The following reagents and authentic standard compounds were obtained
from (suppliers): water, isopropanol, and acetonitrile (Fisher Optima); pyridine
(Acros Organics); C8 – C30 fatty acid methyl esters
[FAMEs], methoxyamine hydrochloride [MeOX],
ethoxyamine hydrochloride [EtOX],
N-methyl-N-(trimethylsilyl)-trifluoroacetamide
[MSTFA],
N-methyl-N-(trimethyl-d9-silyl)-trifluoroacetamide
[MSTFA-d9], ammonium formate, formic acid, and
N-methyl-L-alanine (Sigma-Aldrich);
2′-O-methyluridine-5′-triphosphate,
3′-O-methyluridine-5′-triphosphate,
5-methyluridine-5′-triphosphate (TriLink BioTechnologies);
4-hydroxypropofol-1-O-β-D-glucuronide, and
4-hydroxypropofol-4-O-β-D-glucuronide (Toronto
Research Chemicals).
All metabolites extraction procedures are kept on ice, the quantities
for sample aliquots were 25 μL for blood plasma,
5×106 for cells, 5 mg for tissues, 2 mL for algae
cultures. Metabolites were extracted with 1,000 μL degassed
acetonitrile:isopropanol:water (3:3:2, v/v/v), and then homogenized,
centrifuged, decanted, and evaporated. Extracts were cleaned by 500 μL
degassed acetonitrile:water (1:1, v/v) to remove triglycerides and membrane
lipids, and evaporated again. For GC-MS analysis, internal standards C8
– C30 FAMEs were added to determine the retention index. The dried
samples were derivatized with 10 μL MeOX (or EtOX) in pyridine and
subsequently by 90 μL MSTFA (or MSTFA-d9) for trimethylsilylation of
acidic protons. For LC-MS analysis, the extracted samples were resuspended in 50
μL acetonitrile:water (4:1, v/v) and submitted to instrument.
from (suppliers): water, isopropanol, and acetonitrile (Fisher Optima); pyridine
(Acros Organics); C8 – C30 fatty acid methyl esters
[FAMEs], methoxyamine hydrochloride [MeOX],
ethoxyamine hydrochloride [EtOX],
N-methyl-N-(trimethylsilyl)-trifluoroacetamide
[MSTFA],
N-methyl-N-(trimethyl-d9-silyl)-trifluoroacetamide
[MSTFA-d9], ammonium formate, formic acid, and
N-methyl-L-alanine (Sigma-Aldrich);
2′-O-methyluridine-5′-triphosphate,
3′-O-methyluridine-5′-triphosphate,
5-methyluridine-5′-triphosphate (TriLink BioTechnologies);
4-hydroxypropofol-1-O-β-D-glucuronide, and
4-hydroxypropofol-4-O-β-D-glucuronide (Toronto
Research Chemicals).
All metabolites extraction procedures are kept on ice, the quantities
for sample aliquots were 25 μL for blood plasma,
5×106 for cells, 5 mg for tissues, 2 mL for algae
cultures. Metabolites were extracted with 1,000 μL degassed
acetonitrile:isopropanol:water (3:3:2, v/v/v), and then homogenized,
centrifuged, decanted, and evaporated. Extracts were cleaned by 500 μL
degassed acetonitrile:water (1:1, v/v) to remove triglycerides and membrane
lipids, and evaporated again. For GC-MS analysis, internal standards C8
– C30 FAMEs were added to determine the retention index. The dried
samples were derivatized with 10 μL MeOX (or EtOX) in pyridine and
subsequently by 90 μL MSTFA (or MSTFA-d9) for trimethylsilylation of
acidic protons. For LC-MS analysis, the extracted samples were resuspended in 50
μL acetonitrile:water (4:1, v/v) and submitted to instrument.