Detection method for simultaneously detecting 11 terpene compounds in health care wine

Abstract

The invention relates to the technical field of detection, and provides a detection method for simultaneously detecting 11 terpene compounds in health care wine, which comprises the following steps: removing non-volatile components such as sugar, pigment and the like in the health care wine by solvent-assisted evaporation extraction, performing liquid-liquid micro-extraction on terpene target objects by using a polar and non-polar mixed extraction agent, and determining the content of the terpene compounds in the health care wine. After nitrogen blowing concentration, a gas chromatograph-mass spectrometer is matched with polar and non-polar chromatographic columns to measure the sample. According to the method, interference of irrelevant matrixes is reduced, pollution to a chromatographic column and a detector is reduced, the extraction efficiency of the mixed extraction agent on a target compound is improved, volatile components in a wine sample can be effectively extracted only with a small amount of organic solvent, the use amount of the organic solvent is greatly reduced, and the cost is reduced. Compared with the prior art, the method has the advantages that the cost is reduced, the pollution to the environment is reduced, the response value of a target compound is improved, the detection range is expanded, the method has the advantages of less interference, low detection limit, high response value, wide detection range and high accuracy, and the detection limit is 0.1387 mu g / L to 1.9695 mu g / L.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and in particular to a detection method for simultaneously detecting 11 terpene compounds in health wine. Background Art

[0002] Terpene compounds are a very important functional trace ingredient in health wine. They are generally derived from Chinese herbal medicines. They are a class of natural compounds with isoprene (C5H8) as the molecular skeleton and its oxygen-containing derivatives. According to the length of the carbon chain, they can be divided into monoterpenes (C 10 H 16 ), sesquiterpenes (C 15 H 24 ) and diterpenes (C 20 H 32 ), etc., have multiple physiological activities and medicinal values, such as antibacterial, anti-inflammatory, antiviral, antioxidant, analgesic, digestive, anticancer, antitumor, etc., which are essential to human health. Specifically, such as safranal, it is famous for its multiple functions of protecting cardiovascular, anti-inflammatory, antioxidant and anti-tumor; d-limonene shows the characteristics of choleretic litholysis, antibacterial, anti-inflammatory, antitumor and antioxidant; γ-terpinene and β-elemene are concerned for their antioxidant, antibacterial and antitumor, and antitumor abilities respectively; β-caryophyllene combines anti-inflammatory, anticancer, antibacterial, antioxidant and analgesic effects; and dihydro-β-ionone shows unique advantages in lowering blood lipids and anti-cancer and anti-tumor. In addition, in health wine, terpenes not only play a role in alleviating the side effects of alcohol, regulating physiological balance, preventing diseases and promoting recovery, but also add complex layers of medicinal, spicy, floral and even sweetness to the wine with their unique aroma and sensory properties, giving health wine a unique taste experience and healthy connotation, making it a superior product that combines deliciousness and health.

[0003] Health wine has more complex ingredients than liquor because it contains many medicinal materials. It contains many non-volatile components such as sugar, amino acids, pigments, etc. It is very difficult to accurately quantify trace terpenes. At present, the method for detecting terpenes in wine is mostly headspace solid phase microextraction, but this method has competitive adsorption during the extraction process and is mostly used for qualitative and semi-quantitative research, and cannot be accurately quantified. This study aims to establish a method that can accurately detect trace terpenes in health wine. Summary of the invention

[0004] In view of this, the present invention proposes a detection method for simultaneously detecting 11 terpene compounds in health wine, comprising: using solvent-assisted evaporation extraction for impurity removal, liquid-liquid microextraction combined with nitrogen blowing for extraction and concentration, using a gas spectrometer-gas spectrometer with polar and non-polar chromatographic columns for detection, and using an internal standard curve method for quantification to calculate the content of terpene compounds.

[0005] The technical solution of the present invention is achieved in this way. The present invention provides a detection method for simultaneously detecting 11 terpene compounds in health wine, and the detection method specifically comprises the following steps:

[0006] Wine sample impurity removal: Take 20mL of the wine sample to be tested (50% vol), add 20μL of internal standard and shake well, then remove the non-volatile components under high vacuum by solvent-assisted evaporation extraction (SAFE) device, and collect the volatile components separated by distillation;

[0007] Extraction and concentration: the collected volatile components (20 mL) were placed in a 50 mL centrifuge tube, 10 mL of ultrapure water was added, water and ethanol were mixed in a volume ratio of 2:1, 7.2 g of NaCl (the mass volume ratio of sodium chloride to water was 0.36:1, i.e. 0.36 g of sodium chloride was added to 1 mL of water) was added until the solution was saturated, and then 10 mL of extractant was added, water, ethanol and extractant were mixed in a volume ratio of 2:1:1, oscillated for 5 min, ultrasonicated for 10 min, centrifuged at 8000 rpm for 5 min, refrigerated and stood at 4 ° C overnight, the organic phase was dehydrated with anhydrous Na2SO4, and concentrated to 1 mL at 20 ° C by nitrogen blowing, and the obtained concentrate was detected by gas chromatography-mass spectrometry;

[0008] Quantitative determination by internal standard curve method: prepare a series of gradient standard mixed solutions of terpenes with an ethanol aqueous solution (50% vol) of the same alcohol content as the wine sample to be tested, take 4 mL of each series of gradient standard mixed solutions and place them in a 50 mL centrifuge tube, add 20 μL of internal standard working solution, add 2 mL of ultrapure water, mix water and ethanol in a volume ratio of 2:1, add 1.5 g of NaCl to saturate the solution, then add 2 mL of extractant, mix water, ethanol and extractant in a volume ratio of 2:1:1, oscillate for 5 min, ultrasonicate for 10 min, centrifuge at 8000 rpm for 5 min, refrigerate and stand overnight at 4°C, take the organic phase and dehydrate it with anhydrous Na2SO4, then detect it with gas chromatography-mass spectrometry; draw a standard curve with the ratio of the peak area of ​​terpenes to the peak area of ​​the internal standard as the ordinate and the ratio of the content of terpenes in the series of standard solutions to the content of the internal standard as the abscissa.

[0009] The invention provides a pretreatment method for wine samples, comprising the following steps: taking 20 mL of wine sample to be tested (50% vol), adding 20 μL of internal standard, shaking and oscillating, removing non-volatile components under high vacuum by a solvent-assisted evaporation extraction (SAFE) device, collecting volatile components separated by distillation, placing them in a 50 mL centrifuge tube, adding 10 mL of ultrapure water, mixing water and ethanol in a volume ratio of 2:1, adding 7.2 g of NaCl (the mass volume ratio of sodium chloride to water is 0.36:1, that is, 0.36 g of sodium chloride is added to 1 mL of water) until the solution is saturated, adding 10 mL of extractant, mixing water, ethanol and the extractant in a volume ratio of 2:1:1, oscillating for 5 minutes, ultrasonicating for 10 minutes, centrifuging at 8000 rpm for 5 minutes, refrigerating and standing at 4°C overnight, taking an organic phase for dehydration by anhydrous Na2SO4, and concentrating to 1 mL by nitrogen blowing at 20°C, and detecting the obtained concentrated solution by gas chromatography-mass spectrometry.

[0010] In some embodiments, the method for preparing a standard curve is as follows: a series of gradient standard mixed solutions of terpene compounds are prepared with an ethanol aqueous solution (50% vol) with the same alcohol content as the wine sample to be tested, 4 mL of each series of gradient standard mixed solutions are placed in a 50 mL centrifuge tube, 20 μL of internal standard solution is added, 2 mL of ultrapure water is added, water and ethanol are mixed in a volume ratio of 2:1, 1.5 g of NaCl is added to saturate the solution, and then 2 mL of extractant is added, water, ethanol and extractant are mixed in a volume ratio of 2:1:1, oscillated for 5 minutes, ultrasonicated for 10 minutes, centrifuged at 8000 rpm for 5 minutes, refrigerated and allowed to stand overnight at 4°C, the organic phase is dehydrated with anhydrous Na2SO4 and then detected by gas chromatography-mass spectrometry; the standard curve is drawn with the ratio of the peak area of ​​terpene compounds to the peak area of ​​the internal standard as the ordinate, and the ratio of the content of terpene compounds in the series of standard solutions to the content of the internal standard as the abscissa.

[0011] The internal standard curve method used in the present invention can eliminate the errors caused by changes in operating conditions during sample pretreatment to a certain extent, and can effectively offset the differences in extraction effects caused by differences in sample matrices, making the quantitative results more stable and reliable.

[0012] The present invention uses a gas chromatography-mass spectrometer in combination with polar and non-polar chromatographic columns to analyze and detect terpene compounds in health wine, thereby expanding the detection range of terpene compounds.

[0013] The present invention significantly eliminates the interference of non-volatile impurities such as sugar and pigment in health wine by introducing solvent-assisted evaporation extraction during sample pretreatment, and can improve the accuracy and reliability of terpene compound detection results. At the same time, the technology also effectively prevents the contamination of chromatographic columns and precision detection equipment, thereby extending the service life of these key components.

[0014] The present invention adopts liquid-liquid microextraction technology in the sample pretreatment process, which significantly reduces the consumption of organic solvents compared to liquid-liquid extraction. Only a trace amount of organic solvent is needed to efficiently extract the volatile components in the wine sample, thereby effectively reducing the detection cost and significantly reducing the potential pollution to the environment. Subsequently, further treatment by nitrogen blowing technology is achieved to achieve efficient concentration of terpene compounds in the wine sample, which enhances the signal response value of the target analyte and can improve the detection accuracy to a certain extent.

[0015] The present invention adopts a mixed extractant of n-pentane and anhydrous ether (n-pentane: anhydrous ether = 1:1), which can effectively extract terpene volatile components from wine samples, has good extraction effect, lays a solid foundation for subsequent detection and analysis, and improves the accuracy and reliability of detection results.

[0016] The present invention can detect one or multiple terpene compounds in health wine at the same time. When detecting one or more of the above 11 terpene compounds, the detection repeatability is good, the accuracy is high, and the result is reliable.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention first uses solvent-assisted evaporation extraction to remove non-volatile components such as sugar and pigment in health wine, and then uses polar and non-polar mixed extractants to perform liquid-liquid microextraction on terpenes, which reduces the interference of irrelevant matrices, not only reduces the pollution to the chromatographic column and the detector, but also improves the extraction efficiency of the extractant to the target compound; the liquid-liquid microextraction technology is used, and only a small amount of organic solvent is used to effectively extract the volatile components in the wine sample, which greatly reduces the use of organic solvents, reduces costs and reduces pollution to the environment; and nitrogen blowing can effectively concentrate the terpenes in the wine sample to be tested, improve the response value and detection sensitivity of the target peak, and to a certain extent, can improve the detection accuracy. The gas chromatography-mass spectrometer is used with polar and non-polar chromatographic columns for analysis and detection, which expands the detection range, and uses eugenol-d3, which is closer to the properties of terpenes, as an internal standard, and adopts the internal standard standard curve method for quantification, which eliminates the errors caused by changes in operating conditions during sample pretreatment, and can offset the differences in extraction effects caused by differences in sample matrices, making the quantitative results more stable and reliable. The method of the present invention can simultaneously detect one or more terpene compounds in wine samples, and has the advantages of less interference, low detection limit, high response value, wide detection range and high accuracy, and the detection limit is 0.1387μg / L-1.9695μg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1 This is a chromatogram of a mixed standard solution of 11 terpene compounds and an internal standard in Example 1 of the present invention;

[0021] Figure 2 is a standard curve diagram of d-limonene in Example 1 of the present invention;

[0022] Figure 3 is a standard curve diagram of γ-terpinene in Example 1 of the present invention;

[0023] Figure 4 is a standard curve diagram of p-cymene in Example 1 of the present invention;

[0024] Figure 5 is a standard curve diagram of β-elemene in Example 1 of the present invention;

[0025] Figure 6 is a standard curve diagram of β-caryophyllene in Example 1 of the present invention;

[0026] Figure 7 is a standard curve diagram of safranal in Example 1 of the present invention;

[0027] Figure 8 is a standard curve diagram of α-caryophyllene in Example 1 of the present invention;

[0028] Fig. 9 is a standard curve diagram of α-terpineol in Example 1 of the present invention;

[0029] Fig.10 is a standard curve diagram of Δ-cadinene in Example 1 of the present invention;

[0030] Fig.11 This is a standard curve diagram of dihydro-β-ionone in Example 1 of the present invention;

[0031] Fig.12 is a standard curve diagram of cinnamaldehyde in Example 1 of the present invention;

[0032] Fig.13 is the chromatogram of the wine sample 1 to be tested in Example 1 of the present invention;

[0033] Fig.14 The chromatogram is a recovery rate experiment of the wine sample 1 to be tested in Example 1 of the present invention;

[0034] Fig.15 This is the chromatogram of wine sample 2 to be tested in Example 2 of the present invention. DETAILED DESCRIPTION

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Example 1

[0040] This embodiment provides a method for detecting the concentration of terpene compounds (including d-limonene, γ-terpinene, p-cymene, β-elemene, β-caryophyllene, Δ-cadinene, α-caryophyllene, α-terpineol, dihydro-β-ionone, cinnamaldehyde, and safranal) in health wine, which is as follows:

[0041] 1. Reagents:

[0042] Anhydrous ethanol and anhydrous ether were of chromatographic grade; water was ultrapure water; sodium chloride was of premium grade;

[0043] 2. Samples and standards to be tested:

[0044] Sample 1: Health No. 1, alcohol content 50% vol, provided by Jinpai Co., Ltd.

[0045] Standards of d-limonene, γ-terpinene, p-cymene, β-elemene, β-caryophyllene, safranal, α-caryophyllene, α-terpineol, Δ-cadinene, dihydro-β-ionone, and cinnamaldehyde, with a purity greater than 98.0%, were purchased from Sigma-Aldrich, USA;

[0046] Eugenol-d3 standard product: purity greater than 98.0%, purchased from Shanghai Anpu Laboratory Technology Co., Ltd.

[0047] 3. Preparation of standard solution:

[0048] 3.1 Preparation of terpene compound standard stock solution:

[0049] Accurately weigh the above 11 terpene compound standards into a volumetric flask, add anhydrous ethanol to make up to 10 mL, and obtain 11 single-standard stock solutions of terpene compounds in turn. Store at -20°C in the dark. The specific concentrations are shown in Table 1.

[0050] 3.2 Internal standard solution:

[0051] Accurately weigh 5 mg of eugenol-d3 standard into a 10 mL volumetric flask, dissolve it in anhydrous ethanol to the mark to obtain a 500 mg / L internal standard solution, and store it at -20°C away from light.

[0052] Table 1: Concentrations of single and mixed standards of 11 terpenes

[0053]

[0054] 4. Sample pretreatment:

[0055] (1) Removal of impurities from wine samples: Take 20 mL of the wine sample to be tested (50% vol), add 100 μL of internal standard and shake well, then remove the non-volatile components under high vacuum using a solvent-assisted evaporative extraction (SAFE) device, and collect the volatile components separated by distillation;

[0056] (2) Extraction and concentration: 10 mL of the collected volatile components was placed in a 50 mL centrifuge tube, 5 mL of ultrapure water was added, water and ethanol were mixed in a volume ratio of 2:1, 3.6 g of NaCl was added until the solution was saturated, and then 5 mL of anhydrous ether was added, water, ethanol and anhydrous ether were mixed in a volume ratio of 2:1:1, oscillated for 5 min, ultrasonicated for 10 min, centrifuged at 8000 rpm for 5 min, refrigerated and allowed to stand at 4°C overnight, the organic phase was dehydrated with anhydrous Na2SO4, and concentrated to 1 mL at 20°C by nitrogen blowing. The obtained concentrate was detected by gas chromatography-mass spectrometry.

[0057] 5. Sample testing

[0058] 5.1 Instrument conditions

[0059] Agilent 8890 gas chromatograph-mass spectrometer;

[0060] Chromatographic conditions: nitroterephthalic acid modified polyethylene glycol capillary column (DB-FFAP: 60m×0.25mm×0.25μm); injection port temperature was 250°C; carrier gas was high-purity helium (purity ≥99.999%), non-split, constant flow mode, flow rate 1mL / min; column temperature program: starting temperature 40°C, maintained for 5min, heated to 240°C at a rate of 3°C / min, maintained for 5min ; (5%-phenyl)-methylpolysiloxane capillary chromatographic column (HP-5MS: 30m×0.25mm×0.2μm), heating program: initial temperature 40℃, increased to 200℃ at 3.5℃ / min, maintained for 1min, then increased to 300℃ at 25℃ / min, maintained for 1min; the carrier gas was high-purity helium, the flow rate was 1mL / min, the injection port temperature was 250℃, the injection volume was 1μL, and non-split injection was used.

[0061] Mass spectrometry conditions: ionization mode is electron impact source (EI), energy is 70eV; ion source temperature is 230°C; quadrupole temperature is 150°C; auxiliary channel heating temperature is 280°C; mass spectrometry scanning mass range is 35-350amu, and mass spectrometry analysis uses NIST20 spectral library.

[0062] Detection method: Selected ion scanning acquisition; monitoring ions are shown in Table 2. Scanning method: First, full scan mode (SCAN) was used for qualitative analysis, the instrument parameters were optimized, the retention time and characteristic ions of each active ingredient and internal standard were determined, and then the compounds were grouped and scanned in selected ion mode (SIM), as shown in Table 2.

[0063] Table 2: Mass spectrometry parameters of 11 terpenes

[0064]

[0065]

[0066] 5.2 Preparation of standard curve

[0067] According to "Table 1" in "3.1", accurately transfer appropriate amounts of 11 terpene compound single standard stock solutions into the same 10mL volumetric flask and dilute to volume with anhydrous ethanol to prepare a mixed standard solution. Take an appropriate amount of the mixed standard solution and dilute to volume with an ethanol aqueous solution (50% vol) with the same alcohol content as the wine sample to be tested to obtain standard sample 1, and then dilute it 2.5 times in sequence to obtain a series of mixed standard solutions. Take 4 mL of each series of gradient mixed standard solutions and place them in a 50 mL centrifuge tube, add 20 μL of internal standard solution, add 2 mL of ultrapure water, mix water and ethanol in a volume ratio of 2:1, add 1.5 g of NaCl until the solution is saturated, and then add 2 mL of mixed extractant (anhydrous ether: n-pentane = 1:1), mix water, ethanol and anhydrous ether in a volume ratio of 2:1:1, oscillate for 5 minutes, ultrasonicate for 10 minutes, centrifuge at 8000 rpm for 5 minutes, refrigerate and stand at 4°C overnight, take the organic phase and dehydrate it with anhydrous Na2SO4, and then detect it with a gas chromatography-mass spectrometer; use the ratio of the peak area of ​​terpene compounds to the peak area of ​​the internal standard as the ordinate, and the ratio of the content of terpene compounds in the series of standard solutions to the content of the internal standard as the abscissa to draw a standard curve. The parameters of the standard curves of 11 terpene compounds are shown in Table 3. The standard curves of 11 terpene compounds are as follows: Figure 2-Figure 12 As shown, Figure 1 The chromatographic peaks 1 to 12 are d-limonene, γ-terpinene, p-cymene, β-elemene, β-caryophyllene, safranal, α-caryophyllene, α-terpineol, Δ-cadinene, dihydro-β-ionone, cinnamaldehyde and eugenol-d3 (internal standard), respectively.

[0068] Table 3: Standard curve parameters of 11 terpene compounds

[0069]

[0070]

[0071] 5.3 Sample determination

[0072] The samples to be tested and the standard solution were tested under the same test conditions, and the peak areas A s The peak area A of the internal standard i Substitute the standard curve to calculate the mass concentration of the corresponding terpene compounds in the sample to be tested. The chromatogram of the sample 1 to be tested is as follows: Fig.13 shown.

[0073] 5.4 Calculation

[0074] The contents of 11 terpene compounds in the sample to be tested are calculated according to the following formula:

[0075]

[0076] Where:

[0077] X i ---The content of the i-th terpene compound in the sample to be tested, in micrograms per liter (μg / L);

[0078] X f ---The content of internal standard eugenol-d3 in the sample to be tested, in micrograms per liter (μg / L);

[0079] A i ---The chromatographic peak area of ​​the i-th terpene compound in the sample to be tested;

[0080] A f ---The chromatographic peak area of ​​the internal standard eugenol-d3 in the sample to be tested;

[0081] b---the intercept value of the standard curve;

[0082] k---the slope of the standard curve;

[0083] K---dilution multiple.

[0084] The calculation result is expressed as the arithmetic mean of two independent determination results obtained under repeatability conditions, retained to 2 decimal places.

[0085] 6. Repeatability Experiment

[0086] A total of 6 samples were taken, marked as 1 to 6, and the samples were processed according to the methods of "4. Sample pretreatment" and "5. Sample detection", the content of terpene compounds was determined, and the repeatability of the results of the determination of 11 terpene compounds was calculated. The RSD values ​​were all less than 5%, indicating that the method had good repeatability. The specific results are shown in Table 4.

[0087] Table 4: Repeatability test data results of 11 terpenes in the tested samples

[0088]

[0089]

[0090] 7. Limit of detection (LOD) and limit of quantification (LOQ)

[0091] The limit of detection (LOD) is the sample content corresponding to the signal intensity / baseline noise (S / N) = 3:1; the limit of quantification (LOQ) is the sample content corresponding to the signal intensity / baseline noise (S / N) = 10:1.

[0092] In this example, the detection limits (LOD) of the 11 terpene compounds were 0.1387 μg / L to 1.9695 μg / L, and the quantification limits (LOQ) were 0.4625 μg / L to 6.5651 μg / L. The specific calculation results are shown in Table 5.

[0093] Table 5: Calculation results of the limit of detection (LOD) and limit of quantification (LOQ) of 11 terpenoid compounds in the samples to be tested

[0094] name S / N Concentration / (μg / L) LOD / (μg / L) LOQ / (μg / L) d-Limonene 117.9837 77.4570 1.9695 6.5651 γ-Terpinene 1.6313 0.5041 0.9271 3.0902 p-Cymene 11.9182 0.5512 0.1387 0.4625 β-elemene 1.4318 0.6921 1.4500 4.8334 β-Caryophyllene 2.4934 0.2848 0.3427 1.1424 Safranal 29.8150 11.9015 1.1975 3.9918 α-Caryophyllene 6.3752 0.7083 0.3333 1.1111 α-Terpineol 1.5575 0.6590 1.2693 4.2311 Δ-cadinene 8.7769 0.6554 0.2240 0.7467 Dihydro-β-ionone 2.9897 0.3827 0.3840 1.2800 Cinnamaldehyde 3.7380 1.6638 1.3353 4.4509

[0095] 8. Accuracy Experiment

[0096] Take 6 samples of wine to be tested and divide them into 3 groups, A, B and C, with 2 samples in each group, marked as Group A (Z1-1 and Z1-2), Group B (Z2-1 and Z2-2), and Group C (Z3-1 and Z3-2), respectively; take the 11 kinds of terpene compound mixed standard stock solutions in "3.1", add 10 μL to Group A, 20 μL to Group B, and 30 μL to Group C; add 20 μL of internal standard solution to each group, treat the samples according to the methods of "4. Sample pretreatment" and "5. Sample detection", and determine the content of terpene compounds. The chromatogram of the accuracy experiment of the wine to be tested is shown in the figure. Fig.14 shown.

[0097] The peak areas of terpene compounds and internal standards were substituted into the standard curve to calculate the content and recovery of each terpene compound. The recoveries of the 11 terpene compounds were all between 90% and 105%, and the RSD values ​​were all less than 5%, indicating that the method had good accuracy. The specific calculation results are shown in Table 6.

[0098] Table 6: Accuracy test results of 11 terpenes in wine samples

[0099]

[0100] Example 2

[0101] This embodiment provides a method for detecting the concentration of terpene compounds (including d-limonene, γ-terpinene, p-cymene, β-elemene, β-caryophyllene, safranal, α-caryophyllene, α-terpineol, Δ-cadinene, dihydro-β-ionone and cinnamaldehyde) in health wine, which is as follows:

[0102] 1. Reagents: Same as in Example 1.

[0103] 2. Samples and standards to be tested:

[0104] Test sample 2: Jinbiao Jing Liquor, alcohol content 38% vol, provided by Jingpai Co., Ltd.

[0105] Standard product: Same as Example 1.

[0106] 3. Preparation of standard solution: same as in Example 1.

[0107] 4. Sample pretreatment: same as Example 1.

[0108] 5. Sample testing: Same as Example 1.

[0109] The determination conditions and methods are the same as those in Example 1. The chromatogram of the obtained sample is as follows: Fig.15 The detection results of 11 terpene compounds in the samples to be tested in this example are shown in Table 7.

[0110] Table 7: Detection results of 11 terpenes in samples

[0111]

[0112] Note: “ / ” means not detected.

[0113] 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 simultaneously detecting 11 terpene compounds in health wine, comprising: The steps include: Step 1: Take the terpene compound to be tested, add anhydrous ethanol to dissolve it, and prepare a single standard stock solution; Step 2: Take eugenol-d3, add anhydrous ethanol to dissolve it, and prepare an internal standard solution; Step 3: Take the terpene compound standard stock solution prepared in step 1, prepare a series of gradient standard mixed solutions with an ethanol aqueous solution of the same alcohol content as the wine sample, and then add the internal standard solution prepared in step 2 and mix well; Step 4: Take the wine sample to be tested, add the internal standard solution prepared in step 2 and mix well, remove the non-volatile components in the wine sample by solvent-assisted evaporation extraction, and collect the volatile components in the wine sample; Step 5: Add ultrapure water to the series of standard mixed solutions in step 3 and the volatile components of the wine sample in step 4, so that the water and ethanol in the standard mixed solution to be tested and the wine sample are mixed in a volume ratio of 2:1, and add an appropriate amount of sodium chloride to make the solution reach a saturated state; Step 6: Add the extractant to the solution in step 5, mix the water, ethanol and extractant in the sample in a volume ratio of 2:1:1, oscillate for 5 minutes, sonicate for 10 minutes, centrifuge at 8000 rpm for 5 minutes, and refrigerate and stand at 4°C overnight to separate the aqueous phase and the organic phase; Step 7: Dehydrate the organic phase in step 6 with anhydrous Na2SO4, and concentrate to 1 mL at 20°C with nitrogen blowing. The obtained concentrate is detected by GC-MS to obtain the peak areas of terpene compounds and internal standard substances. Step 8, according to the test results of the series of standard mixed solutions in step 7, a standard curve is drawn with the ratio of the peak area of ​​the terpene compound to the peak area of ​​the internal standard substance as the ordinate and the ratio of the content of the terpene compound in the series of standard mixed solutions to the content of the internal standard substance as the abscissa; Step 9: Substitute the peak areas of the terpene compounds and the internal standard in the wine sample to be tested in step 7 into the standard curve prepared in step 8 to calculate the content of terpene compounds in the wine sample to be tested.

2. The method for simultaneously detecting 11 terpene compounds in health wine according to claim 1, characterized in that: The target terpene compounds to be detected in step 1 include d-limonene, γ-terpinene, p-cymene, β-elemene, β-caryophyllene, safranal, α-caryophyllene, α-terpineol, Δ-cadinene, dihydro-β-ionone and cinnamaldehyde.

3. The method for simultaneously detecting 11 terpene compounds in health wine according to claim 1, characterized in that: The concentration of the internal standard solution used in step 2 is 500 mg / L.

4. The method for simultaneously detecting 11 terpene compounds in health wine according to claim 1, characterized in that: In the step 4, the temperature of the constant temperature water bath and the constant temperature water tank of the solvent-assisted evaporation extraction device is 50°C, liquid nitrogen is added to the two cold traps, and when the vacuum degree is less than 1×10 -3 Pa, slowly open the stopper of the dropping funnel, control the sample flow rate to 1 mL / min, and collect the volatile components. This method is to remove non-volatile components such as sugar and pigment in the health wine under high vacuum, reduce irrelevant matrix interference, and obtain volatile components by distillation separation.

5. The method for simultaneously detecting 11 terpene compounds in health wine according to claim 1, characterized in that: In the step six, the extractant is one or a mixture of two of ethyl acetate, anhydrous ether, dichloromethane, n-hexane and n-pentane.

6. The method for simultaneously detecting 11 terpene compounds in health wine according to claim 5, characterized in that: In step 6, the extractant is a mixture of anhydrous ether and n-pentane in a ratio of 1:

1.

7. The method for simultaneously detecting 11 terpene compounds in health wine according to claim 1, characterized in that: The chromatographic conditions of the gas chromatography-mass spectrometer in step 7 are as follows: a polyethylene glycol capillary column modified with nitroterephthalic acid (DB-FFAP: 60m×0.25mm×0.25μm) is used, the injection port temperature is 250°C, the carrier gas is high-purity helium (purity ≥99.999%), the injection is in splitless constant flow mode, the flow rate is 1mL / min, the injection volume is 1μL, and the heating program is: the starting temperature is 40°C, maintained for 5min, and the temperature is increased to 240°C at a rate of 3°C / min, and maintained for 5min; a (5%-phenyl)-methylpolysiloxane capillary column (HP-5MS: 30m×0.25mm×0. 2μm), injection port temperature was 250℃, carrier gas was high-purity helium (purity ≥99.999%), injection was performed in splitless constant flow mode, flow rate was 1mL / min, injection volume was 1μL, heating program: initial temperature 40℃, increased to 200℃ at 3.5℃ / min, maintained for 1min, then increased to 300℃ at 25℃ / min, maintained for 1min; mass spectrometry conditions were as follows: ionization mode was electron impact source (EI), energy was 70eV; ion source temperature was 230℃; quadrupole temperature was 150℃; auxiliary channel heating temperature was 280℃; mass spectrometry scanning mass range was 35-350amu, and mass spectrometry analysis used NIST20 spectral library.

8. The method for simultaneously detecting 11 terpene compounds in health wine according to claim 1, characterized in that: The calculation formula of terpene compounds in step nine is: In the formula Xi: the content of the i-th terpene compound in the sample to be tested, in micrograms per liter (μg / L); Xf: the content of internal standard eugenol-d3 in the sample to be tested, in micrograms per liter (μg / L); Ai: chromatographic peak area of ​​the i-th terpene compound in the sample to be tested; Af: chromatographic peak area of ​​internal standard eugenol-d3 in the sample to be tested; b: intercept value of the standard curve; k: slope of the standard curve; K: dilution factor.

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