A method for detecting isotopic internal standards of synthetic biological fermentation metabolites

Abstract

The application discloses a kind of synthetic biological fermentation metabolite isotopic internal standard detection method, belong to analytical detection technical field.The method is by to fermentation process carries out multi-point sampling, constructs metabolic fingerprint and monitors chiral evolution, determines the harvest window of target and directional purification strategy;In harvest window period, corresponding stable isotope-labeled substrate of target biosynthesis path is introduced to fermentation system, and biological self-source isotopic-labeled internal standard consistent with target structure is prepared by microorganism metabolism;After the same purification strategy is handled target and internal standard respectively, by mass balance method and isotopic internal standard-quantitative nuclear magnetic resonance method and / or isotopic internal standard-liquid chromatography tandem mass spectrometry method, combined value is determined;Comprehensive evaluation uncertainty obtains standard value.The application is with process monitoring-internal standard preparation-closed loop strategy of combined value to eliminate the system deviation caused by replacement internal standard, applicable to high-precision purity of microbial fermentation product value.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a method for determining the purity of synthetic biological fermentation metabolites using isotope internal standards, which is particularly suitable for the development of standard substances for high-value metabolites produced by microbial fermentation. Background Technology

[0002] The synthetic biology industry utilizes microbial fermentation to produce high-value natural metabolites such as L-ergothioneine, γ-aminobutyric acid (GABA), and resveratrol on a large scale, which are widely used in functional foods, cosmetics, and pharmaceuticals. Purity reference materials are the cornerstone of quality control and metrological traceability for these products, while the methods for determining the values ​​of reference materials are a common key technology for building this cornerstone.

[0003] Currently, the determination of purity standards for fermentation metabolites faces the following technical challenges: First, there is a limitation on the availability of isotopic internal standards. High-precision characterization using isotope dilution mass spectrometry and quantitative nuclear magnetic resonance (NMR) typically relies on stable isotope-labeled analogs of the target analyte as internal standards. However, for a wide variety of novel or uniquely structured fermentation metabolites, commercially available stable isotope-labeled internal standards are often difficult to obtain immediately, and custom synthesis is time-consuming and costly. In practice, chemical structural analogs are often used as surrogate internal standards, but these surrogate standards inherently differ from the target analyte in chromatographic retention behavior, ionization efficiency, and NMR response, introducing biases that are difficult to systematically correct and affecting the accuracy of characterization.

[0004] Second, there is a disconnect between the determination process and the fermentation process. Existing determination methods mostly focus on the purified products after fermentation, failing to utilize the dynamic changes in target compound synthesis and accumulation, byproduct spectrum evolution, and chiral configuration stability during fermentation. This lack of process information leads to purification strategies often relying on experience and lacking data support, making it difficult to accurately remove specific impurities. In particular, it easily overlooks chiral isomer impurities that may arise in the later stages of fermentation, affecting the reliability of the final purity determination.

[0005] Third, an incomplete understanding of the impurity profile limits the accuracy of the mass balance method. The mass balance method is one of the fundamental methods for purity determination, and its accuracy depends on the comprehensive identification and accurate quantification of various impurities in the sample. For metabolites derived from fermentation, the composition of organic impurities is complex, including precursors, intermediates, and byproducts from biosynthetic pathways, other metabolites produced during fermentation, and potentially residues introduced by purification processes. A lack of systematic understanding of the impurity profile leads to incomplete quantification of organic impurities in the mass balance method, affecting the accuracy of the determination results. Summary of the Invention

[0006] The purpose of this invention is to provide an isotope internal standard detection method for synthetic bio-fermentation metabolites, in order to solve the technical problems in the prior art, such as large system bias caused by the use of alternative internal standards due to the lack of commercial isotope internal standards, and incomplete understanding of impurity spectra and lack of data support for purification strategies due to the disconnect between the determination process and the fermentation process.

[0007] To achieve the above objectives, the present invention provides the following technical solution: An isotope internal standard detection method for synthetic bio-fermentation metabolites, used to determine the purity of the synthetic bio-fermentation metabolites, includes the following steps: (1) Fermentation process information acquisition Multi-point sampling is performed during the synthetic bio-fermentation process to construct a metabolic fingerprint profile of the fermentation process to identify impurity profile characteristics and monitor the dynamic changes in the chiral purity of the target analyte. Based on the impurity profile characteristics and the dynamic changes in chiral purity, the harvest window and targeted purification strategy of the target analyte are determined.

[0008] Specifically, the dynamic changes in chiral purity can be monitored by coupling an online polarimeter with a chiral chromatographic column. The metabolic fingerprint can be constructed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF / MS) for non-targeted metabolomics analysis. By analyzing samples taken at different time points, the trends in target concentration with fermentation time, the types and contents of major impurities, and the evolution of target enantiomer excess values ​​with fermentation time can be obtained. Based on the above information, the optimal harvest window can be determined when the target accumulation reaches an acceptable level and the impurity content and chiral impurity ratio are low. Targeted purification strategies can then be developed based on the impurity profile characteristics.

[0009] (2) Preparation of biologically derived isotope-labeled internal standards According to the harvest window determined in step (1), after the fermentation enters the stable period, a stable isotope-labeled substrate corresponding to the biosynthetic pathway of the target substance is introduced into the fermentation system. The stable isotope is integrated into the target substance molecule through microbial metabolism, and a fermentation product containing an isotope-labeled internal standard is obtained. The isotope-labeled internal standard is consistent with the chemical structure and chiral configuration of the target substance.

[0010] The stable isotope-labeled substrates are selected based on the specific biosynthetic pathway of the target analyte. For example, for sulfur-containing amino acid metabolites with histidine as a precursor (such as L-ergothioneine), a combination of L-[¹³C6]histidine and deuterium water (D₂O) can be selected; for amine metabolites with glutamate as a precursor (such as γ-aminobutyric acid), a combination of [¹³C5]glutamate and deuterium water can be selected; and for phenolic metabolites synthesized via the phenylpropane metabolic pathway (such as resveratrol), a combination of [¹³C6] glucose and deuterium water can be selected. This synergistic labeling strategy, combining ¹³C-labeled precursors with deuterium water, allows for the simultaneous labeling of multiple sites and nuclides within the molecule using the microbial metabolic network.

[0011] Preferably, the labeled substrate is slowly added to the fermentation system in the form of a sterile concentrate after the fermentation has entered the stationary phase, in order to reduce the impact on microbial metabolism. During the addition process, the concentration of unlabeled natural substrate in the fermentation system is maintained at no less than 30% of the normal level to ensure the normal growth and metabolic activity of microorganisms and to avoid a significant decrease in the yield of the target product due to excessive substitution of the labeled substrate.

[0012] The obtained isotope-labeled internal standards were confirmed by high-resolution mass spectrometry to have a mass shift between their molecular weight and that of the unlabeled target analytes, corresponding to the number of isotope-labeled atoms.

[0013] (3) Homologous purification The targeted purification strategy determined in step (1) was used to purify the fermentation product containing the target analyte and the fermentation product containing the isotope-labeled internal standard obtained in step (2), respectively. The targeted purification strategy can be designed according to the specific physicochemical properties of the target analyte and impurities, including but not limited to combinations of one or more of the following unit operations: solvent extraction, membrane separation, macroporous adsorption resin chromatography, ion exchange chromatography, reversed-phase preparative chromatography, chiral preparative chromatography, recrystallization, freeze drying, etc.

[0014] Since the target analyte and the labeled internal standard use the same fermentation strain, the same fermentation process, and the same purification procedure, they are highly consistent in chemical structure, chiral configuration, and impurity spectrum characteristics, except for the difference in isotopic composition. This provides a highly consistent internal standard material for subsequent isotopic internal standard determination.

[0015] (4) Joint setting The purified target analyte is subjected to a combined determination. The combined determination includes the mass balance method and at least one method selected from isotope internal standard quantitative nuclear magnetic resonance (qNMR) and isotope internal standard liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0016] Mass balance method: Based on the impurity spectrum characteristics identified in step (1), organic impurities are classified and quantified separately. Specifically, organic impurities can be classified into four categories according to their source and structural characteristics: structural analogs (biosynthetic precursors, intermediates, etc.), chiral isomers, fermentation characteristic impurities (other metabolic byproducts), and process residues. Appropriate analytical methods are used for quantitative determination of each category. Among them, water content can be determined by Karl Fischer method, residual solvents can be determined by headspace gas chromatography, inorganic impurities can be determined by ignition residue method or inductively coupled plasma mass spectrometry, and organic impurities can be determined by high performance liquid chromatography or chromatography-mass spectrometry. The content of chiral isomers is included as an independent item in the purity calculation after chiral chromatography determination. The purity of the target analyte is obtained by subtracting the sum of the contents of all types of impurities from 100%.

[0017] Isotope internal standard-quantitative NMR method: Accurately weigh the target sample and the isotope-labeled internal standard prepared and purified in steps (2)-(3), dissolve them in a suitable deuterated solvent, and collect the 1H NMR spectrum (¹H-NMR). Select the quantitative peaks in the target and labeled internal standards where the chemical shifts do not overlap for independent integration. When ¹³C labeling is used, the ¹H at the labeling site splits into double peaks due to ¹³C-¹H coupling, which can be naturally separated from the corresponding single peak of the unlabeled target, facilitating independent integration. Calculate the purity of the target using the following formula: P_x=P_BLIS×(m_BLIS / m_x)×(I_x / I_BLIS)×(M_x / M_BLIS) In the formula, P_x is the purity of the target analyte, P_BLIS is the purity of the isotope-labeled internal standard (the purity of the internal standard can be predetermined using the mass balance method), m_x and m_BLIS are the sample weights of the target analyte and the internal standard, respectively, I_x and I_BLIS are the integral areas of the selected quantitative peak, and M_x and M_BLIS are the molar masses of the target analyte and the internal standard, respectively (M_BLIS needs to be corrected according to the isotope substitution situation).

[0018] Isotope internal standard-liquid chromatography-tandem mass spectrometry: Detection is performed using multiple reaction monitoring (MRM) mode. Due to the mass difference between the target analyte and the isotope-labeled internal standard, their parent ion-daughter ion transition pairs can be naturally separated during mass spectrometry detection without interference. Calibration curves are established using a series of target analyte standard solutions at different concentrations and isotope-labeled internal standards at constant concentrations, and these calibration curves are used for quantification of the target analyte.

[0019] (5) Uncertainty assessment The uncertainty introduced by each determination method, the uncertainty of batch variation, the uncertainty caused by homogeneity, and the uncertainty caused by stability are comprehensively evaluated to obtain the combined standard uncertainty. Based on the combined standard uncertainty, the expanded uncertainty is obtained to form the final standard value and its expanded uncertainty.

[0020] (6) Application of collaborative verification Using the defined target analyte as a standard, collaborative validation of detection methods for typical matrices is performed. A matrix-matched calibration curve based on the isotope-labeled internal standard is established, and a mechanism for uncertainty transfer from the standard analyte to the detection results is established. Specifically, the standard analyte can be added to a blank matrix containing no target analyte, and matrix-matched calibration is performed using the isotope-labeled internal standard. Pretreatment is optimized to ensure the target analyte recovery meets analytical requirements, validating the applicability of the method. The standard value, expanded uncertainty, and traceability information of each uncertainty component are presented in the form of a digital certificate, realizing the transfer of uncertainty from the standard analyte to the actual sample detection results.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention introduces stable isotope-labeled precursors into the fermentation system and utilizes the microbial metabolic network for bio-autologous synthesis, overcoming the limitations of commercial stable isotope internal standard supply for the development of reference materials for novel or special structured metabolites. It is highly versatile and cost-controllable.

[0022] The labeled internal standard prepared by this invention has a chemical structure and chiral configuration that are completely consistent with the target analyte. Moreover, the preparation and purification processes are from the same source and are synchronized. This effectively eliminates the deviations in chromatographic retention behavior, ionization efficiency, and nuclear magnetic resonance response caused by structural differences in the substitute internal standard, laying a good material foundation for high-precision joint determination.

[0023] This invention provides data support for the comprehensive identification of impurities and the precise formulation of purification strategies through the systematic monitoring of metabolic fingerprinting and chiral evolution during the fermentation process. This gives the formulation of purification strategies a data basis and can effectively avoid the risk of increased chiral impurities due to improper harvesting timing.

[0024] This invention directly uses the impurity spectrum characteristics identified during the fermentation process as the basis for impurity classification in the mass balance method, realizing closed-loop data utilization from process information to value determination operation. This effectively avoids deviations in the mass balance method value determination caused by incomplete impurity recognition, and improves the completeness and accuracy of organic impurity quantification.

[0025] The closed-loop method of process monitoring, internal standard preparation, and joint value determination constructed in this invention is beneficial to the traceability of the determination results and significantly improves the reliability of the standard material.

[0026] The establishment of matrix-based co-validation and uncertainty propagation mechanisms facilitates the application of standard materials in practical testing and helps downstream laboratories achieve metrological uniformity. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are only for explaining the invention and do not constitute any limitation on the scope of protection of the invention. Unless otherwise specified, the raw materials, reagents, and equipment used in the embodiments are all conventional commercially available products, and the specific operating conditions can be appropriately adjusted according to actual conditions.

[0028] Example 1: Detection and determination of L-ergothioneine purity standard material This embodiment uses the fermentation of Yersinia lipolytica to produce L-ergothionein as an example to illustrate the specific implementation of the present invention.

[0029] I. Information Acquisition during Fermentation Process Fermentation was carried out in a fermentation system suitable for the growth of *Yarrowia lipolyticis*. Samples were taken at different time points during the fermentation process.

[0030] Untargeted metabolomics analysis was performed on samples at various time points using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF / MS) to construct a metabolic fingerprint of the fermentation process. Spectral analysis revealed the accumulation trend of L-ergothioneine concentration over fermentation time, as well as the changing trends in the types and contents of major impurities (including the biosynthetic precursor L-histidine, the oxidation byproduct ergothioneine sulfoxide, and residual components of the culture medium).

[0031] Meanwhile, an online polarimeter was coupled with a chiral chromatographic column to monitor the dynamic changes in the ratio of L-ergothioneine to its enantiomer D-ergothioneine in the fermentation broth, and to obtain the evolution of the target enantiomer excess value (ee value) with fermentation time.

[0032] By considering both the accumulation trend of the target compound and the trend of chiral purity changes, the optimal harvest window is determined (e.g., the fermentation time point when the target compound accumulation reaches a plateau and the enantiomeric excess value is at a high level). Based on the impurity profile characteristics identified by the metabolic fingerprint, and combined with the differences in physicochemical properties between the target compound and various impurities (such as molecular size, charge properties, polarity, solubility, etc.), a targeted purification strategy is formulated. For example, the purification strategy may include one or more combinations of the following unit operations: ethanol precipitation to remove large polysaccharide molecules, cation exchange chromatography to remove anionic impurities, reversed-phase preparative chromatography to separate the target compound from histidine and oxidation byproducts, chiral preparative chromatography to remove D-ergothionein (if chiral monitoring shows the accumulation of chiral isomers), ethanol / water recrystallization, and freeze-drying.

[0033] II. Preparation of Biologically Autologous Isotope Labeled Internal Standards Based on the determined harvest window, parallel fermentation batches were designed for preparing labeled internal standards. After fermentation entered the stationary phase, a sterile concentrate of the L-[¹³C6]histidine and deuterium water (D₂O) labeled substrate was slowly added to the fermentation broth under aseptic conditions. The flow rate was controlled within a certain range to avoid drastic disturbance to the fermentation system, while maintaining the concentration of unlabeled native histidine in the fermentation system at no less than 30% of the normal level to ensure normal microbial activity. Fermentation continued until the preset harvest window, and the fermentation broth was collected to obtain the fermentation product containing the isotope-labeled internal standard.

[0034] Labeled batches of fermentation broth were treated with the exact same purification strategy as the target analyte to obtain isotopically labeled L-ergothioneine. This achieved homologous preparation and simultaneous purification of the labeled internal standard and the target analyte, which are chemically identical except for their isotopic composition.

[0035] The labeled product was confirmed using high-resolution mass spectrometry (HRMS): the mass number of the molecular ion peak of the labeled product was detected, and the mass shift between it and the unlabeled L-ergothionein should correspond to the number and type of introduced isotopic labeled atoms (e.g., when 6 ¹³C atoms are introduced, the mass shift is approximately +6 Da). This confirmation result indicates that the labeled product is an isotopically labeled analog of the target analyte.

[0036] III. Homologous Purification Following the defined targeted purification strategy, the unlabeled target analyte fermentation broth and the labeled internal standard fermentation broth were purified separately. Specific conditions for each unit operation (such as column specifications, mobile phase composition, elution gradient, fraction collection range, recrystallization solvent system, etc.) can be optimized and adjusted based on actual separation results.

[0037] IV. Joint setting (a) Mass balance method Based on the characteristics of impurity profiles identified by metabolic fingerprinting, organic impurities are classified into the following categories: structural analogs (such as biosynthetic precursors L-histidine, related intermediates, etc.), chiral isomers (such as D-ergothioneine), fermentation characteristic impurities (such as oxidation byproducts of ergothioneine sulfoxide, other microbial metabolites), and process residues (such as residual solvents that may be introduced during purification, chromatographic media precipitates, etc.).

[0038] Various impurities were quantitatively determined using appropriate methods: moisture was determined using the Karl Fischer method; residual solvents were determined using headspace gas chromatography; inorganic impurities were determined using the residue on ignition method or inductively coupled plasma mass spectrometry; structural analogs and fermentation-characteristic impurities among organic impurities were determined using high-performance liquid chromatography or liquid chromatography-mass spectrometry; the content of chiral isomers was determined by chiral chromatography and included as a separate item in the purity calculation. The purity of the main component was obtained by subtracting the contents of all impurities.

[0039] (II) Isotope internal standard-quantitative nuclear magnetic resonance method Accurately weigh appropriate amounts of the target sample and the labeled internal standard (e.g., 3-8 mg each), dissolve them together in a suitable deuterated solvent (e.g., D₂O), transfer them to an NMR sample tube, and acquire ¹H-NMR spectra on an NMR spectrometer. Select quantitative peaks in the target sample and the labeled internal standard whose chemical shifts do not interfere with each other for independent integration.

[0040] When the internal standard is ¹³C, the ¹H at the labeled site splits into a doublet due to ¹³C-¹H coupling, naturally separating from the singlet of the unlabeled target analyte at that chemical shift, facilitating accurate quantification. The purity of the target analyte is calculated using the aforementioned formula.

[0041] (III) Isotope internal standard-liquid chromatography-tandem mass spectrometry Multiple reaction monitoring (MRM) mode was used for liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis. Due to the mass difference between the target analyte and its isotope-labeled internal standard, their respective parent ion-daughter ion transition pairs could be completely separated during mass spectrometry detection.

[0042] Accurately weigh the target analyte sample and prepare a series of standard solutions of varying concentrations. Add a labeled internal standard solution of constant concentration to each standard solution. Inject the sample for analysis and plot a calibration curve with the peak area ratio of the target analyte to the internal standard as the ordinate and the target analyte concentration as the abscissa. Separately, accurately weigh the target analyte sample, add a labeled internal standard solution of the same concentration as the standard solutions, and perform the same analysis. Substitute the peak area ratio of the target analyte to the internal standard in the sample into the calibration curve to calculate the content of the target analyte in the sample.

[0043] (iv) Summary of the set value results The results obtained from the above-mentioned methods are summarized, and appropriate statistical methods (such as Cochran's method or Grubbs' method for outlier test) are used to test the consistency of the results. The standard value is obtained by weighted average or equal weighted average.

[0044] V. Uncertainty Assessment The uncertainty components are comprehensively evaluated, including: uncertainty introduced by the determination method (the uncertainty of each determination method is evaluated separately and then combined); batch variation uncertainty (obtained by statistical analysis of the determination results of multiple independent fermentation batches); homogeneity uncertainty (obtained by homogeneity test of the packaged samples); and stability uncertainty (obtained by long-term stability test of the samples).

[0045] The combined standard uncertainty is obtained by combining the above components, and the expanded uncertainty is obtained by multiplying it by the coverage factor k=2.

[0046] VI. Application Collaborative Validation The L-ergothioneine standard, after being predefined, was added to a typical blank matrix (such as a cosmetic matrix or food matrix) containing no target analyte. Matrix matching calibration was performed using the isotope-labeled internal standard of this invention to verify the applicability of the method. The accuracy of the method was evaluated through a spike recovery experiment; the matrix effect was assessed by comparing the slope difference between the matrix-matched standard curve and the pure solvent standard curve. Based on this, a mechanism for transferring uncertainty from the standard to the actual sample detection results was established, presenting the standard value, expanded uncertainty, and traceability information of each uncertainty component in an appropriate form (such as a digital certificate) to facilitate traceability for downstream laboratories.

[0047] Example 2: Detection and determination of purity standard material for γ-aminobutyric acid This embodiment uses the production of γ-aminobutyric acid by lactic acid bacteria fermentation as an example to illustrate the applicability of the method of the present invention to different types of metabolites.

[0048] I. Information Acquisition during Fermentation Process Fermentation was carried out in a fermentation system suitable for lactic acid bacteria growth. Samples were taken at different time points during the fermentation process, and untargeted metabolomics analysis was performed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF / MS) to construct a metabolic fingerprint profile, obtaining the accumulation trend of γ-aminobutyric acid (GABA) and the changing trends of major impurities (including residual precursor L-glutamate, lactic acid, and other fermentation byproducts). Simultaneously, an online polarimeter coupled with a chiral column was used to monitor the dynamic changes in the chiral purity of GABA.

[0049] The optimal harvest window is determined by combining the accumulation trend of the target compound with the trend of chiral purity changes. A targeted purification strategy is then developed based on the characteristics of the impurity profile. For example, since γ-aminobutyric acid (GABA) is a small-molecule amino acid, the purification strategy may include one or more combinations of unit operations such as activated carbon decolorization, ion exchange chromatography (e.g., anion exchange to remove lactic acid, cation exchange to separate GABA from glutamic acid using isoelectric point differences), recrystallization, and freeze-drying. If chiral monitoring indicates no risk of racemization throughout the process, the chiral separation step can be omitted from the purification strategy to simplify the process.

[0050] II. Preparation of Biologically Autologous Isotope Labeled Internal Standards Based on a defined harvest window, parallel fermentation batches were designed. After fermentation reached the stationary phase, a combined labeled substrate of [¹³C5]glutamic acid and deuterium water was added to the fermentation system, and fermentation continued until the preset harvest window. The fermentation broth was collected to obtain the fermentation product containing the isotopically labeled internal standard. The labeled batch fermentation broth was treated with the same purification strategy as the target analyte to obtain isotopically labeled γ-aminobutyric acid (GABA). High-resolution mass spectrometry confirmed that its molecular weight showed a mass shift corresponding to the number of introduced isotopic atoms compared to the unlabeled target analyte.

[0051] Sections 3-6: Purification, Joint Value Assignment, Uncertainty Evaluation, and Application Validation Using the same procedure as in Example 1, the target analyte and the labeled internal standard were purified separately according to the targeted purification strategy. The mass balance method and at least one of the isotope internal standard-qNMR and / or isotope internal standard-LC-MS / MS methods were used for joint determination. The uncertainty was comprehensively evaluated and matrix synergistic verification was performed.

[0052] Example 3: Detection and determination of resveratrol purity standard material This embodiment uses the fermentation of Saccharomyces cerevisiae to produce resveratrol as an example to further illustrate the applicability of the method of the present invention to different types of metabolites.

[0053] I. Information Acquisition during Fermentation Process Fermentation was carried out in a fermentation system suitable for the growth of *Saccharomyces cerevisiae*. Samples were taken at different time points during fermentation, and untargeted metabolomics analysis was performed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF / MS) to obtain the accumulation trend of trans-resveratrol and the changing trends of major impurities (such as coumaric acid and cinnamic acid, intermediates in the biosynthetic pathway). Simultaneously, an online polarimeter coupled with a chiral column was used to monitor the dynamic changes in configurational purity (with a focus on monitoring the formation of cis isomers).

[0054] The optimal harvest window is determined by combining the accumulation trend of the target compound and the trend of changes in configuration purity. A targeted purification strategy is developed based on the characteristics of the impurity profile. For example, for resveratrol fermentation broth, the purification strategy may include one or more combinations of unit operations such as organic solvent extraction (e.g., ethyl acetate extraction), silica gel column chromatography, reversed-phase preparative chromatography, recrystallization, and freeze-drying. If configuration monitoring shows that the cis isomer content remains consistently low, the purification strategy can focus on optimizing conditions for the retention of the trans configuration, without the need for additional cis-trans isomer separation steps.

[0055] II. Preparation of Biologically Autologous Isotope Labeled Internal Standards Based on a defined harvest window, parallel fermentation batches were designed. After fermentation reached the stationary phase, a combined labeled substrate of [¹³C6] glucose and deuterium water was added to the fermentation system, and fermentation continued until the preset harvest window. The fermentation broth was collected to obtain the fermentation product containing the isotopically labeled internal standard. The labeled batch fermentation broth was treated with the same purification strategy as the target analyte to obtain isotopically labeled resveratrol. High-resolution mass spectrometry confirmed that its molecular weight showed a mass shift corresponding to the number of introduced isotopic atoms compared to the unlabeled target analyte.

[0056] Sections 3-6: Purification, Joint Value Assignment, Uncertainty Evaluation, and Application Validation Using the same procedure as in Example 1, the target analyte and the labeled internal standard were purified separately according to the targeted purification strategy. The mass balance method and at least one of the isotope internal standard-qNMR and / or isotope internal standard-LC-MS / MS methods were used for joint determination. The uncertainty was comprehensively evaluated and matrix synergistic verification was performed.

[0057] The above three examples, covering metabolites with different structural types of sulfur-containing amino acids, amines, and phenols, demonstrate that the method of the present invention has good universality and can be applied to the purity determination of various microbial fermentation metabolites.

[0058] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for isotopic internal standard detection of synthetic bio-fermentation metabolites, used to determine the purity of the synthetic bio-fermentation metabolites, characterized in that, Includes the following steps: (1) Multiple sampling is performed during the synthetic bio-fermentation process to construct a metabolic fingerprint spectrum of the fermentation process to identify impurity spectrum characteristics and monitor the dynamic changes in the chiral purity of the target compound. Based on the impurity spectrum characteristics and the dynamic changes in chiral purity, the harvest window and targeted purification strategy of the target compound are determined. (2) According to the harvest window, after the fermentation enters the stable period, a stable isotope-labeled substrate corresponding to the biosynthetic pathway of the target substance is introduced into the fermentation system. The isotope is integrated into the target substance molecule through microbial metabolism to obtain a fermentation product containing an isotope-labeled internal standard. The isotope-labeled internal standard is consistent with the chemical structure and chiral configuration of the target substance. (3) Using the targeted purification strategy, the fermentation product containing the target substance and the fermentation product containing the isotope-labeled internal standard obtained in step (2) are purified respectively; (4) The purified target analyte is subjected to a combined determination, the combined determination including the mass balance method and at least one method selected from the isotope internal standard-quantitative nuclear magnetic resonance method and the isotope internal standard-liquid chromatography-tandem mass spectrometry method; wherein, the classification of impurities in the mass balance method is determined based on the impurity spectral characteristics identified in step (1); the isotope internal standard-quantitative nuclear magnetic resonance method and the isotope internal standard-liquid chromatography-tandem mass spectrometry method use the isotope-labeled internal standard prepared and purified in steps (2)-(3) as the internal standard; (5) By comprehensively evaluating the uncertainty of each determination method, the uncertainty of batch variation, the uncertainty caused by homogeneity and the uncertainty caused by stability, the standard value and its expanded uncertainty are obtained. (6) Using the target material after setting the value as a standard material, conduct collaborative verification of detection methods for typical matrices, establish a matrix matching calibration curve based on the isotope-labeled internal standard, and establish a mechanism for the transfer of uncertainty from the standard material to the detection results.

2. The method according to claim 1, characterized in that, In step (1), the dynamic change in chiral purity is monitored by coupling an online polarimeter with a chiral chromatographic column; the metabolic fingerprint is constructed by non-targeted metabolomics analysis using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

3. The method according to claim 1, characterized in that, In step (2), the stable isotope-labeled substrate is selected according to the biosynthetic pathway of the target, specifically from any of the following combinations: (a) a combination of L-[¹³C6]histidine and D2O for L-ergothioneine; (b) a combination of [¹³C5]glutamic acid and D2O for γ-aminobutyric acid; and (c) a combination of [¹³C6] glucose and D2O for resveratrol.

4. The method according to claim 1, characterized in that, In step (2), the labeled substrate is added to the fermentation system in the form of a sterile concentrate after the fermentation enters the stable period, and the concentration of unlabeled natural substrate in the fermentation system is maintained at no less than 30% of the normal level during the addition process.

5. The method according to claim 1, characterized in that, The isotope-labeled internal standard obtained in step (2) was confirmed by high-resolution mass spectrometry to have a mass shift between its molecular weight and that of the unlabeled target analyte, corresponding to the number of isotope-labeled atoms.

6. The method according to claim 1, characterized in that, In step (4), the mass balance method includes: according to the impurity spectrum characteristics identified in step (1), the organic impurities are divided into four categories: structural analogs, chiral isomers, fermentation characteristic impurities and process residues, and quantitative determination is performed on each category; wherein, the content of chiral isomers is included as an independent item in the purity calculation after being determined by chiral chromatography.

7. The method according to claim 1, characterized in that, In step (4), the purity of the target analyte is calculated using the following formula in the isotope internal standard-quantitative nuclear magnetic resonance method: P_x=P_BLIS×(m_BLIS / m_x)×(I_x / I_BLIS)×(M_x / M_BLIS) In the formula, P_x is the purity of the target analyte, P_BLIS is the purity of the isotope-labeled internal standard, m_x and m_BLIS are the sample weights of the target analyte and the labeled internal standard, respectively, I_x and I_BLIS are the integral areas of the selected quantitative peaks, and M_x and M_BLIS are the molar masses of the target analyte and the labeled internal standard, respectively.

8. The method according to claim 1, characterized in that, In step (4), the isotope internal standard-liquid chromatography-tandem mass spectrometry method adopts multiple reaction monitoring mode. Based on the isotope mass difference between the target analyte and the isotope-labeled internal standard, the corresponding parent ion-daughter ion transition pairs are selected for detection. A calibration curve is established with a series of target analyte standard solutions and a constant concentration of the isotope-labeled internal standard. The target analyte is quantified using the calibration curve.

9. The method according to claim 1, characterized in that, In step (6), the collaborative verification includes: adding the standard substance to a blank matrix without the target substance, performing matrix matching calibration using the isotope-labeled internal standard, optimizing the pretreatment to ensure that the target substance recovery rate meets the analytical requirements; and presenting the standard value, expanded uncertainty, and traceability information of each uncertainty component in the form of a digital certificate to realize the transfer of uncertainty from the standard substance to the actual sample detection results.

10. The method according to any one of claims 1 to 9, characterized in that, The synthetic bio-fermentation metabolites are L-ergothioneine, γ-aminobutyric acid, or resveratrol produced by microbial fermentation.