Method for determining squalene content in white spirit

Abstract

The invention relates to the technical field of detection, and particularly provides a method for determining the content of squalene in baijiu, the method utilizes gas chromatography-triple quadrupole mass spectrometry to determine the content of squalene in baijiu with different flavors, and the content of squalene in baijiu with different flavors is determined by adding reagents such as vitamin C and vitamin E and adopting effective treatment measures such as parallel concentration in a sample pretreatment process. The method improves the detection result repeatability and the detection efficiency, reduces the detection limit, has the advantages of good repeatability, high sensitivity, high detection efficiency and small organic solvent dosage, and is beneficial to environmental protection and occupational health and safety.

Description

Technical Field

[0001] The invention relates to the technical field of liquor detection, and in particular to a method for determining the content of squalene in liquor. Background Art

[0002] As the Chinese people pay more attention to healthy living, the traditional concept of liquor is also changing. Squalene is a terpene compound with biological activities such as anti-cancer, anti-tumor, and antioxidant. It can slow down the aging process of the skin, reduce physical fatigue, and strengthen the immune system. It is a typical and practical biologically active substance. Therefore, it has become a development trend for liquor to contain squalene.

[0003] However, the content of squalene in liquor is relatively low, and conventional detection methods require high-multiple concentration. General concentration methods such as water bath evaporation and nitrogen blowing will cause the sample to be exposed for too long, resulting in squalene oxidation loss; rotary evaporation concentration is not only time-consuming and has low detection efficiency, but also affects the repeatability of test results due to the difficulty in controlling the consistency of concentration rate between samples; the liquid-liquid microextraction process has certain requirements on the solvent of the sample body, and organic solvents such as ethanol will affect the extraction efficiency, thereby affecting the repeatability and accuracy of the test.

[0004] Therefore, it is urgent to design a method for determining the squalene content in liquor to ensure that the squalene in the sample is not oxidized during the pretreatment process and to improve the detection repeatability, accuracy and timeliness. Summary of the invention

[0005] In view of this, the present invention proposes a method for determining the content of squalene in liquor, aiming to improve the repeatability and accuracy of the determination of the content of squalene in liquor.

[0006] The technical solution of the present invention is achieved as follows: The present invention provides a method for determining the content of squalene in liquor, the steps of which include:

[0007] Step 1: Mix the liquor sample to be tested with the internal standard solution, concentrate, dry and redissolve to obtain a test solution;

[0008] Step 2, dissolving the squalene standard substance in a solvent to prepare a series of squalene standard solutions, adding an internal standard solution to the series of squalene standard solutions to obtain a standard solution to be tested;

[0009] Step 3: Use gas chromatography-mass spectrometry to detect the test solution and the standard solution to be tested, collect the peak areas of squalene and internal standard respectively, establish a standard curve with the squalene concentration in the standard solution to be tested as the horizontal coordinate and the peak area ratio of squalene to the internal standard as the vertical coordinate, and calculate the content of squalene in the test solution to be tested using the standard curve.

[0010] In some embodiments, the internal standard solution is a squalane solution with a mass concentration of 100-200 mg / L.

[0011] Using a squalane solution in the concentration range of 100-200 mg / L can ensure the detection of the internal standard without excessively interfering with the detection signal of squalene.

[0012] In some embodiments, the preparation method of the squalane solution includes: taking a squalane standard substance, dissolving it with anhydrous ethanol to obtain an internal standard stock solution with a mass concentration of 1000-2000 mg / L, and diluting the internal standard stock solution with n-hexane as a solvent to obtain a squalane solution with a mass concentration of 100-200 mg / L.

[0013] In some embodiments, in step 1, 20 ml of the liquor sample to be tested is mixed with 20 μL of the internal standard solution, concentrated and dried, dissolved with n-hexane and fixed to 2 ml, and filtered with a 0.22 μm organic filter membrane to obtain a test solution.

[0014] In some embodiments, in step 2, 26.76 mg of squalene standard substance is taken in a 10 mL volumetric flask, dissolved with anhydrous ethanol and diluted to the scale to obtain a squalene stock solution with a concentration of 2676 mg / L, and n-hexane is used as a solvent to further dilute the squalene stock solution into a series of standard solutions with a concentration range of 26.76 μg / L-1338 μg / L, and 1 ml of the squalene series standard solution is taken respectively, and 10 μL of the internal standard solution is added to obtain the standard solution to be tested.

[0015] The above concentration range covers the expected squalene concentration range, ensuring the linear response of the detection.

[0016] In some embodiments, in step one, the test solution to be tested also includes at least one of a vitamin C solution and a vitamin E solution.

[0017] In some embodiments, the concentrations of the vitamin C solution and the vitamin E solution are 2000-10000 mg / L, respectively.

[0018] Vitamin C and vitamin E are added to the sample as antioxidants to prevent squalene from being lost due to oxidation during sample processing. The concentration range is set at 2000-10000 mg / L because within this range, the oxidation of squalene can be effectively prevented during sample processing without interfering with subsequent analysis steps.

[0019] In some embodiments, when adding vitamin C solution, 100 μL of vitamin C solution is added to every 20 ml of the liquor sample to be tested, and when adding vitamin E solution, 100 μL of vitamin E solution is added to every 20 ml of the liquor sample to be tested.

[0020] The amount of added vitamin C or vitamin E was optimized to ensure adequate antioxidant protection without affecting the volume and concentration of the sample. Adding 100 μL per 20 ml sample is an experimentally proven effective dose that provides adequate protection.

[0021] In some embodiments, the method of concentration and drying includes: using a parallel evaporation concentrator, the water bath temperature is 50°C, the negative pressure is reduced to 50 psi, and the wine sample is concentrated to dryness, and the negative pressure reduction procedure is: the initial pressure is atmospheric pressure, which is reduced to 800 psi within 2 minutes, and then reduced to 500 psi at a rate of 150 psi / min, maintained for 3 minutes, reduced to 300 psi at a rate of 100 psi / min, maintained for 3 minutes, reduced to 250 psi at a rate of 20 psi / min, maintained for 2 minutes, and reduced to 50 psi at a rate of 2 psi / min before concentrating to near dryness.

[0022] The parallel evaporation concentrator is used to process multiple samples at the same time and improve efficiency. The water bath temperature is 50°C, which is set at a relatively low temperature to reduce the risk of thermal oxidation of squalene. The gradual reduction of negative pressure is to control the evaporation rate, making the sample concentration process more gentle and uniform, and preventing excessive evaporation from causing sample loss or squalene oxidation. This method can improve the repeatability and accuracy of the test while ensuring the integrity of the sample.

[0023] In some embodiments, the instrument for the gas chromatography-mass spectrometry is a gas chromatography-triple quadrupole mass spectrometer, and the analysis conditions are: the stationary phase of the chromatographic column is (5%-phenyl)-methylpolysiloxane, the column length is 30m, the inner diameter is 0.25mm, and the liquid film is 0.25μm; the carrier gas is high-purity He (purity ≥99.999%), constant flow mode, and the flow rate is 1.0mL / min; the heating program is an initial temperature of 80°C, maintained for 1min, increased to 295°C at 10°C / min, maintained for 1min, and then the operating temperature is 320°C, maintained for 5min; the injection port temperature is 250°C, non-divided mode, and the injection volume is 1μL; the mass spectrometry conditions are: electron bombardment ion source (EI), electron energy 70eV, ion source temperature 230°C, transmission line temperature 280°C, quadrupole temperature 150°C, solvent delay 7min, multiple reaction monitoring acquisition mode (MRM).

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The method for determining the content of squalene in liquor provided by the present invention can prepare more than 30 batches of samples at the same time, the method is simple and fast, and at the same time, a better concentration method is adopted, which effectively avoids the loss of squalene that is easily oxidized during the sample concentration process, and improves the accuracy of the detection method for squalene detection. Secondly, the parameters such as temperature, pressure, and time are optimized in the sample pretreatment method, and the repeatability of sample detection is improved. The method can increase the processing volume of samples per unit time, which is conducive to improving processing efficiency, improving detection timeliness, and reducing detection costs. Moreover, the amount of organic reagents used in the detection method is less, which reduces pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 This is a standard curve diagram of squalene in Example 1 of the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with the definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0030] Unless otherwise specified, the methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents and instruments used are all conventional materials, reagents and instruments in the art, and can be obtained by those skilled in the art through commercial channels.

[0031] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including the range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range. In the present specification and claims, range definitions can be combined and / or interchanged, and if not otherwise stated, these ranges include all subranges contained therein.

[0032] Example 1

[0033] This embodiment provides a method for detecting the mass concentration of squalene in liquor, and the specific steps are as follows:

[0034] Preparation of samples to be tested:

[0035] The samples to be tested in this embodiment include Maotai-flavor liquor, Luzhou-flavor liquor, and Light-flavor liquor, all of which are provided by Jinpai Co., Ltd.

[0036] instrument:

[0037] Gas chromatography-triple quadrupole mass spectrometer, model 8890-7000D, purchased from Agilent Technologies;

[0038] Electronic balance: 1 / 100,000 electronic balance, model AB135-S, purchased from Mettler-Toledo;

[0039] Water purifier: Model: Option R7-Flex2, purchased from Veolia Water Treatment Technology (Shanghai) Co., Ltd.;

[0040] Parallel evaporation concentrator: model MPE, purchased from Ruike Group Co., Ltd.

[0041] Reagents and drugs:

[0042] Anhydrous ethanol and n-hexane were of chromatographic grade; squalene standard substance (purity>98%), squalane standard substance (purity>99%), vitamin C (purity>99.9%), and vitamin E (purity>99.9%) were purchased from Shanghai Anpu Laboratory Technology Co., Ltd.

[0043] Vitamin C solution: Accurately weigh an appropriate amount of vitamin C into a volumetric flask, add water to dissolve and make up to volume to prepare a solution containing 5 mg of vitamin C per ml.

[0044] Vitamin E solution: Accurately weigh an appropriate amount of vitamin E into a volumetric flask, add anhydrous ethanol to dissolve and make up to volume to prepare a solution containing 5 mg of vitamin E per milliliter.

[0045] Preparation of squalene solution:

[0046] Accurately weigh 26.76 mg of squalene standard substance into a 10 mL volumetric flask, dissolve it in anhydrous ethanol and dilute to the mark to obtain a squalene stock solution with a concentration of 2676 mg / L. Use n-hexane as the solvent to further dilute it into a series of squalene standard solutions with a concentration range of 26.76 μg / L-1338 μg / L.

[0047] Preparation of squalane internal standard solution

[0048] Accurately weigh 14.63 mg of squalane standard substance into a 10 mL volumetric flask, dissolve it in anhydrous ethanol to the mark, and obtain an internal standard stock solution with a mass concentration of 1463 mg / L. Accurately draw a certain volume of the internal standard stock solution and prepare a squalane internal standard solution with a concentration of 146.3 mg / L using n-hexane as the solvent.

[0049] Preparation of test solution:

[0050] Accurately measure 20ml of Maotai-flavor liquor sample in a 50mL concentrator tube, add 20μl internal standard solution, 100μl vitamin C solution, 100μl vitamin E solution, vortex mix, transfer to parallel evaporation concentrator, concentration temperature is 50℃, negative pressure drop procedure is: initial pressure is atmospheric pressure, drop to 800psi within 2min, then drop to 500psi at a rate of 150psi / min, keep for 3min, drop to 300psi at a rate of 100psi / min, keep for 3min, drop to 250psi at a rate of 20psi / min, keep for 2min, drop to 50psi at a rate of 2psi / min and concentrate to dryness. Take out the concentrator tube and wait for cooling, then re-dissolve with n-hexane, then transfer to a 2mL volumetric flask and make up to volume with n-hexane as solvent, filter in a sample bottle through a 0.22μm filter membrane, and obtain the test solution.

[0051] Preparation of standard solution:

[0052] Accurately pipette 1 mL of squalene series standard working solution into injection vials, accurately add 10 μl of internal standard solution, mix well, and obtain the standard solution to be tested.

[0053] Sample testing:

[0054] Instrument conditions: Agilent 8890 gas chromatograph, triple quadrupole mass spectrometer (GC-MS / MS); the chromatographic column stationary phase is (5%-phenyl)-methylpolysiloxane, the column length is 30m, the inner diameter is 0.25mm, and the liquid film is 0.25μm; the carrier gas is high-purity He (purity ≥99.999%), the flow rate is 1.0mL / min in constant flow mode; the temperature program is an initial temperature of 80℃, maintained for 1min, increased to 295℃ at 10℃ / min, maintained for 1min, and then the operating temperature is 320℃, maintained for 5min; the injection port temperature is 250℃, non-split mode, and the injection volume is 1μL; the mass spectrometry conditions are: electron bombardment ion source (EI), electron energy 70eV, ion source temperature 230℃, transfer line temperature 280℃, quadrupole temperature 150℃, solvent delay 7min, multiple reaction monitoring acquisition mode (MRM).

[0055] Preparation of standard curve: The standard solution to be tested is subjected to gas chromatography-triple quadrupole mass spectrometry analysis. The concentration of squalene in each standard solution is used as the horizontal axis, and the peak area ratio of squalene to squalane is used as the vertical axis to establish a standard curve as shown in the following figure: Figure 1 The concentration of squalene at 3 times the signal-to-noise ratio was taken as the limit of detection (LOD), and the concentration of squalene at 10 times the signal-to-noise ratio was taken as the limit of quantification (LOQ). The following table lists the standard curve equation, linear range, limit of detection, limit of quantification, etc. of squalene.

[0056]

[0057] Calculation: The test solution and the standard solution are measured under the same detection conditions. The squalene content in the liquor sample to be tested is calculated according to the following formula. The calculation result is expressed as the arithmetic mean of the independent measurement results obtained under repeatability conditions, and is retained to 4 decimal places.

[0058]

[0059] Where:

[0060] C x ------Mass concentration of squalene in the sample, in micrograms per liter (μg / L);

[0061] C i ------The mass concentration of squalene in the test solution, in micrograms per liter (μg / L);

[0062] V1------sample sampling volume, in mL;

[0063] V2-------The volume of the test solution, in mL;

[0064] Repeatability experiment:

[0065] Using Maotai-flavor liquor as the repeatability experimental sample, accurately measure 20 ml of the liquor sample to be tested in a 50 ml concentrator tube, a total of 6 portions, marked as S-1 to S-6, treat the sample solution according to the method described in "Preparation of the test solution", determine the squalene content in the test solution, and calculate the repeatability of the squalene determination results in the sample. The repeatability results show that the repeatability RSD value is 2.69%, indicating that the method has good repeatability. The specific results are shown in the following table:

[0066]

[0067] Spike recovery experiment:

[0068] The batch of Maotai-flavor liquor used in the "Repeatability Experiment" was used as the spike recovery experimental sample, and the spike recovery rate was determined at three concentration levels: high, medium, and low. Three parallel samples were prepared for each concentration. The sample solution was treated according to the method described in "Preparation of the Test Solution", the squalene content in the test solution was determined, and the recovery rate of the squalene determination results in the sample was calculated. The recovery rate of squalene was 94.7% to 98.0%, and the RSD value was 1.73%, indicating that the recovery rate of this method was good. The specific calculation results are shown in the following table:

[0069]

[0070] Recovery experiment of spiked light-flavor and strong-flavor liquor:

[0071] According to the method of "spiked recovery experiment" of the above-mentioned Maotai-flavor liquor, spiked recovery experiments were carried out on the light-flavor liquor and the strong-flavor liquor. The squalene content in the samples was first determined. The experimental results showed that the light-flavor liquor sample contained 28.3514μg / L squalene, and the strong-flavor liquor contained 36.6598μg / L squalene. Then, the sample spiked experiment was carried out. 20mL of the light-flavor liquor and the strong-flavor liquor samples were accurately measured in 50mL concentrator tubes, and 20μL of 26.76mg / L squalene standard substance solution was added. Three test samples were prepared for each flavor. The light-flavor liquor samples were named: Q-JB1~Q-JB3, and the strong-flavor liquor samples were named: N-JB1~N-JB3. The sample solutions were treated according to the method described in "Preparation of test sample solutions", the squalene content in the test sample solutions of the spiked recovery samples was determined, and the recovery rate of the squalene determination results in the samples was calculated. The results show that this method is also suitable for the determination of squalene content in light-flavor and strong-flavor liquors. The results of the spike recovery experiments for light-flavor and strong-flavor liquors are shown in the following table:

[0072]

[0073] Example 2

[0074] This embodiment provides a method for detecting the mass concentration of squalene in liquor, and the specific steps are as follows:

[0075] Liquor samples to be tested:

[0076] Maotai-flavor liquor, provided by Jinpai Co., Ltd.

[0077] The difference from Example 1 is that the test solution is prepared using the following steps:

[0078] Accurately measure 20mL of the liquor sample to be tested in a 50mL concentrator tube, add 20μL of squalane internal standard with a concentration of 146.3mg / L, vortex mix, transfer to a parallel evaporation concentrator, the concentration temperature is 50℃, and the negative pressure drop procedure is: the initial pressure is atmospheric pressure, and it is reduced to 800psi within 2min, then reduced to 500psi at a rate of 150psi / min, maintained for 3min, reduced to 300psi at a rate of 100psi / min, maintained for 3min, reduced to 250psi at a rate of 20psi / min, maintained for 2min, and concentrated to 50psi at a rate of 2psi / min. After cooling, remove the concentrator tube, dissolve it in n-hexane, and then transfer it to a 2mL volumetric flask and make up the volume with n-hexane as the solvent. The organic phase is filtered through a 0.22μm filter membrane in a sample bottle to obtain the test solution.

[0079] Repeatability experiment: The test solution was divided into six parts, and the squalene content in the test solution was determined according to the sample detection method of Example 1, and the repeatability of the determination results was calculated.

[0080] The batch of Maotai-flavor liquor used in the above-mentioned "Repeatability Experiment" in this embodiment was used as the spiked recovery experimental sample, and a certain volume of squalene standard solution was added to obtain the experimental sample to be tested. 20 ml of the experimental sample to be tested was accurately measured and placed in a 50 ml concentration tube. The experimental sample to be tested was then treated according to the method described in the "Preparation of Test Solution" in this embodiment, the squalene content in the test solution was determined, and the recovery rate of the squalene determination result in the sample was calculated.

[0081] Example 3

[0082] This embodiment provides a method for detecting the mass concentration of squalene in liquor, and the specific steps are as follows:

[0083] Liquor samples to be tested:

[0084] Maotai-flavor liquor, provided by Jinpai Co., Ltd.

[0085] The difference from Example 1 is that the test solution is prepared using the following steps:

[0086] Accurately measure 20mL of the liquor sample to be tested in a 50mL concentrator tube, add 100μL of 5000mg / L vitamin C and 20μL of 146.3mg / L squalane internal standard, vortex mix, transfer to a parallel evaporation concentrator, the concentration temperature is 50℃, and the negative pressure drop procedure is: the initial pressure is atmospheric pressure, and it is reduced to 800psi within 2min, then reduced to 500psi at a rate of 150psi / min, maintained for 3min, reduced to 300psi at a rate of 100psi / min, maintained for 3min, reduced to 250psi at a rate of 20psi / min, maintained for 2min, and concentrated to 50psi at a rate of 2psi / min. After cooling, remove the concentrator tube, dissolve it in n-hexane, and then transfer it to a 2mL volumetric flask and make up the volume with n-hexane as the solvent. The organic phase is filtered through a 0.22μm filter membrane in a sample bottle to obtain the test solution.

[0087] Repeatability experiment: The test solution was divided into six parts, and the squalene content in the test solution was determined according to the sample detection method of Example 1, and the repeatability of the determination results was calculated.

[0088] The batch of Maotai-flavor liquor used in the above-mentioned "Repeatability Experiment" in this embodiment was used as the spiked recovery experimental sample, and a certain volume of squalene standard solution was added to obtain the experimental sample to be tested. 20 ml of the experimental sample to be tested was accurately measured and placed in a 50 ml concentration tube. The experimental sample to be tested was then treated according to the method described in the "Preparation of Test Solution" in this embodiment, the squalene content in the test solution was determined, and the recovery rate of the squalene determination result in the sample was calculated.

[0089] Example 4

[0090] This embodiment provides a method for detecting the mass concentration of squalene in liquor, and the specific steps are as follows:

[0091] Liquor samples to be tested:

[0092] Maotai-flavor liquor, provided by Jinpai Co., Ltd.

[0093] The difference from Example 1 is that the test solution is prepared using the following steps:

[0094] Accurately measure 20mL of the liquor sample to be tested in a 50mL concentrator tube, add 100μL of 5000mg / L vitamin E and 20μL of 146.3mg / L squalane internal standard, vortex mix, transfer to a parallel evaporation concentrator, the concentration temperature is 50℃, and the negative pressure drop procedure is as follows: the initial pressure is atmospheric pressure, which is reduced to 800psi within 2min, then reduced to 500psi at a rate of 150psi / min, maintained for 3min, reduced to 300psi at a rate of 100psi / min, maintained for 3min, reduced to 250psi at a rate of 20psi / min, maintained for 2min, and concentrated to 50psi at a rate of 2psi / min. After cooling, remove the concentrator tube, dissolve it in n-hexane, and then transfer it to a 2mL volumetric flask and make up the volume with n-hexane as the solvent. The organic phase is filtered through a 0.22μm filter membrane in a sample bottle to obtain the test solution.

[0095] Repeatability experiment: The test solution was divided into six parts, and the squalene content in the test solution was determined according to the sample detection method of Example 1, and the repeatability of the determination results was calculated.

[0096] The batch of Maotai-flavor liquor used in the above-mentioned "Repeatability Experiment" in this embodiment was used as the spiked recovery experimental sample, and a certain volume of squalene standard solution was added to obtain the experimental sample to be tested. 20 ml of the experimental sample to be tested was accurately measured and placed in a 50 ml concentration tube. The experimental sample to be tested was then treated according to the method described in the "Preparation of Test Solution" in this embodiment, the squalene content in the test solution was determined, and the recovery rate of the squalene determination result in the sample was calculated.

[0097] Example 5

[0098] This embodiment provides a method for detecting the mass concentration of squalene in liquor, and the specific steps are as follows:

[0099] Liquor samples to be tested:

[0100] Maotai-flavor liquor, provided by Jinpai Co., Ltd.

[0101] The difference from Example 1 is that the test solution is prepared using the following steps:

[0102] Accurately measure 20mL of the liquor sample to be tested in a 50mL concentrator tube, add 100μL of 5000mg / L vitamin C and vitamin E, 20μL of 146.3mg / L squalane internal standard, vortex mix, transfer to a parallel evaporation concentrator, the concentration temperature is 50℃, and the negative pressure drop procedure is: the initial pressure is atmospheric pressure, and it is reduced to 800psi within 2min, then reduced to 500psi at a rate of 150psi / min, maintained for 3min, reduced to 300psi at a rate of 100psi / min, maintained for 3min, reduced to 250psi at a rate of 20psi / min, maintained for 2min, and concentrated to 50psi at a rate of 2psi / min. After cooling, remove the concentrator tube, dissolve it in n-hexane, and then transfer it to a 2mL volumetric flask and make up the volume with n-hexane as the solvent. The organic phase is filtered through a 0.22μm filter membrane in a sample bottle to obtain the test solution.

[0103] Repeatability experiment: The test solution was divided into six parts, and the squalene content in the test solution was determined according to the sample detection method of Example 1, and the repeatability of the determination results was calculated.

[0104] The batch of Maotai-flavor liquor used in the above-mentioned "Repeatability Experiment" in this embodiment was used as the spiked recovery experimental sample, and a certain volume of squalene standard solution was added to obtain the experimental sample to be tested. 20 ml of the experimental sample to be tested was accurately measured and placed in a 50 ml concentration tube. The experimental sample to be tested was then treated according to the method described in the "Preparation of Test Solution" in this embodiment, the squalene content in the test solution was determined, and the recovery rate of the squalene determination result in the sample was calculated.

[0105] The repeatability results of Examples 2-5 above show that the addition of antioxidants and the type of addition have a significant effect on repeatability. The repeatability RSD is as follows from large to small: no addition > vitamin C addition > vitamin E addition > vitamin E + vitamin C addition. When vitamin C and vitamin E are added at the same time, the repeatability result is the best, with an RSD value of 2.69%. When no antioxidant is added, the repeatability result is the worst, with an RSD value of 27.2%. The specific results are shown in the following table:

[0106]

[0107] At the same time, the results of the spike recovery experiments in Examples 2-5 above show that the addition of antioxidants and the type of addition have a significant effect on the recovery rate. The spike recovery rates are as follows from small to large: no addition > vitamin C addition > vitamin E addition > vitamin E + vitamin C addition. When vitamin C and vitamin E are added at the same time, the spike recovery rate results are the best, which are 92.98%-96.56%. When no antioxidant is added, the spike recovery rate results are the worst, which are 56.89%-79.93%. The specific results are shown in the following table:

[0108]

[0109]

[0110] Comparative Example 1

[0111] This comparative example conducted an experiment to test the effect of different concentration methods on the repeatability of the test results.

[0112] Liquor samples to be tested:

[0113] Maotai-flavor liquor, provided by Jinpai Co., Ltd.

[0114] The difference from Example 1 is that the test solution is prepared using the following steps:

[0115] Accurately measure 20mL of the liquor sample to be tested in a 50mL concentrator tube, add 100μL of 5000mg / L vitamin C and vitamin E, 20μL of 146.3mg / L squalane internal standard solution, vortex mix, transfer to an eggplant-shaped bottle, and concentrate to near dryness by rotary evaporation at 50°C. Remove the eggplant-shaped bottle and wait for cooling, then re-dissolve with n-hexane, and then transfer to a 2mL volumetric flask and make up to volume with n-hexane as solvent. Filter the organic phase through a 0.22μm filter membrane in a sample bottle to obtain the test solution.

[0116] Repeatability experiment: The test solution was divided into six parts, and the squalene content in the test solution was determined according to the sample detection method of Example 1, and the mass concentration of squalene in the sample and the repeatability of the determination result were calculated at the same time.

[0117] The experimental data results of the above comparative examples 1 and 2 are as follows:

[0118]

[0119] The repeatability results showed that the repeatability RSD value of the samples treated by rotary evaporation concentration (11.2%) was significantly higher than that of the samples treated by parallel evaporation concentration (2.69%).

[0120] From the above implementation cases, it can be seen that the innovative addition of antioxidants in the present invention improves the repeatability and accuracy of squalene detection results in liquor, and effectively improves the detection efficiency and sample detection repeatability through parallel concentration. Only a small amount of organic reagents are used in the pretreatment process, which reduces pollution to the environment. It has the advantages of good repeatability, high sensitivity, rapidity and accuracy, and a small amount of organic solvents, and is suitable for popularization and use.

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining the content of squalene in liquor, characterized in that: The method comprises the following steps: Step 1: Mix the liquor sample to be tested with the internal standard solution, concentrate, dry and redissolve to obtain a test solution; Step 2, dissolving the squalene standard substance in a solvent to prepare a series of squalene standard solutions, adding an internal standard solution to the series of squalene standard solutions to obtain a standard solution to be tested; Step 3: Use gas chromatography-mass spectrometry to detect the test solution and the standard solution to be tested, collect the peak areas of squalene and internal standard respectively, establish a standard curve with the squalene concentration in the standard solution to be tested as the horizontal coordinate and the peak area ratio of squalene to the internal standard as the vertical coordinate, and calculate the content of squalene in the test solution to be tested using the standard curve.

2. The method for determining the content of squalene in liquor according to claim 1, characterized in that: The internal standard solution is a squalane solution with a mass concentration of 100-200 mg / L.

3. The method for determining the content of squalene in liquor according to claim 2, characterized in that: The preparation method of squalane solution comprises: taking squalane standard substance, dissolving it with anhydrous ethanol to obtain an internal standard stock solution with a mass concentration of 1000-2000 mg / L, and diluting the internal standard stock solution with n-hexane as solvent to obtain a squalane solution with a mass concentration of 100-200 mg / L.

4. The method for determining the content of squalene in liquor according to claim 1, characterized in that: In step 1, 20 ml of the liquor sample to be tested is mixed with 20 μL of the internal standard solution, concentrated and dried, dissolved with n-hexane and fixed to 2 ml, and filtered to obtain the test solution.

5. The method for determining the content of squalene in liquor according to claim 1, characterized in that: In step 2, 26.76 mg of squalene standard substance was taken into a 10 mL volumetric flask, dissolved with anhydrous ethanol and diluted to the scale to obtain a squalene stock solution with a concentration of 2676 mg / L. The squalene stock solution was further diluted with n-hexane as a solvent to form a series of standard solutions with a concentration range of 26.76 μg / L-1338 μg / L. 1 ml of the squalene series standard solution was taken respectively, and 10 μL of the internal standard solution was added to obtain the standard solution to be tested.

6. The method for determining the content of squalene in liquor according to claim 1, characterized in that: Step one also includes adding at least one of a vitamin C solution and a vitamin E solution to the test sample solution.

7. The method for determining the content of squalene in liquor according to claim 6, characterized in that: The concentrations of the vitamin C solution and the vitamin E solution are 2000-10000 mg / L respectively.

8. The method for determining the content of squalene in liquor according to claim 6, characterized in that: When adding vitamin C solution, add 100 μL of vitamin C solution to every 20 ml of the liquor sample to be tested. When adding vitamin E solution, add 100 μL of vitamin E solution to every 20 ml of the liquor sample to be tested.

9. The method for determining the content of squalene in liquor according to claim 1, characterized in that: The method of concentration and drying includes: using a parallel evaporation concentrator, with a water bath temperature of 50°C and a negative pressure reduced to 50 psi, concentrating the wine sample to dryness, and the negative pressure reduction procedure is as follows: the initial pressure is atmospheric pressure, which is reduced to 800 psi within 2 minutes, then reduced to 500 psi at a rate of 150 psi / min, maintained for 3 minutes, reduced to 300 psi at a rate of 100 psi / min, maintained for 3 minutes, reduced to 250 psi at a rate of 20 psi / min, maintained for 2 minutes, and reduced to 50 psi at a rate of 2 psi / min before concentrating to near dryness.

10. The method for determining the content of squalene in liquor according to claim 1, characterized in that: The instrument for the mass spectrometry was a gas chromatography-triple quadrupole mass spectrometer, and the analysis conditions were as follows: the stationary phase of the chromatographic column was (5%-phenyl)-methylpolysiloxane, the column length was 30 m, the inner diameter was 0.25 mm, and the liquid film was 0.25 μm; the carrier gas was high-purity He, in constant flow mode, with a flow rate of 1.0 mL / min; the temperature program was an initial temperature of 80°C, maintained for 1 min, increased to 295°C at 10°C / min, maintained for 1 min, and then the post-operation temperature was 320°C, maintained for 5 min; the injection port temperature was 250°C, in non-divided flow mode, and the injection volume was 1 μL; the mass spectrometry conditions were: electron impact ion source (EI), electron energy 70 eV, ion source temperature 230°C, transfer line temperature 280°C, quadrupole temperature 150°C, solvent delay 7 min, and multiple reaction monitoring acquisition mode.

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