A method for determining the content of triterpene alcohol in camellia oil

Abstract

The invention relates to the technical field of detection methods, and discloses a method for determining the content of triterpene alcohol in camellia oil, comprising the following steps: (1) subjecting a camellia oil sample to saponification extraction and subjecting it to a test machine; then subjecting it to a silanization derivatization treatment and subjecting it to a test machine again; (2) preparing β-amyrin, butyryl cetyl alcohol and lupeol to obtain standard solutions respectively, wherein the standard solutions of β-amyrin and butyryl cetyl alcohol are directly subject to a test machine, and the standard solution of lupeol is subject to a silanization derivatization treatment and subjecting it to a test machine; (3) gas chromatography detection: a column temperature rising method: maintaining 180-190°C for 1-5min, then heating to 260-300°C at a rate of 10-15°C / min, and maintaining for 20-40min; split ratio: 20-40:1. The invention adopts a gas chromatography external standard method to determine triterpene alcohol, and the result is more scientific, advanced, accurate and reliable.

Description

Technical Field

[0001] The invention relates to the technical field of detection methods, in particular to a method for determining the content of triterpene alcohol in camellia oil. Background Art

[0002] In recent years, people have gradually paid more attention to camellia oil. Studies have found that the fatty acid composition of camellia oil is very similar to olive oil, which is called "liquid gold" by European and American countries. Moreover, camellia oil has less saponification than olive oil, higher vitamin E, and contains special ingredients such as tea polyphenols and triterpenes that are not found in olive oil. It is a special oil that can be compared with olive oil. In addition to being used as a healthy cooking oil, it is also an excellent cosmetic base oil like olive oil.

[0003] Phytosterols usually refer to 4-methyl-free sterols, while triterpenols, also known as 4,4-dimethyl sterols, are a type of phytosterols and a relatively special type of active ingredient in tea oil. It cannot be synthesized in the human body, and is even very scarce in most edible vegetable oils and even in nature. It is only found in a few edible vegetable oils, and also exists in small amounts in grains and fruits.

[0004] Since there are many types of phytosterols, more than 200 types have been found according to reports, and the composition and structure are slightly different and the analysis is relatively complicated, there is no formal method standard for detecting triterpene alcohols in the search of national standards, industry standards, group standards and other standard materials. The detection methods can only be found in a small number of papers and literature. Among them, the detection methods of triterpene alcohols mainly rely on HPLC (ELSD-evaporative light scattering detector, CAD-charged aerosol detector and other detectors), HPLC-MS / MS method, GC-MS method. The HPLC method is limited by the different types of detectors, and the detection conditions and detector types used for different triterpene alcohol substances cannot be unified. At the same time, the detector is not a commonly used detector in the laboratory and does not have good universality. When determining by GC-MS, the main methods are divided into two categories. The first category is thin layer chromatography (mainly for measuring the total amount of methyl-free sterols), which mainly measures the peak area ratio of sterol compounds within the measuring interval, and single quantitative determination is difficult; the second category is the derivatization method (common reagents include: ① a mixed solution of N-methyl-N-trimethylsilane heptafluorobutyramide and 1-methylimidazole; ② a mixed reagent of N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane), but the derivatization method increases the experimental time and cannot separate all triterpene alcohol substances well. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for determining the content of triterpene alcohol in camellia oil. The method adopts gas chromatography external standard method for determination, which has better universality. When there are many types of phytosterols with small differences in composition and structure and the analysis is relatively complicated, the triterpene alcohol can be better separated. The method is an accurate quantitative determination method with strong practicality. The scientificity and advancement of the content determination method and the accuracy and reliability of the determination results are also illustrated by data.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for determining the content of triterpene alcohol in camellia oil, comprising the following steps:

[0008] (1) After the camellia oil sample is subjected to saponification extraction, it is placed on the test machine; then it is subjected to silanization derivatization treatment and placed on the test machine again;

[0009] (2) preparing standard solutions of β-amyrin, butyryl cetyl alcohol and lupeol, respectively, wherein the standard solutions of β-amyrin and butyryl cetyl alcohol are directly tested on the machine, and the standard solution of lupeol is tested on the machine after silylation derivatization;

[0010] (3) Gas chromatography detection: use a capillary column; injection port temperature: 320-340°C; column temperature heating method: maintain at 180-190°C for 1-5 min, then increase the temperature to 260-300°C at a rate of 10-15°C / min, and maintain for 20-40 min; split ratio: 20-40:1.

[0011] At present, the detection methods of triterpene alcohols mainly rely on HPLC (ELSD-evaporative light scattering detector, CAD-charged aerosol detector and other detectors), HPLC-MS / MS, and GC-MS. The HPLC method is limited by the different types of detectors. The detection conditions and detector types used for different triterpene alcohol substances cannot be unified. At the same time, the detectors used are not commonly used in laboratories and do not have good universality. The HPLC-MS / MS and GC-MS methods themselves have high instrument prices and high operating costs. For example, the carrier gas requirements are high, the cost of damage to instrument accessories and replacement of consumables is high, and the electricity cost of the instrument is high. GC (hydrogen flame ionization detector) is the most commonly used detection instrument with low operating and maintenance costs. This method takes into account the universality and operating costs of the instrument and uses gas chromatography for determination.

[0012] Gas chromatography includes internal standard method and external standard method, etc., in the current common vegetable oil detection method, internal standard method is used for quantitative determination, and cholestanol is selected as the internal standard. The internal standard method is used for quantitative determination, and the steps of standard preparation and sample pretreatment are more complicated. At the same time, when the internal standard is used to detect camellia oil samples, there is an obvious interference peak at the retention time position, which does not conform to the selection rule of the internal standard. The present invention adopts gas chromatography external standard method for determination, and the external standard method for quantitative determination of each triterpene alcohol substance is more scientific, advanced, and the determination result is accurate and reliable.

[0013] In addition, most of the current methods for sterols and terpenoids use samples weighed into a conical flask for saponification, and then extracted with n-hexane in a separating funnel. When this type of method uses a conical flask for saponification, whether it is saponified with a magnetic stirrer or saponified with water bath reflux, when processing samples in batches, it is necessary to occupy a large area or a large number of instruments and devices to complete the saponification, and the number of saponifications in a single batch is limited. When extracting samples using a separating funnel, the extraction operation is more cumbersome, the daily detection amount is limited, and the samples cannot be processed in large quantities. The present invention uses a colorimetric tube for saponification, which occupies a small area during saponification, and can perform saponification of large batches of samples. At the same time, there is no need to transfer to a separating funnel for extraction, and the extraction is completed directly in the colorimetric tube, which greatly improves the ability of batch processing when there are more samples, saving time costs. In addition, the saponified solution only requires one-tenth of other methods, and the reagent solution required for extraction only requires one-fifth, saving the cost of reagents.

[0014] Preferably, in step (1), the saponification is carried out in a colorimetric tube, and the specific steps include: adding a camellia oil sample to the colorimetric tube, then adding a potassium hydroxide-ethanol solution, stirring and saponifying at 80-90° C. for 40-60 min, stopping heating, and cooling to obtain a saponified solution.

[0015] Preferably, in step (1), the extraction is performed by extracting the saponified liquid with n-hexane, and the specific steps include: extracting the saponified liquid in the colorimetric tube with n-hexane three times, standing and stratifying, and then centrifuging and washing the extract several times, discarding the lower aqueous solution each time, until the lower aqueous solution is neutral as tested by pH test paper.

[0016] Preferably, the silanization derivatization treatment is as follows: the sample solution is blown dry with nitrogen, a silanization derivatization reagent is added, and then the sample solution is heated at 70-80°C for 20-40 minutes, and after cooling, the sample solution is fixed to volume with n-hexane and then placed on a test machine for testing.

[0017] Preferably, the silylation derivatization reagent comprises a mixture of pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide in a volume ratio of 1:1, and the N,O-bis(trimethylsilyl)trifluoroacetamide mixture comprises N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane in a mass ratio of 99:1.

[0018] Preferably, in step (2), the standard solution is prepared with concentrations of 0, 5, 10, 20, 50, 100, and 200 μg / mL, respectively, and the solvent is a mixed solution of n-hexane and ethyl acetate in a volume ratio of 1:1.

[0019] Preferably, in step (3), the capillary chromatographic column is an HP-5 capillary column having a size of 30 m×0.32 mm×0.25 μm.

[0020] Preferably, detector temperature: 300-320°C; column flow: 1-1.5 mL / min; injection volume: 1 μL.

[0021] Preferably, the air flow rate is 400 mL / min; the hydrogen flow rate is 40 mL / min.

[0022] Preferably, the carrier gas is high-purity nitrogen; the tail gas flow rate is 30 mL / min.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The gas chromatography external standard method is used for determination. The external standard method for quantification of each triterpene alcohol substance is more scientific and advanced, and the determination results are more accurate and reliable;

[0025] (2) The non-derivatized method and the derivatized method are used for simultaneous determination. After the non-derivatized method is used to test and analyze the β-amyrin and butyryl cetyl alcohol in the sample, the sample is subjected to silylation derivatization and the lupeol therein is tested and analyzed to ensure the accuracy and reliability of the final result;

[0026] (3) The saponification method uses a colorimetric tube, which occupies a small area during saponification and can be used to saponify large quantities of samples. At the same time, the extraction can be completed directly in the colorimetric tube, which greatly improves the batch processing capacity when there are many samples and saves time and reagent costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the GC chart of β-amyrin standard solution.

[0028] Figure 2 This is the GC chart of butyryl cetyl alcohol standard solution.

[0029] Figure 3 This is the GC chart of lupeol standard solution.

[0030] Figure 4 This is the GC graph of the camellia oil sample in Comparative Example 2.

[0031] Figure 5 This is the GC graph of the camellia oil sample in Example 2.

[0032] Figure 6 This is the GC graph of the camellia oil sample in Comparative Example 1. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is described below with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0034] Example 1 (standard solution)

[0035] 1. Preparation of Standard Solutions

[0036] Information on triterpene alcohol standards: β-amyrin (CAS No.: 472-28-6), lupeol (CAS No.: 545-47-1), butyryl cetyl alcohol (CAS No.: 559-70-6) were purchased from Shanghai Anpu Laboratory Technology Co., Ltd. All standard curve working solutions were prepared and used immediately.

[0037] 1.1 Preparation of β-amyrin standard solution

[0038] Preparation of β-amyrin standard stock solution: Accurately weigh 5 mg (accurate to 0.1 mg) of β-amyrin standard into a 10 mL volumetric flask, dilute with n-hexane-ethyl acetate solution (1:1, v / v), make up to the mark, shake well, and prepare a final concentration of 500 μg / mL. Store at 0~4°C away from light for future use. The recommended shelf life is 2 months.

[0039] The β-amyrin standard stock solution was diluted into a series of standard solutions with different concentrations, with specific concentrations of 0, 5, 10, 20, 50, 100, and 200 μg / mL, so that the actual detection value of the sample was within the range of the standard curve.

[0040] 1.2 Preparation of butyryl cetyl alcohol standard solution

[0041] Preparation of butyryl cetyl alcohol standard stock solution: Accurately weigh 5 mg (accurate to 0.1 mg) of butyryl cetyl alcohol standard into a 10 mL volumetric flask, dilute with toluene, make up to the mark, shake well, and prepare a final concentration of 500 μg / mL. Store at 0~4°C away from light for future use. The recommended shelf life is 2 months.

[0042] The butyryl cetyl alcohol standard stock solution was diluted into a series of standard solutions with different concentrations, with specific concentrations of 0, 5, 10, 20, 50, 100, and 200 μg / mL, respectively, so that the actual detection value of the sample was within the range of the standard curve.

[0043] 1.3 Preparation of lupeol standard solution

[0044] Preparation of lupeol standard stock solution: Accurately weigh 5 mg (accurate to 0.1 mg) of lupeol standard into a 10 mL volumetric flask, dilute with n-hexane-ethyl acetate solution (1:1, v / v), make up to the mark, shake well, and prepare a final concentration of 500 μg / mL. Store at 0~4°C away from light for future use. The recommended shelf life is 2 months.

[0045] The lupeol standard stock solution was diluted into a series of standard solutions with different concentrations, with specific concentrations of 0, 5, 10, 20, 50, 100, and 200 μg / mL, so that the actual detection value of the sample was within the standard curve range. 200 μL of sample solution was accurately drawn, dried with nitrogen, and then 100 μL of pyridine and 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (containing trimethylchlorosilane) (mass fraction 99% BSTFA + mass fraction 1% TMCS) were added, and placed in a constant temperature drying oven at 70°C for 30 minutes for silanization derivatization. After cooling, 800 μL of n-hexane was accurately added, mixed, and the volume was fixed to 1 mL, ready for testing, and loaded on the machine.

[0046] 2. Gas chromatography detection

[0047] 3. Drawing of standard curve

[0048] Accurately measure 1 μL each of the β-amyrin standard solution, lupeol standard solution, and butyrylcetyl alcohol standard solution, inject them into the gas chromatograph, record the chromatogram, and draw a standard curve with the peak area as the ordinate and the concentration as the abscissa. The results are shown in Table 1.

[0049] Table 1

[0050]

[0051] The detection limit and quantification limit of each triterpene alcohol were determined based on the 3-fold signal-to-noise ratio and 10-fold signal-to-noise ratio. Based on the sample weight of 0.1 g and the fixed volume of 3 mL, the detection limits of β-amyrin, lupeol and butyryl cetyl alcohol were 21, 12 and 12 mg / kg, respectively; the quantification limits of β-amyrin, lupeol and butyryl cetyl alcohol were 81, 39 and 39 mg / kg, respectively, which greatly exceeded the daily sample testing requirements.

[0052] Example 2 (Precision)

[0053] 1. Sample Preparation

[0054] ① Weighing: Accurately weigh 0.1g camellia oil sample (accurate to 0.1mg) in a 25mL colorimetric tube;

[0055] ② Saponification: Add 5 mL of potassium hydroxide-ethanol solution to a 25 mL colorimetric tube, add the colorimetric tube to the rotor and place it on a magnetic stirrer at 80°C for saponification for 50 minutes, stop heating, remove and cool to room temperature, and add 5 mL of pure water;

[0056] ③Extraction: Extract the saponified solution in the colorimetric tube with 20 mL of n-hexane three times, let it stand for stratification, and then combine the extract into a 50 mL centrifuge tube;

[0057] ④ Washing: Wash the n-hexane extract with 10 mL of pure water each time, repeat the washing for two to four times, discard the lower layer of aqueous solution each time, and check with pH test paper until the lower layer of effluent is neutral (shake gently during the first washing to prevent emulsification, and the shaking intensity can be increased each time. If an emulsified layer appears, spray a small amount of saturated salt water);

[0058] ⑤ Concentration and volume adjustment: Put the neutralized n-hexane extract into a nitrogen blower and blow dry, finally dissolve it with n-hexane, ultrasonicate, vortex and adjust the volume to 3mL, filter it with a 0.45μm organic filter (the airflow should not be too large during nitrogen blowing to avoid splashing and loss of sample liquid) and wait for the test, and put it on the machine for the first time (to determine the content of β-amyrin and butyryl cetyl alcohol);

[0059] ⑥ Derivatization: Accurately draw 200μL sample solution from the filtered test solution, blow dry with nitrogen, add 100μL pyridine and 100μL N,O-bis(trimethylsilyl)trifluoroacetamide (containing trimethylchlorosilane) (mass fraction 99% BSTFA + mass fraction 1% TMCS), put it in a constant temperature drying oven and heat it at 70℃ for 30min for silylation derivatization, take it out and cool it, accurately add 800μL n-hexane, mix well, make up to 3mL, and put it on the machine for the second time (to determine the content of lupeol).

[0060] 2. Gas chromatography detection

[0061] The same batch of camellia oil samples were accurately weighed, a total of 6 parts, and a repeatability test was performed according to the operating steps in this embodiment, and the chromatogram was recorded, with the peak area as the ordinate and the concentration as the abscissa. As shown in Table 2, the content results were calculated according to the standard curve, and the RSD of β-amyrin was 0.89%, the RSD of lupeol was 2.63%, and the RSD of butyryl cetyl alcohol was 1.58%, and the RSD results did not exceed 5%, indicating that the precision results were good.

[0062] Table 2

[0063]

[0064] Example 3 (Reproducibility)

[0065] At different times, 6 samples of camellia oil from the same batch were accurately weighed, and a reproducibility test was performed according to the operating steps in Example 2. The chromatogram was recorded with the peak area as the ordinate and the concentration as the abscissa. As shown in Table 3, the content results were calculated according to the standard curve, and the RSD of β-amyrin was 3.11%, the RSD of lupeol was 0.93%, and the RSD of butyryl cetyl alcohol was 2.46%. The RSD results did not exceed 5%, indicating that the reproducibility results were good.

[0066] Table 3

[0067]

[0068] Example 4 (Comparison of different personnel)

[0069] Under the experimental operation of different personnel, the same batch of camellia oil samples were accurately weighed, a total of 6 parts, and the comparison test of different personnel was carried out according to the operating steps in Example 2, and the chromatogram was recorded, with the peak area as the ordinate and the concentration as the abscissa. As shown in Table 4, according to the content results calculated according to the standard curve, the RSD of β-amyrin was 1.50%, the RSD of lupeol was 0.37%, and the RSD of butyryl cetyl alcohol was 1.95%, and the RSD results did not exceed 5%, indicating that the comparison results of different personnel were good.

[0070] Table 4

[0071]

[0072] Example 5 (spiked recovery)

[0073] Camellia oil samples from the same batch were accurately weighed, and 0.5-3 times spike recovery tests were performed on them according to the background contents of β-amyrin, lupeol and butyryl cetyl alcohol in the samples. The test included spiking the background samples at low, medium and high concentrations, and 6 parallel tests were performed for each concentration.

[0074] 1. Preparation of β-amyrin spiked samples

[0075] ① Weighing: Accurately weigh 0.1g of camellia oil sample (accurate to 0.1mg) into a 25mL colorimetric tube, and add different concentrations of β-amyrin standard;

[0076] ② Saponification: Add 5 mL of potassium hydroxide-ethanol solution to a 25 mL colorimetric tube, add the colorimetric tube to the rotor and place it on a magnetic stirrer at 80°C for saponification for 50 minutes, stop heating, remove and cool to room temperature, and add 5 mL of pure water;

[0077] ③Extraction: Extract the saponified solution in the colorimetric tube with 20 mL of n-hexane three times, let it stand for stratification, and then combine the extract into a 50 mL centrifuge tube;

[0078] ④ Washing: Wash the n-hexane extract with 10 mL of pure water each time, repeat the washing two to four times, discard the lower aqueous solution each time, and check with pH paper until the lower effluent is neutral;

[0079] ⑤ Concentrate and fix the volume: Put the neutralized n-hexane extract into a nitrogen blower and blow dry. Finally, dissolve it with n-hexane, sonicate, vortex and fix the volume to 3mL. Filter it with a 0.45μm organic filter to obtain the test solution and put it on the machine.

[0080] 2. Preparation of Lupeol Spiked Samples

[0081] ① Weighing: Accurately weigh 0.1g of camellia oil sample (accurate to 0.1mg) in a 25mL colorimetric tube, and add different concentrations of lupeol standards;

[0082] ② Saponification: Add 5 mL of potassium hydroxide-ethanol solution to a 25 mL colorimetric tube, add the colorimetric tube to the rotor and place it on a magnetic stirrer at 80°C for saponification for 50 minutes, stop heating, remove and cool to room temperature, and add 5 mL of pure water;

[0083] ③Extraction: Extract the saponified solution in the colorimetric tube with 20 mL of n-hexane three times, let it stand for stratification, and then combine the extract into a 50 mL centrifuge tube;

[0084] ④ Washing: Wash the n-hexane extract with 10 mL of pure water each time, repeat the washing two to four times, discard the lower aqueous solution each time, and check with pH paper until the lower effluent is neutral;

[0085] ⑤ Concentrate and fix the volume: Put the neutralized n-hexane extract into a nitrogen blower to dry, dissolve it with n-hexane, sonicate, vortex and fix the volume to 3 mL, and filter it with a 0.45 μm organic filter to obtain the test solution;

[0086] ⑥ Derivatization: Accurately draw 200μL sample solution from the filtered test solution, blow dry with nitrogen, add 100μL pyridine and 100μL N,O-bis(trimethylsilyl)trifluoroacetamide (containing trimethylchlorosilane) (mass fraction 99% BSTFA + mass fraction 1% TMCS), put it in a constant temperature drying oven and heat it at 70℃ for 30min for silylation derivatization, take it out and cool it, add 800μL n-hexane, mix well, make up to 1mL, and put it on the machine.

[0087] 3. Preparation of butyrylcetyl alcohol spiked samples

[0088] ① Weighing: Accurately weigh 0.1g camellia oil sample (accurate to 0.1mg) into a 25mL colorimetric tube, and add different concentrations of butyryl cetyl alcohol standards;

[0089] ② Saponification: Add 5 mL of potassium hydroxide-ethanol solution to a 25 mL colorimetric tube, add the colorimetric tube to the rotor and place it on a magnetic stirrer at 80°C for saponification for 50 minutes, stop heating, remove and cool to room temperature, and add 5 mL of pure water;

[0090] ③Extraction: Extract the saponified solution in the colorimetric tube with 20 mL of n-hexane three times, let it stand for stratification, and then combine the extract into a 50 mL centrifuge tube;

[0091] ④ Washing: Wash the n-hexane extract with 10 mL of pure water each time, repeat the washing two to four times, discard the lower aqueous solution each time, and check with pH paper until the lower effluent is neutral;

[0092] ⑤ Concentrate and fix the volume: Put the neutralized n-hexane extract into a nitrogen blower and blow dry. Finally, dissolve it with n-hexane, sonicate, vortex and fix the volume to 3mL. Filter it with a 0.45μm organic filter to obtain the test solution and put it on the machine.

[0093] 2. Gas chromatography detection

[0094] According to the above steps, a repeatability test was carried out. As shown in Table 5, the overall recovery results were between 88.2% and 100.2%, and the RSD was less than 5%, indicating good test results.

[0095] Table 5

[0096]

[0097] Comparative Example 1 (Internal Standard Method)

[0098] 1. Sample Preparation

[0099] ① Weighing: Accurately pipette the prepared cholestanol internal standard solution into a 25mL colorimetric tube according to the calculation, blow dry it on a nitrogen blower, and accurately weigh 0.1g of sample (accurate to 0.1mg) into a 25mL colorimetric tube;

[0100] ② Saponification: Add 5 mL of potassium hydroxide-ethanol solution to a 25 mL colorimetric tube, add the colorimetric tube to the rotor and place it on a magnetic stirrer at 80°C for saponification for 50 minutes, stop heating, remove and cool to room temperature, and add 5 mL of pure water;

[0101] ③Extraction: Extract the saponified solution in the colorimetric tube with 20 mL of n-hexane three times, let it stand for stratification, and then combine the extract into a 50 mL centrifuge tube;

[0102] ④ Washing: Wash the n-hexane extract with 10 mL of pure water each time, repeat the washing two to four times, discard the lower aqueous solution each time, and check with pH paper until the lower effluent is neutral;

[0103] ⑤ Concentrate and fix the volume: Put the neutralized n-hexane extract into a nitrogen blower and blow dry. Finally, dissolve it with n-hexane, sonicate, vortex and fix the volume to 3mL (the internal standard addition amount is 30μg / mL). Filter it with a 0.45μm organic filter head and wait for testing. Then put it on the machine.

[0104] 2. Gas chromatography detection

[0105] Comparative Example 2 (External standard method, but the sample was not derivatized by silanization)

[0106] 1. Sample Preparation

[0107] ① Weighing: Accurately weigh 0.1g camellia oil sample (accurate to 0.1mg) in a 25mL colorimetric tube;

[0108] ② Saponification: Add 5 mL of potassium hydroxide-ethanol solution to a 25 mL colorimetric tube, add the colorimetric tube to the rotor and place it on a magnetic stirrer at 80°C for saponification for 50 minutes, stop heating, remove and cool to room temperature, and add 5 mL of pure water;

[0109] ③Extraction: Extract the saponified solution in the colorimetric tube with 20 mL of n-hexane three times, let it stand for stratification, and then combine the extract into a 50 mL centrifuge tube;

[0110] ④ Washing: Wash the n-hexane extract with 10 mL of pure water each time, repeat the washing two to four times, discard the lower aqueous solution each time, and check with pH paper until the lower effluent is neutral;

[0111] ⑤ Concentrate and fix the volume: Put the neutralized n-hexane extract into a nitrogen blower and blow dry. Finally, dissolve it with n-hexane, sonicate, vortex and fix the volume to 3mL. Filter it with a 0.45μm organic filter head and put it on the machine.

[0112] 2. Gas chromatography detection

[0113] Comparative Example 3 (External standard method, sample subjected to silanization derivatization)

[0114] 1. Sample Preparation

[0115] ① Weighing: Accurately weigh 0.1g camellia oil sample (accurate to 0.1mg) in a 25mL colorimetric tube;

[0116] ② Saponification: Add 5 mL of potassium hydroxide-ethanol solution to a 25 mL colorimetric tube, add the colorimetric tube to the rotor and place it on a magnetic stirrer at 80°C for saponification for 50 minutes, stop heating, remove and cool to room temperature, and add 5 mL of pure water;

[0117] ③Extraction: Extract the saponified solution in the colorimetric tube with 20 mL of n-hexane three times, let it stand for stratification, and then combine the extract into a 50 mL centrifuge tube;

[0118] ④ Washing: Wash the n-hexane extract with 10 mL of pure water each time, repeat the washing two to four times, discard the lower aqueous solution each time, and check with pH paper until the lower effluent is neutral;

[0119] ⑤ Concentration and volume adjustment: Put the neutralized n-hexane extract into a nitrogen blower and blow dry, then dissolve it with n-hexane, ultrasonicate it, vortex it and adjust the volume to 3 mL, and filter it with a 0.45 μm organic filter head;

[0120] ⑥ Derivatization: Accurately draw 200μL sample solution from the filtered test solution, blow dry with nitrogen, add 100μL pyridine and 100μL N,O-bis(trimethylsilyl)trifluoroacetamide (containing trimethylchlorosilane) (mass fraction 99% BSTFA + mass fraction 1% TMCS), put it in a constant temperature drying oven and heat it at 70℃ for 30min for silylation derivatization, take it out and cool it, accurately add 800μL n-hexane, mix well, make up to 1mL, and put it on the machine.

[0121] 2. Gas chromatography detection

[0122] The above examples and comparative examples all adopted the same gas chromatography analysis conditions as follows:

[0123] Gas chromatography column: HP-5 capillary column, column length 30m, inner diameter 0.32mm, film thickness 0.25μm;

[0124] Column flow rate: 1 mL / min;

[0125] Inlet temperature: 320°C;

[0126] Detector temperature: 300°C;

[0127] Injection volume: 1 μL;

[0128] Split ratio: 20:1;

[0129] Air flow rate: 400mL / min;

[0130] Hydrogen flow rate: 40mL / min;

[0131] Make-up gas flow rate: 30mL / min;

[0132] Carrier gas: high purity nitrogen (99.999%);

[0133] The column temperature was programmed: initially at 180 °C (held for 1 min), then increased from 180 °C to 260 °C at a rate of 15 °C / min and held for 35 min.

[0134] like Figure 1 , 2 3 are GC diagrams of the three standard solutions in Example 1. Comparative Example 1 uses the internal standard method for quantitative determination, and the internal standard substance is selected as cholestanol. The internal standard method for quantitative determination is more complicated in terms of the preparation of the standard substance and the pretreatment of the sample. At the same time, when the internal standard substance is used for the detection of camellia oil samples, Figure 6 As shown, there is an obvious interference peak at the retention time position, which affects the accurate calculation of the final result and does not conform to the selection principle of the internal standard. Therefore, the present invention verifies the determination of each triterpene alcohol substance by the external standard method (see the data of spike recovery and other data for the specific method verification data), and all the results can meet the requirements of the result accuracy. Therefore, the quantitative determination of the external standard method of the present invention is scientific and advanced, and the determination results are accurate and reliable.

[0135] Comparative Example 2: When the content of each triterpene alcohol in the camellia oil sample was determined by the non-derivatization method, Figure 4 As shown, the calculation results for β-amyrin and butyryl cetyl alcohol are accurate, but the result for lupeol is 50% too high. At the same time, the three substances were qualitatively analyzed by Agilent GC-MS. By determining whether the retention time of the target compound in the sample is consistent with the standard, whether both the qualitative ion and the quantitative ion appear in the sample, and whether the relative abundance ratio of the qualitative ion to the quantitative ion in the sample is within the allowable range, under the three necessary conditions, β-amyrin and butyryl cetyl alcohol can meet the qualitative requirements, while lupeol cannot meet the qualitative requirements and may contain other substances that interfere with the final results. In Comparative Example 3, the camellia oil sample was determined by the derivatization method, and the verification and Agilent GC-MS were also performed for qualitative analysis. The calculation results for β-amyrin and lupeol are accurate, while the result for butyryl cetyl alcohol is too high. By verifying the data by the two methods, the present invention finally selected the non-derivative method and the derivatization method for simultaneous determination, such as Figure 5 As shown, after the β-amyrin and butyryl cetyl alcohol in the sample were tested and analyzed by the non-derivatized method, the sample was subjected to silylation derivatization and the lupeol therein was tested and analyzed to ensure the accuracy and reliability of the final result.

[0136] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the specification of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for determining the content of triterpene alcohol in camellia oil, characterized in that: The steps include: (1) The camellia oil sample is subjected to saponification extraction. The camellia oil sample is added to a colorimetric tube, and then potassium hydroxide-ethanol solution is added, and the saponification is stirred at 80-90°C for 40-60 minutes. The heating is stopped, and the saponified solution is obtained after cooling. The saponified solution in the colorimetric tube is extracted with n-hexane three times; then the sample is put on the test machine to test and analyze β-amyrin and butyryl cetyl alcohol in the sample; then the sample is subjected to silanization derivatization treatment, and the sample is put on the test machine again to test and analyze lupeol in the sample; (2) preparing standard solutions of β-amyrin, butyryl cetyl alcohol and lupeol, respectively, wherein the standard solutions of β-amyrin and butyryl cetyl alcohol are directly tested on the machine, and the standard solution of lupeol is tested on the machine after silylation derivatization; (3) Gas chromatography detection: HP-5 capillary column; injection port temperature: 320-340°C; column temperature heating method: 180-190°C for 1-5 min, then increase the temperature to 260-300°C at a rate of 10-15°C / min, and maintain for 20-40 min; detector temperature: 300-320°C; split ratio: 20-40:

1.

2. The method for determining the content of triterpene alcohol in camellia oil according to claim 1, characterized in that: In step (1), the specific steps of extraction include: extracting the saponified liquid in the colorimetric tube with n-hexane three times, standing and stratifying, and then centrifuging and washing the extract several times, discarding the lower aqueous solution each time, until the lower aqueous solution is neutral when tested by pH test paper.

3. The method for determining the content of triterpene alcohol in camellia oil according to any one of claims 1-2, characterized in that: The silanization derivatization treatment is as follows: the sample solution is blown dry with nitrogen, a silanization derivatization reagent is added, and then the sample solution is heated at 70-80°C for 20-40 minutes. After cooling, the sample solution is fixed to volume with n-hexane and then placed on the machine for testing.

4. The method for determining the content of triterpene alcohol in camellia oil according to claim 3, characterized in that: The silanization derivatization reagent is a mixture of pyridine and N,O-bis(trimethylsilyl)trifluoroacetamide in a volume ratio of 1:1, and the N,O-bis(trimethylsilyl)trifluoroacetamide mixture is a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane in a mass ratio of 99:

1.

5. The method for determining the content of triterpene alcohol in camellia oil according to claim 1, characterized in that: In step (2), the standard solutions are prepared with concentrations of 0, 5, 10, 20, 50, 100, and 200 μg / mL, respectively, and the solvent is a mixed solution of n-hexane and ethyl acetate in a volume ratio of 1:

1.

6. The method for determining the content of triterpene alcohol in camellia oil according to claim 1, characterized in that: In step (3), the dimensions of the HP-5 capillary column are: 30 m×0.32 mm×0.25 μm.

7. The method for determining the content of triterpene alcohol in camellia oil according to claim 1, 5 or 6, characterized in that: Column flow rate: 1-1.5mL / min; injection volume: 1μL.

8. The method for determining the content of triterpene alcohol in camellia oil according to claim 1, characterized in that: Air flow rate: 400mL / min; Hydrogen flow rate: 40mL / min.

9. The method for determining the content of triterpene alcohol in camellia oil according to claim 1 or 8, characterized in that: Carrier gas: nitrogen; tail gas flow rate: 30mL / min.

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