Table 1.

Examples of recent studies using LC-MS metabolomics with zebrafish embryos, larvae, and tissues.

Collection Time	Sample per Replicate	Quenching/Storage	Extraction Solvent	Analysis	Reference
96 hpf *	30 pooled individuals	Snap-frozen with liquid nitrogen and stored at −80 °C	1 mL (MeOH/ACN/H2O, 40/40/20, v/v/v)	LC-HRMS Mostly polar metabolites, e.g., amino acids, and sugars	[20]
120 hpf	20 pooled individuals	Snap-frozen with dry ice and stored at −80 °C	1.7 mL (MeOH/H2O/CHCl3, 9/5/3, v/v/v)	LC-HRMS Mostly polar metabolites, e.g., amino acids and organic acids	[15]
168 hpf	50 mg (25 mg for metabolomics and 25 mg for lipidomics)	Not mentioned	Polar metabolites: 800 μL of (MeOH/ACN/H2O, 2/2/1, v/v/v) Lipids: 800 μL of −20 °C CH₂Cl₂/MeOH (3/1, v/v)	LC-HRMS Polar metabolites and lipids	[47]
120 hpf	15 pooled individuals	Snap-frozen and stored at −80 °C	590 μL (MeOH/ H2O/CHCl3, 15/15/29, v/v/v) + 10 uL of SPLASH LIPIDOMIX®)	2D-LC-HRMS Lipids	[70]
52 hpf *	10 pooled individuals	Snap-frozen with liquid nitrogen and stored at −80 °C	250 μL H2O for homogenization. Samples were freeze-dried and extracted with 80% MeOH (volume not specified).	LC-HRMS Mostly polar metabolites, e.g., purine metabolism and some lipids of the arachidonic acid metabolism	[78]
144 hpf	20 pooled individuals	10 μL of 13 mM sodium metabisulfite	450 μL of cold MeOH.	LC-MS/MS Mostly polar metabolites, e.g., kynurenine pathway metabolites, neurotransmitters	[18,19]
48 and 120 hpf	80 pooled individuals	Stored at −80 °C	Each 20 μL sample was extracted with 120 μL of cold 50% MeOH.	LC-HRMS Mostly polar metabolites, e.g., amino acids	[46]
120 hpf *	12 pooled individuals	Not mentioned	Samples were homogenized in 1 mL H2O + unknown amount of CH₂Cl₂	LC-MS/MS Lipids	[34]
24, 48, 72, and 120 hpf	15 pooled individuals	Stored at −80 °C	300 µL (MeOH/H2O, 80/20 v/v)	LC-HRMS Mostly polar metabolites, e.g., choline, betaine, methionine, glucose, and TCA cycle metabolites.	[57]
144 hpf	30 pooled individuals	Snap-frozen in liquid nitrogen	1 mL MeOH	LC-HRMS Polar metabolites, e.g., nucleosides, amino acids, and some lipid classes, e.g., sterol lipids, glycerophospholipids, sphingolipids	[22]
172 hpf *	200 pooled individuals (50 mg)	Snap-frozen in liquid nitrogen	400 µL (MeOH/H2O, 4/1, v/v)	LC-HRMS Polar metabolites, e.g., amino acids lipids, e.g., glycerophospholipids, arachidonic acid metabolism	[69]
24, 48, 72, and 120 hpf	10–15 pooled individuals	Stored at −80 °C	Saponification with alcoholic KOH with 1% ascorbic acid. The pH was adjusted to 2.5 with 12 mol/L HCl. Addition of 2.0 mL of hexane. Removed organic supernatant.	LC-HRMS(/MS) Docosahexaenoic acid, eicosapentaenoic acid, Arachidonic acid, and Linoleic acid	[57]
24 hpf	10 pooled individuals	Snap-frozen in liquid nitrogen and stored at −80 °C	SPE: Added samples to 2 mL 1% ascorbic acid in EtOH and 1 mL H2O. Saponification with 300 µL saturated KOH. Neutralization with 3 mol/L HCl to pH 7.5. Lipids were extracted/separated with Strata-X-A 33 mm Polymeric Strong Anion Exchange cartridges (200 mg/3 mL, Phenomenex) using different combinations of organic solvents: MeOH for Cholesterol, ACN for α-tocopherol, FA/ MeOH/ACN (5/47.5/47.5, v/v/v) for PUFAs.	LC-Single Quadrupole (MS) Free fatty acids Commercial Amplex Red Assay Kit (Life Technologies, Carlsbad, CA) Cholesterol LC-Electrochemical Detection α-tocopherol	[35]
24 and 36 hpf	200 and 100 pooled individuals	Snap-frozen in liquid nitrogen and stored at −80 °C	3 mL 66% MeOH	LC-HRMS(MS) Hydroxy-fatty acids, e.g., 7-HDHA, 10-HDHA, 14-HDHA, and 17-HDHA	[35]
72 and 168 hpf	15 pooled individuals	Stored at −80 °C	8 μL/mg cold MeOH and 3.2 μL/mg H2O. Added remaining solvents (8 μL/mg CHCl3 and 4 μL/mg H2O) to the homogenates. Final ratio: MeOH/H2O/CHCl3 (2/1.8/2, v/v/v). Dilution of upper layer 10-fold and transfer to 1.5 mL vial.	LC-MS/MS 22 amino acids + 22 polar metabolites (e.g., urea, betaine, uridine, inosine, xanthine)	[50]
Adult zebrafish **	Intestines (6 pooled individuals) 50 mg	Not mentioned	400 μL of MeOH/H2O (4/1, v/v)	LC-HRMS Polar metabolites and lipids, e.g., fatty acids, glycerophospholipids, carnitines	[29]
90 dpf	Liver (4 pooled individuals)	Snap-frozen and stored at −80 °C	Homogenized in approximately 1.2 mL of MeOH/H2O (4/1, v/v). Split into two fractions at a ratio of 5/1 (v/v) to analyze metabolites and lipids, respectively. Added MTBE, MeOH, and H2O to a final ratio of MTBE/MeOH/H2O (20/6/7, v/v/v) to the lipid fraction.	LC-HRMS Polar metabolites and lipids	[48]
Adult zebrafish **	Liver (8 pooled individuals)	Snap-frozen in liquid nitrogen and stored at −80 °C	Homogenized with 800 μL of MeOH and 200 μL of H2O. Collected 750 μL after centrifugation. Added another 200 μL H2O and 400 μL CHCl3.	LC-HRMS Mostly glycerophospholipids, amino acids, and fatty acids.	[83]

* Approximated value. ** Exact fish age during collection not specified.