid-liquid extractions with n-hexane (100 mL each time) in a separator funnel along with violent shaking. The n-hexane layer was transferred to heart-shaped flasks and concentrated to near-dryness by rotary evaporation (Shanghai Ailang Instruments, Shanghai, China) at 35°C. The concentrated solutions were then blown to dryness by nitrogen evaporation and the residue was again dissolved in 0.1 mL of isooctane for GC-MS analysis.
Twenty embryos or larvae samples in triplicate per treatment were weighed and homogenized with 0.1 mL isooctane using an electric homogenizer (Tiangen Biotech, China). After 20min ultrasonic extraction, samples were centrifuged at 5000 rpm for 10 min. The supernatant after 0.22-μm fiter was for GC-MS analysis.
An Agilent 7890A/5975C GC-MS system equipped with a Chirasil-Dex capillary column (25 m × 0.25 mm; I.D. 0.25 μm df) from Agilent was used for the purity and concentration determinations. The oven temperature was programmed as follows: 60°C for 2 min, 60–150°C at 10°C·min^-1 (held for 5 min), 150–180°C at 1°C·min^-1 (held for 22 min). SIM ions were m/z 360 (quantification ion), 362, and 358 [29 (link)].