Vitamin E is mainly tocopherol, including four forms of α, β, γ, and δ. Fat-soluble vitamins include retinol, alpha-tocopherol, beta-tocopherol, gamma-tocopherol, and delta-tocopherol. A method for simultaneous detection of 5 fat-soluble vitamins is as follows:
Step 1. Solution configuration and calibration
(1) Preparation of standard products
Take 200 µL of α-tocopherol at a concentration of 5 mg/mL, 20 µL of β-tocopherol at a concentration of 50 mg/mL, 200 µL of γ-tocopherol at a concentration of 1 mg/mL, 20 µL of δ-tocopherol at a concentration of 50 mg/mL, and 560 µL BHT chromatographic methanol with a concentration of 0.1%, mixed evenly to obtain a 1mg/mL vitamin E standard;
Take 50 μL of retinol with a concentration of 1 mg/mL, 500 μL of vitamin E standard with a concentration of 1 mg/mL, and 450 μL of BHT chromatographic methanol with a concentration of 0.1%, vortex and mix well to obtain a solution containing 50 μg/mL vitamin A and 500 μg/mL Mixed standard of vitamin E.
Store at -80°C in the dark for 3 months and avoid repeated freezing and thawing.
(2) Calibration of standard products
Draw 20 μL of a mixed standard substance containing 50 μg/mL vitamin A and 500 μg/mL vitamin E, put it in a 5 mL volumetric flask, and after constant volume with absolute ethanol, calibrate the concentration of the standard substance with an ultraviolet spectrophotometer, respectively in retinol, Measure the UV absorbance value of α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol at the UV absorption wavelength, calculate the actual concentration of the standard according to the formula (1) and (2), and avoid light during the whole process Operation, where C is the actual concentration of the standard to be calibrated in g/100mL, A is the average absorbance of the standard measured at the ultraviolet absorption wavelength, and E is the 1% standard specific absorptivity of the sample to be tested.
C=A/E×dilution factor (1)
Dilution factor=10000×5mL/20μL(2)
(3) Dilution of standard
The standard was serially diluted with 0.1% BHT methanol solution to obtain standard solutions with concentrations of 0.5 μg/mL, 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL and 50 μg/mL. Store at -80°C in the dark for 3 months and avoid repeated freezing and thawing.
(4) Configuration of internal standard working solution
Accurately weigh 2 mg of pentadeuterated retinol, place it in a 10 mL volumetric flask, and dilute to 10 mL with absolute ethanol to obtain a 200 μg/mL pentadeuterated retinol solution; accurately weigh 2.5 mg of hexadeuterated α-tocopherol Phenol was placed in a 5mL volumetric flask, and the volume was adjusted to 5mL with absolute ethanol to obtain a 500 μg/mL hexadeuterio-α-tocopherol solution. The calibration method is the same as that of the standard product.
Step 2. Sample pretreatment
Add the internal standard working solution containing 0.125 μg/mL pentadeuterated retinol and 1.25 μg/mL hexadeuterated α-tocopherol to the sample to be tested, dilute with pure water or aqueous formic acid, add ethanol, methanol or acetonitrile solution, vortex Vortex and mix at high speed to remove protein, add n-hexane for extraction, vortex mix and centrifuge to get the supernatant, dry the supernatant with nitrogen at room temperature, add reconstitution solution, vortex mix and centrifuge to obtain the supernatant is the pretreated sample.
Step 3, liquid chromatography-mass spectrometry analysis
Add 7 μL of the pretreated sample into the injection bottle of the liquid chromatography-mass spectrometry instrument. The chromatographic column uses a Shimadzu C18 column with a specification of 5 μm×2.1 μm×50 mm. The temperature of the chromatographic column is 25 ° C. The chromatographic column adopts a gradient Elution method, the mobile phase of gradient elution adopts ammonium formate-acetonitrile mixed solution, the flow rate of mobile phase is 0.5mL/min, the time of gradient elution is 6min, wherein, the condition of gradient elution is as shown in Table 1, specific It is: within the first 0-0.3min, the mobile phase is constant at 50% ammonium formate and 50% acetonitrile; within the first 0.3-0.6min, the volume percentage of ammonium formate is continuously reduced from 50% to 5%, and the volume percentage of acetonitrile is from 50% % continuously increased to 95%; within 0.6-1.8min, the mobile phase was constant at 5% ammonium formate and 95% acetonitrile; within 1.8-2min, the volume percentage of ammonium formate decreased continuously from 5% to 2%, acetonitrile The volume percentage of ammonium formate continuously increases from 95% to 98%; within the first 2-5min, the mobile phase is constant at 2% ammonium formate and 98% acetonitrile; within the first 5-5.2min, the volume percentage of ammonium formate continuously increases from 2% To 50%, the volume percentage of acetonitrile continuously decreased from 98% to 50%; in the first 5.2-6min, the mobile phase was constant at 50% ammonium formate and 50% acetonitrile.
Step 4. Linear relationship and limit of quantitation
The standard solutions with concentrations of 0.5 μg/mL, 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL and 50 μg/mL were analyzed by liquid chromatography-mass spectrometry, and the chromatographic peak area is the ordinate, with the concentration of the analyte as the abscissa, draw the standard curve, the linear weight is 1/x,
Target Regression equation R
Retinol y=3.45129x+-0.00506 0.999
a-tocopherol y=0.17573x+-0.00284 0.999
β-tocopherol y=0.05136x+0.01238 0.999
γ-tocopherol y=0. 32888x+0.01578 0.996
Delta-tocopherol y = 0.05501x+0.02208 0.993
The results showed that the concentration of retinol, α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol showed a good linear relationship with the corresponding chromatographic peak areas, and the linear range obtained was the same as the clinical reportable range : Linear range and clinically reportable range of 0.05-5 μg/mL for retinol, linear range and clinically reportable range of 0.1-10 μg/mL for γ-tocopherol, α-tocopherol, β-tocopherol and δ-tocopherol The linear and clinically reportable range of tocopherol is 0.5-50 μg/mL.
Step 5. Recovery and precision
The internal standard working solutions of vitamin A and vitamin E were taken respectively, and three concentrations of samples were prepared for the sample recovery and precision experiments.
