Method for determining benzyl alcohol, phenethyl alcohol, phenol and 3-phenylpropanol in wine by internal standard method

Abstract

The invention discloses a method for measuring benzyl alcohol, phenethyl alcohol, phenol and 3-phenylpropanol in wine by an internal standard method, which comprises the following steps: (1) sample pretreatment: weighing 5.0 mL of sample, adding 1.5 g of sodium chloride, and adding 10L of 1.05 g / L 4-chlorobenzyl alcohol as an internal standard substance to obtain a pretreated sample; (2) preparing a standard solution; and (3) determination: detecting and analyzing the pretreated sample by using gas chromatography-mass spectrometry. The detection of benzyl alcohol, phenethyl alcohol, phenol and 3-phenylpropanol in different wines is realized by combining headspace-solid phase microextraction and gas chromatography with an accurate mass spectrum method. The method is simple, convenient and rapid in sample pretreatment and accurate in qualitative and quantitative detection, can meet rapid detection of benzyl alcohol, phenethyl alcohol, phenol, 3-phenylpropanol and the like in the wine, and provides technical support for enhancing quality control of the wine.

Description

Technical Field

[0001] This invention relates to a method for determining benzyl alcohol, phenylethanol, phenol and 3-phenylpropanol in wine using an internal standard method, belonging to the field of food testing technology. Background Technology

[0002] Wine is a beverage obtained through the complete or partial alcoholic fermentation of fresh grapes or grape juice. Its alcohol content is generally between 8% and 15%. It contains a variety of flavor compounds, among which benzyl alcohol is a trace natural flavor compound. Excessive levels can directly affect the taste and quality of the wine, and may even cause dizziness and nausea. In addition, phenethyl alcohol, phenol, and 3-phenylpropanol are also natural flavor compounds in distilled spirits; excessive levels can also affect the taste and quality. Currently, methods for detecting wine often use 4-methyl-2-pentanol and 2-octanol as internal standards, as these have early peak times, similar to short-chain and straight-chain alcohols, and are effective. However, compounds containing benzyl alcohol, phenethyl alcohol, phenol, and 3-phenylpropanol, which contain benzene rings, have later peak times. Therefore, it is necessary to select structurally similar internal standards for more accurate quantification. 4-Chlorobenzol was chosen as the internal standard in this study because of its structural and property similarity to benzyl alcohol. Summary of the Invention

[0003] This application provides an internal standard method for determining benzyl alcohol, phenylethanol, phenol and 3-phenylpropanol in wine. Since 4-chlorobenzyl alcohol has a similar structure and properties to benzyl alcohol, it can be used as an internal standard. Through sample pretreatment and optimization of the detection method, the concentrations of benzyl alcohol, phenylethanol, phenol and 3-phenylpropanol can be accurately detected.

[0004] A method for determining benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol in wine using an internal standard method includes the following steps: (1) Sample pretreatment: Take 5.0 mL of sample, add 1.5 g of sodium chloride, and add 10 µL of 1.05 g / L 4-chlorobenzyl alcohol as an internal standard to obtain a pretreated sample; (2) Preparation of standard solutions: Accurately weigh 0.5 g each of benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol into a 1000 mL volumetric flask, dissolve them with a simulated wine sample and dilute to volume to prepare a mixed standard stock solution with a mass concentration of 0.5 g / L. Store at 4℃ for later use. Dilute the standard stock solution stepwise to prepare a series of standard solutions with concentrations of 0.005, 0.025, 0.05, 0.25, 0.5, 10, 250, and 500 mg / L for later use. (3) Determination: The pretreated samples were detected and analyzed by headspace solid phase microextraction gas chromatography-mass spectrometry.

[0005] Furthermore, the alcohol content of the sample is 12%.

[0006] Furthermore, the headspace solid-phase microextraction conditions are as follows: equilibrium time 10 min; equilibrium temperature 55 ℃; extraction time 25 min; stirring speed: 250 rpm.

[0007] Furthermore, the gas chromatography conditions are as follows: column: RTX-WAX capillary column (30 m × 0.25 μm × 0.25 mm), carrier gas: high-purity helium, injection port temperature: 250℃, column temperature programmed: 50℃ held for 1 min, then increased to 220℃ at 3℃ / min, constant flow mode, flow rate: 1.0 mL / min.

[0008] Beneficial effects This invention establishes a method for the determination of benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol in wine. The method has been optimized in detail to address factors affecting extraction efficiency, overcoming the problem of low extraction efficiency. Through headspace solid-phase microextraction gas chromatography combined with accurate mass spectrometry, the detection of benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol in different wines has been achieved. This method offers simple and rapid sample pretreatment, accurate qualitative and quantitative analysis, and can meet the requirements for rapid detection of benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol in wine, providing technical support for strengthening wine quality control. Attached Figure Description

[0009] Figure 1 A comparison chart of equilibrium temperatures; Figure 2 A time-balanced comparison chart; Figure 3 This is a comparison chart of salt concentrations; Figure 4 A comparison chart of stirring speeds; Figure 5 The TIC chromatogram for the sample is shown. In the chromatogram, 1 represents benzyl alcohol, 2 represents phenylethanol, 3 represents phenol, 4 represents 3-phenylpropanol, and 5 represents 4-chlorobenzyl alcohol. Detailed Implementation

[0010] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0011] Example 1 1. Experimental Section 1.1 Instruments and Reagents Shimadzu GCMS-QP2010 Ultra and workstation (Shimadzu, Japan), AOC-6000 autosampler, electronic balance (Mettler-Toledo Instruments Shanghai Co., Ltd.), 20 mL headspace vials (CTC Analytics AG, Switzerland), sodium chloride (AR), anhydrous ethanol (AR) (all reagents purchased from Sinopharm Chemical Reagent Co., Ltd.), ultrapure water, benzyl alcohol (99.5%), phenylethanol (99.5%), phenol (99.5%), 3-phenylpropanol (99%), 4-chlorobenzyl alcohol (99%) (all purchased from Aladdin Reagent Co., Ltd.), SPME extraction head (50 / 30 µm, DVB / CAR / PDMS, Supelco, Bellefonte, PA, USA). 1.2 Headspace solid-phase microextraction and GC-MS determination conditions Headspace solid-phase microextraction conditions: equilibration time 10 min; equilibration temperature 55 ℃; extraction time 25 min; stirring speed: 250 rpm.

[0012] GC conditions: Column: RTX-WAX capillary column (30 m × 0.25 μm × 0.25 mm), carrier gas: high-purity helium, injection port temperature: 250℃, column temperature programmed: 50℃ held for 1 min, then increased to 220℃ at 3℃ / min, constant flow mode, flow rate: 1.0 mL / min.

[0013] MS conditions: Chromatography-mass spectrometry interface temperature: 250℃, ion source temperature: 230℃.

[0014] 1.3 Preparation of Standard Stock Solution and Simulated Wine Samples Standard stock solution: Accurately weigh 0.5 g each of benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol into a 1000 mL volumetric flask, dissolve and dilute to volume with a simulated wine sample to prepare a mixed standard stock solution with a mass concentration of 0.5 g / L, and store at 4℃ for later use.

[0015] Simulated wine sample: Prepare an ethanol solution with a volume fraction of 12% using anhydrous ethanol and ultrapure water as a simulated wine sample.

[0016] 1.4 Preparation of mixed standard working solutions The standard stock solution was serially diluted with the simulated wine sample prepared in 1.3 to obtain a series of standard solutions with concentrations of 0.005, 0.025, 0.05, 0.25, 0.5, 10, 250, and 500 mg / L, which were then ready for use.

[0017] 1.5 Headspace Solid Phase Microextraction Gas Chromatography-Mass Spectrometry Experimental Procedure Take 5.0 mL of wine sample (pre-added with anhydrous ethanol or diluted to an alcohol content of 12%) or spiked simulated wine sample into a 20.0 mL headspace vial, add 1.5 g of sodium chloride, add 10 µL of 1.05 g / L 4-chlorobenzyl alcohol as an internal standard, seal with a magnetic cap with a silicone rubber gasket, place in the sample tray of an autosampler, set the autosampler speed to 250 rpm, and stir at 55 ℃.

[0018] 2 Results and Analysis 2.1 Optimization of headspace solid-phase microextraction conditions 2.1.1 Optimization of Equilibrium Temperature This study investigated the peak area and relative standard deviation of the target substance at equilibrium temperatures ranging from 40 to 60 °C to select the optimal equilibrium temperature. As shown in Figure 1, the peak area gradually increases with increasing temperature. The peak area reaches its maximum value at 55 °C; if the temperature continues to rise, the peak area decreases. Therefore, 55 °C was determined to be the optimal equilibrium temperature.

[0019] 2.1.2 Selection of extraction time In the extraction process, equilibration time is a crucial factor affecting experimental results. This study investigated the effect of equilibration time (10–30 min) on the peak area and relative standard deviation of the target analyte, thereby selecting the optimal equilibration time. Figure 2 As can be seen, within the range of 10-25 min, the peak areas of benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol gradually increased with the extension of the equilibrium time. When the equilibrium time reached 25 min, the peak areas of each target analyte basically stabilized and no longer changed significantly with the extension of time. Therefore, considering the trend of peak area changes, 25 min was selected as the optimal equilibrium time. At this time, each target analyte can be fully extracted, the peak areas are stable, and the reliability of the experimental results can be guaranteed.

[0020] 2.1.3 Optimization of Salt Concentration Appropriately increasing the ionic strength of the solution during extraction can reduce the solubility of the analyte in the solution, increase the partition coefficient, and enhance the response value of the analyte. This paper discusses the effect of salt on the peak area and relative standard deviation of the target analyte by selecting an addition amount of 0.5-2.0 g of NaCl. Figure 3 As can be seen, within the range of 0.5-1.5 g, the peak areas of benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol gradually increased with the increase of NaCl addition; when the NaCl addition reached 1.5 g, the peak areas of each target compound basically stabilized and no longer changed significantly with the increase of addition.

[0021] Therefore, considering the changing trend of peak area, 1.5 g was selected as the optimal amount of NaCl to be added. At this point, the response values ​​of each target analyte are stable and relatively high, which can effectively enhance the extraction effect and ensure the reliability of the experimental results. 2.1.4 Optimization of stirring speed The effect of stirring speed (100-300 rpm) on the peak area and relative standard deviation of the target analyte was investigated. Figure 4 As can be seen, within the range of 100-250 rpm, the peak areas of benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol gradually increase with the increase of stirring speed; when the stirring speed reaches 250 rpm, the peak areas of each target substance basically stabilize and no longer change significantly with the increase of stirring speed.

[0022] Therefore, considering the changing trend of peak area, 250 rpm was selected as the optimal stirring speed. At this speed, the peak area of ​​each target analyte is stable and relatively high, which can ensure the efficiency of the extraction process and the reliability of the experimental results. 2.2 Development of standard working curves, method detection limits, and quantitative analysis A series of simulated wine samples with specific concentrations of the analytes were prepared, and 10 µL of 1.05 g / L 4-chlorobenzyl alcohol was added as an internal standard. The analytes were determined according to the experimental method in section 1.5, and a standard curve was plotted using the internal standard curve method. The linear equations are shown in Table 1. Benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol were measured at concentrations ranging from 0.025 to 500 mg·L⁻¹. -1 A good linear relationship was observed within the concentration range, with linear correlation coefficients (r) ranging from 0.9996 to 0.9999. The limits of detection for benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol, calculated using a signal-to-noise ratio (S / N) of 3, were 14.72–42.97 µg·L⁻¹. -1 between.

[0023] Table 1. Linear equations, linear ranges, linear correlation coefficients, and limits of detection for benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol. 2.3 Method Accuracy and Precision Three different varieties of wine were sampled and added at four different levels (0.5, 10, 100, and 200 mg / L). -1 Each level was repeated 5 times. The results showed that the recoveries of benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol in the three different wine varieties (1#, 2#, and 3#) ranged from 83.36% to 109.24%, with relative standard deviations (RSD) of ( ). n=5) less than 10%, this method has high accuracy and good stability, and can be used for the detection of benzyl alcohol, phenylethanol, phenol and 3-phenylpropanol in wine.

[0024] Comparative Example 1 Unlike Example 1, 2,2-difluoroethanol was used as the internal standard. The results showed that the detection limit was low and the detection results for benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol were inaccurate.

[0025] Comparative Example 2 Unlike Example 1, the internal standard used was 4-methyl-2-pentanol, and the results showed that the detection results for benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol were inaccurate.

Claims

1. A method for determining benzyl alcohol, phenylethyl alcohol, phenol, and 3-phenylpropanol in wine using an internal standard method, characterized in that, Includes the following steps: (1) Sample pretreatment: Take 5.0 mL of sample, add 1.5 g of sodium chloride, and add 10 µL of 4-chlorobenzyl alcohol as an internal standard to obtain the pretreated sample; (2) Preparation of standard solutions: Accurately weigh 0.5 g each of benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol into a 1000 mL volumetric flask, dissolve them with a simulated wine sample and dilute to volume to prepare a mixed standard stock solution with a mass concentration of 0.5 g / L. Store at 4℃ for later use. Dilute the standard stock solution stepwise to prepare a series of standard solutions with mass concentrations of 0.005, 0.025, 0.05, 0.25, 0.5, 10, 250, and 500 mg / L for later use. (3) Determination: The pretreated samples were detected and analyzed by headspace solid phase microextraction gas chromatography-mass spectrometry.

2. The method for determining benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol in wine using the internal standard method according to claim 1, characterized in that, The alcohol content of the sample was 12%.

3. The method for determining benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol in wine using the internal standard method according to claim 1, characterized in that, The headspace solid-phase microextraction conditions are as follows: equilibrium time 10 min; equilibrium temperature 55 ℃; stirring speed 250 rpm.

4. The method for determining benzyl alcohol, phenylethanol, phenol, and 3-phenylpropanol in wine using the internal standard method according to claim 1, characterized in that, The gas chromatography conditions were as follows: column: RTX-WAX capillary column, size 30 m × 0.25 μm × 0.25 mm; carrier gas: high-purity helium; injection port temperature: 250℃; column temperature was programmed: 50℃ held for 1 min, then increased to 220℃ at 3℃ / min; constant flow mode; flow rate: 1.0 mL / min.

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