(A) Hydrolysis and silylation of sterols for GC-MS analysis

200 µL of crude lipids sample and 50 µg of 5β-cholestan-3α-ol (internal standard dissolved in dichloromethane) were transferred into a 5 mL glass vial fitted with a Teflon-lined screw cap, and contents were evaporated to dryness using a rotary evaporator at 35 °C

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1 mL of 0.5 M methanolic potassium hydroxide (KOH) were added and placed in a water bath at 85 °C for 15 min (for hydrolysis)

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Hydrolyzed samples were immediately cooled in ice and partitioned with 2 mL of cyclohexane and 1 mL of 1 M sodium chloride (NaCl)

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One mL from the upper cyclohexane phase containing sterols was carefully transferred into a new 5 mL Teflon lined glass tube and vacuum-dried in a rotary evaporator at 35 °C

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For silylation of sterols, 1 mL pyridine, and 50 µL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) and added and incubated at 60 °C for 60 min

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After incubation, contents were cooled in ice, filtered through a 0.45 μm PTFE syringe filtered and transferred to a 1.5 mL autosampler vial for gas chromatograph-mass spectrometry (GC-MS) analysis

(B) Preparation of fatty acid methyl esters (FAMEs)

500 µL of crude lipids sample was transferred into a 5 mL glass vial fitted with a Teflon-lined screw cap

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Contents were evaporated to dryness using a rotary evaporator at 35 °C, and 1 mL of boron trifluoride-methanol solution (14% in methanol), and incubated for 10 min at 60 ° in a heat block

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After cooling, the FAMEs were washed with 1 M NaCl and recovered in 2 mL hexane. A pinch of anhydrous sodium sulfate (Na2SO4) was added to the recovered sample (hexane) to absorb the traces of water

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One mL of sample was filtered through a 0.45 μm PTFE syringe filter and transferred to a 1.5 mL autosampler vial for GC-FID analysis and GC-MS analysis