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CN-121994972-A - Qualitative and semi-quantitative integrated analysis method for environmental trace micro-pollutants based on mass spectrum full-scanning mode

CN121994972ACN 121994972 ACN121994972 ACN 121994972ACN-121994972-A

Abstract

The invention provides a qualitative and semi-quantitative integrated analysis method of environmental trace micro-pollutants based on a mass spectrum full-scan mode, which adopts precursor ions without an MS/MS secondary mass spectrum in a full-scan MS mass spectrum to analyze, forms a characteristic table of data of the precursor ions, reserves candidate compounds corresponding to precursor ions with high matching degree and front, and generates a first candidate library; searching data of fragment ions with collision energy less than or equal to 20eV corresponding to the candidate compounds in an open source library, integrating the data of the fragment ions with the data of precursor ions in the first candidate library to construct a target screening library, and performing target retrieval in a full-scan MS mass spectrum, and then performing confidence assessment and semi-quantitative analysis. The method can recover a large amount of lost low-abundance fragment ions, effectively identify trace micro-pollutants, has the minimum detection concentration below 0.5ppt level and has high confidence.

Inventors

  • XUE JINGCHUAN
  • Ku Mengjia
  • ZHU JIAMIN
  • ZHONG XIANG
  • CHEN BOHAN

Assignees

  • 广东工业大学

Dates

Publication Date
20260508
Application Date
20260324

Claims (10)

  1. 1. The qualitative and semi-quantitative integrated analysis method for the environmental trace micro-pollutants based on the mass spectrum full scanning mode is characterized by comprising the following steps of: s1, data preparation, namely acquiring data of an extract of an environmental sample by using a UPLC-HRMS system to obtain a full-scan MS mass spectrum and an MS/MS secondary mass spectrum; s2, primarily screening candidate substances, namely analyzing a full-scan MS mass spectrum and an MS/MS secondary mass spectrum, screening precursor ions without the MS/MS secondary mass spectrum, forming a characteristic table from data of the precursor ions, sorting the matching degree of compounds corresponding to each precursor ion in the characteristic table, and reserving 2-5 candidate compounds with the matching degree ranked at the front for each precursor ion to generate a first candidate library; s3, constructing a target screening library, namely searching data of fragment ions with collision energy less than or equal to 20eV corresponding to candidate compounds in the first candidate library in an open source library, integrating the data of the fragment ions with data of precursor ions in the first candidate library, and constructing the target screening library; S4, verifying and searching, namely, in the full-scanning MS mass spectrum obtained in the step S1, carrying out targeted searching on the mass-to-charge ratio, retention time and mass-to-charge ratio of fragment ions of precursor ions corresponding to the candidate compounds in the targeted screening library, and extracting the mass-to-charge ratio, intensity and chromatographic peak of the fragment ions which exist in the full-scanning MS mass spectrum and are matched with the candidate compounds; s5, evaluating the confidence coefficient, namely screening candidate compounds with highest matching degree according to the number of the matched fragment ions and the similarity of the reverse dot product, namely, the candidate compounds are micro pollutants corresponding to the corresponding precursor ions; S6, semi-quantitative analysis is carried out on the micro pollutants.
  2. 2. The qualitative and semi-quantitative integrated analysis method of environmental trace micro-pollutants according to claim 1, wherein the environmental sample is selected from any one of surface water, river sediment, wastewater from sewage plants or medical wastewater.
  3. 3. The qualitative and semi-quantitative integrated analysis method for environmental trace micro-pollutants according to any one of claims 1 to 2, wherein when the environmental sample is surface water, the extract is obtained by pretreating the surface water, the pretreatment specifically comprises the steps of loading a water sample into a pre-activated polystyrene-divinylbenzene copolymer stationary phase extraction column, vacuum drying after the polystyrene-divinylbenzene copolymer stationary phase extraction column adsorbs target analytes, eluting with acetonitrile or methanol, and collecting an eluent, and the eluent is flowed to a dry state by nitrogen gas to obtain the extract.
  4. 4. The qualitative and semi-quantitative integrated analysis method for environmental trace micro-pollutants according to any one of claims 1 to 2, wherein when the environmental sample is a river sediment, the extract is obtained by pretreating the river sediment, the pretreatment specifically comprises the steps of loading an extracting solution of the river sediment into a pre-activated polystyrene-divinylbenzene copolymer stationary phase extraction column, vacuum drying after the polystyrene-divinylbenzene copolymer stationary phase extraction column adsorbs target analytes, eluting with methanol, and collecting an eluent, wherein the eluent is flowed to a dry state by nitrogen gas, so as to obtain the extract.
  5. 5. The qualitative and semi-quantitative integrated analysis method for the trace amount of micro-pollutants in the environment according to claim 1, the method is characterized in that the parameters of the data acquisition comprise: The chromatographic column is a C18 reversed-phase chromatographic column, the mobile phase is 0.1% formic acid aqueous solution-acetonitrile solution in a positive ion mode, and the mobile phase is 0.5mM ammonium formate aqueous solution-acetonitrile solution in a negative ion mode; The acetonitrile is phase B, and the gradient of the mobile phase is as follows: 1 to 3.0 minutes, 1 to 15% B, 3.0 to 6 minutes, 15-50% B, 6.0-7.5 min, 50-98% B, 7.5-11.5 min, 15-50% B, 6.0-7.5 min, 50-98% B, 7.5-11.5 min.
  6. 6. The qualitative and semi-quantitative integrated analytical method for environmental trace micro-pollutants according to claim 1, wherein the collision energy in step S3 is 0eV, 10eV and 20eV.
  7. 7. The qualitative and semi-quantitative integrated analysis method of environmental trace micro-contaminants according to claim 1, characterized in that the matching tolerance of mass-to-charge ratio of precursor ions or mass-to-charge ratio of fragment ions is ±5ppm, the matching tolerance of retention time is ±0.2min.
  8. 8. The qualitative and semi-quantitative integrated analysis method for environmental trace micro-pollutants according to claim 1, wherein the number of fragment ions matched in the step S5 is more than or equal to 2.
  9. 9. The method of claim 1, wherein the threshold of the inverse dot product similarity in step S5 is 0.4.
  10. 10. The qualitative and semi-quantitative integrated analysis method of environmental trace micro-pollutants according to claim 1, wherein the environmental trace micro-pollutants comprise aromatic compounds, perfluoro and polyfluoroalkyl substances, and phthalate compounds.

Description

Qualitative and semi-quantitative integrated analysis method for environmental trace micro-pollutants based on mass spectrum full-scanning mode Technical Field The invention relates to the technical field of analysis of environmental trace micro-pollutants, in particular to a qualitative and semi-quantitative integrated analysis method of environmental trace micro-pollutants based on a mass spectrum full-scan mode. Background Organic micro-pollutants (OMPs) are widely present in aquatic environments and have long been identified as key human stress factors with a versatile ecological impact. These OMPs often coexist in the environment, possibly triggering adverse biochemical reactions of aquatic organisms by means of nutrient transfer mechanisms, constituting a potential risk to human health even at ultra trace concentrations (ppt grade). It is critical to comprehensively measure the presence of various micro-contaminants in an environment, one of the major challenges faced is filling the detection gap to bring undetected trace levels to a detectable concentration range. Under the rapid development of a chemical mass spectrum library, suspicious screening workflow realized by a High Resolution Mass Spectrum (HRMS) technology thoroughly innovates the field of monitoring micro pollutants. The analysis mode adopts a Data Dependent Acquisition (DDA) strategy to generate an experimental secondary spectrogram (MS/MS), and then the experimental secondary spectrogram is compared with a reference spectrogram library to realize compound identification. This technique has significant success in detecting hundreds of organic contaminants in a complex environmental matrix, even if the contaminants originate from thousands of non-target chromatographic features. However, DDA technology can only generate a fragmentation pattern of high abundance chemicals. Since most micro-contaminants are usually present in an environmental matrix at ultra trace levels, this traditional screening method may lead to significant identification blanks, such that a large number of low abundance feature substances are not identified as "chemically dark substances", for example, patent CN119936286 a discloses a qualitative and quantitative analysis method for autonomous non-targeted identification of halogenated organics for detection of halogenated organics in an original water sample, using a data dependent collection mode, to identify halogenated organics at a concentration of only 10-30 μg/L (ppb level) far from trace levels, whereas patent CN115469036 a discloses a screening and non-targeted analysis method for suspicious substances in food using UPLC-HRMS, also based on a data dependent collection mode, for automatic MS/MS collection of unknown exogenous organisms at low concentrations in complex biological samples, in which (pork samples) 48 veterinary drugs considered to be unknown are co-detected and identified, but which does not take into account that some chemicals may generate in-source cleavage, the relatively low ion abundance of which results in a loss of detection signal. Thus, using only DDA techniques to identify substances in a sample, there is significant one-sidedness. While Data Independent Acquisition (DIA) techniques can generate fragments for all precursor ions in the full scan spectrum, studies have shown that about half of the characteristic signals in the DIA spectrum are missing compared to the full scan data. In addition, full-scan mass spectrometry shows that most of the precursor ions can generate fragments within the source in amounts comparable to secondary fragments generated at low energies in the collision cell. Research proves that the sensitivity of the identification method based on fragments in the source can be more than 10 times higher than that of the traditional targeting analysis, and the method provides a new method for detecting trace chemical substances in the environment. There have been studies based on the in-source fragment identification method (ISFrag), but conventional in-source fragment identification methods (e.g., ISFrag) rely on the feature table generated by MS/MS secondary mass spectrometry to identify precursor ions and their in-source fragment ions. However, MS/MS secondary mass spectrometry feature tables often fail to cover a large number of low abundance fragment ions, resulting in many real fragment-precursor ion pairs being unrecognizable, thereby significantly increasing the false negative rate of analysis. In addition, in complex biological samples, ion signals with low abundance or poor chromatographic behavior are often lost during this pretreatment stage, resulting in a large number of real fragment-precursor ion pairs that cannot be identified, further increasing the false negative rate of the analysis. To address this fundamental limitation, new methods such as EISA-EXPOSOME attempt to bypass feature extraction and directly match the theoretical spectra of the entire