CN115308341A - Method for rapidly determining 5 phytosterols in vegetable oil by non-derivatization-gas chromatography-tandem mass spectrometry - Google Patents
Method for rapidly determining 5 phytosterols in vegetable oil by non-derivatization-gas chromatography-tandem mass spectrometry Download PDFInfo
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- CN115308341A CN115308341A CN202211119646.2A CN202211119646A CN115308341A CN 115308341 A CN115308341 A CN 115308341A CN 202211119646 A CN202211119646 A CN 202211119646A CN 115308341 A CN115308341 A CN 115308341A
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- 238000000034 method Methods 0.000 title claims abstract description 36
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- HCXVJBMSMIARIN-PHZDYDNGSA-N stigmasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@@H](CC)C(C)C)[C@@]1(C)CC2 HCXVJBMSMIARIN-PHZDYDNGSA-N 0.000 claims abstract description 14
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- 238000001514 detection method Methods 0.000 claims description 25
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/06—Preparation
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Abstract
The invention discloses a method for determining 5 phytosterols in vegetable oil by using a non-derivatization-gas chromatography-tandem mass spectrometry, which is characterized in that a pretreatment mode of saponification at room temperature is established based on the enzymolysis of lipase on fat, the combined sterol ester in the vegetable oil is efficiently liberated, and a gas chromatography-tandem mass spectrometer is used for establishing a method for detecting 5 sterols in the vegetable oil, such as campesterol, beta-sitosterol, brassicasterol, stigmasterol and cycloartenol. The chromatographic behavior of 5 sterols on a weak polarity chromatographic column HP-5MS is good; the method has the advantages that the standard recovery rate is 84.7-101.6%, the relative standard deviation is 1.4-4.1%, and the quantitative limit of 5 sterols is 10.0 mg/kg. Compared with the standard method, the method has the characteristics of complex pretreatment operation, harsh saponification conditions, difficult layering during extraction and need of derivatization, is quicker, simpler and more convenient, and is convenient for quickly measuring the sterol in the vegetable oil.
Description
Technical Field
The invention belongs to the technical field of food analysis, and particularly relates to a method for rapidly determining 5 phytosterols in vegetable oil by using a non-derivatization-gas chromatography-tandem mass spectrometry method.
Background
Phytosterol is an important natural active substance, is a compound taking a steroid nucleus as a basic skeleton, has a structural formula shown in figure 1, and contains 4 types of beta-sitosterol, stigmasterol, brassicasterol and brassicasterol in high content. Phytosterols have the functions of preventing coronary atherosclerosis, resisting inflammation, protecting skin, enhancing immunity and the like, can regulate the absorption of human bodies to cholesterol, and are already listed in the category of functional foods. The human body can not synthesize the phytosterol by itself, and only can obtain the phytosterol from food, and the vegetable oil is one of the sources of the dietary sterol. It has been reported that sterols are easily oxidized to form sterol oxides in environments such as heat, light, and metal ions. The oxidation and decomposition of sterol are easily caused in the processing and production links (deodorization and deacidification) of vegetable oil and the refining of vegetable oil, thereby leading to the loss of sterol. Therefore, the development of a detection technology for rapidly and accurately determining the sterol content in the vegetable oil has important significance for evaluating and monitoring the quality of the vegetable oil.
The instrument method for measuring the phytosterol mainly comprises gas chromatography, liquid chromatography, chromatography-mass spectrometry, thin layer chromatography and multidimensional gas chromatography-flight time mass spectrometry. The phytosterol mainly exists in two forms of free sterol and combined sterol ester, and the total sterol amount of the two forms needs to be measured when the sterol content is accurately measured. The pretreatment technology of free state sterol reported at present is mainly a solid phase extraction technology, the detection of the bound state sterol usually adopts potassium hydroxide-ethanol solution to saponify and hydrolyze a sample, and after the sterol is released, derivatization is carried out by utilizing a derivatization reagent N, O-bis (trimethylsilyl) trifluoroacetamide or N-methyl-N-trimethylsilane heptafluorobutanamide. The existing sterol detection standards GB/T23225-2010, animal and vegetable oil sterol composition and sterol total amount determination gas chromatography and NY/T3111-2017, vegetable oil sterol content determination gas chromatography-mass spectrometry are established based on the pretreatment technology. The saponification conditions are harsh (strong alkali solution, high water bath temperature and long saponification time), the extraction operation is complicated, the emulsification is easy during water washing, the layering is difficult, the absolute recovery rate of the target is low, the result reproducibility is poor, and the internal standard needs to be added for correction.
Disclosure of Invention
In order to solve the defects of the background technology, the invention aims to overcome the technical defects and provide a method for rapidly determining 5 phytosterols in vegetable oil by using a non-derivatization-gas chromatography-tandem mass spectrometry method. The method has the advantages of simple and convenient pretreatment, high sensitivity, good reproducibility, high recovery rate and stability, and suitability for the detection of sterol in batch samples.
In order to achieve the purpose, the invention adopts the following technical scheme:
5 the phytosterol is brassicasterol, stigmasterol, beta-sitosterol and cycloartenol.
A method for rapidly determining 5 phytosterol in vegetable oil by non-derivatization-gas chromatography-tandem mass spectrometry,
the method comprises the following steps:
(1) Pretreatment of a sample: accurately weighing a 0.10 g vegetable oil sample into a 50 mL centrifuge tube, adding 5 mL phosphate buffer solution with pH =8.0-10.0 and 0.05g lipase, vortex uniformly mixing for 2min, carrying out enzymolysis in a constant temperature water bath oscillator at 25-45 ℃, and taking out an enzymolysis liquid after 5-20 min; adding 1.5g potassium carbonate after the enzymatic hydrolysate is cooled, then sequentially adding 10ml absolute ethyl alcohol and 10mL water, and uniformly mixing and saponifying for 2-25 min in a vortex manner; adding 10mL n-hexane for extraction for 5min, centrifuging at 6000 r/min for 2min, transferring the supernatant to another 50 mL centrifuge tube, and adding 10mL n-hexane for extraction once; mixing the extractive solutions, and mixing;
(2) Preparing a standard solution: respectively and accurately weighing 10.0 mg brassicasterol, beta-sitosterol, brassicasterol, stigmasterol and cycloartenol standard substances in a 10.0 ml volumetric flask, and fixing the volume of n-hexane to a scale to obtain a single-standard solution with the mass concentration of 1.00 mg/ml; measuring a proper amount of each sterol single-standard solution into the same volumetric flask to prepare a mixed standard solution with the mass concentration of 0.1 mg/ml; respectively sucking and mixing 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 mL of standard solution into a 10mL volumetric flask to prepare standard working solution with mass concentration of 1.0, 2.0, 5.0, 10.0, 20.0 and 50.0 mg/L;
(3) And (4) gas chromatography-mass spectrometry detection.
Wherein, the optimization conditions of the pretreatment of the sample are as follows: accurately weighing a 0.10 g vegetable oil sample into a 50 mL centrifuge tube, adding a phosphate buffer solution 5 mL with pH =9.0 and 0.05g lipase, and uniformly mixing for 2min by vortex; performing enzymolysis in a 37 +/-2 ℃ constant-temperature water bath oscillator, and taking out the enzymolysis liquid after 20 min; adding 1.5g potassium carbonate after the enzymatic hydrolysate is cooled, then sequentially adding 10ml absolute ethyl alcohol and 10mL water, and uniformly mixing and saponifying for 10min in a vortex manner; adding 10mL n-hexane for extraction for 5min, centrifuging at 6000 r/min for 2min, transferring supernatant to another 50 mL centrifuge tube, and adding 10mL n-hexane for extraction once; mixing the extractive solutions, and mixing.
In the detection method, the model of the chromatographic column is HP-5MS, and the size is as follows: 30m × 0.25 mm × 0.25 μm; sample inlet temperature: 250 ℃; ion source temperature: 200 ℃; auxiliary heating temperature: 280 ℃; and (3) sample introduction mode: no-flow sampling, sample injection amount: 1. mu.l; temperature programming: maintaining at 150 deg.C for 1 min, heating to 280 deg.C at 10 deg.C/min, maintaining for 12 min, heating to 300 deg.C at 20 deg.C/min, and maintaining for 8 min; electrons bombard the ionization source, and Multiple Reaction Monitoring (MRM) mode is used for acquisition.
The invention has the beneficial effects that:
the pretreatment process of the existing sterol detection standard is complicated, the saponification condition is harsh, the extraction is difficult to delaminate, the derivatization and the operation are needed, and the method has poor reproducibility. The research develops a mild enzymolysis and saponification pretreatment mode, does not need the steps of high-temperature strong alkali saponification, water washing, solid-phase extraction purification and derivatization, is efficient and convenient, and is suitable for detection of batch samples. Meanwhile, qualitative and quantitative analysis is carried out by utilizing a multi-reaction monitoring mode, so that the interference of a target peak is reduced, and the accuracy and the repeatability of the method are improved. The established method is applied to vegetable oil production enterprises, the sterol content in each link (deodorization and deacidification) of vegetable oil production is detected, and the enterprise is facilitated to optimize the vegetable oil processing and production process by monitoring the change of the sterol content, so that the sterol loss is reduced.
Drawings
FIG. 1 is the structural formula of steroid nucleus;
FIG. 2 is a multi-reaction monitoring chromatogram of 5 sterol standard solutions;
fig. 3 shows the effect of pH of the enzymatic solution and the amount of enzyme on the detection of 5 sterols in corn oil (n = 5);
FIG. 4 is a graph of the effect of base usage on the 5 sterol detection values in corn oil (n = 5);
FIG. 5 is a graph of the effect of an extractant on the recovery of 5 sterols from corn oil;
FIG. 6 is a graph of the effect of extractant volume on recovery of 5 sterols from corn oil.
Detailed Description
In order to make the technical solutions of the present invention better understood and make the above objects, features and advantages of the present invention more comprehensible, the present invention is described in further detail with reference to examples.
Example 1
1. Materials and methods
1.1 Instruments, reagents and materials
TQ-8050 gas chromatography-triple quadrupole tandem mass spectrometer (Shimadzu, japan); one in ten thousand balance (mertler-toledo group, switzerland); milli-Q ultra pure water instruments (Millipore, USA); model SW22 constant temperature water bath shaker (buble ley, germany); centrifuge (SIGMA corporation); vortex (IKA, germany).
Brassicasterol (canada, trc); brassicasterol (japan, tama); stigmasterol (seebaio); beta-sitosterol (seebaio); cycloartenol (ChromaDex, usa); potassium hydroxide, absolute ethyl alcohol, potassium dihydrogen phosphate (national drug group chemical reagent limited); potassium carbonate (Kangde chemical Co., ltd., laiyang city); n-hexane (Fisher, USA); lipase (SIGMA company, USA, enzyme activity is more than or equal to 1208U/mg); the water used is ultrapure water.
1.2 Solution preparation
Preparation of a standard solution: respectively and accurately weighing 10.0 mg brassicasterol, beta-sitosterol, brassicasterol, stigmasterol and cycloartenol standard substances in a 10.0 ml volumetric flask, and fixing the volume of n-hexane to a scale to obtain a single-standard solution with the mass concentration of 1.00 mg/ml. Measuring a proper amount of each sterol single-standard solution into the same volumetric flask to prepare a mixed standard solution with the mass concentration of 0.1 mg/ml. Respectively sucking and mixing 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 mL of the standard solution into a 10mL volumetric flask to prepare the standard working solution with the mass concentration of 1.0, 2.0, 5.0, 10.0, 20.0 and 50.0 mg/L, and storing the standard working solution at 4 ℃ for later use.
Preparation of phosphate buffer (pH = 9.0): weighing a proper amount of potassium hydroxide in a 100 mL beaker, dissolving the potassium hydroxide with water to prepare a solution with the concentration of 400 g/L, and cooling the solution for later use. Weighing 27.0 g monopotassium phosphate into a beaker, dissolving with water, adding a proper amount of the potassium hydroxide solution, adjusting the pH to 9.0, transferring to a 250 mL volumetric flask, and fixing the volume to the scale.
1.3 Sample pretreatment
Accurately weighing 0.10 g vegetable oil sample into a 50 mL centrifuge tube, adding phosphate buffer solution 5 mL with pH =9.0 and 0.05g lipase, and vortexing and mixing for 2min. Carrying out enzymolysis in a constant temperature water bath oscillator at 37 +/-2 ℃, and taking out the enzymolysis liquid after 20 min. Adding 1.5g potassium carbonate after the enzymolysis liquid is cooled, then sequentially adding 10ml absolute ethyl alcohol and 10mL water, and uniformly mixing and saponifying for 10min in a vortex mode. Adding 10mL n-hexane for extraction for 5min, centrifuging at 6000 r/min for 2min, transferring the supernatant to another 50 mL centrifuge tube, and adding 10mL n-hexane for extraction once. Mixing the extractive solutions, mixing, and subjecting to gas chromatography-tandem mass spectrometry.
1.4 Gas chromatography-mass spectrometry conditions
A chromatographic column: model HP-5MS (30 m X0.25 mm X0.25 μm); sample inlet temperature: 250 ℃; ion source temperature: 200 ℃; auxiliary heating temperature: 280 ℃; and (3) sample introduction mode: non-shunt sampling, sample injection amount: 1. mu.l; temperature programming: keeping at 150 deg.C for 1 min, then raising to 280 deg.C at 10 deg.C/min for 12 min, and then raising to 300 deg.C at 20 deg.C/min for 8 min. Electron bombardment ionization source (EI), multiple Reaction Monitoring (MRM) mode. The retention times, monitored ion pairs and corresponding collision energies for the 5 sterols are shown in table 1.
TABLE 5 Retention time, monitoring ion Pair and Collision energy of sterols
* Quantitative ion pair
2. Results and analysis
2.1 Optimization of instrument conditions
2.1.1 Optimization of chromatographic conditions
The target substances are separated and detected by respectively selecting HP-5MS and TG-Innovax MS chromatographic columns, and the results show that 5 kinds of sterols have serious peak tailing on the TG-Innovax MS chromatographic columns, and 5 kinds of sterols on the HP-5MS chromatographic columns are well reserved and have symmetrical peak shapes, so that the detection requirements are met, and therefore, the HP-5MS chromatographic column with the weak polarity is selected for experiments. The multiple reaction monitoring chromatogram of 5 sterols is shown in FIG. 2.
2.1.2 Determination of Mass Spectrometry conditions
Firstly, introducing a standard mixed solution of 5 sterols into a gas chromatography-tandem mass spectrum for full scanning, contrasting with an NIST spectrum library to obtain the retention time and fragment ions of the 5 sterols, selecting the fragment ions with larger mass number and higher strength, obtaining an optimization curve of ion pairs and collision energy by utilizing the Auto SRM function of Shimadzu instrument software, and selecting the optimal ion pairs and collision voltage (see table 1).
2.2 Optimization of pretreatment conditions
Sterol mainly exists in two forms of free sterol and sterol fatty acid ester in vegetable oil, for the free sterol, the presence of fat in vegetable oil influences the extraction efficiency, and for the combined state sterol ester, ester is firstly released into the free sterol, and then extraction determination is carried out. Since the standard substance is not easily purchased due to the phytosterol ester in a conjugated state, a positive corn oil sample is selected for an experiment to explore the optimal pretreatment conditions. The contents of brassicasterol, stigmasterol, beta-sitosterol and cycloartenol in the positive corn oil sample are accurately determined by using a standard NY/T3111-2017 gas chromatography-mass spectrometry for determining the content of sterol in vegetable oil, and the contents are used as standard values to optimize the experimental conditions of the method. The influence of enzymolysis conditions (enzyme dosage, enzymolysis time, enzymolysis pH), saponification conditions (alkali dosage, saponification time and temperature) and extraction conditions (types and volumes of extracting agents) on the detected value are respectively discussed, and the optimal pretreatment conditions are selected by taking the standard recovery rate or the detected value of 5 types of sterols as evaluation indexes.
2.2.1 Optimization of enzymolysis conditions
The bonded phytosterol ester is generally subjected to reflux saponification in a high-temperature strong-alkali environment to be free, and the saponification condition is harsh and long in time. The lipase can degrade fat in the grease into fatty acid and triglyceride, so that the saponification experiment can be carried out under mild conditions. The study examines the dosage of lipase and the optimal activity condition of the lipase, and determines the optimal enzymolysis condition through the detection value of positive sterol.
The research considers the enzymolysis efficiency of lipase under different pH conditions (6.0, 7.0, 8.0, 9.0 and 10.0), optimizes the dosage of the enzyme, and the detection results of 5 sterols including brassicasterol, stigmasterol, beta-sitosterol and cycloartenol in the corn oil are shown in figure 3. As can be seen from fig. 3A, the detection values of 5 sterols were low under acidic (pH = 6.0) and neutral (pH = 7.0) conditions. pH =9.0 was selected as the optimum condition for enzymatic hydrolysis, since the detection value was high at pH 8.0, 9.0, and 10.0, indicating that lipase activity was high under alkaline conditions, and the detection value was not much different as pH increased. As can be seen from FIG. 3B, when the amount of enzyme was 0.05g, the detection values of 5 sterols were the largest, and when the amount of enzyme was 0.01g and 0.02g, the detection values were only 23.2% to 56.5% of that of 0.05g, which may result in incomplete saponification reaction in the following experiment due to incomplete lipolysis. When the amount of the enzyme is more than 0.05g, the detection value of each sterol tends to decrease as the amount of the enzyme increases, and the enzyme may be excessively added to affect the enzymatic hydrolysis efficiency or a certain influence on the subsequent saponification reaction, and this influence may be further confirmed. Therefore 0.05g of enzyme was chosen as the experimental amount.
Enzymolysis is a mild reaction process, and enzymolysis time is an important factor influencing whether fat enzymolysis is complete or not, so that subsequent sterol extraction is influenced. The research investigates the influence of enzymolysis for 5min, 10min, 15min, 20min, 25min and 30min on the detection value. The result shows that the extraction rate of 5 sterols reaches 80% when enzymolysis is carried out for 5min, which indicates that the enzymolysis of the lipase on the vegetable oil is a rapid reaction process, and the highest extraction rate is reached when enzymolysis is carried out for 20 min. When the enzymolysis time is longer than 25min, the sterol content is also reduced, and the loss of the component to be detected is probably caused by overlong enzymolysis time, so that the best enzymolysis time is selected to be 20 min.
2.2.2 Optimization of saponification conditions
The study examined the effect of different amounts of potassium carbonate (0 g, 0.5g, 1.0g, 1.5g, 2.0g, 2.5 g) on saponification, and the content of 5 sterols in corn oil under different amounts of alkali is shown in FIG. 4. The results showed that 5 sterols detected only 50% without potassium carbonate addition and that only free sterols could be extracted without saponification. The sterol content tended to increase with the increase in the amount of potassium carbonate added, and the sterol content tended to be the highest with 1.5g of potassium carbonate added, and the value was stable with the subsequent increase in the amount of potassium carbonate added, indicating that the saponification reaction was complete with 1.5g of alkali, and therefore 1.5g of potassium carbonate was selected for the saponification reaction.
The research also examines the contents of the 5 sterols in the corn oil under different saponification times (2 min, 5min, 10min, 15min, 20min and 25 min) and saponification temperatures (25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃), and the results show that the saponification times and the saponification temperatures have no obvious influence on the saponification reaction results, and the detection values of the 5 sterols are basically consistent. Probably, after the enzymolysis of the sample, the saponification reaction is easier to carry out, and the requirements on temperature and time are not strict, which is also a characteristic that the research is superior to other high-temperature reflux saponification researches. Considering the stability and time cost of saponification reaction, saponification time of 10min and room temperature saponification are selected as the optimal conditions for saponification reaction.
2.2.3 Extraction condition optimization
Carrying out enzymolysis and saponification on the corn oil under selected conditions, and adding 5 sterols of brassicasterol, stigmasterol, beta-sitosterol and cycloartenol with the same content as that in a sample into a solution after the reaction, wherein the adding content is respectively 50mg/kg, 1600mg/kg, 600mg/kg, 5000mg/kg and 150mg/kg. Since sterol is an alcohol substance and can be dissolved in various organic solvents, the present study examined the effect of n-hexane, dichloromethane, toluene, ethyl acetate and petroleum ether-ether (1:1) as extraction agents on the recovery of 5 sterols, respectively, and the results are shown in fig. 5. The result shows that the extraction effect of the toluene and the dichloromethane is poor, and the recovery rate of 5 sterols is 42.6-69.7%; when ethyl acetate and petroleum ether-diethyl ether are used as extracting agents, the recovery rates of campesterol, brassicasterol, stigmasterol and beta-sitosterol are 69.2-91.1%, but the recovery rates of cycloartenol are only 53.6% and 47.7%; the n-hexane extraction effect is optimal, and the recovery rate of 5 sterols is over 80 percent.
The present study also examined the effect of n-hexane volume (5 mL, 10 mL) and number of extractions (one and two) on recovery, and the results are shown in fig. 6. When the extraction volume is 10ml, the extraction is more sufficient, the recovery rate of 5 sterols is more than 90%, and therefore 10ml of n-hexane is finally selected for extraction twice as the extraction condition for experiments.
2.3 Investigation of matrix Effect
Since vegetable oil Matrix is complicated and the variety of vegetable oil is large, it may cause a certain Matrix Effect in the measurement, and it is necessary to evaluate the Matrix Effect (ME). ME = (slope of substrate matching calibration curve/slope of solvent standard curve-1) × 100%. When | ME | is less than 20%, it means that the weak matrix effect is negligible; when the < I > ME </I > is 20% -50%, a medium stroma effect is represented; if ME is greater than 50%, a strong matrix effect is indicated, and a matrix effect correction is required. The matrix solutions of peanut oil, corn oil, soybean oil and sesame oil were respectively selected as solvents to prepare 5 kinds of sterol standard solutions, and compared with the solvent standard solution prepared with n-hexane, the matrix effect was calculated, and the results are shown in table 2. The result shows that the ME values of 5 sterols in the 4 vegetable oils are 0.8-16.3%, and are less than 20%, the sterols are expressed as weak matrix effects, and matrix effect correction is not needed, so that the experiment adopts a solvent standard solution for quantitative analysis.
TABLE 2 matrix Effect of the sterols
2.4 Evaluation of methodology
2.4.1 Standard curve and quantitative limit
Under the optimized conditions, 5 kinds of sterol standard series solutions are measured to obtain the linear relation between mass concentration and peak area response. Fitting a linear equation to obtain a correlation coefficient (R) by taking the peak area of the mass concentration as the abscissa (x, mg/L) as the ordinate (y) 2 ），The linear range of the beta-sitosterol is 0.05 to 50.0 mg/L, the linear range of the other 4 sterols is 0.05 to 20.0 mg/L, and the quantitative limit of the 5 sterols is 10.0 mg/kg (see Table 3).
TABLE 3 calibration curves, correlation coefficients, quantitation limits, spiked recovery and relative standard deviation of 5 sterols
2.4.2 Experiment on recovery rate and precision of added standard
Three levels of spiking experiments were performed on corn oil samples, with the spiking amounts divided into two groups based on the level of each sterol in the sample. The addition amounts of brassicasterol, stigmasterol and beta-sitosterol were 200mg/kg, 500mg/kg and 1000mg/kg, and the addition amounts of brassicasterol and cycloartenol were 50mg/kg, 100mg/kg and 200mg/kg, and each addition level was measured in parallel 6 times, and the addition standard recovery rate and Relative Standard Deviation (RSD) of each sterol were calculated, and the results are shown in table 3. As can be seen from Table 3, the average recovery rate of sterol is between 84.7% and 101.6% and the RSD is between 1.4% and 4.1% within the range of the addition mass concentration.
2.5 Comparison of the present research method with the Standard method
The content of 5 sterols including brassicasterol, stigmasterol, beta-sitosterol and cycloartenol in corn oil is respectively measured by an established method and a standard method NY/T3111-2017 gas chromatography-mass spectrometry for measuring the content of the sterols in vegetable oil, and the difference of internal and external standard quantification of the method and the standard method is examined. Each method was performed 6 times in parallel, and the average value of each sterol measured by each method and the relative standard deviation thereof were calculated, and the results are shown in Table 4.
TABLE 4 variability of the results of the 5 sterols determined by the present research and standard methods
The result shows that the content of the 5 sterols measured by the research method is consistent with that measured by the standard method internal standard method, the relative standard deviation of the method is between 0.8% and 3.0%, the relative standard deviation of the standard method internal standard method is between 2.9% and 3.4%, and the two methods have good reproducibility. When the standard method is used for quantifying, the detection result is low, the detection value is only 73.1-76.9% of that of the internal standard method, the relative standard deviation is as high as 40.4-43.3%, the reproducibility is poor, and the sample cannot be accurately quantified. The standard method uses strong alkaline solution for saponification, the water washing step is easy to emulsify, the layering is difficult, and the recovery rate of the target is low, so the standard method needs to adopt an internal standard method for quantification to correct errors caused by fussy pretreatment on detection, and the use of the internal standard increases the cost of the experiment. Compared with the standard method, the method has the characteristics of harsh saponification conditions, difficult layering during extraction and need of derivation and internal standard correction, is faster, simpler, more convenient and more accurate, and is convenient for batch determination of sterol in vegetable oil.
Claims (4)
1. A method for rapidly determining 5 phytosterol in vegetable oil by using a non-derivatization-gas chromatography-tandem mass spectrometry method is characterized by comprising the following steps:
(1) Pretreatment of a sample: accurately weighing a 0.10 g vegetable oil sample into a 50 mL centrifuge tube, adding a phosphate buffer solution 5 mL with the pH =8.0-10.0 and 0.05g of lipase, whirling and uniformly mixing for 2min, carrying out enzymolysis in a constant-temperature water bath oscillator at the temperature of 25-45 ℃, and taking out an enzymolysis solution after 5-20 min; adding 1.5g potassium carbonate after the enzymatic hydrolysate is cooled, then sequentially adding 10ml absolute ethyl alcohol and 10mL water, and uniformly mixing and saponifying for 2-25 min in a vortex manner; adding 10mL n-hexane for extraction for 5min, centrifuging at 6000 r/min for 2min, transferring the supernatant to another 50 mL centrifuge tube, and adding 10mL n-hexane for extraction once; mixing the extractive solutions, and mixing;
(2) Preparing a standard solution: respectively and accurately weighing 10.0 mg brassicasterol, beta-sitosterol, brassicasterol, stigmasterol and cycloartenol standard substances in a 10.0 ml volumetric flask, and fixing the volume of n-hexane to a scale to obtain a single-standard solution with the mass concentration of 1.00 mg/ml; measuring a proper amount of each sterol single-standard solution into the same volumetric flask to prepare a mixed standard solution with the mass concentration of 0.1 mg/ml; respectively sucking and mixing 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 mL of standard solution into a 10mL volumetric flask to prepare standard working solution with mass concentration of 1.0, 2.0, 5.0, 10.0, 20.0 and 50.0 mg/L;
(3) And (4) gas chromatography-mass spectrometry detection.
2. The method for rapidly determining 5 phytosterols in vegetable oil by non-derivatization-gas chromatography-tandem mass spectrometry as claimed in claim 1, wherein the optimization conditions of the pretreatment of the sample are as follows: accurately weighing a 0.10 g vegetable oil sample into a 50 mL centrifuge tube, adding a phosphate buffer solution 5 mL with pH =9.0 and 0.05g lipase, and uniformly mixing for 2min by vortex; performing enzymolysis in a 37 +/-2 ℃ constant-temperature water bath oscillator, and taking out the enzymolysis liquid after 20 min; adding 1.5g potassium carbonate after the enzymatic hydrolysate is cooled, then sequentially adding 10ml absolute ethyl alcohol and 10mL water, and uniformly mixing and saponifying for 10min in a vortex manner; adding 10mL n-hexane for extraction for 5min, centrifuging at 6000 r/min for 2min, transferring supernatant to another 50 mL centrifuge tube, and adding 10mL n-hexane for extraction once; mixing the extractive solutions, and mixing.
3. The method for rapidly determining 5 phytosterols in vegetable oil by non-derivatization-gas chromatography-tandem mass spectrometry according to claim 1, wherein the chromatographic column: model number HP-5MS, size: 30m × 0.25 mm × 0.25 μm; sample inlet temperature: 250 ℃; ion source temperature: 200 ℃; auxiliary heating temperature: 280 ℃; and (3) sample introduction mode: no-flow sampling, sample injection amount: 1. mu.l; temperature programming: keeping at 150 deg.C for 1 min, then raising to 280 deg.C at 10 deg.C/min, keeping for 12 min, then raising to 300 deg.C at 20 deg.C/min, keeping for 8 min; electrons bombard the ionization source, and the collection is carried out in a multi-reaction monitoring (MRM) mode.
4. The method for rapidly determining 5 phytosterols in vegetable oil by using a non-derivatization-gas chromatography-tandem mass spectrometry method according to claim 1, wherein the 5 phytosterols are brassicasterol, stigmasterol, beta-sitosterol and cycloartenol.
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