5-HT and 5-HIAA measurement in male tilapia brain area 2 and 3 (control = 8, defeat = 8) was carried out using liquid chromatography-mass spectrometry (the Agilent Technologies 6410 Triple Quad Liquid chromatography-mass spectrometry equipped with a ZORBAX SB- C18 column). The procedures were as described previously (4 (link)). Briefly, the brain tissues were homogenized with 350 μL of 20 ng/mL isoproterenol (internal standard, Nacalai Tesque) in 50% acetonitrile (Fisher Scientific) with 0.1% formic acid (Sigma). The supernatant was filtered by Cosmospin filter G (Nacalai Tesque). A standard solution of 5-HT, 5-HIAA (Sigma), 5-HIAA, and 20 ng/mL isoproterenol was prepared in 50% acetonitrile with 0.1% formic acid in the range of 1.25 to 160 ng/mL. The production of 5-HT, 5-HIAA, and isoproterenol was observed for further analysis (m/z 177.1–160 for 5-HT, m/z 192.07–146 for 5-HIAA, and m/z 212.1–194 for isoproterenol). Data acquisition software Agilent Masshunter Quantitative Analysis software (RRID: SCR_015040, Agilent Technologies) was used for data analysis.
Protocol detail
Quantifying 5-HT and 5-HIAA in Tilapia Brain
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5-HIAA
SB- C18 column
isoproterenol
formic acid
Cosmospin filter G
acetonitrile
Agilent Masshunter Quantitative Analysis software
A 5 ht
Acetonitrile
Brain
Brain 2
Formic acid
Hiaa
Isoproterenol
Liquid chromatography
Male
Mass spectrometry
Tilapia
Tissues
Corresponding Organization :
Other organizations : Monash University Malaysia
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Variable analysis
independent variables
- Defeat condition (control = 8, defeat = 8)
- 5-HT measurement in male tilapia brain area 2 and 3
- 5-HIAA measurement in male tilapia brain area 2 and 3
- Brain tissue homogenization with 350 μL of 20 ng/mL isoproterenol (internal standard, Nacalai Tesque) in 50% acetonitrile (Fisher Scientific) with 0.1% formic acid (Sigma)
- Filtration of supernatant using Cosmospin filter G (Nacalai Tesque)
- Preparation of standard solution of 5-HT, 5-HIAA (Sigma), and 20 ng/mL isoproterenol in 50% acetonitrile with 0.1% formic acid in the range of 1.25 to 160 ng/mL
- Observation of the production of 5-HT, 5-HIAA, and isoproterenol (m/z 177.1–160 for 5-HT, m/z 192.07–146 for 5-HIAA, and m/z 212.1–194 for isoproterenol)
- Use of Agilent Masshunter Quantitative Analysis software (RRID: SCR_015040, Agilent Technologies) for data analysis
- Positive control: Standard solution of 5-HT, 5-HIAA (Sigma), and 20 ng/mL isoproterenol in the range of 1.25 to 160 ng/mL
- Negative control: Not explicitly mentioned
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