Reagent and method for molecular structure analysis of branched chain fatty acid

Abstract

The invention relates to a reagent and a method for molecular structure analysis of branched chain fatty acid, and relates to the field of analysis and detection technologies, the reagent comprises benzo-12-crown-4-ether, 18-crown-6-ether and a compound containing a metal ion ligand; the isomer molecule is any one of 2-branched chain fatty acid and 4-branched chain fatty acid; the method comprises the following steps: mixing at least one branched chain fatty acid sample of 2-methyl caprylic acid, 4-methyl caprylic acid, 2-ethyl caprylic acid and 4-ethyl caprylic acid, which needs to be subjected to positional isomer analysis, with a reagent to prepare a mixed aqueous solution of 2- / 4-branched chain fatty acid-benzo-12-crown-4-ether / 18-crown-6-ether-metal ions; performing ion source ionization operation on the basis of the mixed aqueous solution to generate 2- / 4-branched fatty acid-B-12C4 / 18C6-metal ion type monovalent positive ions; and measuring the ion mobility spectrometry of the monovalent positive ions, and identifying heterogeneous structure information according to the obtained ion mobility spectrometry. The method has the effect of rapidly and accurately detecting the structural information of the branched chain fatty acid isomer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection technology, and in particular to a reagent for branched-chain fatty acid molecular structure analysis and a method thereof. BACKGROUND

[0002] Branched-chain fatty acids (BCFA) as bioactive lipids with potential nutritional properties have recently received increasing attention for their wide application in human health. BCFA is composed of a variety of fatty acids with alkyl branching of methyl groups, which can have an even or odd number of carbons. The most common type of BCFA has a methyl group on the penultimate carbon (iBCFA) or the antepenultimate carbon (aiBCFA), and these fatty acids are mainly saturated. The most abundant and most studied BCFA mainly comes from dairy products and ruminant foods, or from microbial cell membranes and human secretions. Some of these branched-chain fatty acids are volatile and associated with aroma, for example, 4-alkyl branched-chain fatty acids are associated with "mutton flavor", and two BCFA, 4-methyloctanoic acid and 4-ethyloctanoic acid, are the main compounds causing "mutton flavor". However, the structure of these compounds is Figure 2 shows isomers composed of different carbon chain isomers, and the difference in structure can cause differences in properties and activities.

[0003] With the development of instrument technology, researchers have developed techniques to identify and characterize 2 / 4-alkyl branched-chain fatty acid isomers, enabling accurate analysis and elucidation of their structural differences. Current methods for identifying branched-chain fatty acid isomers include gas chromatography-mass spectrometry, gas-phase infrared spectroscopy, supercritical fluid chromatography, capillary electrophoresis-mass spectrometry, ultra-high performance liquid chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy.

[0004] In view of the above related art, although the method in the prior art shows certain separation capacity, factors such as chemical derivatization, long time required for separation, complex sample pretreatment, and low sensitivity limit its wide application. SUMMARY

[0005] In order to quickly and accurately detect the structural information of branched-chain fatty acid isomers, the present application provides a reagent for branched-chain fatty acid molecular structure analysis and a method thereof.

[0006] In a first aspect, the present application provides a reagent for branched-chain fatty acid molecular structure analysis, which adopts the following technical solution: A reagent for branched-chain fatty acid molecular structure analysis, comprising benzene-12-crown-4-ether, 18-crown-6-ether, and a compound containing a single metal ion ligand, for 2- / 4-branched-chain fatty acid isomer molecular structure analysis.

[0007] Optionally, the benzene-12-crown-4-ether has a molecular formula of C12H16O4 and a molecular weight of 224.25, and the 18-crown-6-ether has a molecular formula of C12H24O6 and a molecular weight of 264.32.

[0008] Optionally, the metal ion is any one of Co²⁺, Ca²⁺, Ba²⁺, or Mn²⁺.

[0009] Optionally, the mixed solution for mixing the benzene-12-crown-4-ether, the 18-crown-6-ether, and the compound containing a single metal ion ligand has pure water as a solvent.

[0010] By using the above technical solution, the specific coordination system formed by the benzene-12-crown-4-ether, the 18-crown-6-ether, and the metal ion can effectively interact with the branched fatty acid, thereby providing a good reaction basis and condition for subsequent analysis of the molecular structure of the 2- / 4-branched fatty acid isomer. Pure water as a solvent has the advantages of environmental friendliness, low cost, and stable chemical properties, which can ensure the stable existence of each component in the reagent, reduce the interference of impurities introduced by the solvent on the analysis results, and ensure the accuracy and reliability of the subsequent analysis.

[0011] In a second aspect, the present application provides a branched fatty acid molecular structure analysis method, which uses the following technical solution: A branched fatty acid molecular structure analysis method, comprising: Step S1: mixing at least one branched fatty acid sample of 2-methyloctanoic acid, 4-methyloctanoic acid, 2-ethyloctanoic acid, and 4-ethyloctanoic acid, which need to be analyzed for positional isomer, with a reagent for branched fatty acid molecular structure analysis to prepare a 2- / 4-branched fatty acid-benzene-12-crown-4-ether / 18-crown-6-ether-metal ion mixed aqueous solution; Step S2: performing ion source ionization operation based on the mixed aqueous solution to generate 2- / 4-branched fatty acid-benzene-12-crown-4-ether / 18-crown-6-ether-metal ion type monovalent positive ions; Step S3: using an ion mobility spectrometry-mass spectrometry instrument to measure the ion mobility spectrum of the monovalent positive ions, and identifying the positional isomer structure information of the branched fatty acid according to the drift time, collision cross-section value, and peak separation degree of the obtained ion mobility spectrum.

[0012] By using the above technical scheme, the ion mobility spectrometry-mass spectrometry combination instrument is used for measurement, the high sensitivity detection characteristics of ion mobility spectrometry on ion mobility rate difference and the advantages of mass spectrometry on ion mass-to-charge ratio are fully utilized. According to the drift time obtained by the ion mobility spectrometry, the migration of the ions in the electric field can be accurately reflected, and the migration time of the ions formed by the branched fatty acid isomers after reacting with the reagent is different. The collision cross section value can provide the area information of the interaction of the ions in the migration process with other molecules, which is helpful to further distinguish the isomers. The peak separation degree reflects the separation degree between different ion peaks, and good peak separation degree can ensure accurate identification of each isomer. By comprehensively considering these parameters, the positional isomer structure information of the branched fatty acid can be quickly and accurately identified, and the problems of long time required for chemical derivatization and separation, complex sample pretreatment and low sensitivity in the prior art are overcome, and an efficient and reliable method for structure analysis of branched fatty acid isomers is provided.

[0013] Optionally, the ion source capable of generating ions is an electrospray ionization ion source.

[0014] Optionally, the method further comprises a compensation method for the mixed aqueous solution before performing the ion source ionization operation, and the method comprises: Step S4: acquiring the absolute concentration and the complex component ratio in the mixed aqueous solution according to a preset interval period; Step S5: fitting a complex decomposition kinetics curve based on the absolute concentration, and determining a complex decomposition rate; Step S6: calculating a predicted compensation time and a predicted concentration loss amount based on the absolute concentration, the complex component ratio, the complex decomposition rate and a preset absolute concentration threshold value; Step S7: performing a compensation operation based on the predicted compensation time and the predicted concentration loss amount, and determining a modified mixed aqueous solution.

[0015] By using the above technical scheme, the mixed aqueous solution is compensated before performing the ion source ionization operation, which can effectively avoid the decrease of ionization efficiency caused by the change of solution components, thereby improving the accuracy of subsequent ion mobility spectrometry measurement. Specifically, by periodically acquiring the absolute concentration and the complex component ratio in the mixed aqueous solution, the state change of the solution can be mastered in real time. Based on these data, the complex decomposition kinetics curve can be fitted to accurately predict the decomposition rate of the complex, and then the predicted compensation time and the concentration loss amount can be calculated. According to the calculation results, the compensation operation is performed, which can ensure that the mixed aqueous solution maintains a stable component ratio before ionization, thereby providing reliable guarantee for subsequent ion mobility spectrometry measurement.

[0016] Optionally, the method further comprises an ion mobility spectrometry pre-scanning method performed after the compensation operation of the mixed aqueous solution, and the method comprises: Step S8: determining a characteristic drift time interval based on the ion mobility spectrometry pre-scan of the mixed aqueous solution; Step S9: determining an actual drift time based on the ion mobility spectrometry pre-scan of the modified mixed aqueous solution; Step S10: determining a drift time deviation value based on the characteristic drift time interval and the actual drift time; Step S11: performing steps S2 to S3 when the drift time deviation value falls within a preset stable relative deviation range; Step S12: obtaining a placement duration corresponding to the modified mixed aqueous solution when the drift time deviation value does not fall within the stable relative deviation range; Step S13: obtaining a modified absolute concentration and a modified complex component proportion in the modified mixed aqueous solution when the placement duration is less than a preset stable window period; Step S14: re-determining a modified expected concentration loss amount based on the modified absolute concentration and the modified complex component proportion; Step S15: repeatedly performing a compensation operation according to the modified expected concentration loss amount when the modified expected concentration loss amount exists, and performing steps S9 to S15, and accumulating a compensation number of times; Step S16: outputting a waste sample signal when the placement duration is greater than the stable window period or the compensation number of times exceeds a preset maximum compensation number of times.

[0017] By using the above technical solution, by comparing the characteristic drift time interval and the actual drift time, the drift time deviation value can be calculated. When the drift time deviation value falls within a preset stable relative deviation range, it indicates that the state of the mixed aqueous solution is stable, and at this time, steps S2 to S3 can be performed for formal ion mobility spectrometry measurement. If the drift time deviation value does not fall within the stable relative deviation range, the placement duration corresponding to the modified mixed aqueous solution is further obtained. If the placement duration is less than a preset stable window period, it indicates that the state of the mixed aqueous solution may not be completely stable, at which time the modified absolute concentration and the modified complex component proportion in the modified mixed aqueous solution are obtained, and the modified expected concentration loss amount is re-determined based on these data. If the modified expected concentration loss amount exists, the compensation operation is repeatedly performed according to the loss amount, and steps S9 to S15 are performed again, while the compensation number of times is accumulated. Such a design can ensure that when the state of the mixed aqueous solution is unstable, the solution state is gradually adjusted through multiple compensation and pre-scanning until the stable condition is reached. However, if the placement duration is greater than the stable window period or the compensation number of times exceeds a preset maximum compensation number of times, it indicates that the mixed aqueous solution may have been unable to reach a stable state through the compensation operation, at which time a waste sample signal is outputted to avoid measuring invalid samples, thereby saving time and resources.

[0018] Optionally, the method further comprises determining an estimated concentration based on the predicted compensation time and the predicted concentration loss amount before performing the compensation operation, and the method comprises: Step S61: disassembling the predicted concentration loss amount to obtain a to-be-mixed component and a corresponding theoretical to-be-mixed amount of each component; Step S62: determining a predicted volume of the added reagent according to the to-be-mixed component and the theoretical to-be-mixed amount, and obtaining a current mixed aqueous solution volume; Step S63: calculating an estimated mixed liquid concentration based on the to-be-mixed component, the theoretical to-be-mixed amount, the predicted volume of the added reagent, the current mixed aqueous solution volume, and a preset complex combination rate; Step S64: if the estimated mixed liquid concentration does not reach an absolute concentration threshold, calculating a concentration deviation value; Step S65: reversely deducing an adjusted to-be-mixed amount based on the concentration deviation value, the complex combination rate, and a concentration of the added reagent, and accumulating an adjusted to-be-mixed amount number; Step S66: superimposing the theoretical to-be-mixed amount and the adjusted to-be-mixed amount to obtain a corrected to-be-mixed amount, and returning to Step S62 to recalculate the estimated mixed liquid concentration; Step S67: repeatedly performing Steps S62 to S66 until the estimated mixed liquid concentration reaches the absolute concentration threshold, and then performing Step S7; Step S68: outputting a waste sample signal when the adjusted to-be-mixed amount number is greater than a preset maximum adjustment number.

[0019] By adopting the above technical solution, before performing the compensation operation, the to-be-mixed component and its theoretical amount that need to be supplemented can be determined by disassembling the predicted concentration loss amount, thereby providing accurate guidance for subsequent compensation operations. According to the to-be-mixed component and the theoretical to-be-mixed amount, the predicted volume of the added reagent can be accurately determined, and the estimated mixed liquid concentration can be further calculated in combination with the current mixed aqueous solution volume. If the estimated mixed liquid concentration does not reach the preset absolute concentration threshold, the concentration deviation value is calculated, and the adjusted to-be-mixed amount is reversely deduced based on the deviation value, the complex combination rate, and the concentration of the added reagent. During this process, the adjusted to-be-mixed amount number is accumulated to ensure the accuracy and controllability of the adjustment. The theoretical to-be-mixed amount and the adjusted to-be-mixed amount are superimposed to obtain the corrected to-be-mixed amount, and the estimated mixed liquid concentration is recalculated, thereby forming a closed-loop adjustment mechanism. The above steps are repeatedly performed until the estimated mixed liquid concentration reaches the absolute concentration threshold, and then the subsequent compensation operation is performed. However, if the adjusted to-be-mixed amount number exceeds the preset maximum adjustment number, it indicates that the mixed aqueous solution may have failed to reach a stable state through simple adjustment, and a waste sample signal is outputted to avoid invalid operation and resource waste.

[0020] Optionally, the method further comprises adjusting the complex combination rate before performing the compensation operation, and the method comprises: Step S601: obtaining the current temperature of the mixed aqueous solution; Step S602: if the current temperature is higher than the preset optimal temperature range, obtaining the high-temperature binding rate corresponding to the current temperature; Step S603: re-determining the complex binding rate based on the high-temperature binding rate and outputting.

[0021] By adopting the above technical scheme, the influence of environmental temperature change on the analysis result can be effectively coped with, and the stability and accuracy of branched fatty acid molecular structure analysis are improved.

[0022] In summary, the present application has at least one of the following beneficial technical effects: Realize the rapid and accurate separation and identification of branched fatty acid positional isomers: the present application forms a ternary complex with 2- / 4-branched fatty acids by innovative design of benzene-12-crown-4-ether / 18-crown-6-ether-metal ion reagent, and the structural differences of the ternary complex are caused by the spatial steric hindrance and crown ether ring matching difference caused by the different positions of the branched fatty acids. The slight structural differences of the fatty acids are converted into significant differences in the collision cross section of the complex. Combined with ion mobility spectrometry-mass spectrometry technology, baseline separation of isomers can be achieved without chemical derivatization and complex pretreatment. The pain points of complex pretreatment and long time in the prior art are solved, and the sensitivity and accuracy of detection are ensured. It is suitable for rapid analysis of standard samples and actual samples such as goat milk; By fitting the decomposition kinetics curve to predict the concentration loss and dynamically compensate, combining ion mobility spectrometry pre-scanning to verify the stability of the complex, and adjusting the binding rate according to the temperature, the detection deviation caused by the fluctuation of the sample state is avoided. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the chemical structure diagram of benzene-12-crown-4-ether and 18-crown-6-ether in the embodiment of the present application; Figure 2 is the structure diagram of the ternary complex formed by 2- / 4-branched fatty acids and benzene-12-crown-4-ether / 18-crown-6-ether, metal ions in the embodiment of the present application; Figure 3 is the mass spectrum and extracted ion mobility spectrum diagram in the negative ion mode in the embodiment of the present application, wherein: a is the mass spectrum diagram of [MA-H]-, b is the extracted ion mobility spectrum diagram of [2 / 4-MA-H]-, c is the mass spectrum diagram of [EA-H]-, and d is the extracted ion mobility spectrum diagram of [2 / 4-EA-H]-; Figure 4 is the mass spectrum and extracted ion mobility spectrum diagram in the positive ion mode in the embodiment of the present application Figure 1Wherein: a is the mass spectrum of [MA+B-12C4+Co-H]+, b is the extracted ion mobility spectrum of [2 / 4-MA+B-12C4+Co-H]+, c is the mass spectrum of [EA+B-12C4+Ca-H]+, and d is the extracted ion mobility spectrum of [2 / 4-EA+B-12C4+Ca-H]+. Figure 5 is a mass spectrum and an extracted ion mobility spectrum in positive ion mode in the embodiment of the present application Figure 2 Wherein: a is the mass spectrum of [MA+18C6+Mn-H]+, b is the extracted ion mobility spectrum of [2 / 4-MA+18C6+Mn-H]+, c is the mass spectrum of [EA+18C6+Ca-H]+, and d is the extracted ion mobility spectrum of [2 / 4-EA+18C6+Ca-H]+. Figure 6 is a flow chart of a branched fatty acid molecular structure analysis method in the embodiment of the present application. DETAILED DESCRIPTION

[0024] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0025] The embodiment of the present application discloses a reagent and a method for branched fatty acid molecular structure analysis. Referring to Figures 1 to 5 A reagent for branched fatty acid molecular structure analysis, the reagent comprising a mixed aqueous solution of benzo-12-crown-4-ether, 18-crown-6-ether and a compound containing a single metal ion ligand. Wherein, the molecular formula of benzo-12-crown-4-ether is C12H16O4, and the molecular weight is 224.25, the molecular formula of 18-crown-6-ether is C12H24O6, and the molecular weight is 264.32. The compound containing a single metal ion ligand can be a salt corresponding to Co²⁺, Ca²⁺, Ba²⁺ or Mn²⁺ (such as CoCl2, etc.). The mixed aqueous solution is an aqueous system containing B-12C4 / 18C6 and the above metal ions, and the solvent can be selected from pure water, or a mixture of water and liquid such as methanol, ethanol, acetonitrile, etc. Herein, B-12C4 is benzo-12-crown-4-ether, and 18C6 is 18-crown-6-ether. The weight ratio of B-12C4, 18C6 and the metal ion compound in the mixed aqueous solution can be adjusted according to the detection requirements.

[0026] Further, for 2-methyloctanoic acid and 4-methyloctanoic acid isomers, the best separation effect of the complex system is 2 / 4-MA-B-12C4-Co²⁺. Here, 2-MA is 2-methyloctanoic acid, and 4-MA is 4-methyloctanoic acid. For 2-ethyloctanoic acid and 4-ethyloctanoic acid isomers, high-efficiency separation can be achieved by using a 2 / 4-EA-18C6-Ca²⁺ complex system. Here, 2-EA is 2-ethyloctanoic acid, and 4-EA is 4-ethyloctanoic acid. The above systems can make the separation degree (Rp-p) of isomers reach 0.622 and 0.560, respectively, meeting the baseline separation requirement.

[0027] Based on the same inventive concept, the present application also provides a branched fatty acid molecular structure analysis method.

[0028] Referring to Figure 6 A branched fatty acid molecular structure analysis method comprises: Step S1: based on at least one branched fatty acid sample of 2-methyloctanoic acid, 4-methyloctanoic acid, 2-ethyloctanoic acid and 4-ethyloctanoic acid which needs to be analyzed for positional isomers, mixed with a reagent for branched fatty acid molecular structure analysis to prepare a mixed aqueous solution of 2- / 4-branched fatty acid-B-12C4 / 18C6-metal ion.

[0029] Since the positional isomerism of branched fatty acids (such as 2-MA and 4-MA) only reflects the difference in the position of the branched chain on the carbon chain (2 and 4), such subtle structural differences are difficult to distinguish by ion mobility spectrometry or mass spectrometry if directly detected.

[0030] The crown ether (B-12C4, 18C6) and metal ion in the analysis reagent can form a branched fatty acid-crown ether-metal ion ternary complex with the branched fatty acid through non-covalent interaction.

[0031] Due to the spatial steric hindrance of the 2- / 4-position branched chain and the difference in matching with the crown ether ring, the spatial conformation of the ternary complex formed by different positional isomers and the crown ether and metal ion is different, which converts the subtle differences of the fatty acids themselves into significant structural differences of the ternary complex, creating a distinguishable basis for subsequent detection.

[0032] Step S2: ionizing the mixed aqueous solution through an ion source to generate 2- / 4-branched fatty acid-B-12C4 / 18C6-metal ion type monovalent positive ions.

[0033] The detection object of ion mobility spectrometry-mass spectrometry is gas-phase ions, so the neutral ternary complex in the solution must be converted into charged gas-phase ions.

[0034] By using soft ionization techniques such as electrospray ionization, the non-covalent bond structure of the ternary complex can be preserved (if hard ionization is used, the complex may be destroyed and the original structure cannot be reflected), and the complex in the solution can form a stable monovalent positive ion (such as [2-MA+B-12C4+Co-H] +).

[0035] Only after ionization, the complex can enter the drift tube (ion mobility spectrometry part) and mass analyzer (mass spectrometry part) of the ion mobility spectrometry-mass spectrometry, so this step is the key bridge connecting the solution reaction and instrument detection.

[0036] Step S3: Using the ion mobility spectrometry-mass spectrometry instrument, the ion mobility spectrum of the monovalent positive ion is measured, and the positional isomer structure information of the branched fatty acid is identified according to the drift time, collision cross section value and peak separation degree of the obtained ion mobility spectrum.

[0037] The role of ion mobility spectrum: ions are driven to migrate in the drift tube by an electric field, and the mobility is directly related to the collision cross section (CCS) of the ion, because the collision cross section is determined by the spatial structure of the ion. Because the spatial conformation of the ternary complex formed by 2- / 4-position isomers is different, the collision cross section is different, so there is a difference in the drift time in the ion mobility spectrum. At the same time, the peak separation degree (Rp-p) can quantify the separation effect between isomers, and judge whether baseline separation can be achieved.

[0038] The role of mass spectrometry: the chemical composition of the ion can be confirmed by the mass-to-charge ratio, verifying that the branched fatty acid-crown ether-metal ion ternary complex is detected, rather than impurity ions, providing composition-level verification for the separation results of ion mobility spectrometry.

[0039] Finally, by combining drift time, collision cross section and peak separation degree, the positional isomers of 2- / 4-branched fatty acids can be accurately distinguished from the ion mobility spectrum, and the isomer structure information can be identified.

[0040] Among them, the ion source that can generate ions can be an electrospray ionization ion source, a laser-assisted desorption ionization ion source, a desorption electrospray ionization ion source, or other types of ion sources that can generate B-12C4 / 18C6-2 / 4-MA / EA-metal ion-containing monovalent positive ions.

[0041] Among them, it also includes a compensation method for mixing the aqueous solution before performing the ion source ionization operation, which includes: Step S4: Obtain the absolute concentration and complex component ratio in the mixed aqueous solution according to the preset interval period.

[0042] The interval period refers to a time interval for periodic detection, which can be set according to actual experimental requirements and solution stability, for example, detection is performed once every 30 minutes, 1 hour or longer. The absolute concentration refers to the specific concentration value of each component in the mixed aqueous solution, which can be measured by professional concentration detection instruments or methods, such as high performance liquid chromatography. The compound component ratio refers to the proportion of branched fatty acids, crown ethers and metal ions in the mixed aqueous solution, which can be obtained by chemical analysis combined with calculation, which is a common technical means in the art and will not be described here.

[0043] Step S5: fitting the compound decomposition kinetics curve based on the absolute concentration and determining the compound decomposition rate.

[0044] The compound decomposition kinetics curve refers to the curve obtained by fitting the change of the absolute concentration in the mixed aqueous solution with time by a mathematical model. The curve can directly reflect the decomposition degree of the compound at different time points. The horizontal coordinate is usually the time variable, and the vertical coordinate is the absolute concentration of the compound. By using numerical analysis methods such as nonlinear regression or difference equation, a kinetic model can be constructed based on the measured concentration data, and then the rate constant of compound decomposition can be determined. The compound decomposition rate refers to the proportion or amount of the compound in the mixed aqueous solution decomposed per unit time.

[0045] Step S6: calculating the predicted compensation time and the predicted concentration loss based on the absolute concentration, the compound component ratio, the compound decomposition rate and the preset absolute concentration threshold.

[0046] The absolute concentration threshold refers to the minimum concentration standard that each component in the mixed aqueous solution should reach, which is determined according to experimental requirements and compound stability. The predicted compensation time refers to the time node at which reagents need to be supplemented or the solution components need to be adjusted according to the difference between the current solution state and the absolute concentration threshold, combined with the compound decomposition rate. The predicted concentration loss refers to the total amount of concentration decrease of each component due to continuous decomposition of the compound within the compensation time, which is calculated by integrating the absolute concentration, the decomposition rate and the time parameter by a mathematical model.

[0047] Step S7: performing compensation operation based on the predicted compensation time and the predicted concentration loss, and determining the modified mixed aqueous solution.

[0048] The compensation operation refers to adding an appropriate amount of reagents (such as crown ethers, metal ion salts or branched fatty acid samples) or adjusting the solution volume (such as dilution or concentration) to the mixed aqueous solution according to the predicted compensation time and the predicted concentration loss, so that the concentration of each component in the solution is restored to the expected range. The modified mixed aqueous solution refers to the mixed aqueous solution after the compensation operation.

[0049] The method further comprises performing ion mobility spectrometry pre-scanning after performing the compensation operation on the mixed aqueous solution, and the method comprises the following steps: Step S8: determining a characteristic drift time interval based on the ion mobility spectrometry pre-scanning performed on the mixed aqueous solution.

[0050] The ion mobility spectrometry pre-scanning refers to a rapid targeted screening mode of ion mobility spectrometry, and specifically refers to a targeted scanning performed on the mixed aqueous solution immediately after preparation and before decomposition, without performing full-range drift time scanning, but focusing on the expected signal interval of the complex (2- / 4-branched fatty acid-B-12C4 / 18C6-metal ion), and rapidly capturing the characteristic drift time range of the complex in a stable state without calculating complex parameters such as collision cross section and peak separation. The characteristic drift time interval refers to a narrow range of drift time intervals of the complex (2- / 4-branched fatty acid-B-12C4 / 18C6-metal ion) in a stable conformation in the mixed aqueous solution immediately after preparation and before decomposition, which is captured by ion mobility spectrometry pre-scanning and meets the effective detection requirement of signal intensity, and is a landmark time reference range indicating the structural stability of the complex.

[0051] Step S9: determining an actual drift time based on the ion mobility spectrometry pre-scanning performed on the modified mixed aqueous solution.

[0052] The actual drift time refers to the actual migration time of the complex (2- / 4-branched fatty acid-B-12C4 / 18C6-metal ion) measured in the ion mobility spectrometry pre-scanning process of the modified mixed aqueous solution after the compensation operation.

[0053] Step S10: determining a drift time deviation value based on the characteristic drift time interval and the actual drift time.

[0054] The drift time deviation value refers to the difference obtained by comparing the characteristic drift time interval with the actual drift time, which can directly reflect the deviation degree of the actual migration time of the complex in the modified mixed aqueous solution from the expected migration time in a stable state.

[0055] Step S11: performing steps S2 to S3 when the drift time deviation value falls within a preset stable relative deviation range.

[0056] The stable relative deviation range refers to an interval within which the drift time deviation value is allowed to fluctuate, which is set based on a large amount of experimental data accumulation and complex stability analysis. When the drift time deviation value falls within the stable relative deviation range, it indicates that the deviation of the structure of the complex in the modified mixed aqueous solution from the structure in the initial stable state is within an acceptable range, and at this time, the solution state meets the requirements of subsequent ion source ionization and ion mobility spectrometry-mass spectrometry detection, and steps S2 to S3 can be continued.

[0057] Step S12: Obtain the placement duration of the modified mixed aqueous solution when the drift time deviation value does not fall within the stable relative deviation range.

[0058] The placement duration refers to the length of time elapsed from the completion of the preparation of the mixed aqueous solution to the current time. A dedicated timing device is provided to time the placement duration of each mixed aqueous solution in the sample. When the drift time deviation value does not fall within the stable relative deviation range, it indicates that the structure of the complex in the modified mixed aqueous solution has changed significantly compared to the initial stable state, resulting in a large deviation between the actual migration time and the expected migration time. At this time, the placement duration of the solution corresponding to the solution is obtained to analyze the change of the solution stability over time.

[0059] Step S13: Obtain the modified absolute concentration and the modified complex component ratio in the modified mixed aqueous solution when the placement duration is less than the preset stable window period.

[0060] The stable window period refers to the time range within which the mixed aqueous solution can maintain a relatively stable complex structure. This range is determined based on a large amount of experimental data and reflects the longest duration of maintaining the stability of the complex structure of the solution under specific conditions.

[0061] The modified absolute concentration refers to the actual concentration values of the components in the mixed aqueous solution after compensation and placement for a period of time. These values can be determined by the same concentration detection instrument or method as in step S4.

[0062] The modified complex component ratio refers to the proportion of branched fatty acids, crown ethers, and metal ions in the mixed aqueous solution after placement for a period of time. This ratio can be obtained through the same chemical analysis means as in step S4 combined with calculations.

[0063] When the placement duration is less than the stable window period, it indicates that the solution is still in a relatively stable state. At this time, the complex in the solution may have some deviation from the initial stable state, but has not yet undergone irreversible decomposition or structural changes. Therefore, the modified absolute concentration and the modified complex component ratio in the modified mixed aqueous solution can be further obtained.

[0064] Step S14: Redetermine the modified expected concentration loss based on the modified absolute concentration and the modified complex component ratio.

[0065] The modified expected concentration loss refers to the total amount of concentration decrease of each component that may be caused by the continuous decomposition of the complex, which is recalculated through a mathematical model based on the modified absolute concentration and the modified complex component ratio when the placement duration is less than the stable window period.

[0066] Step S15: When the correction predicted concentration loss amount exists, repeat the compensation operation according to the correction predicted concentration loss amount, and execute steps S9 to S15, and accumulate the compensation times.

[0067] The compensation times refer to the number of times of compensation accumulated in the compensation operation of the correction mixed aqueous solution.

[0068] Step S16: When the placement duration is greater than the stable window period or the compensation times exceed the preset maximum compensation times, output the waste sample signal.

[0069] The maximum compensation times refer to the maximum number of times of compensation allowed for the correction mixed aqueous solution, which is based on the experimental cost, solution stability and detection efficiency. Here, the number of times is twice. The waste sample signal refers to a warning information for prompting the technician that the sample has serious stability problems or the compensation is invalid. When the placement duration of the mixed aqueous solution exceeds the preset stable window period, it means that the structure of the complex in the solution has been irreversibly changed, so the waste sample signal is output.

[0070] The method further comprises determining the estimated concentration based on the predicted compensation time and the predicted concentration loss amount before the compensation operation, and the method comprises: Step S61: Disassemble the predicted concentration loss amount to obtain the to-be-mixed components and the theoretical to-be-mixed amount of each component.

[0071] The to-be-mixed components refer to specific substances involved in the predicted concentration loss amount that need to be added to the mixed aqueous solution in the compensation operation, mainly including branched fatty acids, crown ethers and metal ions, and the like. The theoretical to-be-mixed amount is the specific amount of each component calculated according to the predicted concentration loss amount, which is required to add to restore the solution to the expected concentration range.

[0072] Step S62: Determine the predicted volume of the added reagent according to the to-be-mixed components and the theoretical to-be-mixed amount, and obtain the current mixed aqueous solution volume.

[0073] The predicted volume of the added reagent refers to the original solution volume of the required reagent calculated by the volume conversion formula according to the theoretical to-be-mixed amount of the to-be-mixed components, combined with the purity, solvent ratio and density parameters of each reagent. The current mixed aqueous solution volume refers to the actual volume value of the current mixed aqueous solution measured by professional measuring tools or instruments before the compensation operation.

[0074] Step S63: Calculate the estimated mixed liquid concentration based on the to-be-mixed components, the theoretical to-be-mixed amount, the predicted volume of the added reagent, the current mixed aqueous solution volume and the preset complex binding rate.

[0075] The complex combination rate refers to the efficiency of the combination of branched fatty acids, crown ethers and metal ions to form ternary complexes in the mixed aqueous solution, which is affected by the ratio of reagents, solution environment (such as pH value, ionic strength) and reaction conditions (such as temperature, time), and is determined by pre-experiment. The estimated mixed liquid concentration refers to the expected concentration of each component in the solution after compensation operation, which is calculated according to the theoretical addition amount of the mixed components, the expected volume of the added reagent, the current solution volume and the complex combination rate. The specific formula is: estimated mixed liquid concentration = (current absolute concentration × current solution volume + theoretical mixed amount × complex combination rate) / (current solution volume + expected volume of added reagent).

[0076] Step S64: If the estimated mixed liquid concentration does not reach the absolute concentration threshold, calculate the concentration deviation value.

[0077] The concentration deviation value refers to the difference obtained by comparing the estimated mixed liquid concentration with the preset absolute concentration threshold, which reflects the deviation degree of the expected concentration of each component in the solution after compensation operation from the target concentration.

[0078] Step S65: Based on the concentration deviation value, the complex combination rate and the concentration of the added reagent, the mixed amount adjustment value is reversely deduced, and the mixed amount adjustment times are accumulated.

[0079] The mixed amount adjustment value refers to the specific amount of the mixed component that needs to be adjusted, which is obtained by reverse calculation according to the concentration deviation value, the complex combination rate and the concentration of the added reagent when the estimated mixed liquid concentration does not reach the absolute concentration threshold. The specific formula is: mixed amount adjustment value = concentration deviation value × (current solution volume + expected volume of added reagent) / complex combination rate. The mixed amount adjustment times refer to the number of times of adjusting the theoretical mixed amount of the mixed component during the compensation operation of the expected concentration loss.

[0080] Step S66: Superimpose the theoretical mixed amount and the mixed amount adjustment value to obtain the corrected mixed amount, and return to step S62 to recalculate the estimated mixed liquid concentration.

[0081] The corrected mixed amount refers to the new amount of each component that needs to be added to the mixed aqueous solution after adjustment based on the concentration deviation value.

[0082] Step S67: Repeat steps S62 to S66 until the estimated mixed liquid concentration reaches the absolute concentration threshold, and then execute step S7.

[0083] Step S68: When the mixed amount adjustment times are greater than the preset maximum adjustment times, output the waste sample signal.

[0084] The maximum number of adjustments refers to the maximum number of adjustments allowed for the theoretical mixed amount of the components to be mixed, which is determined based on experimental efficiency, cost considerations, and solution stability. When the number of adjustments exceeds this preset value, it means that after multiple adjustments, the estimated mixed liquid concentration still cannot reach the expected absolute concentration threshold. At this time, the waste sample signal is output, prompting the technician that the sample is not suitable for further analysis.

[0085] The method also includes adjusting the complex binding rate before performing the compensation operation, which comprises: Step S601: Obtain the current temperature of the mixed aqueous solution.

[0086] The current temperature refers to the actual temperature of the mixed aqueous solution to be compensated before performing the compensation operation, rather than the temperature of the external environment or the temperature of the experimental bench. This temperature is directly measured by an infrared temperature measuring instrument.

[0087] Step S602: If the current temperature is higher than the preset optimal temperature range, obtain the high-temperature binding rate corresponding to the current temperature.

[0088] The optimal temperature range refers to the temperature range verified by pre-experiments, which can make the binding rate of 2- / 4-branched fatty acid-B-12C4 / 18C6-metal ion complex in the mixed aqueous solution be in a high stable interval, and the complex conformation does not change significantly. The high-temperature binding rate refers to the actual binding rate of 2- / 4-branched fatty acid-B-12C4 / 18C6-metal ion complex in the mixed aqueous solution when the current temperature is higher than the preset optimal temperature range. This value is determined in advance by pre-experiments and stored as the core basis for adjusting the mixed amount in high-temperature environments.

[0089] Step S603: Redetermine the complex binding rate based on the high-temperature binding rate and output.

[0090] Based on the above, the present embodiment carries out detailed experimental verification, and the specific steps are as follows: Step E1: Standard sample analysis experiment: Select 2-MA, 4-MA, 2-EA, and 4-EA standard samples, respectively, and prepare standard stock solutions with a concentration of 10⁻³ mol・L⁻¹; mix the stock solution with B-12C4 solution and CoCl2 solution in equal volumes, and dilute to a final concentration of 10⁻³ mol・L⁻¹; use the standard sample analysis method to analyze the standard sample, and obtain the standard sample analysis result. 5mol L-1, and the reaction was carried out at room temperature for 15 min, during which vortex was used to ensure uniform mixing. The prepared mixed solution was injected into a Bruker TIMS-TOF MS ion mobility spectrometry-mass spectrometry instrument for detection, and the detection conditions were set as follows: positive ion mode, capillary voltage 4.5 kV, dry gas temperature 200 °C, and drift separation voltage gradient 0.7-1.0 V s cm-2. The detection results showed that the collision cross section (CCS) of [2-MA+B-12C4+Co-H]+ and [4-MA+B-12C4+Co-H]+ was significantly different (ΔCCS>5 Å2), and the peak separation degree Rp-p was 0.622, achieving baseline separation of the two isomers.

[0091] Step E2: Analysis of actual goat milk sample: 2 mL of commercially available goat milk sample was taken and placed in a 5 mL centrifuge tube, and an equal volume (2 mL) of cold acetone was added. After vortexing for 1 min, centrifugation was performed at 8000 rpm for 5 min. The supernatant was taken and filtered through a 0.22 μm organic phase filter membrane, and the filtered solution was placed in a nitrogen evaporator (45 °C, 0.15 MPa) and dried. 1 mL of a methanol / water solution (volume ratio of methanol to water 1:1) containing B-12C4 and Co2+ was added to the residue, and vortexed for 30 s to obtain the sample solution to be detected. Ion mobility spectrometry-mass spectrometry detection was performed according to the detection conditions of step E1, and quantitative analysis was performed by standard curve method. The 4-MA content in the sample was detected to be 85.2 ng mL-1. To verify the accuracy, different concentrations of 4-MA standard were added to the goat milk sample with known content, and the recovery rate was measured to be 92.7%-106.3%, indicating that the method is suitable for accurate detection of actual samples.

[0092] Step E3: Quantitative method verification experiment: a series of concentration gradient 2-MA / 4-MA mixed standard solutions (concentration ratio 1:20-20:1, corresponding concentration range 0.3 ng mL-1-200 ng mL-1) were prepared, and 3 samples were prepared for each concentration gradient. According to the method of step E1, the samples were processed and detected, and the drift peak area corresponding to each concentration standard was taken as the vertical coordinate, and the standard concentration was taken as the horizontal coordinate. The standard curve was fitted by least squares method, and the correlation coefficient R2 was greater than 0.99, indicating good linear relationship. At the same time, the detection limit and the limit of quantification of the method were calculated by multiple detection of the blank sample, and the results showed that the LOD was 0.1 ng mL-1, and the LOQ was 0.3 ng mL-1, meeting the requirements of ng-level trace quantification.

[0093] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A reagent for branched fatty acid molecular structure analysis, characterized by: The reagent includes benzene-12-crown-4-ether, 18-crown-6-ether and a compound containing a single metal ion ligand, and is used for analyzing the molecular structure of 2- / 4-branched fatty acid isomers.

2. A reagent for the analysis of branched fatty acid molecular structure according to claim 1, characterized by: The benzene-12-crown-4-ether has a molecular formula of C12H16O4 and a molecular weight of 224.25, and the 18-crown-6-ether has a molecular formula of C12H24O6 and a molecular weight of 264.

32.

3. A reagent for the analysis of branched fatty acid molecular structure according to claim 1, characterized by: The metal ion is any one of Co²⁺, Ca²⁺, Ba²⁺ or Mn²⁺.

4. A reagent for the structural analysis of branched fatty acid molecules according to any one of claims 1 to 2, characterized in that: The mixed solution for mixing the benzene-12-crown-4-ether, 18-crown-6-ether and the compound containing the single metal ion ligand has pure water as a solvent.

5. A method for analyzing a branched fatty acid molecular structure, characterized by, It comprises: Step S1: mixing a branched fatty acid sample based on at least one of 2-methyloctanoic acid, 4-methyloctanoic acid, 2-ethyloctanoic acid and 4-ethyloctanoic acid in need of position isomer analysis with the reagent for branched fatty acid molecular structure analysis according to any one of claims 1 to 4 to prepare a 2- / 4-branched fatty acid-benzene-12-crown-4-ether / 18-crown-6-ether-metal ion mixed aqueous solution; Step S2: performing ion source ionization operation based on the mixed aqueous solution to generate 2- / 4-branched fatty acid-benzene-12-crown-4-ether / 18-crown-6-ether-metal ion type monovalent positive ions; Step S3: measuring the ion mobility spectrum of the monovalent positive ions by using an ion mobility spectromass spectrometer, and identifying the position isomer structure information of the branched fatty acid according to the drift time, collision cross section value and peak separation degree of the obtained ion mobility spectrum.

6. The method of claim 5, wherein: The ion source capable of generating ions is an electrospray ionization ion source.

7. The method of claim 5, wherein the branched fatty acid molecular structure analysis method is characterized by, It further comprises a compensation method for the mixed aqueous solution before performing the ion source ionization operation, and the method comprises: Step S4: acquiring the absolute concentration and the complex component ratio in the mixed aqueous solution at a preset interval period; Step S5: fitting a complex decomposition kinetics curve based on the absolute concentration and determining a complex decomposition rate; Step S6: calculating a predicted compensation time and a predicted concentration loss amount based on the absolute concentration, the complex component ratio, the complex decomposition rate and a preset absolute concentration threshold value; Step S7: performing a compensation operation based on the predicted compensation time and the predicted concentration loss amount, and determining a revised mixed aqueous solution.

8. The method of claim 7, wherein the branched fatty acid molecular structure analysis method is characterized by, It further comprises a method for performing ion mobility spectrum pre-scanning after performing the compensation operation on the mixed aqueous solution, and the method comprises: Step S8: determining a characteristic drift time interval based on the ion mobility spectrum pre-scanning of the mixed aqueous solution; Step S9: determining an actual drift time based on the ion mobility spectrum pre-scanning of the revised mixed aqueous solution; Step S10: determining a drift time deviation value based on the characteristic drift time interval and the actual drift time; Step S11: performing steps S2 to S3 when the drift time deviation value falls within a preset stable relative deviation range; Step S12: acquiring a placement duration corresponding to the revised mixed aqueous solution when the drift time deviation value does not fall within the stable relative deviation range. Step S13: obtaining a corrected absolute concentration and a corrected compound component ratio in the corrected mixed aqueous solution when the placement duration is less than the preset stable window period; Step S14: re-determining a corrected expected concentration loss amount based on the corrected absolute concentration and the corrected compound component ratio; Step S15: repeating the compensation operation according to the corrected expected concentration loss amount when the corrected expected concentration loss amount exists, performing steps S9 to S15, and accumulating the compensation times; Step S16: outputting a waste sample signal when the placement duration is greater than the stable window period or the compensation times exceed the preset maximum compensation times.

9. The method of claim 7, wherein the branched fatty acid molecular structure analysis method is characterized by, The method further comprises determining an estimated concentration based on the expected compensation time and the expected concentration loss amount before performing the compensation operation, and the method comprises: Step S61: decomposing the expected concentration loss amount to obtain a to-be-mixed component and a corresponding theoretical to-be-mixed amount of each component; Step S62: determining a reagent expected volume according to the to-be-mixed component and the theoretical to-be-mixed amount, and obtaining a current mixed aqueous solution volume; Step S63: calculating an estimated mixed liquid concentration based on the to-be-mixed component, the theoretical to-be-mixed amount, the reagent expected volume, the current mixed aqueous solution volume, and a preset compound binding rate; Step S64: calculating a concentration deviation value if the estimated mixed liquid concentration does not reach an absolute concentration threshold value; Step S65: inversely deducing a to-be-mixed amount adjustment value based on the concentration deviation value, the compound binding rate, and a reagent concentration, and accumulating a to-be-mixed amount adjustment times; Step S66: superimposing the theoretical to-be-mixed amount and the to-be-mixed amount adjustment value to obtain a corrected to-be-mixed amount, and returning to step S62 to recalculate the estimated mixed liquid concentration; Step S67: repeating steps S62 to S66 until the estimated mixed liquid concentration reaches the absolute concentration threshold value, and performing step S7; Step S68: outputting a waste sample signal when the to-be-mixed amount adjustment times are greater than a preset maximum adjustment times.

10. The method of claim 7, wherein the branched fatty acid molecular structure analysis method is characterized by, The method further comprises adjusting the compound binding rate before performing the compensation operation, and the method comprises: Step S601: obtaining a current temperature of the mixed aqueous solution; Step S602: obtaining a high-temperature binding rate corresponding to the current temperature if the current temperature is higher than a preset optimal temperature range; Step S603: re-determining the compound binding rate based on the high-temperature binding rate, and outputting.

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