A method for hplc-mrms non-targeted analysis of water-soluble natural monomeric fragrance raw materials and application thereof

Abstract

This invention belongs to the field of analytical technology for water-soluble natural monomeric fragrance raw materials in tobacco products, and relates to a non-targeted HPLC-MRMS analysis method for water-soluble natural monomeric fragrance raw materials and its application. The method includes: 1. Using liquid chromatography-magnetic resonance mass spectrometry (LC-MRMS), a fast analytical column is selected. After the sample is separated by the chromatographic column, two chromatographic peaks with retention time periods appear in the chromatogram, separating sugar-related interfering substances; 2. Segmentation processing is used to extract chromatograms with characteristic components or non-sugar retention time periods, reconstructing the chromatogram and the average primary mass spectrum. The average primary mass spectrum of this time period is used to identify and analyze the water-soluble natural monomeric fragrance raw materials in the fragrance base. This invention achieves the separation of interfering components such as sugars from the characteristic components of water-soluble natural monomeric fragrance raw materials by systematically optimizing the chromatographic column, injection volume, flow rate, and gradient elution conditions, thereby reducing matrix interference and ion inhibition effects.

Description

Technical Field

[0001] This invention belongs to the field of analytical technology of water-soluble natural monomeric fragrance raw materials for tobacco, specifically relating to an HPLC-MRMS non-targeted analysis method for water-soluble natural monomeric fragrance raw materials and its application. Background Technology

[0002] Water-soluble natural single-component aroma compounds in tobacco products refer to aromatic substances extracted from natural plants that are soluble in water or water-based solvents (such as ethanol-water solutions, propylene glycol, etc.). These substances are widely used as additives in cigarettes and other tobacco products to improve and enrich their aroma and taste. Water-soluble natural single-component aroma compounds in tobacco products are extracted through processes such as extraction and cold pressing. They are easily affected by factors such as the origin, type, and processing of the raw materials. Their chemical composition is complex and diverse, with a relatively high content of large molecular compounds. Moreover, most water-soluble natural single-component aroma compounds contain sugars, especially those from fruits and vegetables, which have a high sugar content. Conventional gas chromatography and liquid chromatography provide limited information during these analyses. Magnetic resonance mass spectrometry (MRMS), however, offers advantages in analyzing complex and large molecular compounds due to its high resolution, high sensitivity, and precision. MRMS analysis techniques are divided into direct injection and coupled analysis techniques.

[0003] However, while direct injection analysis offers advantages such as speed, accuracy, and comprehensive sample delivery with rich spectral information, some high-content components or interfering substances (such as sugars) can compete with characteristic or target components during ionization, inhibiting their ionization efficiency. This results in lower characterization responses and less prominent characterization of characteristic components in water-soluble natural single-component fragrance raw materials, and they are also prone to forming adduct ions. Furthermore, the identification and resolution rate of water-soluble natural single-component fragrance raw materials is low due to the large number of shared components among them.

[0004] Therefore, given the problems with direct sample injection analysis and the properties of water-soluble natural monomeric fragrance raw materials, it is necessary to explore a magnetic resonance mass spectrometry analysis technique that can reduce ion suppression effects and effectively separate sugar substances to solve the above-mentioned technical problems. Summary of the Invention

[0005] The specific technical solution of this invention is as follows: A non-targeted HPLC-MRMS method for water-soluble natural monomeric fragrance raw materials, comprising the following steps: Step S1: Liquid chromatography-magnetic resonance mass spectrometry is used for analysis. A fast analytical column is selected. After the sample is separated by the chromatographic column, two chromatographic peaks with retention time periods are displayed in the chromatogram, separating the sugar interfering substances from the characteristic components or target components.

[0006] Step S2: Using segmented slicing, chromatograms with characteristic components or non-sugar retention time periods are extracted, and the chromatograms and average primary mass spectra are reconstructed. The average primary mass spectra of the extracted time period are used to identify and analyze the water-soluble natural monomeric fragrance raw materials in the fragrance base.

[0007] Rapid analysis columns include: 3cm-5cm C18 rapid analysis columns.

[0008] Preferably, the rapid analysis column is a 5cm C18 rapid analysis column.

[0009] Preferably, in step S1, the chromatographic analysis conditions include: Injection volumes include 3 μL, 5 μL, and 10 μL; mobile phase flow rates include 0.5 mL / min, 0.7 mL / min, and 1.0 mL / min.

[0010] More preferably, the chromatographic analysis conditions specifically include: mobile phase A is 0.1% formic acid water, mobile phase B is 0.1% formic acid acetonitrile, the flow rate is 0.5 mL / min, and the injection volume is 5 μL.

[0011] Gradient elution procedures include: Equilibrium phase: 0 min - 3 min, 95% A phase + 5% B phase.

[0012] Rapid elution: 3-5 minutes, phase B increases from 5% to 90%.

[0013] Strong elution: 5-10 minutes, phase B increases from 90% to 100%.

[0014] Cleaning column: 10 min - 19.99 min, maintaining 100% B phase.

[0015] Rapid equilibration: within 20 minutes, it instantly returns to 5% phase.

[0016] Post-run balancing: Maintain 5% B phase for 5 minutes.

[0017] This invention also discloses an application of a non-targeted analysis method for water-soluble natural monomeric fragrance raw materials using HPLC-MRMS. The application of this non-targeted analysis method includes: The core workflow is "extracted ion chromatogram construction - baseline correction - extraction of EIC peaks from extracted ion chromatograms - mass spectrometry reconstruction". Based on the non-targeted analysis method of HPLC-MRMS, data on water-soluble natural single fragrance raw materials and fragrance bases are collected and a total ion current chromatogram (TIC) is constructed. An adaptive ion linkage algorithm is used to construct the extracted ion chromatogram (EIC), a local minimum algorithm is used to correct the baseline, and a dynamic window smoothing strategy is used to extract effective peaks. HPLC-MRMS data preprocessing is completed, and the EIC peak signals of the extracted ion chromatograms are segmented according to retention time. The information is integrated to generate segmented mass spectra, and a common and characteristic ion library of water-soluble natural single fragrance raw materials is constructed. The Jaccard coefficient is used to quantify the overlap of characteristic ions to achieve the identification of water-soluble natural single fragrance raw materials.

[0018] Preferably, the application further includes: identifying water-soluble natural monomeric fragrance raw materials in the fragrance base module; constructing a common ion library by identifying ions that can be identified in all similar fragrance raw materials for each water-soluble natural monomeric fragrance raw material; based on the common ions, identifying ions with a strength greater than all other fragrance raw materials and unique to that specific water-soluble natural monomeric fragrance raw material as characteristic ions, and constructing a corresponding characteristic ion library; and selecting ions with a signal-to-noise ratio (S / N) greater than 50 and a mass-to-charge ratio close to each other, according to a ratio of 0.001. Ions with similar mass-to-charge ratios are selected based on their response intensity using the Da tolerance fusion method. These ions are then preprocessed. Finally, the Jaccard coefficient is used as the core matching index. The overlap of characteristic ions is quantified using the formula J(A,B)=|A∩B| / |A∪B|, where |A∩B| is the number of ions in A and B with an m / z deviation ≤ 0.001Da and a response intensity that meets the threshold, and |A∪B| is the total number of ions in A and B. The coefficient ranges from 0 to 1, with a higher degree of matching indicating a closer match to 1.

[0019] The beneficial effects of this invention are: 1. This invention can effectively reduce ion suppression effects and improve the ionization efficiency and response of characteristic or target components. At the same time, this invention has the advantages of short analysis time and high analysis efficiency. Moreover, it retains the peaks of water-soluble components and also takes into account the peaks of alcohol-soluble components and column cleaning in a short time, preventing cross-contamination between samples.

[0020] 2. This invention provides rapid analysis in a short time, avoiding the interference of retention time drift on the analysis in conventional analysis. It also saves memory space (the normal data size of a sample analyzed by liquid chromatography-magnetic resonance mass spectrometry is about 2GB, while the data size of a sample analyzed by the method of this invention is 700MB), improves the running speed of the software for identifying and analyzing water-soluble natural monomeric fragrance raw materials, reduces the requirements of the equipment hardware operating environment, and is conducive to expanding applications.

[0021] 3. Compared with existing direct sample injection analysis technology using magnetic resonance mass spectrometry, this invention significantly improves the identification and resolution rate of water-soluble natural monomeric fragrance raw materials. The method of this invention provides a rapid liquid chromatography-magnetic resonance mass spectrometry (LC-MS / MS) analysis method for quality control, characteristic analysis, and identification and analysis of water-soluble natural monomeric fragrance raw materials in tobacco flavorings. Attached Figure Description

[0022] Figure 1 This invention provides a non-targeted HPLC-MRMS analysis method for water-soluble natural monomeric fragrance raw materials and its application. The HPLC-MRMS analysis spectra of 30mm and 50mm columns are compared, with the upper image representing 30mm and the lower image representing 50mm. Figure 2 The HPLC-MRMS analysis spectra of the present invention at flow rates of 0.5, 0.7, and 1.0 mL / min are compared; the upper figure represents 0.5 mL / min, the middle figure represents 0.7 mL / min, and the lower figure represents 1.0 mL / min. Figure 3 The mass spectra of the present invention are compared after 4 min of HPLC-MRMS analysis at flow rates of 0.5, 0.7, and 1.0 mL / min; the upper spectrum is at 0.5 mL / min, the middle spectrum is at 0.7 mL / min, and the lower spectrum is at 1.0 mL / min. Figure 4 This is a comparison of HPLC-MRMS analytical spectra at chromatographic gradient elution times of 15 min and 20 min, as presented in this invention; the upper figure represents 15 min and the lower figure represents 20 min. Figure 5 This invention provides blank HPLC-MRMS analysis of positive ion mode BPC spectra and mass spectra; Figure 6 This invention provides blank HPLC-MRMS analysis of negative ion mode BPC spectra and mass spectra; Figure 7 The image shows the BPC chromatogram of the monk fruit extract analyzed by magnetic resonance mass spectrometry (HPLC-MRMS) in this invention. Figure 8 This invention provides an analysis of the mass spectra of monk fruit extract at different time points using HPLC-MRMS. Figure 9 This invention provides a comparison of the magnetic resonance mass spectrometry (HPLC-MRMS) and direct injection analysis mass spectra of the monk fruit extract. Figure 10 This is a schematic diagram of the HPLC-MRMS data analysis process of the present invention. Detailed Implementation

[0023] The relevant technologies of this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0024] like Figures 1-10 As shown, the HPLC-MRMS non-targeted analysis method for water-soluble natural single-component fragrance raw materials in this embodiment employs liquid chromatography-magnetic resonance mass spectrometry (LC-MRMS) with a 5 cm C18 rapid analytical column. After separation by the chromatographic column, the chromatogram displays two peaks with specific retention time periods, effectively separating interfering substances (such as sugars) from characteristic or target components, reducing the ion inhibition effect of interfering substances on characteristic or target components, and significantly improving the chromatographic and mass spectrometric response of characteristic or target components. Simultaneously, the identification and resolution method for water-soluble natural single-component fragrance raw materials uses segmented slicing, extracting chromatograms with retention time periods for characteristic components or non-sugars, reconstructing the chromatogram and average primary mass spectrum, and using the average primary mass spectrum of this segment to identify and resolve water-soluble natural single-component fragrance raw materials in the fragrance base. This significantly improves the identification and resolution rate of water-soluble natural single-component fragrance raw materials.

[0025] The chromatographic analysis conditions in this embodiment include: Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Flow rate: 0.5 mL / min; Injection volume: 5 μL; Gradient elution program: 0 min-3 min 5% B, 3 min-5 min 5% B-90% B, 5 min-10 min 90% B-100% B, 10 min-19.99 min 100% B, 20 min 5% B, followed by a 5 min run.

[0026] The mass spectrometry analysis conditions in this embodiment include: Ion source: ESI source.

[0027] Mass spectrometry conditions: FTMS; Scan range: 100-1600 Da; Scan mode: positive and negative ion mode; Number of scans: 1; Size: 1 M; Cumulative time (s): 0.1 s; Capillary voltage: 4500 V; Dryer flow rate: 8.0 mL / min; Drying temperature: 200 °C; Nozzle pressure: 1.0 bar; Time of flight: 1.0 s. Radio frequency: 2 MHz; Frequency: 2 MHz.

[0028] Optimization of liquid phase separation conditions in this embodiment: To rapidly separate characteristic substances from interfering components and to facilitate the elution of substances such as sugars, the experiment investigated and optimized the chromatographic column, injection volume, flow rate, gradient elution, chromatographic mass spectrometry linkage mode, and matrix blank.

[0029] Column selection: To achieve rapid separation of interferences, two rapid analytical columns of different lengths, Eclipse Plus C18 (4.6×50mm, 3.5um) and Eclipse Plus C18 (4.6×30mm, 3.5um), were compared. The analytical results are shown in [Figure number missing]. Figure 1 As shown in the figure, under the same chromatographic conditions, the separation of the chromatographic peaks of the two columns is basically the same. The peak of the 30 mm column eluted slightly earlier than that of the 50 mm column. Taking all factors into consideration, the Eclipse Plus C18 50 mm column was finally selected for sample analysis.

[0030] Optimization of injection volume: To examine the effect of injection volume on analytical results, the same sample was analyzed with injection volumes of 3 μL, 5 μL, and 10 μL. The results showed that the spectral response increased with the increase of injection volume. When the injection volume was 3 μL, some samples had low responses. When the injection volume was 10 μL, some compounds with relatively high content were difficult to elute, resulting in column residue and cross-contamination between samples. Therefore, considering all factors, the injection volume was determined to be 5 μL.

[0031] Optimization of mobile phase flow rate: To investigate the effect of flow rate on the analytical results, the effects of flow rates of 0.5 mL / min, 0.7 mL / min, and 1.0 mL / min on the analytical results were compared. The analytical results are shown in [Figure number missing]. Figure 2 and Figure 3 The analysis results show that as the flow rate increases, the target analyte elutes earlier, and the sample peaks are more concentrated. Figure 3 Analysis shows that when the flow rate is 0.5 mL / min, the abundance of mass spectrometry ion response and the number of characteristic ions after 4 min are better than those at 0.7 mL / min and 1.0 mL / min. Taking into account both the abundance of mass spectrometry ion response and the number of characteristic ions, the final chromatographic flow rate is determined to be 0.5 mL / min.

[0032] Optimization of gradient elution conditions: Based on the established separation conditions, since some water-soluble natural monomeric fragrance raw materials did not elute completely within 15 minutes, the elution time of the 100% organic phase was optimized by extending it from 2 minutes to 10 minutes, and the data acquisition time was extended to 20 minutes. This ensured complete elution of the water-soluble natural monomeric fragrance raw materials and avoided cross-contamination between samples. A comparison of the analytical spectra of some water-soluble natural monomeric fragrance raw materials at 15 minutes and 20 minutes is shown below. Figure 4By extending the collection time of the organic phase, the water-soluble natural monomeric fragrance raw materials elute completely. Therefore, the gradient elution conditions were finally determined as follows: Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Flow rate: 0.5 mL / min; Injection volume: 5 μL; Gradient elution program: 0 min-3 min 5% B, 3 min-5 min 5% B-90% B, 5 min-10 min 90% B-100% B, 10 min-19.99 min 100% B, 20 min 5% B, followed by a 5 min run.

[0033] Matrix blank in this embodiment: During the method optimization process, the impact of sample blanks on the analytical results was investigated experimentally. The analytical results are shown below. Figure 5 and Figure 6 As shown in the figure, the positive ion mode of matrix blank analysis mainly affects ions 403, 425, and 827, while the ion response of the negative ion mode is low and has little impact on the analysis.

[0034] Methodological examination of this implementation method: Intra-day precision: Five water-soluble natural monomeric fragrance raw material samples were prepared in parallel and analyzed five times consecutively using HPLC-MRMS. Characteristic ions with relatively high response rates (453, 471, 579, 647, 823, 839, 941, 1235, and 1293) were selected. The mass number deviation of the characteristic ions in the five measurements was calculated, and the results are shown in Table 1. The results show that the maximum difference in the mass number of the characteristic ions among the five measurements was 0.00484 Da, and the intra-day relative standard deviation (RSD) of the mass number of the characteristic ions was <0.00014%, indicating that the method has good precision and accuracy. The intra-day precision results of the magnetic resonance mass spectrometry HPLC-MRMS analysis are shown in Table 1 below.

[0035] Inter-day precision: Six water-soluble natural monomeric fragrance raw material samples were prepared in parallel and analyzed continuously for six days using HPLC-MRMS. Characteristic ions with relatively high response rates (453, 471, 579, 647, 823, 839, 941, 1235, and 1293) were selected. The mass number deviation of characteristic ions over the six days was calculated, and the results are shown in Table 2. The results show that the maximum difference in the mass number of characteristic ions over the six days was 0.00213 Da, and the inter-day relative standard deviation (RSD) of the mass number of characteristic ions was <0.00006%, indicating good instrument stability and method precision. The inter-day precision results of HPLC-MRMS analysis are shown in Table 2 below.

[0036] Analysis and application of water-soluble natural monomeric fragrance raw materials in this embodiment: HPLC-MRMS data analysis method: This method uses "extracted ion chromatogram construction - baseline correction - EIC peak extraction - mass spectrometry reconstruction" as its core process (see...). Figure 10 Based on HPLC-MRMS, data on water-soluble natural single-component fragrance raw materials and fragrance bases were collected and TICs were constructed. An EIC was constructed using an adaptive ion linkage algorithm, a local minimum algorithm was used to correct the baseline, and a dynamic window smoothing strategy was used to extract effective peaks. HPLC-MRMS data preprocessing was completed, and the EIC peak signals were segmented according to retention time. The information was integrated to generate segmented mass spectra, and a common and characteristic ion library of water-soluble natural single-component fragrance raw materials was constructed. The overlap of characteristic ions was quantified using the Jaccard coefficient to achieve the identification of water-soluble natural single-component fragrance raw materials.

[0037] In the fragrance base module, the identification of water-soluble natural monomeric fragrance raw materials is performed. Ions that can be identified in all similar fragrance raw materials for each water-soluble natural monomeric fragrance raw material are designated as common ions, and a corresponding common ion library is constructed. Based on these common ions, ions with intensities greater than all other fragrance raw materials and unique to that specific water-soluble natural monomeric fragrance raw material are identified as characteristic ions, and a corresponding characteristic ion library is constructed. Ions with a signal-to-noise ratio (S / N) greater than 50 and similar mass-to-charge ratios are fused. Ions with similar mass-to-charge ratios are then fused with a tolerance of 0.001 Da. The top 100 ions based on response intensity are selected for preprocessing. Finally, the Jaccard Index (JCD) is used as the core matching index. The overlap of characteristic ions is quantified by the formula J(A,B)=|A∩B| / |A∪B|, where |A∩B| is the number of ions in A and B with an m / z deviation ≤0.001Da and a response intensity that meets the threshold, and |A∪B| is the total number of ions in A and B. The coefficient takes a value of 0-1, and the closer it is to 1, the higher the matching degree.

[0038] This analytical algorithm effectively separates interfering components from characteristic components, thereby solving the problems of insufficient targeting, weak interference elimination ability, ion suppression effect, and low identification accuracy in the analysis of water-soluble natural monomeric fragrance raw materials in direct injection.

[0039] Example This embodiment applies the established HPLC-MRMS analytical method to analyze 59 water-soluble natural monomeric fragrance raw materials. The aim is to separate interfering substances from characteristic components, addressing the issue of reduced response of characteristic components due to ionization effects of some interfering substances (such as sugars) during direct injection in magnetic resonance mass spectrometry. Taking monk fruit extract as an example, the BPC spectrum acquired by HPLC-MRMS of monk fruit is shown below. Figure 7 Mass spectra at different time points (0-4 min and 4-20 min) are shown below. Figure 8During the 0-4 min period, most water-soluble substances (such as sugars with 543 Da, 723 Da, 885 Da, and 1047 Da) appear. During the 4-20 min period, the medium polarity and macromolecular characteristic components of water-soluble monomeric fragrance raw materials (801 Da, 963 Da, and 1125 Da) are present. This method effectively separates sugars from characteristic components, reduces the ion inhibition effect of sugars on characteristic components, and highlights the characterization of characteristic components. It provides an efficient analytical method for the analysis and quality control of water-soluble natural monomeric fragrance raw materials in fragrance bases. The analysis of 59 water-soluble natural monomeric fragrance raw materials provides efficient data support for the identification and analysis of water-soluble natural monomeric fragrance raw materials in functional fragrance bases.

[0040] HPLC-MRMS identification and analysis of water-soluble natural monomeric fragrance raw materials: First, an HPLC-MRMS analysis database of 59 natural single-component fragrance raw materials was established, and the data format was converted to ASCII or CDF format to facilitate spectral analysis by data processing software.

[0041] Secondly, the loading and preprocessing of data for water-soluble natural monomeric fragrance raw materials and fragrance base modules are performed, such as segmentation. Since the chromatographic peak information before 4 minutes is mainly composed of sugars, and most water-soluble natural monomeric fragrance raw materials contain sugars, there are many common sugar components before 4 minutes, which interfere with the identification and analysis of water-soluble natural monomeric fragrance raw materials in the fragrance base. This interference is already evident in direct injection analysis. This method extracts the chromatographic peaks after 4 minutes, extracts and reconstructs the average mass spectrometry characteristic ions of this time period, and uses them for HPLC-MRMS identification and analysis of water-soluble natural monomeric fragrance raw materials in the fragrance base. The purpose is to reduce common interfering ions, highlight characteristic or target component ions, and improve the identification and analysis rate of water-soluble natural monomeric fragrance raw materials.

[0042] Finally, the recognition resolution rate is calculated. This invention selects the top 10 fragrance raw materials in the water-soluble natural monomer fragrance raw materials and counts the proportion of fragrance raw materials in the fragrance base formula identified in the top 10 fragrance raw materials. That is, the number of fragrance raw materials in the fragrance base formula is used as the denominator, and the number of fragrance raw materials in the fragrance base identified in the top 10 fragrance raw materials is used as the numerator. Then, multiply by 100% to get the recognition resolution rate.

[0043] P=(N / M)*100% Where P is the recognition resolution rate, N is the number of fragrance raw materials identified in the fragrance base among the top 10 fragrance raw materials, and M is the number of fragrance raw materials in the fragrance base formula.

[0044] Comparison of identification and analysis results between direct injection and HPLC-MRMS: Using the established database of 59 natural monomeric fragrance raw materials, direct injection and HPLC-MRMS analysis were conducted to identify and analyze the fragrance raw materials in 30 functional fragrance base modules. Both methods of identification and analysis statistically analyzed the top 10 fragrance raw materials. The results are shown in Table 3. The results indicate that the direct injection ESI source had a minimum identification resolution of 0% and a maximum of 60%, with positive ion mode accounting for 13.3% and negative ion mode accounting for 23.3% of the identification resolution above 50%. The APCI source had a minimum identification resolution of 0% and a maximum of 100%, accounting for 3.3%, with 33.3% of the 50% resolution. The resolution of HPLC-MRMS for the ESI source positive ion mode ranged from a minimum of 44.0% to a maximum of 100%, accounting for 20% of the total, with a resolution of over 50% accounting for 93.3%. For the ESI source negative ion mode, the resolution ranged from a minimum of 30.0% to a maximum of 100%, accounting for 13.3%, with a resolution of over 50% accounting for 66.7%. For the APCI source negative ion mode, the resolution ranged from a minimum of 30.0% to a maximum of 100%, accounting for 13.3%, with a resolution of over 50% accounting for 86.7%. Based on the resolution analysis of fragrance raw materials, HPLC-MRMS analysis showed a better resolution than direct injection analysis, and the resolution of the ESI source positive ion mode was superior to both the negative ion mode and the APCI source.

[0045] Therefore, the established HPLC-MRMS non-targeted analysis method for water-soluble natural monomeric fragrance raw materials solves the problem of ion inhibition effect of sugar components on characteristic or target components during direct sample injection analysis, and improves the ion response of characteristic or target components. Secondly, this method uses a 5cm short column for rapid analysis, dividing the sample retention in the chromatographic column into two segments. The first segment mainly consists of sugars, while the second segment mainly consists of characteristic or target components. Ignoring peak separation efficiency, this method removes interfering substances such as sugars, significantly reducing the ion inhibition effect of interfering substances on characteristic or target components and improving their ionization efficiency. Thirdly, in data analysis, segmented processing is used, extracting chromatographic peaks over a specific time period to reconstruct chromatograms and mass spectra. The average mass spectrum of the extracted time period is used for the identification and analysis of water-soluble natural single-component fragrance raw materials. Compared to the methods in the literature that combine chromatographic peaks with retention time or secondary mass spectrometry fragments for identifying and analyzing fragrance raw materials, this method uses the precise mass number of the primary average mass spectrum peak over the extracted time period for identification and analysis, ignoring retention time and avoiding retention time drift problems in conventional analysis. This provides a new analytical method for the identification and analysis of water-soluble natural single-component fragrance raw materials, especially for these materials.

[0046] In summary, the HPLC-MRMS non-targeted analysis method for water-soluble natural monomeric fragrance raw materials established in this invention effectively separates interfering components such as sugars from the characteristic components of water-soluble natural fragrance raw materials by systematically optimizing chromatographic separation conditions such as column, injection volume, flow rate, and gradient elution, thereby reducing matrix interference and ion inhibition effects. Furthermore, methodological validation demonstrates good intra-day and inter-day precision, ensuring the accuracy and stability of analytical results. In practical applications, a reliable database was constructed for the analysis of 59 natural monomeric fragrance raw materials. Combined with a dedicated data analysis algorithm, the identification and resolution rate of water-soluble natural monomeric fragrance raw materials in functional fragrance bases was significantly improved, with performance significantly superior to direct injection analysis. This method provides a novel and practical technical means for the efficient analysis, quality control, and formulation analysis of water-soluble natural monomeric fragrance raw materials and functional fragrance bases, possessing significant theoretical value and application prospects.

[0047] It should be emphasized that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for the non-targeted analysis of water-soluble natural monomeric fragrance raw materials by HPLC-MRMS, characterized in that, Includes the following steps: Step S1: Liquid chromatography-magnetic resonance mass spectrometry is used for analysis. A fast analytical column is selected. After the sample is separated by the chromatographic column, two chromatographic peaks with retention time periods are displayed in the chromatogram, separating the sugar interfering substances from the characteristic components or target components. Step S2: Using segmented slicing, chromatograms with characteristic components or non-sugar retention time periods are extracted, and the chromatograms and average primary mass spectra are reconstructed. The average primary mass spectra of the extracted time period are used to identify and analyze the water-soluble natural monomeric fragrance raw materials in the fragrance base. The rapid analysis column includes a 3cm-5cm C18 rapid analysis column.

2. A method of HPLC-MRMS non-targeted analysis of water soluble natural monomeric flavor materials according to claim 1, characterized in that, The rapid analysis column is a 5cm C18 rapid analysis column.

3. The HPLC-MRMS non-targeted analysis method for water-soluble natural monomeric fragrance raw materials according to claim 1, characterized in that, In step S1, the chromatographic analysis conditions include: Injection volumes include: 3 μL, 5 μL, and 10 μL; The mobile phase flow rates include: 0.5 mL / min, 0.7 mL / min, and 1.0 mL / min.

4. The HPLC-MRMS non-targeted analysis method for water-soluble natural monomeric fragrance raw materials according to claim 3, characterized in that, The specific chromatographic analysis conditions include: mobile phase A is 0.1% formic acid water, mobile phase B is 0.1% formic acid acetonitrile, flow rate is 0.5 mL / min, and injection volume is 5 μL; Gradient elution procedures include: Equilibrium phase: 0-3 minutes, 95% Phase A + 5% Phase B; Rapid elution: 3-5 minutes, phase B increases from 5% to 90%; Strong elution: 5-10 minutes, phase B increases from 90% to 100%; Cleaning column: 10-19.99 min, maintaining 100% B phase; Rapid equilibration: within 20 minutes, it instantly returns to 5% phase; Post-run balancing: Maintain 5% B phase for 5 minutes.

5. An application of a non-targeted HPLC-MRMS method for the analysis of water-soluble natural monomeric fragrance raw materials, characterized in that, The application employs the non-targeted analysis method according to any one of claims 1 to 4, and the application includes: The core workflow is "extracted ion chromatogram construction - baseline correction - extraction of EIC peaks from extracted ion chromatograms - mass spectrometry reconstruction". Based on the non-targeted analysis method of HPLC-MRMS, data on water-soluble natural single fragrance raw materials and fragrance bases are collected and a total ion current chromatogram (TIC) is constructed. An adaptive ion linkage algorithm is used to construct the extracted ion chromatogram (EIC), a local minimum algorithm is used to correct the baseline, and a dynamic window smoothing strategy is used to extract effective peaks. HPLC-MRMS data preprocessing is completed, and the EIC peak signals of the extracted ion chromatograms are segmented according to retention time. The information is integrated to generate segmented mass spectra, and a common and characteristic ion library of water-soluble natural single fragrance raw materials is constructed. The Jaccard coefficient is used to quantify the overlap of characteristic ions to achieve the identification of water-soluble natural single fragrance raw materials.

6. The application of the HPLC-MRMS non-targeted analysis method for water-soluble natural monomeric fragrance raw materials according to claim 5, characterized in that, The application also includes: In the fragrance base module, the identification of water-soluble natural monomeric fragrance raw materials is performed by identifying ions that can be identified in all similar fragrance raw materials for each water-soluble natural monomeric fragrance raw material as common ions, and constructing a corresponding common ion library. Based on the common ions, ions with a strength greater than all other fragrance raw materials and unique to that specific water-soluble natural monomeric fragrance raw material are identified as characteristic ions, and constructing a corresponding characteristic ion library. Ions with a signal-to-noise ratio (S / N) greater than 50 and a mass-to-charge ratio close to each other are fused together with a tolerance of 0.001 Da. The top 100 ions based on response intensity are selected and preprocessed. Finally, the Jaccard coefficient is used as the core matching index, and the overlap of characteristic ions is quantified by the formula J(A, B) = |A∩B| / |A∪B|, where |A∩B| is the number of ions in A and B with an m / z deviation ≤ 0.001 Da and a response intensity that meets the threshold, and |A∪B| is the total number of ions in A and B. The coefficient ranges from 0 to 1, with a higher degree of matching indicating a closer value to 1.

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