CN-121994970-A - Dual-mode cooperative detection method for unsaturated fatty acid in white spirit

CN121994970ACN 121994970 ACN121994970 ACN 121994970ACN-121994970-A

Abstract

The invention discloses a dual-mode cooperative detection method for unsaturated fatty acid in white spirit, and relates to the technical field of food detection. Aiming at the detection pain points with high alcohol ester matrix characteristics of white spirit, easy oxidization of unsaturated fatty acid and large content span, the method comprises the steps of selectively enriching and purifying white spirit samples through a silanization modified C18 solid phase extraction small column, completing low-temperature anti-oxidization derivatization through a composite derivatization reagent and an anti-oxidization system, quantifying palmitic acid, oleic acid and linoleic acid by adopting a gas chromatography-mass spectrometry combined method, directly detecting alpha-linolenic acid by adopting a high performance liquid chromatography-quadrupole time-of-flight mass spectrometry combined method, and finally calibrating and outputting results by combining an internal standard method and an external standard method. The invention realizes the efficient purification of the white spirit matrix, controls the oxidation loss rate of alpha-linolenic acid to be within 3%, can synchronously realize the high-precision quantification of constant components and the high-sensitivity detection of trace components, is suitable for the detection of the white spirit with full fragrance and full alcohol degree, has excellent stability and universality, and provides reliable technical support for the quality evaluation of the white spirit.

Inventors

SUN XIZHEN
YANG QIANG
FENG YUAN
YANG YEHUI
XIONG XIAOTONG
XIONG YAQING
Ni Xingting
YAN LING
YANG FENG
LI QIANG

Assignees

劲牌有限公司

Dates

Publication Date: 20260508
Application Date: 20260313

Claims (10)

1. The dual-mode collaborative detection method for unsaturated fatty acid in white spirit comprises the steps of pretreatment enrichment, derivatization, instrument detection and content quantification of target unsaturated fatty acid in white spirit samples, and is characterized in that the target unsaturated fatty acid comprises palmitic acid, oleic acid, linoleic acid and alpha-linolenic acid, and the method comprises the following steps: S1, selectively enriching and purifying a matrix, namely diluting a white spirit sample, loading the diluted white spirit sample to a modified C18 solid phase extraction column modified by a silanization reagent for enriching, eluting by a formic acid aqueous solution to remove impurities, eluting by an ethyl acetate-n-hexane mixed solution, and concentrating to obtain a target component crude extract; s2, performing low-temperature anti-oxidation derivatization, namely taking part of the crude extract of the target component, adding a composite derivatization reagent and an anti-oxidation auxiliary agent, performing low-temperature derivatization reaction under the protection of inert gas, and stopping the reaction in an ice water bath after the reaction is finished to obtain a derivatization product; S3, dual-mode collaborative detection, namely detecting the derivative product by adopting a gas chromatography-mass spectrometry method, and using the derivative product for quantifying palmitic acid, oleic acid and linoleic acid; And S4, calibrating and outputting results, namely calibrating an internal standard method result detected by combining gas chromatography-mass spectrometry and an external standard method result detected by combining high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, and calculating and outputting the content of each target unsaturated fatty acid in the white spirit.
2. The method of claim 1, wherein in the step S1, the modified C18 solid phase extraction column is a 500mg/3mL C18 column modified by 0.5% -1% silanization reagent, the activation process of the column is that 2.5-3mL methanol and 4-6mL ultrapure water are sequentially used for flushing at a flow rate of 1mL/min, the column is kept in a wet state after activation, the loading condition is that 1mL white spirit sample is taken and diluted by 8-10mL ultrapure water, then 40-60 mu L0.1mg/mL heptadecanoic acid n-hexane internal standard solution is added, the sample is loaded after uniform mixing, the column is kept stand for 8-12min, the conditions of leaching and elution are that 4-6mL4% -6% formic acid aqueous solution is used for leaching, leaching is discarded, then 4-6mL ethyl acetate-n-hexane mixed solution with a volume ratio of 1:8-1:10 is used for eluting, and the target component crude extract is obtained after collecting the eluent and concentrating to 1-2 drops under the condition of nitrogen at 38-42 ℃ in a dark state.
3. The method of claim 1, wherein in the step S2, the compound derivatization reagent is formed by mixing 12% -16% of boron trifluoride-methanol solution and 5% -8% of boron trifluoride diethyl ether solution according to a volume ratio of 3:1, the antioxidation auxiliary agent is 0.01% -0.03% of tert-butyl hydroquinone methanol solution, the addition amount is 5% -10% of the volume of the compound derivatization reagent, the condition of the low-temperature derivatization reaction is that the air in a reaction system is replaced by high-purity nitrogen, then the reaction system is sealed, the reaction system is placed in a water bath with the temperature of 65-70 ℃ for 25-35min, and the reaction is terminated by cooling in an ice water bath for 4-6min immediately after the reaction is finished.
4. The method according to claim 1, wherein in the step S3, parameters of gas chromatography-mass spectrometry combined detection are DB-WAX polar chromatographic column, 30m×0.25mm×0.25 μm, sample inlet temperature 245-255 ℃, split ratio 8:1-12:1, sample inlet amount 1 μL, carrier gas of high purity helium with constant flow rate 0.9-1.1mL/min, heating program of 60 ℃ for 2min, heating to 240 ℃ at 4-6 ℃ for 12-18min, ion source of EI source, temperature of 220-240 ℃ and scanning mode of SCAN combined with SIM selective ion monitoring, solvent delay of 4-6min, and internal standard quantification of heptadecanoic acid as internal standard; The parameters of the high performance liquid chromatography-quadrupole time-of-flight mass spectrometry combination detection are that a C18 reversed phase chromatographic column is adopted, the column temperature is 2.1mm multiplied by 100mm multiplied by 1.8 mu m, the column temperature is 28-32 ℃, the flow rate is 0.28-0.32mL/min, the mobile phase A is 0.08-0.12% formic acid aqueous solution, the mobile phase B is acetonitrile, the gradient elution program is that the phase A is 40% at 0min, the phase B is 60% at 3min, the phase A is 10% and the phase B is 90% at 3min, the initial proportion is recovered and balanced to 7min when the temperature is kept to 5min and 6min, the mass spectrum source is ESI anion source, the spray voltage is 3.3-3.7kV, the desolventizing gas temperature is 340-360 ℃, the scanning range is 100-500m/z, and the quantification is carried out by an external standard method.
5. The method of claim 1, wherein the modified C18 solid phase extraction column modified by the silylation reagent is modified by a stepwise gradient silylation and fluorinated end group plugging process, and specifically comprises the steps of thoroughly plugging the end group of a silica gel matrix carrier by using small-molecule trimethylchlorosilane, eliminating residual silicon hydroxyl active sites, and functionally modifying the plugged filler by using tridecafluorooctyltrichlorosilane.
6. The method according to claim 3, wherein the antioxidant auxiliary agent is a compound methanol solution of tert-butylhydroquinone and vitamin E, the mass ratio of the tert-butylhydroquinone to the vitamin E is 3:1, and the total addition amount is 5% -10% of the volume of the compound derivatization reagent.
7. The method according to claim 3, wherein the composite derivatizing agent is further added with tetrabutylammonium bromide serving as a phase transfer catalyst, and the addition amount is 0.01% -0.03% of the total mass of the derivatizing reaction system.
8. A method according to claim 3, wherein the low temperature derivatization reaction is carried out in a microwave-assisted heating device, and the reaction temperature is controlled to be 60-70 ℃ and the total reaction time is 15-20min by adopting a batch heating mode of heating for 10 seconds and a batch heating mode of 20 seconds.
9. The method according to claim 1, wherein in step S4, the calibration is specifically performed by performing data fusion and cross-validation on the results of the two detection methods, wherein the content of palmitic acid, oleic acid and linoleic acid is based on the result of gas chromatography-mass spectrometry combined detection, and the content of α -linolenic acid is based on the result of high performance liquid chromatography-quadrupole time of flight mass spectrometry combined detection.
10. The method according to claim 1, wherein the method is suitable for detecting Maotai-flavor, luzhou-flavor, fen-flavor, oryza Glutinosa, double-flavor, phoenix-flavor, fagopyrum-flavor distilled spirit, and the alcoholic strength of the adapted distilled spirit is in the range of 28-68% vol.

Description

Dual-mode cooperative detection method for unsaturated fatty acid in white spirit Technical Field The invention relates to the technical field of food quality safety detection and food functional component analysis, in particular to a dual-mode collaborative detection method for unsaturated fatty acid in white spirit, which is particularly suitable for synchronous and accurate detection of free palmitic acid, oleic acid, linoleic acid and alpha-linolenic acid in white spirit with different flavors and different alcoholicity. Background Unsaturated fatty acid is an important flavor precursor substance and functional trace component in white spirit, wherein the content and proportion of palmitic acid, oleic acid, linoleic acid and alpha-linolenic acid are directly related to the flavor quality, drinking comfort and health value of the white spirit, especially alpha-linolenic acid is used as essential fatty acid for human body, and the trace occurrence state is one of core indexes for evaluating the high-end quality of the white spirit. Therefore, the method for synchronously and accurately measuring the constant mg/L-level to trace mug/L-level unsaturated fatty acid in the white spirit is established, and has important significance for quality control, production process optimization, product authenticity identification and health value excavation of the white spirit. The detection of unsaturated fatty acid in the existing white spirit is mainly carried out in a single detection mode, wherein a gas chromatography-mass spectrometry (GC-MS) is a conventional means for fatty acid analysis, but the method has the bottleneck that the method is difficult to break when being applied to a complex matrix of the white spirit, firstly, the high-content alcohol esters such as ethanol, ethyl acetate and the like in a white spirit system can generate strong matrix interference, the conventional pretreatment method can not realize the effective separation of target fatty acid and interfering substances, the chromatographic separation effect and the mass spectrum quantitative accuracy are seriously influenced, secondly, the double bond activity of polyunsaturated fatty acid, particularly alpha-linolenic acid, is high, the oxidative degradation is extremely easy to occur in the conventional high-temperature derivatization process, the conventional derivatization process has no targeted oxidation protection measures, the loss of the target is serious, and trace component quantitative results are obviously lower or even cannot be detected. The high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF) can directly detect free fatty acid without derivatization, so that the oxidation risk in the derivatization process is avoided, the detection sensitivity to trace components is high, but an obvious short plate exists when the method is applied to white spirit detection, the chromatographic column and a mass spectrum ion source can be seriously polluted by directly injecting white spirit high alcohol ester matrix, the long-term use stability of an instrument is influenced, meanwhile, the detection of constant fatty acid is easily interfered by the inhibition effect of matrix ions, the quantitative precision cannot meet the accurate quantitative requirement of constant components. In addition, the prior pretreatment technology has obvious limitations, the conventional C18 solid phase extraction filler has insufficient purification selectivity on extremely complex matrixes of white spirit, high content of alcohol ester interferents are difficult to thoroughly remove, and the conventional antioxidant means only singly adds phenolic antioxidants, so that the full-process effective protection of trace components with high unsaturation degree can not be realized in a high-temperature microenvironment of a derivative reaction. At present, the technical contradiction of the core which cannot be effectively solved in the field is that the high-fidelity quantification of the easily oxidized constant components and the high-sensitivity detection of the trace easily oxidized components cannot be simultaneously realized in the same white spirit sample and the same pretreatment process, and even if two instruments of GC-MS and HPLC-QTOF are simply used in parallel, the same sample is required to be pretreated respectively, so that the process is complicated, the sample consumption is high, and systematic errors which cannot be calibrated can be introduced due to inconsistent pretreatment processes, so that the actual requirements of batch and standardized detection in the white spirit industry cannot be completely met. Disclosure of Invention In view of the above, the invention provides a dual-mode collaborative detection method for unsaturated fatty acid in white spirit. The technical scheme of the invention is realized by providing a dual-mode collaborative detection method for unsaturated fatty acid in whi