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Protocol for Analyses of Cholesterol Metabolites of Optic Nerve Using GC-MS Methods

Carry out all procedures at room temperature unless otherwise specified.

1 Tissue Preparation and Lipid Extraction

a) Dissect the optic nerve and trim off the fatty layer that surrounds the optic nerve. Add the optic nerve tissue sample to a cryovial, and determine the total weight of the tissue using an electric balance.

b) Break the tissue membrane with five freeze/thaw cycles by alternating between -80°C (or liquid nitrogen) and 40 °C (with water bath).

c) Mince the optic nerve tissue with scissors for 60 s. After mincing, flush the vial with argon gas and keep the tissue sample aside.

d) Add a 400 μL of methanol/chloroform (1:1) to the optic nerve tissue sample.

e) Apply a homogenizer to the tissue for 120 s.

f) Add 350 μL of chloroform to the sample and apply homogenizer for another 30 s. Close tube tightly and vortex for 45 s.

g) Centrifuge the samples twice for 15 min at 10,000 × g. Observe the sample and the two layers—upper (aqueous) and lower (organic). If the two layers are not apparent, vortex the tube for 60 s and centrifuge again for 15 min.

h) Pre-weigh two empty vials and label them as proteins and lipids. Pipette out the top fraction into the vial-labelled proteins, and pipette out the bottom fraction into the vial-labelled lipids. Set proteins vial aside. Flush the lipids tube with argon gas.

i) Put the chloroform tubes into speed vacuum. SpeedVac at room temperature for 90 min. Observe the tube after 90 s, and if liquid is still present, SpeedVac until all are dry with 15 min intervals. Weigh the tube. Flush with argon gas and store at -80°C until use for mass spectrometer.

2 Gas Chromatography Mass Spectrometry Analysis

Use an Agilent CP-Sil 8 CB, column with ultrahigh purity helium as the carrier gas with a flow rate of 1.2 mL/min. The conditions are as follows: injector temperature and injection volume of 1 μL in splitless mode. The initial oven temperature is 100 °C for 4 min, ramped to 318 °C at a rate of 10 °C/min, and held at 318 °C for 6 min for a total run time of 31.8 min.

2.1 Preparation of Lipid Standards

a) Lipid standards such as cholesterol and cholesterol d7 are run separately to confirm efficiency of the GC to obtain proper spectra. Usually, the concentration of these standards is between 50 and 100 μL in chloroform.

b) These standard solutions are run five times through the GC-MS to ensure reproducibility.

c) For quantification purposes, a calibration curve is obtained by running solutions with varying concentrations of cholesterol and cholesterol-d7.

2.2 Preparation of Lipid Samples

a) The dried lipid samples from Subheading 1, step i are resuspended in 100 μL of chloroform.

b) 50 μL internal standard (100 picoM of Cholesterol d7) is added to the lipid samples for quantification purposes. Quantification is determined by comparing the peak area of the cholesterol d7 in the spectra with the rest of the peaks.


  1. Sanjoy K. Bhattacharya. (2019). Metabolomics: Methods and Protocols. Springer-Verlag.
* For Research Use Only. Not for use in diagnostic procedures.
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