2.1 Instruments and devices
SHIMADZU GCMS-TQ8040 triple quadrupole gas chromatography-mass spectrometry (SHIMADZU technology co., LTD.), ASE 350 accelerated solvent extraction (Thermo mercer technology co., LTD.), AutoEVA30Plus automatic nitrogen blowing concentrator (Reco Group Co., LTD.), IKA T25 high-speed tissue homogenizer (Germany Aika Instrument Equipment Co., LTD.), SK8200H ultrasonic cleaning machine (Shanghai Keguide Ultrasonic Instrument Co., LTD.), VORTEX 2 VORTEX (SI DIGITAL, USA), Mili-q type pure water meter (Merck Millebo Company), Dk-8d Type Thermostatic electric water bath (Shanghai Yiheng Technology Co., LTD.), Gnp-9050 Water-proof Constant temperature Incubator (Shanghai Precision Scientific Instrument Co., LTD.), Model AL104 Electronic analytical balance (Mettler Toledo, Switzerland).
2.2 Standards and reagents
Cholesterol (≥99%, CHER), cholestanol (≥95%, CHAN), brassicasterol (≥95%, BRER), ergosterol (≥95%, ERER), campesterol (≥95%, CAER), stigmasterol (≥95%, STER), β-sitosterol (≥95%, SIER), stigmasterol (≥95%, SIER), stigmastanol(≥95%, STAN), all the above standard substances were purchased from Tianjin Alta Technology Co., LTD. 1-methylimidazole (≥99%), and N-Methyl-N-triMethylsilylheptafluorobutyraMide (≥99%) was purchased from (Shanghai Aladdin Biochemical Technology Co., LTD.), Potassium hydroxide and petroleum ether (30-60 °C) were purchased from Tianjin Kermeo Reagent Co., LTD.), dichloromethane ethanol purchased from (Shanghai Anpu Experimental Technology Co., LTD.) rapeseed samples purchased from Hailar area. dichloromethane ethanol purchased from Shanghai Anpu Experimental Technology Co., LTD.) rapeseed samples purchased from Hailar area.
2.3 Pretreatment method
2.3.1 Fat extraction
Weighed 5.00g (accurate to 0.01g) of rapeseed in a small beaker, added 3-5 g of quartz sand, stirred evenly, then poured into a 22 mL extraction tank, the extraction solvent was petroleum ether 30-60℃, extraction temperature 100℃, extraction pressure 10.0 MPa, static extraction 6 Min, rinsed volume 30%, cycle twice, purge for 100 s, dry the extraction tube, bake to constant weight (the extraction tube needs to be roast to constantly weight in advance), the difference between the two weights is the fat content in the sample.
2.3.2 Total sterol extraction
Quantify and weighed 50 mg (accurate to 0.1 mg) of the oil extract in a centrifuge tube, added 4 mL of potassium hydroxide ethanol solution (2 mol/L) and 0.5 mL of dichloromethane, vortex, and mixed well, then centrifuge for 5 min at 30 ℃ in a shaker water bath for 18 h at 10,000 r/min. Added 5 mL dichloromethane and 3 mL water, mixed them gently, centrifuged them at 10000 r/min for 5 min, removed the upper water phase, and washed the organic phase with 5 mL water three times until clarified the lower solution. The organic phase obtained was blown with nitrogen in the water bath at 40~50℃ and stopped when the remaining 1 mL was left. And transfer the organic phase to the reaction flask for use.
2.3.3 Derivatization procedure
Under the terms of 40℃ , slowly blow out the solvent in the reaction flask with nitrogen gas, and then 100 μL of silanization reagent (N-Methyl-N-triMethylsilylheptafluorobutyraMide: 1-methylimidazole 950:50) was added. The reaction vials were tightly capped and placed on a thermostat at 75 °C for 20 min. Cool the flask to room temperature, add n-ethane and allow to reflux to 1 mL, standard solution derivatization method is the same as the sample derivatization procedure and supplied to a gas chromatography-tandem mass spectrometer for detection.
2.4 Test conditions
2.4.1 Chromatographic Conditions
Carrier gas control mode: Constant column flow (1.2 mL/min) Chromatographic column :DB-5MS (30 m 0.25 mm 0.25 μm) or equivalent column, Injector temperature :290, Injection method: split injection split ratio 10:1, Flow rate: 1.2 mL/min, Column temperature programmed: the initial temperature was 100 (for 1 min), increased to 220 at 50 ℃/min, and increased to 290 at 5 ℃/min (for 8 min).
2.4.2 Mass spectrometry conditions
Electron bombardment (EI) ion source, Voltage energy:70 eV, Transmission line temperature 300, Ion source temperature 250, Selective ion acquisition mode (SIM).
2.4.3 Preparation of standard sample and preparation of standard curve
2.4.3.1 Reserve solution of the standard curve
To make the stock solutions of -sitosterol and the other seven sterols, accurately weigh 5 mg -sitosterol into a PTFE tube and 1 mg each of cholesterol, cholestanol, rapeseed sterol, ergosterol, campesterol, stigmasterol, and stigmasterol into another PTFE tube, respectively, adding 100 L of silanization reagent, derivatized in an oven at 75 °C
2.4.3.2 Standard curve working solution
The concentration gradients of β -sitosterol were 5.0, 50.0, 5.0×102, 2.5×103, 1.0×104 mg/L by diluting the reserve solution with n-hexane. The concentration gradients of the standard sample solutions of other substances mixed are 2.0, 20.0, 2.0×102, 1.0×103, 2.5×103 mg/L and are now available for use.