A method for analyzing methyl mercury methyl carbon in a complex matrix

Abstract

The application discloses a method for analyzing methyl mercury methyl carbon in a complex matrix, which comprises the following steps: taking a methyl mercury-containing sample to be measured, adding sodium tetraphenylborate to perform a phenyl derivatization reaction; performing headspace solid-phase microextraction enrichment on the derivatized sample; introducing the enriched solid-phase microextraction fiber into a gas chromatograph mass spectrometer to perform thermal desorption, separation and detection; and qualitatively or quantitatively analyzing the methyl mercury methyl carbon according to the retention time, mass spectrum response signal and isotope characteristics. The application is aimed at the problems of low methyl mercury content and strong matrix interference in a complex matrix such as a microbial culture system, and combines phenyl derivatization, solid-phase microextraction enrichment and gas chromatograph mass spectrometry detection, so that the enrichment efficiency and detection sensitivity are improved, the matrix interference is reduced, the method has the characteristics of high sensitivity, good matrix adaptability and stable repeatability, and is suitable for trace methyl mercury methyl carbon analysis in a complex culture matrix and isotope characteristic determination of a 13C labeled system.

Description

Technical Field

[0001] This invention relates to the field of environmental analysis and detection technology, and in particular to an analytical method for methylmercury methyl carbon in complex matrices. Background Technology

[0002] Methylmercury is one of the most toxic and biomagnified organic mercury forms in the environment, exhibiting strong neurotoxicity and bioaccumulation. It can be amplified step-by-step through the food chain, ultimately posing a persistent threat to ecosystem safety and human health. Existing research indicates that the formation of methylmercury is closely related to microbial metabolic activities, particularly in anaerobic or microaerobic environments. Some microorganisms can convert inorganic mercury into methylmercury, significantly altering the migration, transformation, and toxicity of mercury in the environment. Studies on the mechanisms of methylmercury formation show that different exogenous carbon sources and their transformation during intracellular metabolism not only affect the growth status and metabolic activity of microorganisms but may also directly or indirectly influence the source of methyl carbon in methylmercury. Therefore, establishing analytical methods for methyl carbon in methylmercury is crucial for revealing the methylmercury formation process and its carbon source in microbial culture systems, and provides necessary technical support for further understanding the microbial-mediated mercury methylation mechanism.

[0003] Currently, analytical studies on methylmercury primarily focus on the total amount and speciation analysis of environmental, biological, or contaminated samples. Commonly used methods include gas chromatography-mass spectrometry (GC-MS) with element-specific detectors (ESDs), GC-MS with atomic fluorescence spectrometry (AFI), inductively coupled plasma mass spectrometry (ICP-MS), and related techniques. These methods have a solid foundation in routine quantitative analysis of methylmercury, enabling relatively good qualitative and quantitative detection of the target analyte, and have been widely applied in environmental monitoring, risk assessment, and pollution research. However, when the research objective extends further to the source apportionment of methyl carbon in methylmercury, existing methods often struggle to simultaneously address requirements such as trace levels of the target analyte, interference from complex matrices, and the retention of methyl carbon information. In particular, some element-response-based detection techniques essentially detect mercury itself, making it difficult to directly retain molecular-level information related to the source of methyl carbon, thus exhibiting significant limitations in methyl carbon source analysis.

[0004] In microbial culture systems, methylmercury is typically present at trace levels, while the culture medium itself is a complex system. This system usually contains high concentrations of inorganic salts, unreacted substrates, vitamins, reducing agents, buffer salts, and metabolites, among other components. These coexisting substances can not only alter the state of methylmercury within the system but also introduce significant interference during sample pretreatment and detection. For example, during derivatization, different types of organic components and soluble metabolites may affect the efficiency of the derivatization reaction, leading to incomplete conversion of the target analyte. During enrichment, organic matter and salts in the complex matrix may affect the partitioning behavior of the target analyte among the liquid phase, headspace, and extraction fibers, causing fluctuations in extraction efficiency. In subsequent instrument detection, issues such as co-eluting components, matrix background, and signal suppression can further reduce the detection response and repeatability of the target analyte. The combined effect of these factors makes the analysis of methyl carbon in methylmercury from complex culture media significantly more challenging than that of conventional environmental samples.

[0005] Furthermore, methylmercury is typically present at low concentrations in microbial culture systems, and sample volumes are limited, often resulting in insufficient effective target amounts for analysis, further increasing the difficulty of method development. While some existing analytical methods can detect methylmercury, they suffer from problems such as cumbersome pretreatment steps, limited enrichment efficiency, insufficient sensitivity, and poor adaptability to complex culture media when applied to complex substrates. On the other hand, some elemental analysis methods, although possessing high detection sensitivity, often destroy organic structural information during detection, making them difficult to use directly for methyl carbon analysis and failing to meet the requirements of stable isotope tracing studies for preserving molecular structural information. For research subjects that require reliable detection at trace levels, maximizing the preservation of methyl carbon information, and adaptability to interference from complex culture media, current technology still lacks an analytical method that is highly sensitive, relatively simple to operate, suitable for complex culture systems, and applicable to methyl carbon source research.

[0006] With the development of stable isotope tracing technology, studying the source of methyl carbon in methylmercury using ¹³C-labeled carbon sources has become a research direction with clear scientific significance. However, to reliably determine the isotopic composition of methyl carbon in methylmercury in microbial culture systems, not only is sufficient sensitivity and resolution required from the detection platform, but also the minimization of target analyte loss during pretreatment and the avoidance of reaction inhibition, enrichment bias, and signal interference caused by complex matrices. Therefore, it is practically necessary to establish a method that simultaneously considers derivatization efficiency, enrichment capacity, detection sensitivity, and isotope signal recognition capability for the analysis of trace methyl carbon in complex culture media.

[0007] Therefore, it is necessary to establish a method for the analysis of trace methylmercury methyl carbon in complex culture media such as microbial culture systems. By optimizing the target derivatization method, enrichment method and detection conditions, the enrichment efficiency and detection response of the target can be improved, the interference of complex matrices on the analysis process can be reduced, and a reliable, stable and applicable analytical method can be provided for the study of the source of methylmercury methyl carbon. Summary of the Invention

[0008] To address the problems of low target analyte content, complex culture medium composition, strong matrix interference, limited enrichment efficiency during pretreatment, and insufficient detection response in the analysis of methylmercury methyl carbon in complex culture media such as microbial culture systems, existing technologies also struggle to meet the requirements of trace analysis and methyl carbon information interpretation. This invention provides an analytical method for methylmercury methyl carbon in complex matrices to achieve stable detection, reliable determination, and isotopic characterization of trace methylmercury methyl carbon in complex culture media.

[0009] To address the aforementioned technical problems, this invention provides an analytical method for methylmercury methyl carbon in complex matrices, comprising the following steps:

[0010] S1 Take the test sample containing methylmercury, add a phenyl derivatizing reagent to carry out a derivatization reaction, and convert methylmercury into a phenyl derivative;

[0011] S2 enriches the derivatized sample obtained from S1 by headspace solid-phase microextraction, thereby enriching the phenylated derivative on the solid-phase microextraction fiber.

[0012] S3 introduces the enriched solid-phase microextraction fiber into the injection port of a gas chromatography-mass spectrometry (GC-MS) instrument for thermal desorption, allowing the target analytes to enter the GC-MS instrument for separation and detection;

[0013] S4 performs qualitative or quantitative analysis of methylmercury methyl carbon based on the retention time, mass spectrometry response signal, and isotopic characteristics of the target analyte.

[0014] As a preferred technical solution, the phenyl derivative reagent in S1 is sodium tetraphenylborate.

[0015] As a preferred technical solution, the derivatization reaction in S1 is carried out in a brown headspace vial, and the reaction system includes ultrapure water, sodium acetate buffer, and the sample to be tested.

[0016] As a preferred technical solution, the derivatization reaction is carried out in a 40 mL brown headspace vial, and the reaction system includes 15 mL of ultrapure water, 2 mL of sodium acetate buffer, and the sample to be tested.

[0017] As a preferred technical solution, headspace solid-phase microextraction is used in S2, and the solid-phase microextraction fiber used is a 100 μm PDMS extraction column.

[0018] As a preferred technical solution, the headspace solid-phase microextraction in S2 has an extraction temperature of 65 ℃ and an extraction time of 45 min.

[0019] As a preferred technical solution, the injection port temperature in S3 is 200 ℃ and the carrier gas flow rate is 1.4 mL / min.

[0020] As a preferred technical solution, the complex matrix is ​​a microbial culture medium sample, which contains inorganic salts, unreacted substrates, metabolites and other organic components.

[0021] As a preferred technical solution, the method is applicable to the analysis of methyl carbon composition and isotope characterization of methylmercury in ¹³C-labeled microbial culture systems.

[0022] As a preferred technical solution, the ¹³C labeled microbial culture system is prepared by adding labeled carbon sources and unlabeled carbon sources to the culture medium in a certain proportion.

[0023] Compared with the prior art, this application has at least the following beneficial effects:

[0024] This invention employs a phenyl derivative strategy to convert methylmercury into a derivative more suitable for subsequent enrichment and gas chromatography-mass spectrometry detection. This improves the volatility, hydrophobicity, and detection response of the target analyte, thereby enhancing the sensitivity of trace methylmercury methyl carbon analysis in complex culture media.

[0025] This invention combines phenyl derivatization with solid-phase microextraction enrichment, which can improve the enrichment efficiency of target substances while reducing the complexity of pretreatment steps and reducing the interference of salts, organic substrates and metabolites in complex culture media on the analysis process.

[0026] This invention improves the thermal desorption efficiency, chromatographic separation effect, and mass spectrometry response stability of target analytes by optimizing solid-phase microextraction conditions and gas chromatography-mass spectrometry detection conditions, which is beneficial for achieving stable analysis of trace methylmercury methyl carbon in complex culture media.

[0027] This invention is applicable to complex culture media samples such as microbial culture systems, and can be used for qualitative or quantitative analysis of trace methylmercury methyl carbon, providing a reliable analytical method for the study of the source of methylmercury methyl carbon.

[0028] This invention constructs an analytical route that combines phenyl derivatization, solid-phase microextraction enrichment, and gas chromatography-mass spectrometry detection. It takes into account the adaptability to complex matrices, the needs of trace analysis, and the requirements for methyl carbon information resolution, and has good practicality and application value. Attached Figure Description

[0029] Figure 1 This is an experimental flowchart of the present invention for the analysis of trace methylmercury methyl carbon in complex culture media;

[0030] Figure 2 The method of the present invention is applied to 13 Figure showing the analytical results of the methyl carbon incorporation ratio of methylmercury in the C-labeled sodium pyruvate culture system. Detailed Implementation

[0031] To further illustrate the technical solution of the present invention, the embodiments of the present invention are described below in conjunction with specific examples. It should be noted that these embodiments are for illustrative purposes only and do not constitute a limitation on the scope of protection of the present invention. Equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should all fall within the scope of protection of the present invention.

[0032] This invention addresses the challenges of low methylmercury content, strong matrix interference, insufficient sensitivity of existing methods, and difficulty in retaining methyl carbon information in complex culture media. It provides an analytical scheme combining phenyl derivatization, solid-phase microextraction enrichment, and gas chromatography-mass spectrometry detection. Compared to existing technologies, this invention features high sensitivity, good matrix adaptability, and stable reproducibility, making it particularly suitable for the qualitative and quantitative analysis of trace methylmercury methyl carbon in complex samples such as microbial culture systems. 13 Isotope characterization in C-labeled systems.

[0033] like Figure 1 As shown, this invention provides a method for analyzing the methyl carbon of methylmercury in a complex matrix, comprising the following steps:

[0034] Take the test sample containing methylmercury, add a phenyl derivatizing reagent to carry out a derivatization reaction, and convert methylmercury into a phenyl derivative.

[0035] Preferably, sodium tetraphenylborate is used as the phenyl derivatization reagent. To prevent photodegradation of the sample, the derivatization reaction is carried out in a brown headspace vial, and the reaction system includes ultrapure water, sodium acetate buffer, and the sample to be tested.

[0036] As an optional implementation, a 40 mL brown headspace vial is used. The reaction system includes 15 mL of ultrapure water, 2 mL of sodium acetate buffer, and the sample to be tested. Phenylation derivatization can improve the volatility and hydrophobicity of the target analyte, thereby enhancing subsequent enrichment efficiency and detection response.

[0037] The derivatized sample was enriched by headspace solid-phase microextraction, so that the phenylated derivative was enriched on the solid-phase microextraction fiber.

[0038] Preferably, a 100-micron PDMS extraction column is used. Extraction temperature and time significantly affect the enrichment effect; as preferred conditions, the extraction temperature is controlled at 65 degrees Celsius, and the extraction time is 45 minutes. Solid-phase microextraction enrichment can improve the enrichment efficiency of target analytes while reducing pretreatment steps, effectively reducing the interference of salts, organic substrates, and metabolites in complex culture media on the analytical process.

[0039] The enriched solid-phase microextraction fiber is introduced into the injection port of a gas chromatography-mass spectrometry (GC-MS) instrument for thermal desorption, allowing the target analyte to enter the GC-MS instrument for separation and detection.

[0040] As preferred conditions, the injection port temperature was set to 200 degrees Celsius, and the carrier gas flow rate was 1.4 mL / min. Under these conditions, the target analyte exhibited good thermal desorption efficiency, chromatographic peak shape, and mass spectrometric response.

[0041] Based on the retention time, mass spectrometry response signal, and isotopic characteristics of the target analyte, qualitative or quantitative analysis of methylmercury methyl carbon is performed.

[0042] The above method is applicable to the analysis of methylmercury methyl carbon in complex culture media. The complex culture media sample is a microbial culture medium sample containing inorganic salts, unreacted substrates, metabolites, and other organic components. This method can also be used for... 13 Analysis of methyl carbon composition and isotopic characterization of methylmercury in C-labeled microbial culture system 13 The C-labeled microbial culture system is prepared by adding labeled and unlabeled carbon sources to the culture medium in a specific ratio.

[0043] The present invention will be further described below with reference to specific embodiments.

[0044] Example 1

[0045] In this embodiment, the analyte used in the method optimization and condition establishment stage is a methylmercury standard solution. As a standard sample with a clearly defined source of the target analyte and a relatively simple matrix composition, the methylmercury standard solution can be used for derivatization reagent screening, solid-phase microextraction condition optimization, gas chromatography-mass spectrometry analysis condition optimization, and methodological performance verification.

[0046] During the method optimization process, methylmercury standard solution was used as the object to compare the effects of different derivatization reagents on the methylmercury derivatization response. Key parameters such as solid-phase microextraction temperature, extraction time, injection port temperature and carrier gas flow rate were optimized to establish phenyl derivatization-solid-phase microextraction-gas chromatography-mass spectrometry analysis conditions suitable for the analysis of trace methyl carbon of methylmercury.

[0047] After establishing and optimizing the method, it was further applied to the analysis of methylmercury methyl carbon in complex culture substrate samples. The complex culture substrate samples were microbial culture media; specific application examples are further described below.

[0048] Example 2

[0049] To prevent photodegradation of the sample during pretreatment, the derivatization reaction was carried out in a 40 mL brown headspace vial. The reaction system consisted of 15 mL ultrapure water, 2 mL sodium acetate buffer, and a certain amount of the sample to be tested. Sodium tetraphenylborate was added to the reaction system as a derivatization reagent to convert methylmercury in the sample into a phenyl derivative for subsequent headspace solid-phase microextraction and gas chromatography-mass spectrometry detection.

[0050] During the screening of derivatization reagents, sodium tetraethylborate, sodium tetrapropylborate, and sodium tetraphenylborate were compared. The results showed that sodium tetraphenylborate-derived phenylmethylmercury exhibited higher chromatographic response and better enrichment, with the extraction efficiency of the phenylated product being approximately 5.2 times that of the conventional propylation system. Therefore, sodium tetraphenylborate was selected as the core derivatization reagent for the method of this invention.

[0051] After derivatization, headspace solid-phase microextraction (SPE) was used to enrich the target product. The SPE apparatus used was a manual handle with a 100 μm PDMS extraction column. Extraction time and temperature significantly affected the distribution equilibrium of the analyte among the sample matrix, headspace, and fiber coating phases. Selected ion monitoring (SIM) mode was employed, and the extraction conditions were optimized using the peak height of the molecular ion (m / z 294) of phenylmethylmercury as the response index. The results showed that extraction at 65 °C and a extraction time of 45 min yielded better enrichment and analytical response; therefore, this condition was selected as the SPE conditions for this embodiment.

[0052] After the above steps, the methylmercury in the sample is converted into a phenylated derivative suitable for headspace enrichment and gas chromatography-mass spectrometry detection, and enriched on the surface of the solid-phase microextraction fiber, providing a sample basis for subsequent gas chromatography-mass spectrometry analysis.

[0053] Example 3

[0054] The phenylmethylmercury derivative, enriched by solid-phase microextraction, was thermally desorbed at the injection port of a gas chromatography-mass spectrometry (GC-MS) system before being separated and detected by a mass spectrometer. To ensure high desorption efficiency and good peak shape, the injection port temperature and carrier gas flow rate were optimized. Results showed that the chromatographic response of the target analyte significantly improved when the injection port temperature increased from 180 °C to 200 °C; however, the response decreased with further temperature increases. Therefore, the injection port temperature was determined to be 200 °C. Further investigation was conducted on the effect of carrier gas flow rate on detection sensitivity. Results indicated that the peak area of ​​phenylmethylmercury reached its maximum at a carrier gas flow rate of 1.4 mL / min, while the retention time remained stable. Therefore, 1.4 mL / min was selected as the standard carrier gas flow rate for this method.

[0055] In the method validation, a series of methylmercury standard solutions of different concentrations were prepared to investigate the linear range and quantitative performance of this method. The results showed that within the absolute mass range of methylmercury (30–2000 ng), there was a good linear relationship between the quantitative ion signal and mass of the phenylated product, with a correlation coefficient R² reaching 0.9993. Further, considering the repeatability of the characteristic ion ratio (I295 / I294), the limit of quantitation for this method was determined to be 30 ng.

[0056] To evaluate the resolving power of this method for methylmercury methyl carbon isotope signals, the characteristic ion ratio (I295 / I294) was investigated within the aforementioned linear range. The results show that this ratio maintains good stability within the effective detection range, and the method can resolve approximately 0.3% of isotope enrichment changes; combined with natural... 13 C background abundance, when in the sample 13 Reliable quantification can be achieved when the carbon abundance is higher than approximately 1.4%. These results demonstrate that this method is not only suitable for the detection of trace methylmercury but also meets the requirements for subsequent methyl carbon isotope analysis.

[0057] Example 4

[0058] After completing the method establishment and optimization, the method of the present invention is applied to... 13 Analysis of methylmercury methyl carbon in C-labeled microbial culture system samples. The culture medium was prepared using a partial labeling method to ensure the detection of carbon isotope signals in methylmercury while maintaining normal strain growth. Taking the experimental group with a total carbon source concentration of 50 mM as an example, an unlabeled common carbon source was first added to the basal medium to reach a concentration of 45 mM; after high-temperature autoclaving, the high-concentration carbon source was added before inoculation. 13The C-labeled carbon source stock solution was aseptically added to the culture medium through a 0.22 μm filter membrane to achieve a total carbon source concentration of 50 mM, thereby making the molar ratio of labeled carbon source to unlabeled carbon source 1:9, i.e., the labeling abundance 10%. All isotope tracing experimental groups were prepared according to the above ratio.

[0059] In a single carbon source tracer system, sodium pyruvate was used as the experimental substrate, and different carbon sites were set up. 13 C-labeled group. All labeling experiments were conducted at a standard mercury exposure concentration of 0.1 mg / L. Parallel experimental groups of sodium pyruvate labeled at C1, C2, and C3 sites were set up for subsequent analysis of the methyl carbon composition of methylmercury. After cultivation, samples from the culture system were taken and analyzed according to the phenyl derivatization, solid-phase microextraction enrichment, and gas chromatography-mass spectrometry detection conditions described above in this invention. The carbon isotopic composition of methylmercury was calculated based on the characteristic ion ratios of the target analyte.

[0060] Figure 2 This invention demonstrates the application of the method of the present invention. 13 Analysis results of the methyl carbon incorporation ratio of methylmercury in the C-labeled sodium pyruvate culture system.

[0061] In the experiment, sodium pyruvate was used as the sole carbon source, and three experimental groups were set up with C1 site labeling, C2 site labeling, and C3 site labeling, with a labeling abundance of 10% in each group. After analyzing the culture system samples according to the method described in this invention, the proportion of methyl carbon in methylmercury from the labeled carbon source was calculated based on the characteristic ion ratio of methylmercury (295 / 294).

[0062] These results demonstrate that the method of the present invention can effectively distinguish the differences in isotopic signals at different carbon sites in complex microbial culture media, providing a feasible analytical means for elucidating the specific source of methyl carbon in methylmercury.

[0063] Using the above embodiments, the method of the present invention can identify the isotopic characteristics of methylmercury methyl carbon in complex microbial culture media and can be used to analyze the differences in the contribution of different carbon sites to the formation of methylmercury methyl carbon.

[0064] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for analyzing the methyl carbon of methylmercury in a complex matrix, characterized in that, Includes the following steps: S1 Take the test sample containing methylmercury, add a phenyl derivatizing reagent to carry out a derivatization reaction, and convert methylmercury into a phenyl derivative; S2 enriches the derivatized sample obtained from S1 by headspace solid-phase microextraction, thereby enriching the phenylated derivative on the solid-phase microextraction fiber. S3 introduces the enriched solid-phase microextraction fiber into the injection port of a gas chromatography-mass spectrometry (GC-MS) instrument for thermal desorption, allowing the target analytes to enter the GC-MS instrument for separation and detection; S4 performs qualitative or quantitative analysis of methylmercury methyl carbon based on the retention time, mass spectrometry response signal, and isotopic characteristics of the target analyte.

2. The analytical method according to claim 1, characterized in that, The phenyl derivative reagent in S1 is sodium tetraphenylborate.

3. The analytical method according to claim 1, characterized in that, The derivatization reaction in S1 is carried out in a brown headspace vial, and the reaction system includes ultrapure water, sodium acetate buffer, and the sample to be tested.

4. The analytical method according to claim 1, characterized in that, The derivatization reaction was carried out in a 40 mL brown headspace vial, and the reaction system included 15 mL of ultrapure water, 2 mL of sodium acetate buffer, and the sample to be tested.

5. The analytical method according to claim 1, characterized in that, In S2, headspace solid-phase microextraction is used, and the solid-phase microextraction fiber used is a 100 μm PDMS extraction column.

6. The analytical method according to claim 1, characterized in that, The headspace solid-phase microextraction in S2 is performed at an extraction temperature of 65 °C for 45 min.

7. The analytical method according to claim 1, characterized in that, The injection port temperature in S3 is 200 ℃, and the carrier gas flow rate is 1.4 mL / min.

8. The analytical method according to claim 1, characterized in that, The complex matrix is ​​a microbial culture medium sample, which contains inorganic salts, unreacted substrates, metabolites, and other organic components.

9. The analytical method according to claim 8, characterized in that, The method is applicable to the analysis of methyl carbon composition and isotopic characterization of methylmercury in ¹³C-labeled microbial culture systems.

10. The analytical method according to claim 9, characterized in that, The ¹³C labeled microbial culture system is prepared by adding labeled carbon sources and unlabeled carbon sources to the culture medium in a certain proportion.

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