Method for detecting higher alcohols in serum and application thereof

Abstract

The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a method for detecting higher alcohols in serum and application of the method in metabolic kinetic analysis of alcoholic beverages. In order to realize the effective separation and synchronous detection of fatty alcohol, aromatic alcohol and isomers thereof, reduce the volatile loss of higher alcohol and the use amount of a sample and reduce the serum matrix effect, the method comprises the following steps: performing pretreatment extraction and derivatization on drunk serum; high performance liquid chromatography-triple quadrupole mass spectrometry is adopted to detect higher alcohols in serum after drinking and the content of the higher alcohols. Results prove that the detection method disclosed by the invention has the advantages of low sample amount, high precision and accuracy, low matrix effect and low detection limit. The detection method disclosed by the invention is used for detecting the content of ethanol and higher alcohol in serum in the metabolic kinetics analysis of the alcoholic beverage, so that the alcoholic beverage with excellent metabolic efficiency can be screened, and data support can be provided for the evaluation of slow drunkenness, fast sobering and light hangover.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of chemical analysis and detection, and particularly relates to a method for detecting high alcohols in serum and application thereof in metabolic kinetics analysis of alcoholic beverages. BACKGROUND

[0002] High alcohols are key flavor components of alcoholic beverages and also core factors affecting the severity of hangover. There is a competitive metabolic relationship between ethanol and high alcohols in blood, and the two inhibit each other's metabolic process, resulting in prolonged metabolic residence time in the body. Therefore, accurate detection of the content and metabolic law of high alcohols in serum is crucial for hangover evaluation.

[0003] The existing methods for detecting high alcohols in the body mainly include gas chromatography-mass spectrometry (GC-MS), headspace sampling-gas chromatography-flame ionization detection (HS-GC-FID), diethyl ether extraction-capillary chromatography, etc. However, these methods have significant limitations: (1) aromatic alcohols such as benzyl alcohol and 1-phenylethanol cannot be detected, and aromatic alcohols may affect the severity of hangover; (2) the content of high alcohols in serum is low, and a large amount of sample is required, usually >1 mL to be detected; (3) high alcohols are highly volatile, and even if stored at -80℃, they are still easy to lose, and the loss is further aggravated during extraction and sampling; (4) the matrix effect is obvious, which interferes with the accuracy of the detection signal.

[0004] Therefore, it is urgent to develop a method for detecting high alcohols in serum with less sample amount, wide detection range (including aromatic alcohols and isomers), high sensitivity and good stability, and to realize accurate analysis of the metabolic kinetics of high alcohols in the body after alcohol intake, thereby providing technical support for metabolic differences and hangover evaluation of alcoholic beverages. SUMMARY

[0005] The existing technology has the following core problems: (1) how to break through the detection range limitation and realize the synchronous detection of straight-chain and branched-chain aliphatic alcohols (C2-C12) and aromatic alcohols and effective separation of isomers; (2) how to optimize the sample pretreatment and derivatization conditions to reduce the loss of high alcohol volatility, reduce the serum matrix effect, and at the same time reduce the sample amount to ≤500 μL; (3) how to establish a metabolic kinetics analysis scheme suitable for different alcoholic beverages to accurately compare the metabolic differences of high alcohols in the body of different alcoholic beverages and provide a basis for hangover risk evaluation. In order to solve the above problems, the present application optimizes the sample pretreatment and derivatization conditions, uses isotopic chemical derivatization labeling, and uses high performance liquid chromatography-triple quadrupole mass spectrometry to detect high alcohols in serum and their content.

[0006] To achieve the above application purposes, the technical solutions adopted by the present application are as follows: In a first aspect, the present application provides a method for detecting high alcohols in serum, comprising the following steps: (1) Serum sample pretreatment: extract the serum with saturated sodium chloride solution and organic extractant; (2) Serum sample derivatization: add pyridine solution and SOCI2 solution to the extracted organic extractant for derivatization reaction, then dry and redissolve after standing and quenching to obtain the derivatized serum sample; (3) Preparation of internal standard solution: take 21 kinds of higher alcohols respectively in organic solvent to prepare standard stock solution mixture, obtain mixed standard solution, use pyridine-d5 solution instead of pyridine solution to refer to the derivatization operation of step (2) to obtain higher alcohol internal standard solution after derivatization of the mixed standard solution; (4) Preparation of higher alcohol standard curve: use blank serum instead of organic solvent in step (3) to obtain mixed standard solution with serum as the matrix, then dilute with blank serum gradient, and then perform the same extraction and derivatization treatment, add the internal standard solution of step (3) as an internal standard, obtain the peak area ratio by liquid chromatography-mass spectrometry, and then construct a standard curve based on the corresponding concentration ratio; the peak area ratio and the concentration ratio refer to the ratio of the mixed standard solution after derivatization treatment with serum as the matrix and the internal standard solution prepared in step (3); (5) Detection and quantification of higher alcohols in serum: after detecting the derivatized serum sample in step (2) by liquid chromatography-mass spectrometry, calculate the content of higher alcohols in serum combined with the standard curve; In steps (4) and (5), the liquid chromatography-mass spectrometry is detected by high performance liquid chromatography-triple quadrupole mass spectrometry, and the chromatographic conditions are as follows: ChromCore phenyl ether chromatographic column; The mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is pure acetonitrile; The gradient program is: 0-10 min, 10% B; 10-20 min, linearly increased to 20% B; 20-30 min, linearly increased to 30% B; 30-40 min, linearly increased to 40% B; 45 min, linearly increased to 100% B; 50 min, return to 10% B; The mass spectrometry conditions of the alcohol derivative are as follows:

[0007] The higher alcohols include: ethanol, benzyl alcohol, 1-phenylethanol, 2-phenylethanol, propanol, isopropanol, butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol, pentanol, 2-pentanol, isopentanol, active pentanol, hexanol, 1-heptanol, 2-heptanol, 1-octanol, 3-octanol, 2-nonanol, 1-decanol.

[0008] In step (1), the serum is collected after drinking.

[0009] Preferably, the wine is at least one of distilled wine, fermented wine or prepared wine.

[0010] Further preferably, the wine comprises at least one of baijiu, brandy, whiskey, vodka, rum, gin, tequila, fruit distillate, yellow rice wine, beer, grape wine, fruit wine, sake or milk wine.

[0011] More preferably, the wine is baijiu, selected from at least one of Jiangxiang type, Nongxiang type, Qingxiang type or Mixiang type.

[0012] In step (1), the organic extractant is dichloromethane.

[0013] In step (1), the extraction is that equal volume of saturated sodium chloride solution and organic extractant are added into serum, oscillated at 1500 rpm for 1-5 min, centrifuged at 10000 rpm for 5-15 min, and the upper organic phase is obtained.

[0014] In step (2), the solvent of pyridine solution and SOCI2 solution is consistent with the organic extractant.

[0015] In step (2), the derivatization conditions meet at least one of the following: The molar ratio of pyridine to SOCI2 is 1:1-1:2; preferably, the molar ratio of pyridine to SOCI2 is 1:1.5; The molar ratio of pyridine to hydroxyl functional group is 1:10-20; preferably, the molar ratio of pyridine to hydroxyl functional group is 1:15; The reaction temperature is 20-25℃; preferably, the reaction temperature is 25℃ The reaction time is 1-1.5 h; preferably, the reaction time is 1 h.

[0016] In step (2), the standing is at -80℃ for 2 hours.

[0017] In step (2), both the quenching and the redissolving use 50% v / v acetonitrile aqueous solution.

[0018] In the chromatographic conditions, the column temperature is 25-28℃; the injection volume is 5 μL.

[0019] In the mass spectrometry conditions, the positive electrospray ionization mode is used, the ion spray voltage is 5.5 kV, the ion source temperature is 500℃, and the quantification is performed in the multiple reaction monitoring mode.

[0020] In a second aspect, the application provides the use of the above-mentioned detection method in the analysis of alcohol beverage metabolic kinetics.

[0021] The application is to collect serum samples at different time points, detect the content of ethanol and higher alcohol in the serum, draw a concentration-time curve, and analyze the metabolic kinetics parameters.

[0022] Beneficial effects: After the serum after drinking is pretreated, extracted and derivatized, the higher alcohol and content thereof in the serum after drinking are detected by using high performance liquid-triple quadrupole mass spectrometry. Through optimizing the pretreatment extraction conditions, the derivatization reaction conditions, and combining the optimized chromatography-mass spectrometry detection conditions, the sample amount of only 400 μL can realize the accurate separation and detection of 21 kinds of higher alcohol containing aromatic alcohol and isomers. The experimental results prove that the detection limit of the detection method is 0.17-15.14 ng / mL, the quantitative limit is 0.56-50.47 ng / mL, which indicates that the detection method can detect and quantify the analyte with a concentration as low as sub-nanokel. At the same time, the intra-day and inter-day precision CV% are less than 10%, the recovery rate is 85-120%, and the matrix effect ME% is 80-120%. It can be seen that the detection method has the advantages of low sample amount, high precision and accuracy, low matrix effect, and low detection limit.

[0023] The detection method of the application is used in the metabolic kinetics analysis of alcoholic beverages to detect the content of ethanol and higher alcohol in the serum, which not only can screen alcoholic beverages with excellent metabolic efficiency, but also can provide data support for the evaluation of "slow intoxication, fast sobering, and light hangover". BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 C-T curve graph of the ethanol and higher alcohol concentration in SD rats in the body of four kinds of liquor in Example 2 of the application with time; Figure 2 Efficiency comparison graph of different salt-assisted extraction of higher alcohol in Comparative Example 1 of the application; Figure 3 Graph of experimental results of different derivatization conditions screening in Comparative Example 2 of the application: (a) reaction time, (b) temperature of derivatization reaction, (c) molar ratio of pyridine to SOCl2, and (d) molar ratio of pyridine to hydroxyl functional group. DETAILED DESCRIPTION

[0025] In order to make the technical problems, technical solutions and beneficial effects of the present application more clear, the present application will be further described in detail in combination with the embodiments. Unless otherwise defined, all technical terms used in this text have the same meaning as understood by those of ordinary skill in the art.

[0026] In one embodiment of the application, a detection method of higher alcohol in serum is provided, comprising the following steps: (1) Serum sample pretreatment: the serum is extracted with saturated sodium chloride solution and organic extractant; (2) Serum sample derivatization: pyridine solution and SOCI2 solution are added to the organic extract after extraction for derivatization reaction, then after standing, quenching and drying, the derivatized serum sample is obtained; (3) Preparation of internal standard solution: 21 kinds of higher alcohols are taken respectively in organic solvent to prepare standard stock solution, then mixed and diluted to obtain mixed standard solution, pyridine-d5 solution is used instead of pyridine solution to refer to the derivatization operation of step (2) to derive the mixed standard solution, and then the higher alcohol internal standard solution is obtained; (4) Preparation of higher alcohol standard curve: the mixed standard solution with serum as the matrix is obtained by replacing the organic solvent of step (3) with blank serum, then the same extraction and derivatization treatment is carried out after gradient dilution with blank serum, the internal standard solution of step (3) is added as an internal standard, the peak area ratio is obtained by liquid chromatography-mass spectrometry, and then the standard curve is constructed based on the corresponding concentration ratio; the peak area ratio and concentration ratio refer to the ratio of the mixed standard solution after derivatization treatment with serum as the matrix to the internal standard solution prepared in step (3); (5) Detection and quantification of higher alcohols in serum: the derivatized serum sample in step (2) is detected by liquid chromatography-mass spectrometry, and the content of higher alcohols in serum is calculated by combining the standard curve.

[0027] In some specific embodiments of the present application, before the serum sample is detected, an equal volume of saturated sodium chloride solution and organic extractant is added for pretreatment, which can reduce the water content in the organic extractant, reduce the solubility of higher alcohols in serum, and improve the extraction efficiency. Compared with other salts, saturated sodium chloride solution has the highest extraction efficiency for higher alcohols.

[0028] In some specific embodiments of the present application, pyridine solution is used to derivatize the serum under anhydrous solvent and catalyst SOCI2 solution, which can improve the content and ionization efficiency of higher alcohols and reduce the detection difficulty. Through screening and optimization of derivatization conditions, it is found that when the molar ratio of pyridine to SOCI2 is 1:1-1:2, the molar ratio of pyridine to hydroxyl functional group is 1:10-20, the reaction temperature is 20-25°C, and the reaction time is 1-1.5 h, the detection efficiency is high.

[0029] In some preferred embodiments of the present application, when the derivatization reaction temperature is 25°C, the peak area of the higher alcohol derivative is the largest; the reaction time is 1 hour, and the reaction is complete, and there is no significant gain by prolonging the time; the molar ratio of pyridine to SOCI2 is 1:1.5, which has the best catalytic effect; and the molar ratio of pyridine to hydroxyl functional group is 1:15, which is completely derivatized.

[0030] In some specific embodiments of the present application, standing for 2 hours and quenching after derivatization can prevent liquid from boiling.

[0031] In some specific embodiments of the present application, the derivatization is followed by drying, re-dissolving and constant volume, which can be more accurate in quantification.

[0032] In some specific embodiments of the present application, the high performance liquid chromatography-triple quadrupole mass spectrometry is used for detection, and the chromatographic conditions are as follows after pre-optimization: ChromCore phenyl ether chromatographic column is used. The mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is pure acetonitrile. The gradient program is as follows: 0-10 min, 10% B; 10-20 min, linearly increased to 20% B; 20-30 min, linearly increased to 30% B; 30-40 min, linearly increased to 40% B; 45 min, linearly increased to 100% B; 50 min, returned to 10% B. The mass spectrometry conditions of the alcohol derivatives are shown in Table 1.

[0033] The above detection method can accurately separate and detect 21 kinds of high alcohols containing aromatic alcohols and isomers, including ethanol, benzyl alcohol, 1-phenyl ethanol, 2-phenyl ethanol, propanol, isopropyl alcohol, butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol, pentanol, 2-pentanol, isopentyl alcohol, active pentanol, hexanol, 1-heptanol, 2-heptanol, 1-octanol, 3-octanol, 2-nonyl alcohol, 1-decanol.

[0034] In an embodiment of the present application, the above detection method is also used for analyzing the metabolic kinetics of alcoholic beverages to screen alcoholic beverages with the best metabolic performance, and to provide data support for the evaluation of "slow intoxication, fast sobering, and light hangover".

[0035] In some specific embodiments of the present application, serum samples are collected from experimental animals ingesting alcoholic beverages at different time points, the content of high alcohols in the serum is detected by the method, a concentration-time (C-T) curve is drawn, the metabolic rate of ethanol and high alcohols in the serum corresponding to the target alcoholic beverage is determined by C-T curve analysis, the residual amount of high alcohols in the serum at a specific time point is determined, and alcoholic beverages with excellent metabolic efficiency are screened.

[0036] The following specific embodiments will be used to explain the scheme of the present application. Those skilled in the art will understand that the following embodiments are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific technology or condition is not specified in the embodiments, it is carried out according to the technology or condition described in the literature in the art or according to the product instruction. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.

[0037] The SPF level SD rats used in the following examples are 6-8 weeks old, weighing 280-320 g, and in good health.

[0038] The 21 higher alcohols include: ethanol, benzyl alcohol, 1-phenylethanol, 2-phenylethanol, propanol, isopropanol, butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol, pentanol, 2-pentanol, isopentanol, active pentanol, hexanol, 1-heptanol, 2-heptanol, 1-octanol, 3-octanol, 2-nonyl alcohol, 1-decanol.

[0039] The high performance liquid-triple quadrupole mass spectrometry detection conditions are as follows: ChromCore phenyl ether chromatographic column: 4.6x150 mm, 5 μm; The mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is pure acetonitrile; The gradient program is: 0-10 min, 10% B is kept, 10-20 min, linearly increased to 20% B, 20-30 min, linearly increased to 30% B, 30-40 min, linearly increased to 40% B, 45 min, linearly increased to 100% B, 50 min, returned to 10% B; The syringe reservoir temperature is 4°C to prevent sample degradation; The column oven temperature is 25°C; The injection volume is 5 μL.

[0040] The mass spectrometry adopts positive electrospray ionization mode, the ion spray voltage is 5.5 kV, the ion source temperature is 500°C, and the quantification is performed in multiple reaction monitoring (MRM) mode.

[0041] Example 1: Detection of higher alcohols in serum 1. Reagent preparation: saturated sodium chloride solution (dissolve sodium chloride to saturation at 20°C), 50% v / v pyridine-d5 solution (50 mL pyridine-d5+50 mL dichloromethane), 50% v / v pyridine solution (50 mL pyridine+50 mL dichloromethane), 75% v / v SOCI2 solution (75 mL SOCI2+25 mL dichloromethane), 0.1% formic acid aqueous solution (1 mL formic acid+999 mL ultrapure water); 2. Sample pretreatment: take 400 μL of thawed post-drinking rat blank serum, add 400 μL of saturated sodium chloride solution and 400 μL of dichloromethane, oscillate at 1500 rpm for 2 min, centrifuge at 10000 rpm for 5 min, and take 200 μL of the upper organic phase; 3. Sample derivatization: The specific operation is as follows: 160 μL of pyridine solution (50 % v / v) and 160 μL of SOCI2 solution (75 % v / v) are added to the organic layer, which is reacted at 25 °C for 1 hour, stored at -80 °C for 2 hours, quenched with 50 % v / v aqueous acetonitrile solution to prevent liquid boiling, dried in a vacuum concentrator, redissolved with 50 % v / v aqueous acetonitrile solution, and stored at -80 °C for analysis; 4. Preparation of internal standard solution: 21 kinds of higher alcohols are respectively dissolved in dichloromethane to prepare 50 mg / mL standard stock solution, and then 200 μL of each of the 21 kinds of higher alcohol standard stock solution is mixed and diluted to 10 mL with dichloromethane, so that the final concentration of each higher alcohol is 1 mg / mL. Pyridine-d5 solution is used instead of pyridine solution to refer to the derivatization operation in step 3, and the standard solution is derivatized, dried in a vacuum concentrator, redissolved with 50 % v / v aqueous acetonitrile solution, and stored at -80 °C. When the sample is measured, the external standard sample and the measured sample are added as internal standards. 5. Preparation of standard curve: 21 kinds of higher alcohols are respectively dissolved in blank rat serum to prepare 50 mg / mL standard serum stock solution, and then 200 μL of each of the 21 kinds of higher alcohol standard serum stock solution is mixed and diluted to 10 mL with blank rat serum, so that the final concentration of each higher alcohol in the serum is 1 mg / mL. Then, the blank rat serum is gradiently diluted to 10, 25, 50, 100, 250, 500, and 1000 ng / mL, and the sample extraction and derivatization are performed according to steps 2 and 3. After the derivatized liquid is vacuum dried, the internal standard sample in step 4 is added as an internal standard, diluted to a certain volume, and detected according to the above high-performance liquid chromatography-triple quadrupole mass spectrometry detection conditions. The concentration ratio of the serum as a matrix and the derivatized mixed standard solution prepared in step 4 to the internal standard solution is the abscissa, and the peak area of the corresponding ratio is the ordinate. The standard curve is drawn, and the linear range, detection limit, quantification limit, and accuracy of the standard curve are shown in Table 3. 6. Detection and quantification: The standard solution prepared in step 4 is added to the serum sample derivatized in step 3, and then injected into the liquid chromatograph-mass spectrometer. The MRM detection signal of the sample is detected according to the above high-performance liquid chromatography-triple quadrupole mass spectrometry detection conditions, and the content of higher alcohols in the serum is calculated according to the standard curve.

[0042] 7. Results and discussion: (1) In order to accurately detect the content of higher alcohols in serum, the specific parent ion is selected for collision-induced dissociation during mass spectrometry MRM detection, the interference of other daughter ions is removed, and only the selected specific daughter ion is subjected to mass spectrometry signal collection to obtain the MRM and collision energy (CE) of each higher alcohol derivative, as shown in Table 1.

[0043] Table 1: Mass spectrometry analysis information of 21 kinds of alcohol derivatives

[0044] In order to investigate the accuracy and precision of the detection method of the present application, the standard addition recovery experiment was carried out in the detection process.

[0045] As shown in Table 2, the intra-day and inter-day precision (CV%) of the detection method of the present application is less than 10%, which indicates that the operation process of the detection method of the present application is stable and has high reproducibility. Whether in a short time or over several days, the method can produce consistent and reliable results, and the operation of the method is little affected by small changes in personnel, date, etc. Moreover, the matrix effect (ME%) of the detection method of the present application is 80-120%, which indicates that the matrix components in the sample do not significantly interfere with the detection of the target analyte. It can be seen that the detection method of the present application has good selectivity, can separate the analyte to be measured from the matrix, accurately measure, and ensure the accuracy of the quantitative results, and the detection signal truly reflects the concentration of the analyte to be measured without being excessively enhanced or inhibited by the matrix.

[0046] As shown in Table 3, the detection limit of the detection method of the present application is 0.17-15.14 ng / mL, and the quantification limit is 0.56-50.47 ng / mL, which indicates that the detection method of the present application has high sensitivity and can detect and quantify the analyte to be measured with a concentration as low as nanogram level.

[0047] Table 2: Precision and matrix effect of 21 alcohol derivatives

[0048] Table 3: Linear range, detection limit, quantification limit and accuracy of 21 alcohol derivatives

[0049] Example 2: Comparison of metabolic kinetics of different alcoholic beverages 1. Animal grouping and administration: 128 SPF SD rats were divided into 4 groups (n=32 / group), and were administered 10 mL / kg of Baijiu 1, Baijiu 2, Baijiu 3 and Baijiu 4 by gavage, respectively; 2. Serum collection: 4 rats were taken from each group to collect blood at 15, 30, 60, 90, 120, 180, 240 and 360 min after administration, and the serum was obtained by centrifugation; 3. Detection and analysis: the content of ethanol and higher alcohols in the serum was detected by the method of Example 1, and the concentration-time C-T curve was drawn (as shown in Figure 1 ), and the results are as follows: 1. Ethanol metabolism: the ethanol peak value (30-90 min) of samples A and B was 4000-6000 μg / mL, and that of samples C and D was 2000-3000 μg / mL; at 360 min, sample D had the lowest content (252 μg / mL); 2. Higher alcohol metabolism: sample B had the highest peak value of propanol (15-30 min), which was 2 times that of other groups; the peak values of butanol, 2-butanol and pentanol (15-60 min) had no significant difference among the four groups, but sample D had the fastest metabolism rate, and the pentanol content at 360 min was only 1 / 3 of that of sample B; 3. Conclusion: the ethanol and higher alcohol metabolism rate of sample D was significantly higher than those of the other three samples, which could reduce the risk of hangover.

[0050] Comparative Example 1: Comparison experiment of extraction efficiency of higher alcohols assisted by different salts The saturated sodium chloride solution of Example 1 was replaced with potassium sulfate, sodium sulfate, potassium chloride, ammonium sulfate and ammonium chloride, respectively, and the other operation steps were consistent, and the peak areas of higher alcohols extracted by different salts were compared. As shown in Table 1, when sodium chloride was used for extraction, the extraction efficiency of the remaining higher alcohols was the highest (single factor variance analysis P<0.05) except for propanol and 2-butanol. Figure 2

[0051] Comparative Example 2: Derivatization condition screening experiment Single factor experiment design was carried out by changing the time (1.0, 1.5, 2.0, 2.5, 3.0 h), temperature (20, 25, 30, 35℃), molar ratio of pyridine to SOCI2 (1:0.1, 1:0.5, 1:1, 1:1.5, 1:2) and molar ratio of pyridine to hydroxyl functional group (1:1, 1:5, 1:10, 1:15, 1:20) of the derivatization reaction in step 3 of Example 1, respectively. The samples were detected according to the steps of Example 1, and the results are shown in Table 2. Figure 3 As shown in Table 2, when the derivatization reaction temperature was 25℃, the peak area of the higher alcohol derivative was the largest; the reaction was complete in 1 hour, and there was no significant gain by prolonging the time; the catalytic effect was best when the molar ratio of pyridine to SOCI2 was 1:1.5; and the derivatization was complete when the molar ratio of pyridine to hydroxyl functional group was 1:15.​

Claims

1. A method for the detection of higher alcohols in serum, characterized in that, The method comprises the following steps: (1) serum sample pretreatment: extract the serum with saturated sodium chloride solution and organic extractant; (2) serum sample derivatization: add pyridine solution and SOCl2 solution to the extracted organic extractant for derivatization reaction, then dry and redissolve after standing and quenching to obtain the derivatized serum sample; (3) preparation of internal standard solution: take 21 kinds of higher alcohols respectively in an organic solvent to prepare a standard stock solution mixture, obtain a mixed standard solution, and use pyridine-d5 solution instead of pyridine solution to refer to the derivatization operation of step (2) to derive the mixed standard solution to obtain a higher alcohol internal standard solution; (4) preparation of higher alcohol standard curve: use blank serum instead of the organic solvent of step (3) to obtain a mixed standard solution with serum as the matrix, then dilute with blank serum gradient, and perform the same extraction and derivatization treatment, add the internal standard solution of step (3) as an internal standard, obtain the peak area ratio by liquid chromatography-mass spectrometry, and then construct a standard curve based on the corresponding concentration ratio; the peak area ratio and the concentration ratio refer to the ratio of the mixed standard solution with serum as the matrix and after derivatization treatment to the internal standard solution prepared in step (3); (5) detection and quantification of higher alcohols in serum: after detecting the derivatized serum sample in step (2) by liquid chromatography-mass spectrometry, calculate the content of higher alcohols in serum by combining the standard curve; The liquid chromatography-mass spectrometry is detected by high performance liquid chromatography-triple quadrupole mass spectrometry, and the chromatographic conditions are as follows: ChromCore phenyl ether chromatographic column; Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is pure acetonitrile; The gradient program is: 0-10 min, 10% B; 10-20 min, linearly increased to 20% B; 20-30 min, linearly increased to 30% B; 30-40 min, linearly increased to 40% B; 45 min, linearly increased to 100% B; 50 min, return to 10% B; The mass spectrometry conditions of the alcohol derivative are as follows: 。 2. The method of claim 1, wherein the serum is from a human. In step (1), the serum is collected after drinking; preferably, the alcohol is at least one of distilled alcohol, fermented alcohol or prepared alcohol; further preferably, the alcohol includes at least one of baijiu, brandy, whiskey, vodka, rum, gin, tequila, fruit distillate, yellow rice wine, beer, grape wine, fruit wine, sake or milk wine; more preferably, the alcohol is baijiu selected from at least one of Jiangxiang type, Nongxiang type, Qingxiang type or rice flavor type.

3. The method of claim 1, wherein the serum is from a human. In step (1), the organic extractant is dichloromethane.

4. The method of claim 1, wherein the serum is from a human. In step (1), the extraction is adding equal volume of saturated sodium chloride solution and organic extractant into serum, oscillating at 1500 rpm for 1-5 min, centrifuging at 10000 rpm for 5-15 min to obtain the upper organic phase.

5. The method of claim 1, wherein the serum is from a human. In step (2), the solvents of pyridine solution and SOCl2 solution are consistent with the organic extractant.

6. The method of claim 1, wherein the serum is from a human. In step (2), the derivatization conditions meet at least one of the following: The molar ratio of pyridine to SOCl2 is 1:1-1:2; preferably, the molar ratio of pyridine to SOCl2 is 1:1.

5. The molar ratio of pyridine to hydroxyl functional group is 1:10-20; preferably, the molar ratio of pyridine to hydroxyl functional group is 1:15; The reaction temperature is 20-25℃; preferably, the reaction temperature is 25℃. The reaction time is 1-1.5 h; preferably, the reaction time is 1 h.

7. The method of claim 1, wherein the serum is from a human. In step (2), the standing is at -80℃ for 2 hours.

8. The method of claim 1, wherein the serum is from a human. In step (2), both the quenching and the redissolving use 50% v / v acetonitrile aqueous solution.

9. The method of claim 1, wherein the serum is from a human. In the chromatographic conditions, the column temperature is 25-28℃; the injection volume is 5 μL; the mass spectrometric conditions are that the positive electrospray ionization mode is used, the ion spray voltage is 5.5 kV, the ion source temperature is 500℃, and the quantification is performed in the multiple reaction monitoring mode.

10. The use of the detection method according to any one of claims 1-9 in the analysis of alcohol beverage metabolic kinetics.

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