CN-121994951-A - Method for rapidly screening chemical components of traditional Chinese medicine based on UHPLC-HRMS multidimensional data filtering technology

CN121994951ACN 121994951 ACN121994951 ACN 121994951ACN-121994951-A

Abstract

A method for rapidly screening chemical components of traditional Chinese medicine based on UHPLC-HRMS multidimensional data filtering technology relates to the technical field of mass spectrum data analysis and the technical field of component analysis of traditional Chinese medicine complex systems. The method comprises the steps of obtaining mass spectrum data of a sample, carrying out format conversion on the data, leading the data into an analysis system, setting polygonal mass loss filtering windows for different types of targets to realize primary screening of the data, carrying out secondary fine screening through diagnosis ion filtering and neutral loss filtering, classifying and identifying screening results, carrying out fine analysis on complex data areas by adopting an area segmentation strategy, and finally associating primary and secondary mass spectrum information to finish identification of target compounds. The method remarkably improves the screening efficiency and accuracy of the target compounds in the complex matrix through multistage filtration and visual interaction, and can be widely used for rapid discovery and accurate identification of the target compounds in the fields of traditional Chinese medicine complex systems, natural products, metabonomics and the like.

Inventors

XIONG ZHILI
DU ZHEN
DU HAILING
WANG YAJING
REN MENGXIN
WANG LUYAO
QIN FENG
ZHAO LONGSHAN

Assignees

沈阳药科大学

Dates

Publication Date: 20260508
Application Date: 20260122

Claims (10)

1. A method for rapidly screening chemical components of traditional Chinese medicine based on UHPLC-HRMS multidimensional data filtering technology is characterized by comprising the following steps of obtaining original mass spectrum data of a traditional Chinese medicine sample through UHPLC-HRMS, conducting format conversion on the original mass spectrum data and guiding the data into an analysis system, setting polygonal mass loss filtering windows aiming at different types of targets to achieve primary screening, conducting secondary fine screening on the basis of primary screening through diagnosis ion filtering and neutral loss filtering, conducting classification identification differentiation on the results, conducting fine analysis on complex data areas through area segmentation strategies, and associating primary and secondary mass spectrum information to finish identification of target compounds.
2. The method according to claim 1, characterized in that it comprises the steps of: Sample pretreatment and mass spectrum detection pretreatment, namely carrying out component extraction, centrifugation and filtration treatment on a traditional Chinese medicine sample to be detected to obtain a sample liquid to be detected which meets the detection requirements of UHPLC-HRMS; Constructing a target compound database, namely collecting molecular formula, accurate molecular mass, characteristic secondary mass spectrum fragment information and possible cracking paths of target compound types to be screened, and constructing a special database; Raw data acquisition, namely selecting a chromatographic column adapting to a target compound, setting chromatographic conditions matching a UHPLC-HRMS detection system, constructing a chromatographic method, measuring a sample, and acquiring and generating a raw data file; Data conversion and preliminary screening, namely converting the original data into a mat format, then importing MDFocus + software, setting one or more polygonal mass loss filtering windows based on mass loss characteristics of target compounds in a database, screening and retaining precursor ions meeting requirements, and marking the precursor ions as a first color; secondary fine screening, namely, on the basis of primary screening, realizing fine screening through double targeting screening: a. Diagnostic ion filtration, namely matching the accurate quality and the quality error margin of diagnostic ions according to the characteristics of target compounds in a database, and marking the precursor ions meeting the conditions as a second color; b. Neutral loss filtering, namely matching based on the accurate mass and mass error tolerance of the characteristic neutral loss group of the target compound, and marking the precursor ions meeting the conditions as a third color; aiming at the ion point dense region, setting a specific mass-to-charge ratio range, a mass-to-loss range and a segmentation step length according to mass-to-charge ratio and mass-loss distribution characteristics of a target compound, and realizing visualized separation of subintervals and accurate positioning of target ions by a segmentation focusing technology, wherein the mass-to-charge ratio range extends +/-5 Da towards two ends based on minimum and maximum nominal mass values of the target ions, and the mass-loss range extends +/-5 mDa towards two ends based on minimum and maximum values of fractional parts of the mass-to-charge ratio of the target ions; And (3) identifying and verifying the compound, namely selecting a target ion signal, calling a secondary mass spectrogram of the target ion signal, and comparing the target ion signal with database information or standard data by combining the primary accurate mass, the chromatographic retention time and the secondary mass spectrum fragment characteristics to finish the identification of the target compound.
3. The method of claim 2, wherein in the pretreatment of the sample and the pretreatment of mass spectrometry, the component extraction is performed by one or more methods selected from the group consisting of solvent extraction, ultrasonic-assisted extraction, microwave-assisted extraction, and supercritical fluid extraction, wherein the solvent used in the solvent extraction comprises water, absolute ethanol, or 30% -95% aqueous ethanol, the solvent extraction method comprises standing, stirring, or reflux extraction, wherein the number of times of reflux extraction is 1-3, each time is 1 h-10 h, the rotational speed in centrifugation is 8000 rpm-13000 rpm, the time is 10 min-60 min, and the filtration is performed by using a 0.22 μm filter membrane.
4. The method according to claim 2, wherein in the original data acquisition link, the chromatographic column is a chromatographic column adapting to an HPLC system or a UHPLC system, the chromatographic column packing particle size adapting to the HPLC system is 3 μm-5 μm, the column length is 100 mm-250 mm, the inner diameter is 2.1 mm-4.6 mm, the chromatographic column packing particle size adapting to the UHPLC system is 1.7 μm-2.5 μm, the column length is 50 mm-150 mm, the inner diameter is 2.1 mm, the pH application range of the chromatographic column covers the pH value of the mobile phase, the chromatographic column is required to meet the conditions that the chromatographic peak separation degree of target active components is more than or equal to 1.5, the theoretical plate number is more than or equal to 3000, the tailing factor is 0.9-1.2, and the column temperature is controlled to be 25-40 ℃.
5. The method of claim 2, wherein in the data conversion and preliminary screening step, the polygonal mass loss filter window is composed of at least four vertex coordinates [ nominal mass Da, mass loss value mDa ], each target compound class corresponds to an independent screening window, and the format conversion is that the array format is converted into the text format by MS conversion software, and then converted into the mat format by MDFocus + software.
6. The method of claim 2, wherein in the secondary fine screening step, the mass error margins are all ± 5 mDa, which are adjustable according to instrument accuracy.
7. The method of claim 2, wherein in the step of segment focusing and screening, the segment step size is set according to the ion distribution density, the whole is 10 mDa, and the dense area is 5 mDa.
8. The method of claim 2, wherein in the step of identifying and verifying the compound, the evidence of consistency of the identification of the compound comprises that the primary accurate mass error and the chromatographic retention time are both in high agreement with standard information, and the secondary mass spectrogram is systematically related to the reference spectrogram on the aspects of characteristic fragment ion occurrence law, relative abundance distribution trend and neutral loss mode.
9. The use of the method according to any one of claims 1-8 in the screening of chemical components of traditional Chinese medicine, wherein the chemical components of traditional Chinese medicine comprise salvianolic acid compounds including salvianolic acid monomers, dimers, trimers and tetramers.
10. The use of the method of any one of claims 1-8 for rapid discovery and accurate identification of target compounds in the fields of traditional Chinese medicine complex systems, natural products and metabonomics.

Description

Method for rapidly screening chemical components of traditional Chinese medicine based on UHPLC-HRMS multidimensional data filtering technology Technical Field The invention relates to the technical field of mass spectrum data analysis and the technical field of traditional Chinese medicine complex system component analysis, in particular to a method for rapidly screening and identifying target compounds in complex samples, which is particularly suitable for the technical field of mass spectrum data processing and traditional Chinese medicine complex system analysis. Background In the fields of modern research of traditional Chinese medicine, metabonomics analysis, food safety monitoring and the like, the method has important significance in rapid and accurate identification and characterization of chemical components in a complex system. The method not only fundamentally clarifies the drug effect substance basis of the medicinal materials, reveals biological markers and evaluates the scientific preconditions of product safety, but also promotes the research and development of related innovative medicaments, the formulation of accurate diagnosis and treatment schemes and the establishment of quality control standards. In modern analytical chemistry, ultra-high-resolution liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) technology has become a core tool for analyzing and identifying compound structures in complex systems (such as traditional Chinese medicine extracts, metabonomics samples, biological fluid samples and the like) because of its capability of providing retention time, accurate mass number and multi-stage fragment ion information of the compounds. However, the data generated by this technique is large-scale and complex in background, both containing the target compound signal and being doped with a large amount of matrix interference and instrument noise, making rapid and accurate screening and identification of target components therefrom a great challenge. To address the processing challenges presented by the vast amount of data, mass loss filtration (MDF) techniques are often used to initially screen compounds with similar core backbones. However, the MDF function in existing commercial software is usually based on rectangular or parallelogram filter windows, which have wider coverage, are prone to introducing more false positive results, and commercial software is costly, limiting its popularity. Although the Excel platform-based self-defined polygonal MDF method can define more accurate windows, the method is severely dependent on complex manual formula editing and data processing, has complicated flow, low efficiency and low fault tolerance, and cannot realize parallel screening of multi-class compounds and integrated visualization and interactive analysis of primary and secondary mass spectrum data. Diagnostic ion filtration (IDF) and Neutral Loss Filtration (NLF) are important auxiliary fine screening means, and often lack of automation and visual integration with MDF screening procedures, so that synergistic effect is difficult to play. Therefore, the integrated polygonal MDF primary screening, IDF/NLF fine screening, visual interaction and sectional focusing functions are integrated, the operation is simple and convenient, the source is opened or the cost is low, and the special data processing platform has urgent needs and important significance for accelerating the rapid discovery and accurate identification of complex components in traditional Chinese medicines such as red sage root and the like and promoting the basic research of traditional Chinese medicine substances and the development of new medicines. Disclosure of Invention Aiming at the defects existing in the prior art, the invention provides a method for rapidly screening chemical components of traditional Chinese medicines based on a UHPLC-HRMS multidimensional data filtering technology, which is realized based on a mass spectrum data analysis method of multidimensional data filtering and visual interaction and a special software system (MDFocus +). The software has been registered for computer software copyright registration, accession number 2025R11L3552525. The method realizes the targeted primary screening, depth verification and accurate positioning of target compounds (such as salvianolic acid components) in complex mass spectrum data by integrating a multidimensional collaborative screening strategy of self-defined polygonal mass loss window filtering, ion/neutral loss fine screening diagnosis and regional sectional focusing. The method aims to solve the problems of low manual screening efficiency and high false positive rate of the traditional screening method, and remarkably improves the automation degree, accuracy and overall efficiency of data analysis by means of a visual interactive interface of software. The method for rapidly screening chemical components of traditional Chinese medicine