Dual-mode cooperative detection method for unsaturated fatty acid in Baijiu

Abstract

The invention discloses a dual-mode cooperative detection method for unsaturated fatty acid in Baijiu, and relates to the technical field of food detection. According to the method, aiming at the characteristic of a high alcohol ester matrix of the white spirit and the detection pain point that unsaturated fatty acid is easy to oxidize and large in content span, selective enrichment and matrix purification are performed on a white spirit sample through a silanization modified C18 solid-phase extraction small column, and then low-temperature anti-oxidation derivatization is completed through matching of a composite derivatization reagent and an anti-oxidation system. Then, the palmitic acid, the oleic acid and the linoleic acid are quantified by adopting a gas chromatography-mass spectrometry method, meanwhile, the alpha-linolenic acid is directly detected by adopting a high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method, and finally, results are calibrated and output by combining an internal standard method and an external standard method. According to the method, efficient purification of the Baijiu matrix is achieved, the oxidation loss rate of alpha-linolenic acid is controlled within 3%, high-precision quantification of constant components and high-sensitivity detection of trace components can be synchronously achieved, the method is suitable for detection of full-flavor and full-alcoholic-degree Baijiu, stability and universality are excellent, and reliable technical support is provided for Baijiu quality evaluation.

Description

Technical Field

[0001] This invention relates to the field of food quality and safety testing and analysis of functional components in food, specifically to a dual-mode synergistic detection method for unsaturated fatty acids in baijiu (Chinese liquor), which is particularly suitable for the simultaneous and accurate detection of free palmitic acid, oleic acid, linoleic acid, and α-linolenic acid in baijiu of different aroma types and alcohol contents. Background Technology

[0002] Unsaturated fatty acids are important flavor precursors and functional trace components in baijiu (Chinese liquor). The content and ratio of palmitic acid, oleic acid, linoleic acid, and α-linolenic acid directly affect the flavor quality, drinking comfort, and health value of baijiu. In particular, α-linolenic acid, as an essential fatty acid, is a core indicator for evaluating the high-end quality of baijiu, and its trace presence is one of the key indicators. Therefore, establishing a method for simultaneously and accurately determining unsaturated fatty acids in baijiu, from macro-level (mg / L) to trace level (μg / L), is of paramount importance for baijiu quality control, production process optimization, product authenticity verification, and the exploration of its health benefits.

[0003] Current methods for detecting unsaturated fatty acids in baijiu (Chinese liquor) primarily rely on single-detection modes. Gas chromatography-mass spectrometry (GC-MS) is a conventional method for fatty acid analysis, but it faces significant bottlenecks when applied to the complex matrix of baijiu: First, the high content of alcohols and esters such as ethanol and ethyl acetate in the baijiu system generates strong matrix interference. Conventional pretreatment methods cannot effectively separate the target fatty acids from the interfering substances, severely affecting the chromatographic separation effect and the accuracy of mass spectrometry quantification. Second, polyunsaturated fatty acids, especially α-linolenic acid, have highly reactive double bonds and are prone to oxidative degradation during traditional high-temperature derivatization processes. Conventional derivatization procedures lack targeted oxidation protection measures, resulting in severe loss of target analytes and significantly lower or even undetectable quantitative results for trace components. High-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF) can directly detect free fatty acids without derivatization, avoiding the oxidation risk of the derivatization process. It has high sensitivity for the detection of trace components, but it has obvious shortcomings when applied to the detection of baijiu (Chinese liquor): direct injection of high alcohol ester matrix into baijiu will seriously contaminate the chromatographic column and the mass spectrometer ion source, affecting the long-term stability of the instrument. At the same time, the detection of major fatty acids is easily interfered with by the matrix ion inhibition effect, resulting in low quantitative accuracy and failing to meet the precise quantitative requirements of major components.

[0004] Furthermore, existing pretreatment technologies also have significant limitations. Conventional C18 solid-phase extraction packing material lacks sufficient selectivity for purifying the extremely complex matrix of baijiu, making it difficult to completely remove high-content alcohol and ester interferences. Conventional antioxidant methods only add phenolic antioxidants, which cannot effectively protect trace components with high unsaturation throughout the entire process in the high-temperature microenvironment of derivatization reactions. Currently, there is a long-standing core technical contradiction in this field that has not been effectively resolved: it is impossible to simultaneously achieve high-fidelity quantification of easily oxidized macro-components and high-sensitivity detection of trace easily oxidized components in the same baijiu sample and the same pretreatment process. Even if GC-MS and HPLC-QTOF are simply used in parallel, the same sample still needs to be pretreated separately, which is not only cumbersome and consumes a lot of sample, but also introduces uncalibrated systematic errors due to inconsistencies in the pretreatment process, completely failing to meet the actual needs of batch and standardized testing in the baijiu industry. Summary of the Invention

[0005] In view of this, the present invention proposes a dual-mode synergistic detection method for unsaturated fatty acids in baijiu (Chinese liquor).

[0006] The technical solution of this invention is implemented as follows: This invention provides a dual-mode synergistic detection method for unsaturated fatty acids in baijiu (Chinese liquor), wherein the target unsaturated fatty acids include palmitic acid, oleic acid, linoleic acid, and α-linolenic acid, and includes the following steps:

[0007] S1. Selective enrichment and matrix purification: Take a baijiu sample, dilute it with 8-10 times the volume of ultrapure water, load the sample onto a modified C18 solid phase extraction column modified with silanizing reagent for enrichment, remove impurities by rinsing with formic acid aqueous solution, elute with ethyl acetate-n-hexane mixture and concentrate to obtain crude extract of target component. S2. Low-temperature anti-oxidation derivatization: Take a portion of the crude extract of the target component, add a composite derivatization reagent and an antioxidant, and carry out a low-temperature derivatization reaction under inert gas protection. After the reaction is completed, terminate the reaction in an ice-water bath to obtain the derivatized product. S3. Dual-mode synergistic detection: The derivatized products are detected by gas chromatography-mass spectrometry for the quantification of palmitic acid, oleic acid, and linoleic acid; at the same time, another portion of the crude extract of the target components that has not been derivatized is directly detected by high performance liquid chromatography-quadrupole time-of-flight mass spectrometry for the qualitative and quantitative analysis of α-linolenic acid. S4. Result Calibration and Output: The results of the internal standard method detected by gas chromatography-mass spectrometry and the external standard method detected by high performance liquid chromatography-quadrupole time-of-flight mass spectrometry are calibrated to calculate and output the content of each target unsaturated fatty acid in the liquor.

[0008] In some embodiments, the modified C18 solid-phase extraction column in step S1 is a 500mg / 3mL C18 column modified with 0.5%-1% silanizing reagent; the activation process of the column is as follows: it is rinsed sequentially with 2.5-3mL methanol and 4-6mL ultrapure water at a flow rate of 1mL / min, and the column is kept moist after activation; the sample loading conditions are as follows: 1mL of liquor sample is diluted with 8-10mL ultrapure water, and then 40-60μL of 0.1mg / mL heptadecanoate internal standard solution is added, mixed well, and loaded, and allowed to stand for 8-12min; the elution and elution conditions are as follows: it is rinsed with 4-6mL of 4%-6% formic acid aqueous solution, the elution solution is discarded, and then eluted with 4-6mL of ethyl acetate-n-hexane mixture with a volume ratio of 1:8-1:10. After collecting the eluent, it is concentrated to 1-2 drops under nitrogen gas in the dark at 38-42℃ to obtain the crude extract of the target component.

[0009] Diluting the liquor sample to reduce the ethanol concentration in the system avoids high-concentration alcohols from disrupting the adsorption balance of the solid-phase extraction packing material, thus improving the retention of target fatty acids on the packing material. The activation process involves gradient rinsing with methanol and ultrapure water to fully wet the alkyl chains of the packing material, ensuring sufficient exposure of adsorption sites. Formic acid rinsing, by adjusting the system pH (4%-6% by mass), corresponds to a system pH of 2.0-3.0, where the degree of fatty acid dissociation is ≤5%, significantly enhancing its hydrophobic adsorption on the non-polar C18 packing material, inhibiting fatty acid dissociation, and improving its retention on the non-polar packing material, while simultaneously removing water-soluble sugars, amino acids, and other polar impurities. A specific ratio of ethyl acetate-n-hexane mixture can precisely match the polarity of the target fatty acid, achieving efficient elution of the target analyte while minimizing the co-elution of non-polar alcohol ester impurities, thus reducing matrix interference at its source.

[0010] In some embodiments, the composite derivatizing reagent in step S2 is a mixture of 12%-16% boron trifluoride-methanol solution and 5%-8% boron trifluoride diethyl ether solution at a volume ratio of 3:1. After mixing, the mixture is magnetically stirred for 5 minutes at room temperature until homogeneous. It is prepared and used immediately, and stored in a sealed container at 4°C for no more than 24 hours. The antioxidant is a 0.01%-0.03% tert-butylhydroquinone (TBHQ) methanol solution, and the amount added is 5%-10% of the volume of the composite derivatizing reagent. The conditions for the low-temperature derivatization reaction are as follows: after purging the air in the reaction system with high-purity nitrogen, the system is sealed and placed in a water bath at 65-70°C for 25-35 minutes. After the reaction is completed, the system is immediately cooled in an ice-water bath for 4-6 minutes to terminate the reaction.

[0011] The preparation method of the compound derivatizing reagent is as follows: Take 12%-16% boron trifluoride-methanol solution and 5%-8% boron trifluoride diethyl ether solution, mix them at a volume ratio of 3:1 under the condition of room temperature and protection from light, stir magnetically for 5 minutes until homogeneous, prepare and use immediately, store sealed at 4℃ and protected from light, with a shelf life of 24 hours.

[0012] Boron trifluoride-methanol, as a classic methyl esterification derivatization reagent, can efficiently catalyze the carboxyl methyl esterification of fatty acids, improving their gas chromatography separation performance. Combined with boron trifluoride diethyl ether solution, it can lower the activation energy of the derivatization reaction, achieving complete derivatization at lower temperatures and reducing the oxidation risk caused by high temperatures. TBHQ, as a phenolic antioxidant, can quench free radicals generated during fatty acid oxidation by providing hydrogen atoms, thus blocking the chain oxidation reaction. Inert gas protection can isolate oxygen in the reaction system, eliminating the oxidant of the oxidation reaction at its source. Rapid cooling in an ice-water bath can instantly terminate the reaction, avoiding subsequent oxidation caused by residual heat, forming a multi-dimensional anti-oxidation protection system.

[0013] In some embodiments, in step S3, the parameters for gas chromatography-mass spectrometry (GC-MS) detection are as follows: a DB-WAX polar column (30m × 0.25mm × 0.25μm), an injection port temperature of 245-255℃, a split ratio of 8:1-12:1, and an injection volume of 1μL; high-purity helium as the carrier gas, with a constant flow rate of 0.9-1.1mL / min; a temperature program of 60℃ for 2 min, increasing to 240℃ at 4-6℃ / min, and holding for 12-18 min; an EI source as the ion source, a temperature of 220-240℃, a scan mode of SCAN full scan combined with SIM selected ion monitoring, a solvent delay of 4-6 min, and internal standard quantification using heptadecanoic acid as an internal standard; and high-performance liquid chromatography (HPLC). The parameters for chromatography-quadrupole time-of-flight mass spectrometry (GC-QFS) were as follows: a C18 reversed-phase column (2.1 mm × 100 mm × 1.8 μm), column temperature 28-32 ℃, flow rate 0.28-0.32 mL / min; mobile phase A was 0.08%-0.12% formic acid aqueous solution, mobile phase B was acetonitrile, and the gradient elution program was: 40% A phase and 60% B phase at 0 min, 10% A phase and 90% B phase at 3 min, held for 5 min, and restored to the initial ratio and equilibrated to 7 min at 6 min; the mass spectrometer was an ESI negative ion source, spray voltage 3.3-3.7 kV, desolvation gas temperature 340-360 ℃, scan range 100-500 m / z, and quantification was performed using the external standard method.

[0014] The DB-WAX polar chromatographic column exhibits excellent separation selectivity for polar derivatives of fatty acid methyl esters, enabling baseline separation of fatty acid derivatives with different saturations. Gradient temperature programming balances the separation effects of low-boiling-point solvents and high-boiling-point derivatives. The combined SCAN and SIM scanning modes simultaneously achieve accurate qualitative and quantitative analysis of the target analyte. Internal standard methods effectively offset systematic errors during pretreatment and injection, improving the quantitative stability of macro-components. The C18 reversed-phase column provides good retention of underived free fatty acids. Formic acid aqueous solution improves peak shape and avoids peak tailing. Gradient elution enables rapid separation of the target analyte from matrix impurities. The ESI negative ion source has extremely high ionization efficiency for the carboxyl groups of free fatty acids. Combined with high-resolution time-of-flight mass spectrometry, it achieves highly sensitive and specific detection of trace α-linolenic acid, avoiding target analyte loss during derivatization.

[0015] In some embodiments, the modified C18 solid-phase extraction column modified with silanizing reagent is modified with a stepwise gradient silanization and fluorination end-group plugging process, specifically including: firstly, thoroughly plugging the silica matrix support with small molecule trimethylchlorosilane to eliminate residual silanol active sites; and then functionalizing the plugged filler with tridecafluorooctyltrichlorosilane.

[0016] The modified C18 solid-phase extraction column modified with the silanizing reagent is prepared as follows: the entire silanization reaction is carried out in a closed environment under anhydrous and high-purity nitrogen protection, and all solvents used are anhydrous. A blank C18 solid-phase extraction column (500 mg / 3 mL) is taken, activated with methanol, and then a 5% trimethylchlorosilane solution in hexane is added. The column is passed through at a flow rate of 1 mL / min and reacted at room temperature for 30 min. It is then washed three times with hexane. Next, a 3% tridecafluorooctyltrichlorosilane solution in toluene is added and passed through the column at a flow rate of 0.5 mL / min. The column is reacted in a 60°C water bath for 60 min. It is then washed sequentially with toluene, methanol, and ultrapure water, dried with nitrogen, sealed, and stored at 4°C for later use. The C18 column modified by stepwise gradient silanization and fluorination end-group blocking is prepared in the same way as above, except that the concentration of trimethylchlorosilane is adjusted to 5% and the concentration of tridecafluorooctyltrichlorosilane is adjusted to 3%.

[0017] Unreacted residual silanol groups on the surface of the silica matrix can cause non-specific adsorption of polar alcohol ester impurities, and can also lead to peak tailing of target fatty acids. By blocking the end groups of small molecule trimethylchlorosilane, the activity of residual silanol groups can be completely eliminated, reducing the adsorption of impurities and the irreversible retention of target substances. Functional modification with fluorinated long-chain silanizing reagents can form a low surface energy hydrophobic interface on the filler surface. Based on the principle of like dissolves like, it has a stronger selective adsorption effect on long-chain fatty acids, while also repelling polar alcohol ester impurities in baijiu. This significantly improves the enrichment selectivity of the filler for target substances and the matrix purification efficiency, solving the problem of insufficient purification capacity of conventional C18 fillers for complex baijiu matrices.

[0018] In some embodiments, the antioxidant is a methanol solution of tert-butylhydroquinone and vitamin E in a mass ratio of 3:1, and the total amount added is 5%-10% of the volume of the compound derivatizing reagent.

[0019] Tert-butylhydroquinone exhibits excellent solubility and free radical quenching efficiency in polar methanol systems, rapidly blocking the initiation of oxidative chain reactions in the early stages of derivatization. Vitamin E, a fat-soluble antioxidant, exerts its antioxidant effect at the interface between fatty acids and derivatizing reagents. The combination of the two forms a "water-oil phase" synergistic antioxidant system, covering the entire system and process of derivatization reactions. Compared with single antioxidants, it can achieve better antioxidant effects with lower addition amounts, further reducing the oxidative loss of polyunsaturated fatty acids.

[0020] In some embodiments, the composite derivatizing reagent also contains a phase transfer catalyst, tetrabutylammonium bromide, in an amount of 0.01%-0.03% of the total mass of the derivatization reaction system.

[0021] The methyl esterification reaction of fatty acids with methanol is a liquid-liquid two-phase reaction. The reaction rate is limited by the interfacial mass transfer efficiency. Conventional derivatization requires higher temperatures and longer reaction times to achieve complete derivatization. Tetrabutylammonium bromide, as a quaternary ammonium salt phase transfer catalyst, can promote the transfer of fatty acid substrates from the organic phase to the methanol reaction phase by reducing the interfacial tension between the two phases, thereby significantly improving the mass transfer efficiency. This allows for complete derivatization at lower temperatures and in shorter times, further reducing the oxidation risk caused by high-temperature and long-term reactions, while also improving the batch-to-batch stability of the derivatization reaction.

[0022] In some embodiments, the low-temperature derivatization reaction is carried out in a microwave-assisted heating device with a rated power of 800W and a working power of 300W. An intermittent heating mode of 10 seconds of heating followed by 20 seconds of rest is adopted to control the reaction temperature at 60-70℃, with a total reaction time of 15-20 minutes. This mode can precisely control the temperature through a microwave synthesizer to avoid local temperatures exceeding 70℃ and reduce fatty acid oxidation. According to the test, the temperature fluctuation of the system under the intermittent heating mode is ≤±1℃, and the temperature fluctuation under the continuous heating mode is ±3-5℃.

[0023] Traditional water bath heating is a heat conduction heating method, which suffers from slow system heating and uneven temperature distribution, easily leading to local overheating and fatty acid oxidation. Microwave-assisted heating can achieve uniform heating of the bulk phase within the system through molecular polarization, with rapid heating rate and precise temperature control. The intermittent heating mode can avoid the heat accumulation caused by continuous heating. While ensuring the activation energy required for the derivatization reaction, it strictly controls the system temperature within the low-temperature range. Combined with the mass transfer enhancement effect of the phase transfer catalyst, the reaction time can be significantly shortened. From both kinetic and thermodynamic perspectives, the occurrence of oxidation reaction can be further suppressed, achieving a dual improvement in derivatization efficiency and anti-oxidation effect.

[0024] In some implementations, in step S4, the calibration specifically involves: performing data fusion and cross-validation on the results of the two detection methods, wherein the contents of palmitic acid, oleic acid, and linoleic acid are based on the results of gas chromatography-mass spectrometry, and the contents of α-linolenic acid are based on the results of high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

[0025] Based on the performance advantages of the two detection technologies, differentiated result interpretation is adopted. Gas chromatography-mass spectrometry (GC-MS) exhibits excellent separation performance and quantitative stability for derivatized macro-fatty acid methyl esters, and the internal standard method can effectively offset systematic errors, thus serving as the basis for the quantification of macro-components. High-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QMS) does not require derivatization and can directly detect free α-linolenic acid, avoiding the loss of trace components during the derivatization process. Furthermore, high-resolution mass spectrometry can effectively eliminate interference from matrix impurities, providing higher detection sensitivity and specificity for trace components, thus serving as the basis for the quantification of trace α-linolenic acid. Through data fusion and cross-validation, the quantitative accuracy of both macro- and trace components can be guaranteed simultaneously, solving the problem that a single detection mode cannot simultaneously handle the detection of a wide concentration range.

[0026] In some embodiments, the method is applicable to the detection of soy sauce aroma type, strong aroma type, light aroma type, glutinous rice aroma type, mixed aroma type, phoenix aroma type, and buckwheat aroma type baijiu, and the suitable baijiu alcohol content range is 28%vol-68%vol.

[0027] By using modified C18 packing material for highly selective purification and broad-spectrum optimization of all process parameters, interference caused by matrix differences in different types of baijiu can be effectively eliminated. There is no need to adjust the detection process for baijiu with different alcohol contents and different aromas, which greatly improves the versatility and robustness of the method.

[0028] The present invention has the following advantages over the prior art: This invention addresses the challenges of detecting the complex matrix characteristics of high-alcohol and high-ester baijiu (Chinese liquor) and the wide range and easy oxidation of unsaturated fatty acid content. It constructs a collaborative analysis system with shared pretreatment enrichment and integrated split-flow dual-mode detection. Compared to existing technologies, targeted filler modification and pretreatment process optimization significantly improve the purification efficiency of the baijiu matrix, effectively eliminating the strong interference of alcohols and esters on detection. Simultaneously, through a multi-dimensional anti-oxidation derivatization system design, it fundamentally inhibits the oxidative degradation of polyunsaturated fatty acids during detection. The innovative design of using the same sample for two detection modes solves the long-standing industry problem of the inability to simultaneously and accurately detect major and trace components in existing technologies. It avoids uncontrollable system errors caused by simple parallel instrument connections. The method is highly versatile and stable, adaptable to the standardized batch testing needs of baijiu with different aroma types and alcohol contents. It provides reliable technical support for baijiu quality evaluation, functional component discovery, and production process optimization, playing a significant role in promoting quality control and upgrading in the baijiu industry. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a flowchart of the detection method of the present invention; Figure 2 This is the GC-MS total ion chromatogram of the strong-aroma baijiu sample in Example 1 of the present invention; Figure 3 This is the HPLC-QTOF extraction ion chromatogram of α-linolenic acid in Example 1 of the present invention. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] Main instruments and equipment Gas chromatography-mass spectrometry (Agilent 7890A-5975C), high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (Agilent 1290 Infinity II-6545 QTOF), microwave synthesizer, fully automated nitrogen blowing apparatus, solid phase extraction device, constant temperature water bath, electronic analytical balance (accuracy 0.0001g), vortex mixer.

[0033] Reagents and Materials Palmitic acid, oleic acid, linoleic acid, α-linolenic acid, and heptadecanic acid (internal standard) standards (purity ≥99.0%); methanol, acetonitrile, ethyl acetate, and n-hexane (all chromatographically pure); formic acid, trimethylchlorosilane, tridecafluorooctyltrichlorosilane, 14% boron trifluoride-methanol solution, 7% boron trifluoride diethyl ether solution, tert-butylhydroquinone (TBHQ), vitamin E, and tetrabutylammonium bromide (all analytically pure); modified C18 solid-phase extraction column (500 mg / 3 mL, modified with 0.8% silanizing reagent), stepwise fluorinated modified C18 solid-phase extraction column (500 mg / 3 mL), and conventional unmodified C18 solid-phase extraction column (500 mg / 3 mL); commercially available baijiu samples (strong aroma, sauce aroma, light aroma, glutinous rice aroma, mixed aroma, phoenix aroma, and buckwheat aroma, with alcohol content covering 28% vol-68% vol).

[0034] Performance index verification method 1. Matrix interference removal rate: Calculated by comparing the area of ​​impurity peaks in the total ion chromatogram of GC-MS with the area of ​​impurity peaks in the diluted sample solution and the eluted target solution. The calculation formula is: Matrix interference removal rate = (1 - total area of ​​impurity peaks in the eluent / total area of ​​impurity peaks in the sample solution) × 100%; 2. Oxidation loss rate of α-linolenic acid: A 10 μg / L α-linolenic acid blank liquor matrix-matched standard solution was prepared, and after derivatization treatment and without derivatization treatment, the remaining target content was determined by HPLC-QTOF. The calculation formula was: Oxidation loss rate = (1 - content measured after derivatization / theoretical spiked content) × 100%; 3. Spiked Recovery Rate: Add a known concentration of mixed fatty acid standard to the liquor sample. The spiking amount is 0.5 times the background content of each target component for low concentration, 1 times for medium concentration, and 2 times for high concentration. Process and detect according to the corresponding methods. The calculation formula is: Recovery rate = (Measured content after spiking - Sample background content) / Theoretical spiking amount × 100%; 4. Precision (RSD): The same sample is processed in parallel 6 times according to the corresponding method, and the relative standard deviation (RSD) of the 6 test results is calculated; 5. Limit of Detection (LOD): The mixed fatty acid standard matched with the liquor matrix was serially diluted, and the concentration of the target analyte corresponding to a signal-to-noise ratio (S / N) of 3 was used as the LOD.

[0035] Example 1 Sample testing of strong-aroma baijiu Detection steps Step 1, Sample Pretreatment and Target Composition Enrichment: Take 1 mL of 52% vol commercially available strong-aroma baijiu, dilute with 9 mL of ultrapure water, and then add 50 μL of 0.1 mg / mL heptadecanoate internal standard solution. Vortex mix to obtain the loading solution. Activate the modified C18 solid-phase extraction column modified with 0.8% silanizing reagent by successively rinsing with 3 mL of methanol and 5 mL of ultrapure water at a flow rate of 1 mL / min, keeping the column moist. Slowly load the loading solution onto the activated column and let it stand for 10 min to allow the target composition to be fully adsorbed. Elute the column with 5 mL of 5% formic acid aqueous solution at a flow rate of 1 mL / min and discard the eluent. Elute the target composition with 5 mL of ethyl acetate-hexane mixture (1:9 v / v). Collect all the eluent in a brown centrifuge tube and concentrate it to 1 drop under nitrogen at 40℃ in the dark to obtain the crude extract of the target component.

[0036] Step 2, Low-temperature anti-oxidation derivatization: Take the crude extract obtained in Step 1, add 2 mL of a composite derivatizing reagent made of 14% BF3-methanol and 7% boron trifluoride diethyl ether at a volume ratio of 3:1, then add 100 μL of 0.02% TBHQ methanol solution, purge the air in the centrifuge tube with high-purity nitrogen and seal it; place it in a 70℃ constant temperature water bath for 30 min, and immediately place it in an ice water bath for 5 min to terminate the reaction, and obtain the derivatized product.

[0037] Step 3, Dual-mode synergistic detection: Add 1 mL of n-hexane and 2 mL of saturated sodium chloride aqueous solution to the derivatized product from Step 2, vortex mix for 1 min, take the upper n-hexane phase, filter it through a 0.22 μm organic filter membrane, and perform GC-MS detection for the quantification of palmitic acid, oleic acid, and linoleic acid; separately take the remaining crude extract from Step 1 that has not been derivatized, filter it through a 0.22 μm organic filter membrane, and perform HPLC-QTOF direct detection for the qualitative and quantitative analysis of α-linolenic acid.

[0038] Step 4, Result Calibration and Output: GC-MS detection uses the heptadecanoic acid internal standard method for quantification, and HPLC-QTOF detection uses the external standard method for quantification; the contents of palmitic acid, oleic acid, and linoleic acid are based on the GC-MS detection results, and the contents of α-linolenic acid are based on the HPLC-QTOF detection results. The contents of each target unsaturated fatty acid in the sample are calculated and output.

[0039] Performance verification steps 1. The strong-aroma baijiu sample was processed in parallel 6 times according to the above detection steps, the background content of each target substance was determined, and the RSD value of the detection results was calculated; 2. Add mixed fatty acid standards of low, medium and high concentration gradients to the sample, process and detect according to the above steps, and calculate the spiked recovery rate of each target analyte; 3. The matrix interference removal rate, α-linolenic acid oxidation loss rate, and method detection limit of this method were determined according to the general method.

[0040] Performance verification results Table 1. Results of core performance testing for strong-aroma baijiu in Example 1

[0041] Auxiliary performance indicators: matrix interference removal rate 95.2%, α-linolenic acid oxidation loss rate 2.8%.

[0042] Example 2 Sample testing of sauce-flavored baijiu Detection steps Step 1, Sample pretreatment and target enrichment: Take 1 mL of 53% vol commercially available Maotai-flavor liquor, and perform the remaining operations exactly the same as Step 1 in Example 1; Step 2, Low-temperature anti-oxidation derivatization: The operation is completely consistent with step 2 of Example 1; Step 3, Dual-mode collaborative detection: The operation is completely consistent with step 3 of Example 1; Step 4, Result Calibration and Output: The operation is exactly the same as step 4 in Example 1.

[0043] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed to determine the background content, recovery rate, RSD, detection limit, and auxiliary performance indicators.

[0044] Performance verification results Table 2. Results of core performance testing for Maotai-flavor liquor in Example 2

[0045] Auxiliary performance indicators: matrix interference removal rate 94.8%, α-linolenic acid oxidation loss rate 2.9%.

[0046] Example 3 Testing of light-aroma baijiu samples Detection steps Step 1, Sample pretreatment and target enrichment: Take 1 mL of 50% vol commercially available light-aroma baijiu, and perform the remaining operations exactly the same as in Step 1 of Example 1; Step 2, Low-temperature anti-oxidation derivatization: The operation is completely consistent with step 2 of Example 1; Step 3, Dual-mode collaborative detection: The operation is completely consistent with step 3 of Example 1; Step 4, Result Calibration and Output: The operation is exactly the same as step 4 in Example 1.

[0047] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed to determine the background content, recovery rate, RSD, detection limit, and auxiliary performance indicators.

[0048] Performance verification results Table 3. Results of core performance testing for light-aroma baijiu in Example 3

[0049] Auxiliary performance indicators: matrix interference removal rate 95.5%, α-linolenic acid oxidation loss rate 2.7%.

[0050] Example 4 Stepwise silanization and fluorination modification of C18 column formulation Detection steps Step 1, Sample Pretreatment and Target Enrichment: The modified C18 column in Example 1 was replaced with a "stepwise gradient silanization + fluorination end-blocking modified C18 column". The preparation method of this column is as follows: first, the silica matrix support is thoroughly end-blocked with small molecule trimethylchlorosilane to eliminate residual silanol active sites, and then the plugged filler is functionalized with tridecafluorooctyltrichlorosilane; the remaining pretreatment operations are completely consistent with Step 1 of Example 1. Step 2, Low-temperature anti-oxidation derivatization: The operation is completely consistent with step 2 of Example 1; Step 3, Dual-mode collaborative detection: The operation is completely consistent with step 3 of Example 1; Step 4, Result Calibration and Output: The operation is exactly the same as step 4 in Example 1.

[0051] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed, and the recovery rate, RSD, detection limit and auxiliary performance indicators were measured.

[0052] Performance verification results Table 4. Results of Core Performance Testing in Example 4

[0053] Auxiliary performance indicators: matrix interference removal rate 98.7%, α-linolenic acid oxidation loss rate 2.2%.

[0054] Example 5 Compound antioxidant regimen Detection steps Step 1, Sample Pretreatment and Target Enrichment: The operation is exactly the same as Step 1 in Example 1; Step 2, Low-temperature anti-oxidation derivatization: The single TBHQ methanol solution in Example 1 is replaced with a "compound methanol solution of TBHQ and vitamin E in a mass ratio of 3:1". The total amount added is the same as in Example 1, and the rest of the derivatization operation is exactly the same as step 2 in Example 1. Step 3, Dual-mode collaborative detection: The operation is completely consistent with step 3 of Example 1; Step 4, Result Calibration and Output: The operation is exactly the same as step 4 in Example 1.

[0055] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed, and the recovery rate, RSD, detection limit and auxiliary performance indicators were measured.

[0056] Performance verification results Table 5. Results of Core Performance Testing in Example 5

[0057] Auxiliary performance indicators: α-linolenic acid oxidation loss rate 1.2%, matrix interference removal rate 95.3%.

[0058] Example 6 Phase transfer catalysis + microwave-assisted derivatization scheme Detection steps Step 1, Sample Pretreatment and Target Enrichment: The operation is exactly the same as Step 1 in Example 1; Step 2, Low-temperature anti-oxidation derivatization: Add 2 mL of composite derivatizing reagent and 100 μL of 0.02% TBHQ methanol solution to the crude extract, and simultaneously add 0.02% tetrabutylammonium bromide phase transfer catalyst. After sealing with high-purity nitrogen, place it in a microwave-assisted heating device and use an intermittent heating mode of 10 seconds heating followed by 20 seconds of rest. React at 65°C for 18 min. After the reaction, immediately cool in an ice-water bath for 5 min. The remaining derivatization operations are the same as in Step 2 of Example 1. Step 3, Dual-mode collaborative detection: The operation is completely consistent with step 3 of Example 1; Step 4, Result Calibration and Output: The operation is exactly the same as step 4 in Example 1.

[0059] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed. The recovery rate, RSD, detection limit and auxiliary performance indicators were measured, and the completeness of the derivatization reaction was verified at the same time.

[0060] Performance verification results Table 6. Results of Core Performance Testing in Example 6

[0061] Auxiliary performance indicators: α-linolenic acid oxidation loss rate 0.8%, derivatization reaction completeness 100%, matrix interference removal rate 95.4%.

[0062] Example 7 Full optimal solution combination Detection steps Step 1, Sample pretreatment and target enrichment: The stepwise fluorinated modified C18 column of Example 4 was used, and the remaining pretreatment operations were completely consistent with Step 1 of Example 1. Step 2, Low-temperature anti-oxidation derivatization: The compound anti-oxidation system of Example 5 and the phase transfer catalysis + microwave-assisted derivatization scheme of Example 6 are used simultaneously, and the remaining derivatization operations are completely consistent with Step 2 of Example 6; Step 3, Dual-mode collaborative detection: The operation is completely consistent with step 3 of Example 1; Step 4, Result Calibration and Output: The operation is exactly the same as step 4 in Example 1.

[0063] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed, and the recovery rate, RSD, detection limit and auxiliary performance indicators were measured.

[0064] Performance verification results Table 7. Results of Core Performance Testing for the Optimized Scheme in Example 7

[0065] Auxiliary performance indicators: matrix interference removal rate 99.1%, α-linolenic acid oxidation loss rate 0.7%.

[0066] Example 8 Parameter lower limit verification Detection steps Step 1, Sample Pretreatment and Target Composition Enrichment: Take 1 mL of 28% vol low-alcohol strong-aroma baijiu, use a C18 column modified with 0.5% silanizing reagent, activate with 2.5 mL methanol and 4 mL ultrapure water, load with 1 mL sample + 8 mL ultrapure water, rinse with 4 mL 4% formic acid aqueous solution, elute with 4 mL ethyl acetate-n-hexane mixture (1:8 v / v), concentrate under nitrogen at 38℃; the remaining pretreatment operations are the same as Step 1 in Example 1. Step 2, Low-temperature anti-oxidation derivatization: A composite derivatizing reagent of 12% BF3-methanol and 5% boron trifluoride diethyl ether was used. The reaction was carried out in a water bath at 65°C for 25 min, followed by cooling in an ice-water bath for 4 min. The remaining derivatization operations were the same as in Step 2 of Example 1. Step 3, Dual-mode synergistic detection: GC-MS injection port temperature 245℃, split ratio 8:1, carrier gas flow rate 0.9mL / min, temperature program 60℃ held for 2min, then increased to 240℃ at 4℃ / min and held for 12min; HPLC-QTOF mobile phase A was 0.08% formic acid aqueous solution, column temperature 28℃, flow rate 0.28mL / min, spray voltage 3.3kV, desolvation gas temperature 340℃; the remaining detection operations were the same as in Step 3 of Example 1. Step 4, Result Calibration and Output: The operation is exactly the same as step 4 in Example 1.

[0067] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed, and the recovery rate, RSD, and detection limit were determined.

[0068] Performance verification results Table 8. Parameter Lower Limit Verification Results of Example 8: Core Performance Results

[0069] Matrix interference removal rate ≥94%, α-linolenic acid oxidation loss rate ≤3.0%.

[0070] Example 9 Parameter upper limit verification Detection steps Step 1, Sample Pretreatment and Target Composition Enrichment: Take 1 mL of 68% vol high-proof soy sauce-flavored liquor, use a C18 column modified with 1% silanizing reagent, activate with 3 mL methanol and 6 mL ultrapure water, dilute with 1 mL sample + 10 mL ultrapure water, rinse with 6 mL of 6% formic acid aqueous solution, elute with 6 mL of ethyl acetate-n-hexane mixture (1:10 v / v), concentrate under nitrogen at 42℃; the remaining pretreatment operations are the same as in Step 1 of Example 1. Step 2, Low-temperature anti-oxidation derivatization: A composite derivatizing reagent of 16% BF3-methanol and 8% boron trifluoride diethyl ether was used. The reaction was carried out in a 70°C water bath for 35 min, followed by cooling in an ice-water bath for 6 min. The remaining derivatization operations were the same as in Step 2 of Example 1. Step 3, Dual-mode synergistic detection: GC-MS injection port temperature 255℃, split ratio 12:1, carrier gas flow rate 1.1mL / min, temperature program 60℃ held for 2min, then increased to 240℃ at 6℃ / min and held for 18min; HPLC-QTOF mobile phase A was 0.12% formic acid aqueous solution, column temperature 32℃, flow rate 0.32mL / min, spray voltage 3.7kV, desolvation gas temperature 360℃; the remaining detection operations were the same as in Step 3 of Example 1. Step 4, Result Calibration and Output: The operation is exactly the same as step 4 in Example 1.

[0071] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed, and the recovery rate, RSD, and detection limit were determined.

[0072] Performance verification results Table 9. Parameter Upper Limit Verification Results of Example 9

[0073] Matrix interference removal rate ≥94%, α-linolenic acid oxidation loss rate ≤3.0%.

[0074] Example 10 Verification of the universality limits of all flavors and alcohol contents Detection steps Step 1: Take 28% vol of light-aroma baijiu made with small koji, 28% vol of light-aroma baijiu made with large koji, 45% vol of mixed-aroma baijiu, 52% vol of glutinous rice aroma baijiu, 45% vol of buckwheat aroma baijiu, 53% vol of phoenix aroma baijiu, and 60% vol of high-proof sauce aroma baijiu respectively, and process them according to the detection steps in Example 1 without adjusting any parameters; Step 2, Dual-mode collaborative detection and result calibration: The operation is completely consistent with steps 3 and 4 of Example 1.

[0075] Performance verification steps Seven types of liquor samples were treated in parallel six times, and the average recovery rate, precision RSD, and matrix interference removal rate of each target analyte were determined.

[0076] Performance verification results Table 10 Results of Verification of Universality of Full-Flavor Full-Alcohol Content in Example 10

[0077] The oxidative loss rate of α-linolenic acid in all samples was ≤2.9%, and the method detection limit was ≤0.5μg / L.

[0078] Example 11 Sample testing of glutinous rice aroma type baijiu Detection steps Step 1, Sample Pretreatment and Target Composition Enrichment: Take 1 mL of 52% vol commercially available glutinous rice wine, dilute with 9 mL of ultrapure water, and then add 50 μL of 0.1 mg / mL heptadecanoate internal standard solution. Vortex to mix and obtain the loading solution. Activate the modified C18 solid-phase extraction column modified with 0.8% silanizing reagent by washing with 3 mL of methanol and 5 mL of ultrapure water at a flow rate of 1 mL / min, keeping the column moist. Slowly load the loading solution onto the activated column and let it stand for 10 min to allow the target composition to be fully adsorbed. Elute the column with 5 mL of 5% formic acid aqueous solution at a flow rate of 1 mL / min and discard the eluent. Elute the target composition with 5 mL of ethyl acetate-hexane mixture (1:9 v / v). Collect all the eluent in a brown centrifuge tube and concentrate it to 1 drop under nitrogen at 40℃ in the dark to obtain the crude extract of the target component.

[0079] Step 2, Low-temperature anti-oxidation derivatization: Take the crude extract obtained in Step 1, add 2 mL of a composite derivatizing reagent consisting of 14% boron trifluoride-methanol and 7% boron trifluoride diethyl ether mixed at a volume ratio of 3:1, then add 100 μL of 0.02% tert-butylhydroquinone (TBHQ) methanol solution, purge the air in the centrifuge tube with high-purity nitrogen and seal it; place it in a 70℃ constant temperature water bath for 30 min, and immediately place it in an ice water bath for 5 min to terminate the reaction, obtaining the derivatized product.

[0080] Step 3, Dual-mode synergistic detection: Add 1 mL of n-hexane and 2 mL of saturated sodium chloride aqueous solution to the derivatized product from Step 2, vortex mix for 1 min, take the upper n-hexane phase, filter it through a 0.22 μm organic filter membrane, and perform GC-MS detection for the quantification of palmitic acid, oleic acid, and linoleic acid; separately take the remaining crude extract from Step 1 that has not been derivatized, filter it through a 0.22 μm organic filter membrane, and perform HPLC-QTOF direct detection for the qualitative and quantitative analysis of α-linolenic acid.

[0081] Step 4, Result Calibration and Output: GC-MS detection uses the heptadecanoic acid internal standard method for quantification, and HPLC-QTOF detection uses the external standard method for quantification; the contents of palmitic acid, oleic acid, and linoleic acid are based on the GC-MS detection results, and the contents of α-linolenic acid are based on the HPLC-QTOF detection results. The contents of each target unsaturated fatty acid in the sample are calculated and output.

[0082] Performance verification steps The glutinous rice aroma type liquor sample was processed in parallel 6 times according to the above detection steps, the background content of each target substance was determined, and the RSD value of the detection results was calculated. Add mixed fatty acid standards of low, medium and high concentration gradients to the sample, process and detect according to the above steps, and calculate the spiked recovery rate of each target analyte; The matrix interference removal rate, α-linolenic acid oxidation loss rate, and method detection limit of this method were determined using a general method.

[0083] Performance verification results Table 11 Results of core performance testing for glutinous rice-flavored Baijiu in Example 11

[0084] Auxiliary performance indicators: matrix interference removal rate 96.2%, α-linolenic acid oxidation loss rate 2.3%.

[0085] Performance gradient description: The core detection indicators of this embodiment, such as recovery rate, precision, matrix interference removal rate, oxidation loss control, and detection limit, are all superior to the detection effect of light-aroma baijiu in the original embodiment 3, and are also significantly superior to the detection effect of buckwheat-aroma baijiu, fully meeting the inventor's performance requirements.

[0086] Comparative Example 1 Detection steps Step 1, Sample pretreatment: Take the same 52% vol strong-aroma baijiu as in Example 1, and use a conventional unmodified C18 solid phase extraction column. The other loading, rinsing, and elution parameters are completely consistent with Step 1 of Example 1. Step 2, Derivatization: Add 2 mL of a single 14% BF3-methanol derivatizing reagent to the crude extract. Without antioxidants or inert gas protection, react in a 75°C water bath for 40 min. Terminate the step without an ice-water bath. Step 3, Detection: All derivatized products were processed and detected using only GC-MS, without HPLC-QTOF split detection; Step 4, Quantification of Results: All results were quantified using the external standard method.

[0087] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed and each index measured.

[0088] Performance verification results Table 12 Results of testing the core performance of the existing technical solution in Comparative Example 1

[0089] Auxiliary performance indicators: matrix interference removal rate 68.2%, α-linolenic acid oxidation loss rate 32.5%.

[0090] Comparative Example 2 Removal of the "selective enrichment of modified C18 columns" feature Detection steps The only difference was that the modified C18 column in Example 1 was replaced with a conventional unmodified C18 column; all other testing steps and parameters were exactly the same as in Example 1.

[0091] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed and each index measured.

[0092] Performance verification results Table 13 Results of core performance testing in Comparative Example 2

[0093] Auxiliary performance indicators: matrix interference removal rate 71.5%, α-linolenic acid oxidation loss rate 18.5%.

[0094] Comparative Example 3 Remove the "integrated split-mode collaborative detection" feature Detection steps The split design was cancelled, and all crude extracts from Example 1 were derivatized according to step 2. After derivatization, the extracts were divided into two parts and detected by GC-MS and HPLC-QTOF respectively. All other parameters were completely consistent with those in Example 1.

[0095] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed and each index measured.

[0096] Performance verification results Table 14 shows the core performance test results for Comparative Example 3.

[0097] Auxiliary performance indicators: α-linolenic acid oxidation loss rate 20.3%.

[0098] Comparative Example 4 Remove the characteristics of the "multi-dimensional anti-oxidation derivative system" Detection steps Only three features of Example 1—the addition of TBHQ antioxidant, high-purity nitrogen protection, and ice-water bath to terminate the reaction—were removed; all other derived parameters and all detection steps were completely consistent with Example 1.

[0099] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed and each index measured.

[0100] Performance verification results Table 15 shows the core performance test results for Comparative Example 4.

[0101] Auxiliary performance indicators: α-linolenic acid oxidation loss rate 31.2%.

[0102] Comparative Example 5 Remove the "internal standard method + external standard method stratified calibration" feature Detection steps Both GC-MS and HPLC-QTOF used the external standard method for quantification, and the rules for adding heptadecanoic acid internal standard and stratification calibration were eliminated. All other detection steps and parameters were completely consistent with those in Example 1.

[0103] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed and each index measured.

[0104] Performance verification results Table 16 shows the core performance test results for Comparative Example 5.

[0105] Auxiliary performance indicators: matrix interference removal rate 85.2%, α-linolenic acid oxidation loss rate 2.9%.

[0106] Comparative Example 6 Detection steps Step 1: Take the same strong-aroma baijiu as in Example 1, and divide it into two equal portions. The first portion is subjected to independent pretreatment, derivatization, and GC-MS detection of major components according to the method in Comparative Document 1. The second portion is subjected to independent pretreatment and HPLC-QTOF detection of α-linolenic acid according to the method in Comparative Document 2. Step 2: The two samples were quantified using the external standard method, and the remaining reagents and parameters were consistent with the conventional methods of existing technology.

[0107] Performance verification steps The performance verification steps were exactly the same as in Example 1, with the same sample being treated in parallel 6 times and each index measured.

[0108] Performance verification results Table 17 shows the core performance results of the existing parallel scheme in Comparative Example 6.

[0109] Auxiliary performance indicators: The relative deviation of 6 parallel test results for the same sample is ≥10%.

[0110] Comparative Example 7 Detection steps Based on Comparative Example 1, only two modifications were made: the modified C18 column was replaced and TBHQ antioxidant was added. The remaining derivatization and detection schemes followed the existing technical methods of Comparative Example 1 (high temperature derivatization, single GC-MS detection, no inert gas protection, and no ice-water bath termination).

[0111] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed and each index measured.

[0112] Performance verification results Table 18 shows the core performance results of the combination of existing technology and common knowledge in Comparative Example 7.

[0113] Auxiliary performance indicators: matrix interference removal rate 88.3%, α-linolenic acid oxidation loss rate 18.7%.

[0114] Comparative Example 8 Detection steps The only difference was that the stepwise fluorination-modified C18 column in Example 4 was replaced with a conventional single-step silanization-modified C18 column; all other detection steps and parameters were exactly the same as in Example 4.

[0115] Performance verification steps The performance verification steps were exactly the same as in Example 4, with six parallel treatments performed and each index measured.

[0116] Performance verification results Table 19 shows the core performance test results for Comparative Example 8.

[0117] Auxiliary performance indicators: Matrix interference removal rate 92.1%.

[0118] Comparative Example 9 Detection steps The only difference was that the TBHQ + Vitamin E compound antioxidant in Example 5 was replaced with an equal mass of single TBHQ methanol solution; all other detection steps and parameters were exactly the same as in Example 5.

[0119] Performance verification steps The performance verification steps were exactly the same as in Example 5, and the process was repeated 6 times in parallel to measure each index.

[0120] Performance verification results Table 20 shows the core performance test results for Comparative Example 9.

[0121] Auxiliary performance indicators: α-linolenic acid oxidation loss rate 2.6%.

[0122] Comparative Example 10 Detection steps The only difference is that the microwave-assisted + phase transfer catalytic derivatization in Example 6 is replaced with the conventional water bath derivatization in Example 1. All other detection steps and parameters are completely consistent with those in Example 6.

[0123] Performance verification steps The performance verification steps were exactly the same as in Example 6, with six parallel treatments performed and each index measured.

[0124] Performance verification results Table 21 Results of Core Performance Testing in Comparative Example 10

[0125] Auxiliary performance indicators: completeness of derivatization reaction 82%, α-linolenic acid oxidation loss rate 2.7%, reaction time 30 min.

[0126] Comparative Example 11 Detection steps Take the same strong-aroma baijiu as in Example 1, divide it into two equal portions, and perform solid-phase extraction enrichment twice independently according to the pretreatment method of Example 1. The first crude extract was derivatized and then detected by GC-MS, while the second crude extract was not derivatized and was directly detected by HPLC-QTOF. All other reagents and parameters were completely consistent with those in Example 1.

[0127] Performance verification steps The performance verification steps were exactly the same as in Example 1, with the same sample being treated in parallel 6 times and each index measured.

[0128] Performance verification results Table 22 shows the core performance test results for Comparative Example 11.

[0129] Auxiliary performance indicators: The relative deviation of 6 parallel test results for the same sample is ≥11%.

[0130] Comparative Example 12 Detection steps The only difference is that the high-purity nitrogen protection for the derivatization reaction in Example 1 is removed; all other detection steps and parameters are exactly the same as in Example 1.

[0131] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed and each index measured.

[0132] Performance verification results Table 23 shows the core performance test results for Comparative Example 12.

[0133] Auxiliary performance indicators: α-linolenic acid oxidation loss rate 21.7%.

[0134] Comparative Example 13 Detection steps The only difference is that the ice-water bath cooling step after the reaction in Example 1 was omitted, and the derivatization reaction was completed and the mixture was allowed to cool naturally to room temperature. All other detection steps and parameters were exactly the same as in Example 1.

[0135] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed and each index measured.

[0136] Performance verification results Table 24 shows the core performance test results for Comparative Example 13.

[0137] Auxiliary performance indicators: α-linolenic acid oxidation loss rate 12.3%.

[0138] Comparative Example 14 Detection steps The only difference was that the 5% formic acid aqueous solution rinse in Example 1 was replaced with an equal volume of ultrapure water rinse; all other detection steps and parameters were completely consistent with Example 1.

[0139] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed and each index measured.

[0140] Performance verification results Table 25 shows the core performance test results for Comparative Example 14.

[0141] Auxiliary performance indicators: matrix interference removal rate 78.5%, GC-MS spectrum shows a large number of polar impurity interference peaks.

[0142] Comparative Example 15 Detection steps Based on Comparative Example 1, all conventional optimization methods in the field were added: replacing with a modified C18 column, adding TBHQ antioxidant, inert gas protection, 70℃ water bath derivatization, and GC-MS+HPLC-QTOF detection respectively. Only the core innovations of the present invention, namely "integrated split design, composite derivatization reagent, ice-water bath termination, and stratified calibration system", were not adopted. All other methods adopted were the best existing technologies.

[0143] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed and each index measured.

[0144] Performance verification results Table 26 shows the test results of the core performance of the best existing technology solution in Comparative Example 15.

[0145] Auxiliary performance indicators: matrix interference removal rate 89.2%, α-linolenic acid oxidation loss rate 11.8%.

[0146] Comparative Example 16 Detection steps Using 53% vol Kunsha sauce-flavored baijiu (the most complex type of baijiu in terms of matrix) as a sample, parallel tests were conducted using the method of Example 1 and the conventional method of existing technology in Comparative Example 1.

[0147] Performance verification steps The performance verification steps were exactly the same as in Example 1, with six parallel treatments performed and each index measured.

[0148] Performance verification results Table 27 Comparison of detection performance in extremely complex matrices (Comparative Example 16)

[0149] The verification results of the above embodiments and comparative examples demonstrate that the selective enhanced enrichment and purification-ultra-low loss derivatization and transformation-dual-mode adaptive detection synergistic analysis system constructed in this invention can effectively solve the industry pain point of detecting unsaturated fatty acids in the complex matrix of baijiu. Compared with existing conventional detection methods, this invention improves the matrix interference removal rate to over 94% through a dedicated purification design using modified C18 solid-phase extraction; through a multi-dimensional anti-oxidation derivatization system, the oxidation loss rate of α-linolenic acid is stably controlled within 3%, with the optimal scheme reaching as low as 0.7%. The synergistic combination of these core technical features enables simultaneous high-precision quantification of macro-components (RSD≤5%) and high-sensitivity detection of trace α-linolenic acid (detection limit as low as 0.3 μg / L), adapting to baijiu samples of all aroma types and alcohol content ranges without parameter adjustment. The stability, versatility, and detection performance of the method in this invention are significantly superior to existing technologies and simple combinations of conventional methods, providing reliable technical support for baijiu quality evaluation and functional component analysis.

[0150] 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 within the protection scope of the present invention.

Claims

1. A dual-mode synergistic detection method for unsaturated fatty acids in baijiu (Chinese liquor), comprising pretreatment enrichment, derivatization, instrumental detection, and content quantification of target unsaturated fatty acids in baijiu samples, characterized in that, The target unsaturated fatty acids include palmitic acid, oleic acid, linoleic acid, and α-linolenic acid, and the method includes the following steps: S1. Selective enrichment and matrix purification: After diluting the liquor sample, the sample was loaded onto a modified C18 solid-phase extraction column modified with silanizing reagent for enrichment. After rinsing with formic acid aqueous solution to remove impurities, and eluting with ethyl acetate-n-hexane mixture, the sample was concentrated to obtain the crude extract of the target component. S2. Low-temperature anti-oxidation derivatization: Take a portion of the crude extract of the target component, add a composite derivatization reagent and an antioxidant, and carry out a low-temperature derivatization reaction under inert gas protection. After the reaction is completed, terminate the reaction in an ice-water bath to obtain the derivatized product. S3. Dual-mode synergistic detection: The derivatized products are detected by gas chromatography-mass spectrometry for the quantification of palmitic acid, oleic acid, and linoleic acid; at the same time, another portion of the crude extract of the target components that has not been derivatized is directly detected by high performance liquid chromatography-quadrupole time-of-flight mass spectrometry for the qualitative and quantitative analysis of α-linolenic acid. S4. Result Calibration and Output: The results of the internal standard method detected by gas chromatography-mass spectrometry and the external standard method detected by high performance liquid chromatography-quadrupole time-of-flight mass spectrometry are calibrated to calculate and output the content of each target unsaturated fatty acid in the liquor.

2. The method according to claim 1, characterized in that, In step S1, the modified C18 solid-phase extraction column is a 500mg / 3mL C18 column modified with 0.5%-1% silanizing reagent. The activation process of the column is as follows: it is rinsed sequentially with 2.5-3mL methanol and 4-6mL ultrapure water at a flow rate of 1mL / min, and the column is kept moist after activation. The sample loading conditions are as follows: 1mL of liquor sample is diluted with 8-10mL ultrapure water, and then 40-60μL of 0.1mg / mL heptadecanoate internal standard solution is added. After mixing, the sample is loaded and allowed to stand for 8-12min. The elution and elution conditions are as follows: it is rinsed with 4-6mL of 4%-6% formic acid aqueous solution, the elution is discarded, and then eluted with 4-6mL of ethyl acetate-hexane mixture with a volume ratio of 1:8-1:

10. After collecting the eluent, it is concentrated to 1-2 drops under nitrogen gas in the dark at 38-42℃ to obtain the crude extract of the target component.

3. The method according to claim 1, characterized in that, In step S2, the composite derivatizing reagent is a mixture of 12%-16% boron trifluoride-methanol solution and 5%-8% boron trifluoride diethyl ether solution at a volume ratio of 3:1; the antioxidant is a 0.01%-0.03% tert-butylhydroquinone methanol solution, added at 5%-10% of the volume of the composite derivatizing reagent; the conditions for the low-temperature derivatization reaction are: after purging the air in the reaction system with high-purity nitrogen, the system is sealed and placed in a water bath at 65-70℃ for 25-35 minutes, and the reaction is terminated by immediately cooling in an ice-water bath for 4-6 minutes after the reaction is completed.

4. The method according to claim 1, characterized in that, In step S3, the parameters for gas chromatography-mass spectrometry (GC-MS) detection are as follows: DB-WAX polar column, 30m×0.25mm×0.25μm, injection port temperature 245-255℃, split ratio 8:1-12:1, injection volume 1μL; carrier gas is high-purity helium, constant flow rate 0.9-1.1mL / min; temperature program is: 60℃ for 2min, then increased to 240℃ at 4-6℃ / min, held for 12-18min; ion source is EI source, temperature 220-240℃, scanning mode is SCAN full scan combined with SIM selected ion monitoring, solvent delay 4-6min, and heptadecanoic acid is used as internal standard for quantification using the internal standard method. The parameters for high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTF-MS) were as follows: a C18 reversed-phase column (2.1 mm × 100 mm × 1.8 μm), column temperature 28-32 °C, and flow rate 0.28-0.32 mL / min; mobile phase A was 0.08%-0.12% formic acid aqueous solution, and mobile phase B was acetonitrile. The gradient elution program was as follows: at 0 min, phase A 40% and phase B 60%; at 3 min, phase A 10% and phase B 90%; hold for 5 min; at 6 min, restore the initial ratio and equilibrate to 7 min; the mass spectrometer was an ESI negative ion source with a spray voltage of 3.3-3.7 kV, a desolvation gas temperature of 340-360 °C, and a scan range of 100-500 m / z. Quantification was performed using the external standard method.

5. The method according to claim 1, characterized in that, The modified C18 solid-phase extraction column modified with silanizing reagent is modified by a stepwise gradient silanization and fluorination end-group plugging process, specifically including: first, thoroughly plugging the silica matrix support with small molecule trimethylchlorosilane to eliminate residual silanol active sites; and then functionalizing the plugged filler with tridecafluorooctyltrichlorosilane.

6. The method according to claim 3, characterized in that, The antioxidant is a methanol solution of tert-butylhydroquinone and vitamin E in a mass ratio of 3:1, and the total amount added is 5%-10% of the volume of the compound derivatizing reagent.

7. The method according to claim 3, characterized in that, The composite derivatizing reagent also contains a phase transfer catalyst, tetrabutylammonium bromide, in an amount of 0.01%-0.03% of the total mass of the derivatization reaction system.

8. The method according to claim 3, characterized in that, The low-temperature derivatization reaction is carried out in a microwave-assisted heating device, using an intermittent heating mode of 10 seconds of heating followed by 20 seconds of rest, controlling the reaction temperature at 60-70℃, with a total reaction time of 15-20 minutes.

9. The method according to claim 1, characterized in that, In step S4, the calibration specifically involves: performing data fusion and cross-validation on the results of the two detection methods, wherein the contents of palmitic acid, oleic acid, and linoleic acid are based on the detection results of gas chromatography-mass spectrometry, and the contents of α-linolenic acid are based on the detection results of high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

10. The method according to claim 1, characterized in that, The method is applicable to the detection of soy sauce aroma type, strong aroma type, light aroma type, glutinous rice aroma type, mixed aroma type, phoenix aroma type and buckwheat aroma type of baijiu, and is suitable for baijiu alcohol content range of 28%vol-68%vol.

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