CN111751477B - Method for determining content of squalene and beta-sitosterol in vegetable oil - Google Patents

Abstract

The invention discloses a method for determining the content of squalene and beta-sitosterol in vegetable oil, which comprises the following steps: s1, saponification treatment of vegetable oil: placing the uniformly mixed vegetable oil sample in a glass test tube with a plug, adding an internal standard and a potassium hydroxide-methanol solution, and then placing the mixture in a constant-temperature water bath kettle for saponification; s2, SPE small column purification: diluting the saponified sample solution with 80% methanol solution, passing through a solid phase extraction column, eluting impurities with 80% methanol, eluting the target compound with ethyl acetate, collecting all eluents, concentrating to a constant volume, and passing through a membrane to obtain the sample to be detected. The method has simple detection steps and less solvent consumption, and is suitable for batch detection of the content of squalene and beta-sitosterol.

Description

A kind of method for measuring squalene and beta-sitosterol content in vegetable oil

technical field

The present invention relates to a sample processing method, in particular to a method for determining the content of squalene and beta-sitosterol in vegetable oil.

Background technique

Edible vegetable oil is an important source of human energy and fatty acid intake. In addition, vegetable oil also contains various trace components such as squalene, phytosterol, and vitamin E. These fat concomitants are also an important part of people's daily diet. . In the early years, oil nutrition research mostly focused on the composition of fatty acids in oil, one-sided emphasis on the role of fatty acids, and insufficient understanding of the importance of trace fat concomitants in oil. In recent years, with the improvement of living standards, consumers have gradually begun to pay attention to the intake of these beneficial trace components in vegetable oils, in order to achieve a healthy diet and dietary balance.

Squalene is a highly unsaturated natural terpenoid, which belongs to lipid unsaponifiables. It has various physiological functions such as improving the activity of superoxide dismutase in the body, enhancing the immunity of the body, anti-aging, anti-fatigue, and anti-tumor. . Phytosterols are a class of natural alcohol compounds with cyclopentane polyhydrophenanthrene as the skeleton. They have the functions of lowering cholesterol, anti-cancer, and preventing and treating cardiovascular diseases. They are known as "the key to life". The most important phytosterols in vegetable oils are brassicasterol, stigmasterol, campesterol and β-sitosterol.

Both squalene and phytosterols in vegetable oils belong to the unsaponifiable components of lipids. Currently, the methods for the determination of squalene or phytosterols in vegetable oils mainly include gas chromatography, gas chromatography-mass spectrometry, high performance liquid chromatography, liquid Chromatography-mass spectrometry, etc., and most of these reported literatures still use the classic saponification-liquid-liquid extraction as the pretreatment method. The traditional liquid-liquid extraction method has cumbersome operation steps, long time consumption, high consumption of flammable, explosive and toxic reagents, and easy emulsification, which leads to poor reproducibility of the method.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for measuring the content of squalene and β-sitosterol in vegetable oil, so as to solve the problem that when measuring squalene and β-sitosterol in vegetable oil, the operation steps are cumbersome, time-consuming, flammable and explosive. The consumption of toxic reagents is high, and the easy emulsification leads to the problem of poor method reproducibility.

In order to achieve the above object, the present invention provides the following technical scheme: a method for measuring the content of squalene and β-sitosterol in vegetable oil, which is characterized in that comprising the steps:

S1, vegetable oil saponification treatment: take the mixed vegetable oil sample, put it in a stoppered glass test tube, add internal standard and potassium hydroxide-methanol solution, and then put it into a constant temperature water bath for saponification;

S2, SPE cartridge purification: the saponified sample solution is diluted with 80% methanol solution, passed through a solid phase extraction cartridge, rinsed with 80% methanol for impurities, and finally eluted with ethyl acetate to elute the target compound, collect all washes The sample to be tested is obtained after deliquoring, concentrating to constant volume and passing through the membrane.

Preferably, the step S2 is specifically:

S21. Activation: pipette 5 mL of methanol solution and 5 mL of 80% methanol solution, take the SPE cartridge, and use 5 mL of methanol solution and 5 mL of 80% methanol solution to wash the cartridge in turn;

S22, sample loading: the saponified sample solution in the step S1 is loaded onto the SPE small column;

S23, rinsing: rinse the small column with 5 mL of 80% methanol, rinse twice, and use a vacuum pump to completely dry the liquid;

S24. Elution: place a centrifuge tube under the SPE column, add ethyl acetate to elute the sample, and drip dry naturally to obtain a sample eluent;

S25. Concentration: put it into a nitrogen blower, and blow the sample eluent until it is close to drying and stop;

S26, dilute to volume: dilute to 1 mL with ethyl acetate;

S27. Filtration: filter the sample solution after constant volume through a membrane to prepare a sample to be tested.

Preferably, adding the internal standard in the step S1 is specifically: adding squalane and 5α-Cholestan-3β-ol as the internal standard respectively, and the concentrations are both 1 mg/mL.

Preferably, in the step S1, the constant temperature of the water bath is 70°C, and the saponification reaction time is 60min.

Preferably, the concentration of potassium hydrochloride-methanol solution in the step S1 is 2 mol/L.

Preferably, a 0.22 μm organic filter membrane is used as the filter membrane in the step S2.

Compared with the prior art, the present invention has the following beneficial effects: the present invention adopts the solid phase extraction small column purification of reversed-phase polymer filler as a means to purify the squalene and β-sitosterol in the vegetable oil simultaneously, and is purified by GC-FID. Detecting, using the internal standard method to accurately quantify the content of the two, the invention adopts solid-phase extraction column purification instead of the traditional liquid-liquid extraction purification method, the detection steps are simple, the solvent consumption is small, and it is suitable for squalene and squalene in vegetable oil samples. Batch detection of β-sitosterol content, environmental protection, safety, high efficiency, complete purification, basically no impurity interference; high detection sensitivity, while achieving higher sensitivity and lower detection limit, good reproducibility and stability .

Description of drawings

Fig. 1 is a kind of flow chart of the method for measuring squalene and β-sitosterol content in vegetable oil provided by the invention;

Fig. 2 is the gas chromatogram of the determination of squalene and β-sitosterol in the standard sample in the embodiment;

Fig. 3 is the gas chromatogram of the determination of squalene and β-sitosterol in the Camellia oleifera seed oil sample in the embodiment;

Figure 4 is a gas chromatogram for the determination of squalene and β-sitosterol in Camellia oleifera seed oil samples in Comparative Example.

Detailed ways

The present invention will be further elaborated below with reference to specific embodiments, which are only used to explain the present invention, but not to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used, without special instructions, are reagents and materials that can be obtained from commercial channels:

The following are the instruments and reagents used in the examples

Agilent 7890A gas chromatograph with FID detector (Agilent, USA);

Solid phase extraction device (Agilent, USA);

DC-12-DA nitrogen blowing instrument (Zhejiang Scientific Equipment Import and Export Co., Ltd.);

IKA MS3 vortex mixer (IKA, Germany);

Milli-Q Gradient ultrapure water system (Millipore, USA);

HP-5 capillary gas chromatography column, 30m×0.32mm×0.25μm (Agilent, USA);

Methanol (chromatographically pure, Merck, Germany);

Potassium hydroxide, dichloromethane, ethyl acetate (analytical grade, Sinopharm Chemical Reagent Co., Ltd.);

Oasis HLB solid phase extraction cartridge (500mg/6mL, Waters, USA);

Squalene standard (purity ≥99.0%, Sigma-Aldrich, USA);

Squalane internal standard (purity ≥99.5%, Dr. Ehrenstorfer, Germany);

β-Sitosterol (purity ≥98.0%, Shanghai Anpu Experimental Technology Co., Ltd.);

5α-Cholestan-3β-ol standard product (purity ≥95.0%, Sigma-Aldrich, USA); preparation of standard solution: accurately weigh 25mg squalene and 25mg squalane standard product in a 25mL volumetric flask, respectively use n-hexane Dilute the alkane to the mark, and prepare a 1 mg/mL squalene standard stock solution and a 1 mg/mL internal standard squalane standard solution. Accurately weigh 25mg β-sitosterol and 25mg 5α-Cholestan-3β-ol standard substance in a 25mL volumetric flask, and dilute to the mark with ethyl acetate, respectively, to prepare 1mg/mL β-sitosterol standard stock solution and 1mg/mL β-sitosterol standard stock solution. mL of the internal standard 5α-Cholestan-3β-ol standard solution.

A method for determining the content of squalene and β-sitosterol in vegetable oil, wherein the pretreatment process of the sample comprises the following steps:

1. Saponification of vegetable oil

Pipette 50 μL of squalane and 50 μL of 5α-Cholestan-3β-ol internal standard respectively into a 10 mL stoppered glass test tube, dry the solvent with a nitrogen blower, and weigh 0.15g (accurate to 0.0001g) oil sample in this stopper. Add 2.5mL 2mol/L potassium hydroxide-methanol solution to a glass test tube, and saponify in a water bath at 70°C for 60min. After taking it out while hot, it was diluted to 10 mL with 80% methanol, and it was to be purified.

2. Solid phase extraction cartridge cleanup

Load the HLB (500mg, 6mL) cartridge on the solid phase extraction device, add 5mL of methanol first, then add 5mL of 80% methanol to activate the cartridge, then put the saponified sample solution on the cartridge, and then rinse the cartridge with 5mL of 80% methanol , rinse twice, and use a vacuum pump to completely dry the liquid, use a 10 mL centrifuge tube at the bottom to receive the eluent, add 7 mL of ethyl acetate to elute the sample, and drip dry naturally to obtain the sample eluent. Put it into a nitrogen blower, blow the sample eluent until it is nearly dry, and then make up to 1 mL with ethyl acetate, and pass through a 0.22 μm organic filter for GC-FID detection.

3. GC-FID internal standard method for detection process

The GC conditions are:

Chromatographic column: HP-5 capillary column (30m×0.32mm×0.25μm);

Chromatographic conditions: inlet temperature: 300 °C, splitless; injection volume: 1.0 μL, column flow: 1.5 mL/min;

Column temperature: 160 °C for 1 min, 15 °C/min to 280 °C, hold for 5 min, then 5 °C/min to 300 °C, and finally held for 7 min.

Detector: FID, 300°C, temperature 300°C, hydrogen flow rate 30mL/min, air flow rate 400mL/min, makeup gas flow rate 30mL/min;

Methodological evaluation

With reference to GB/T 27417-2017 "Guidelines for Confirmation and Validation of Chemical Analysis Methods for Conformity Assessment", the evaluation experiments were carried out in terms of linearity range, detection limit, precision and accuracy (matrix spike recovery). Standard curves of squalene and β-sitosterol corrected with squalane and 5α-Cholestan-3β-ol internal standard, respectively, refer to Figure 2 and Figure 3.

1. Linear range of the method, linear equation, correlation coefficient

To prepare a squalene working solution of 50 μg/mL squalane internal standard: dilute the 1 mg/mL squalene standard stock solution with ethyl acetate to the mass concentrations of 5, 10, 20, 50, 100, 200 μg/mL, respectively. Standard working solution, ensure that the mass concentration of squalane internal standard in each working solution is 50 μg/mL.

Prepare 50μg/mL 5α-Cholestan-3β-ol internal standard β-sitosterol working solution: Dilute 1 mg/mL β-sitosterol standard stock solution with ethyl acetate to the mass concentrations of 10, 20, 40, 100, 200, respectively , 400 μg/mL standard working solution, ensure that the mass concentration of 5α-Cholestan-3β-ol internal standard in each working solution is 50 μg/mL.

Standard curve preparation: Under the given chromatographic conditions, the above standard working solutions were injected, and the mass ratio of squalene to squalane was taken as the abscissa, and the peak area ratio of squalene to squalane was the ordinate. The internal standard calibration curve of squalene was obtained; taking the mass ratio of β-sitosterol to 5α-Cholestan-3β-ol as the abscissa, and the peak area ratio of β-sitosterol to 5α-Cholestan-3β-ol as the ordinate, the obtained β-sitosterol internal standard calibration curve, the specific results are shown in Table 1.

Table 1. Linear range, linear equation and correlation coefficient between squalene and β-sitosterol

2. Method recovery and precision

Since the contents of squalene and β-sitosterol in different vegetable oil samples vary greatly, and the contents of squalene and β-sitosterol in camellia oil are relatively low, the camellia oil was selected as the sample for the spike recovery experiment, and the Three levels of 10, 50, and 200 mg/kg, low temperature and dark, allow the standard to be fully balanced in the sample, saponify, purify and detect according to the aforementioned method for determining the content of squalene and β-sitosterol in vegetable oil, The recoveries and relative standard deviations of squalene and β-sitosterol at different spiked levels (same level sample group n=6) were calculated respectively, and the specific results are shown in Table 2.

Table 2. Spike recoveries and relative standard deviations for squalene and β-sitosterol (n=6)

3. The detection limit and quantification limit of the method

Under the aforementioned chromatographic conditions, the mixed standard solution was diluted step by step, and the detection limits of squalene and β-sitosterol were calculated to be 0.5 mg/kg and 1.0 mg/kg with the signal-to-noise ratio S/N=3, respectively. The limits of quantification for squalene and β-sitosterol were calculated to be 2.0 mg/kg and 4.0 mg/kg, respectively, with a signal-to-noise ratio S/N=10.

4. Determination of actual samples

In this experiment, 4 kinds of edible vegetable oil samples (Camellia oleifera seed oil, olive oil, corn oil and sesame oil) were selected and operated according to the steps in the Examples (the results are shown in Table 3). The results showed that the content of squalene in olive oil was significantly higher than that of other vegetable oils, and it was a good source of vegetable squalene. For the β-sitosterol component, the content of squalene in corn oil was the highest, followed by sesame oil, olive oil and camellia oil. Oil, this result is consistent with the results reported in related literature.

Table 3. Squalene and β-sitosterol in 4 vegetable oils

Comparative ratio

The same Camellia oleifera seed sample was selected for sample pretreatment with reference to the currently commonly used liquid-liquid extraction method. The specific process is as follows: weigh about 1.0g Camellia oleifera seed oil sample, put it into a 250mL ground conical flask, add 50mL of 2mol/L hydrogen Potassium oxide-ethanol solution, saponified in a constant temperature water bath at 85°C for 1 hour, removed and cooled, transferred to a 500mL separatory funnel, added 50mL saturated sodium chloride solution, then added 50mL petroleum ether, shaken for extraction for 2min, and allowed to stand , after layering, transfer the upper organic phase into a 250mL separating funnel, add 50mL and 30mL petroleum ether to the lower saponification solution respectively, extract twice, combine the three petroleum ether layers in a 250mL separating funnel, add 50mL each time The organic phase was washed with deionized water until it was neutral. After the organic phase was dehydrated through anhydrous sodium sulfate, it was concentrated to near dryness by rotary evaporation in a water bath at 35°C, dissolved in n-hexane and made up to 10 mL, passed through a 0.22 μm organic filter membrane, and filtered. Gas chromatograph (GC-FID) measurement, the gas chromatographic conditions completely adopt the chromatographic conditions mentioned above, and the test results are shown in Figure 4.

Comparing Figure 3 and Figure 4, it can be seen that this method can significantly reduce impurities in the sample and avoid contamination of the chromatographic sample introduction system. At the same time, the method is easy to operate, can be processed in batches, requires less organic reagents, and is calibrated by the internal standard method. It can better ensure the accuracy of continuous test samples, and has superior economy.

The above are only examples of the present invention, and common knowledge such as well-known specific technical solutions and/or characteristics in the solutions are not described too much here. It should be pointed out that for those skilled in the art, some modifications and improvements can be made without departing from the technical solution of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation of the present invention. effect and the applicability of the patent. The scope of protection claimed in this application shall be based on the content of the claims, and the descriptions of the specific implementation manners in the description can be used to interpret the content of the claims.

Claims (6)

1. a method for measuring squalene and β-sitosterol content in vegetable oil, is characterized in that, comprises the steps: S1, vegetable oil saponification treatment: take the mixed vegetable oil sample, put it in a stoppered glass test tube, add internal standard and potassium hydroxide-methanol solution, and then put it into a constant temperature water bath for saponification; S2, SPE cartridge purification: the saponified sample solution was diluted with 80% methanol solution, passed through the HLB solid phase extraction cartridge, rinsed with 80% methanol, and finally eluted the target compound with ethyl acetate, and collected all the The eluate, concentrated to a constant volume and passed through the membrane, is the sample to be tested. 2. a kind of method for measuring squalene and β-sitosterol content in vegetable oil according to claim 1, is characterized in that, described step S2 is specially: S21. Activation: pipette 5 mL of methanol solution and 5 mL of 80% methanol solution, take the SPE cartridge, and use 5 mL of methanol solution and 5 mL of 80% methanol solution to wash the cartridge in turn; S22, sample loading: the saponified sample solution in the step S1 is loaded onto the SPE small column; S23, rinsing: rinse the small column with 5 mL of 80% methanol, rinse twice, and use a vacuum pump to completely dry the liquid; S24. Elution: place a centrifuge tube under the SPE column, add ethyl acetate to elute the sample, and drip dry naturally. get the sample eluent; S25. Concentration: put it into a nitrogen blower, and blow the sample eluent until it is close to drying and stop; S26, dilute to volume: dilute to 1 mL with ethyl acetate; S27. Filtration: filter the sample solution after constant volume through a membrane to prepare a sample to be tested. 3. a kind of method for measuring squalene and β-sitosterol content in vegetable oil according to claim 2, is characterized in that, adding internal standard in described step S1 is specially: add squalane and 5α-Cholestan respectively -3β-ol was used as the internal standard, and the concentration was 1 mg/mL. 4. a kind of method for measuring squalene and β-sitosterol content in vegetable oil according to claim 3, is characterized in that: in described step S1, the constant temperature of water bath is 70 ℃, and the saponification reaction time is 60min. 5. a kind of method for measuring squalene and β-sitosterol content in vegetable oil according to claim 4, is characterized in that: in described step S1, the concentration of potassium hydroxide-methanol solution is 2mol/L. 6 . The method for measuring the content of squalene and β-sitosterol in vegetable oil according to claim 5 , wherein the filter membrane in the step S2 adopts a 0.22 μm organic filter membrane. 7 .

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