Mass spectrometry method and system

Abstract

In mass spectrometry, a deuterium-resolved mass spectrum of a gas sample that determines the composition of volatile metabolites excreted by a human or animal subject after ingestion of deuterated water. The deuterium-resolved mass spectrum is used to determine the presence or amount of at least one deuterated metabolite. In addition, a corresponding mass spectrometry system is also disclosed.

Description

Technical Field

[0001] The present invention relates to a mass spectrometry method and a mass spectrometry system. Background Art

[0002] The elucidation of biochemical and metabolic pathways is crucial for a deeper understanding of biological systems. Stable isotopes, such as deuterium ( 2 H), are powerful tools for achieving this goal. Deuterium ([[]] 2 H) has been used as a metabolic tracer, combined with different mass spectrometry methods, such as hydrogen / deuterium exchange (HDX) mass spectrometry, to explore dynamic protein structures or protein interactions, and to study fatty acid / lipid synthesis.

[0003] Recently, it has been proposed to monitor the content of deuterium isotopes ([[]] 2 H 2 O) in metabolites after ingestion of heavy water ([[]] 2 H). For example, X. Fu et al. disclosed a work on studying de novo lipogenesis in vivo by monitoring the content of deuterium isotopes in fatty acids in mice (Nature Communications, 12:3756, 2021, doi: 10.1038 / s41467-021-23958-4). In the study, after administering deuterium-labeled water to mice, blood samples were collected, and the plasma was processed as follows: Proteins in the plasma sample (5 μL) were precipitated using 2 mL of a mixed solvent (methanol / dichloromethane volume ratio 1:1), and triglycerides were extracted. After the sample was shaken for 1 minute, it was centrifuged at an acceleration of 1635×g for 5 minutes. The dichloromethane layer was transferred to a new container and dried under nitrogen. The sample was saponified with 1 mL of 0.5 mol / L KOH solution in methanol at 80 °C for 1 hour. After obtaining fatty acids, they were extracted with a dichloromethane / aqueous solution with a volume ratio of 1:1 for 1 minute, and then the solvent was evaporated under nitrogen. The dried lipid extract was resuspended in 50 μL of a mixed solvent of 1% triethylamine / acetone and reacted with 50 μL of 1% perfluorobenzylbromide / acetone at room temperature for 30 minutes. Before mass spectrometry analysis, 1 mL of isooctane was added to the previous sample. In addition, liver extracts were also processed in a similar manner. The processed plasma or liver extracts were then analyzed by deuterium-labeled mass spectrometry. This method requires invasive collection of blood samples or liver extracts, and the sample preparation process is very complex and time-consuming.

[0004] EP3559675A1 discloses a method for analyzing exhaled breath condensate by liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS / MS) methods to monitor amino acid levels. Deuterated leucine is used as an internal standard.

[0005] US2010255598A1 discloses the detection of 2 H-labeled markers in exhaled breath by Fourier transform infrared (FTIR) spectroscopy. In one embodiment, the parent therapeutic agent is labeled with 2 H-labeled markers. After the therapeutic agent is metabolized (e.g., by enzymatic action), the marker becomes a volatile or semi-volatile metabolite and is present in exhaled breath. In another embodiment, the therapeutic agent is combined with 2 H-labeled taggants, which generate markers in exhaled breath that are easily detectable. Since 2 H-labeled therapeutic agents require specific synthesis, 2 H-labeled therapeutic agents are difficult to obtain and may be very expensive.

[0006] L. Shi et al. in the article "Optical imaging of metabolic dynamics in animals" (Nature Communications, 2018, 9(1): 2995) combined deuterium oxide ( 2 H 2 O) probes with stimulated Raman scattering microscopy for in-situ imaging of metabolic activity.

[0007] S. Davies et al. in the article "Rapid measurement of deuterium content of breath following oral ingestion to determine body water" (Physiol. Meas. 2001 Nov; 22(4): 651-9, doi:10.1088 / 0967-3334 / 22 / 4 / 301) disclosed a method for measuring the deuterium content of water in exhaled breath after oral ingestion of deuterated water using flowing afterglow mass spectrometry. Only the deuterium content in the water signal was analyzed. The same is true for the following publication: P. Spanel et al. in "Coordinated FA-MS and SIFT-MS analyses of breath following ingestion of D 2In the article "O and ethanol: total body water, dispersal kinetics and ethanol metabolism" (Physiol. Meas. 2005 Aug; 26(4): 447 - 57, doi: 10.1088 / 0967 - 3334 / 26 / 4 / 011); C. Chan et al. in the article "Anon - invasive, on - line deuterium dilution technique for the measurement of total body water in haemodialysis patients" (Nephrol. Dial. Transplant. 2008 Jun; 23(6): 2064 - 70, doi: 10.1093 / ndt / gfn045); D. Smith et al. in the article "Comparative measurements of total body water in healthy volunteers by online breath deuterium measurement and other near - subject methods" (Am. J. Clin. Nutr. 2002 Dec; 76(6): 1295 - 301, doi: 10.1093 / ajcn / 76.6.1295). Summary of the Invention

[0008] An object of the present invention is to provide a non - invasive method for studying the metabolic pathways of a subject, which can be carried out in real - time without complex sample preparation procedures or the synthesis of labeled therapeutic agents.

[0009] A mass spectrometry method, comprising:

[0010] Obtaining a gas sample containing volatile metabolites excreted by a human or animal subject after ingestion of deuterated water;

[0011] Determining a mass spectrum of the deuterium - resolved mass spectrometry of the gas sample, wherein the deuterium - resolved mass spectrometry is a mass spectrometry technique capable of resolving deuterium - labeled volatile metabolites; and

[0012] Processing the mass spectrum of the deuterium - resolved mass spectrometry to determine the presence or content of at least one deuterium - labeled metabolite in the gas sample.

[0013] According to the present invention, a living human or animal subject ingests deuterated water. During or after the ingestion of deuterated water, a gas sample is obtained from the subject, and a deuterium-resolved mass spectrum of the gas sample is determined. A deuterium-resolved mass spectrum is a mass spectrum having sufficient resolution to distinguish deuterated compounds from isotopes having the same integer mass but containing no deuterium or less deuterium, the isotope comprising at least one carbon with natural abundance ( 13 C), nitrogen ( 15 N), or oxygen ( 17 O and 18 O) heavy isotopes. The deuterium-resolved mass spectrum is processed to determine the presence or amount of at least one deuterium-labeled metabolite in the gas sample. Specifically, the deuterium-resolved mass spectrum can be processed to identify at least one mass spectrometric feature associated with at least one deuterium-labeled metabolite in the gas sample and calculate the intensity of the mass spectrometric feature to determine an indicator of the amount of the deuterium-labeled metabolite.

[0014] By this method, a highly valuable in-depth understanding of the subject's metabolic pathways can be obtained. The method can be performed in real time, i.e., the deuterium-resolved mass spectrum is determined immediately after the gas sample is obtained from the subject, avoiding possible deterioration of the sample due to gas sample storage. The method does not require any complex sample preparation, such as in the case of blood samples. Nor does it require specially synthesized isotope-labeled therapeutic agents. Therefore, the method can be carried out at a relatively low cost.

[0015] In a preferred embodiment, the gas sample comprises the gas exhaled by the subject. Exhaled samples from humans are particularly easy to obtain, and exhaled samples can be easily transmitted to the ion source in real time. Additionally, or alternatively, the gas sample may contain volatile metabolites excreted through the skin.

[0016] Processing the deuterium-resolved mass spectrum may include a mass spectrometric separation step for separating the mass spectrometric features associated with deuterium-labeled metabolites from the mass spectrometric features of metabolites isotopes having the same integer mass but containing no deuterium or less deuterium. Due to the presence of natural abundance heavy isotopes of carbon ( 13 C), nitrogen ( 15 N), and oxygen ( 17 O and 18 O), these isotopes inevitably occur naturally and should be distinguished from the deuterium-labeled metabolites of interest. The mass spectrometric separation step specifically uses a kernel density function for separation, and the specific steps and principles are as follows: In high-resolution mass spectrometry techniques, high resolution (resolution > 10000) can distinguish very close mass number peaks, such as isotope peaks or the peak shapes of complex samples, which may be merged or blurred in traditional low-resolution mass spectrometry. In this case, using kernel density estimation (KDE) helps to more accurately identify and analyze these high-resolution data, thus enabling more accurate quantitative and qualitative analysis.

[0017]

[0018] Among them, f(x) represents the density estimation value at position x; n represents the total number of data points; h represents the bandwidth, and the bandwidth is used to control the width of the kernel function; x i represents the position of the i-th data point; K represents the kernel function. In this embodiment, x i represents the measured value of the i-th m / z; x represents the standard m / z value, that is, the kernel density function at the standard m / z value is to be calculated to achieve the separation of characteristic peaks.

[0019] To ensure the smoothness of the characteristic peaks, the kernel function K in this embodiment adopts a Gaussian kernel function, specifically:

[0020] The bandwidth h of the kernel density function can be adjusted according to the resolution of the mass analyzer in the mass spectrometry. The specific steps are as follows:

[0021] (1) Calculate the corresponding bandwidth h using the following formula:

[0022]

[0023] Among them, m represents the m / z of the measured substance; corresponding to the previous x, that is, the standard m / z value, R is the resolution of the mass spectrometer; the bandwidth h of the kernel density function can be obtained using the above formula to ensure that the kernel density estimation matches the resolution of the mass spectrometer.

[0024] (2) Adjust the bandwidth h:

[0025] Adjust the bandwidth h by combining the traditional bandwidth selection method with the resolution of the mass spectrometer: specifically including:

[0026]

[0027] Among them, σ is the standard deviation of all measured m / z, n is the number of samples, and the bandwidth h can also be calculated in combination with Δm;

[0028] h adjusted = max(h, Δm).

[0029] Among them,

[0030] Processing deuterium-resolved mass spectra may include determining the isotope ratio of deuterium-labeled metabolites to a metabolite isotopologue as a reference in a gas sample. The reference may be a fully protonated isotope, i.e., an isotope in which all deuterium nuclei in the deuterium-labeled metabolite are replaced by protons. Calculating the isotope ratio only considers protonated compounds (positive ion mode) and deprotonated compounds (negative ion mode) that contain only the elements C, H, N, and O and conform to the "seven golden rules". First, perform possible molecular formula matching on the exact mass numbers obtained by a high-resolution mass spectrometer, find the molecular formula of the reference and calculate the mass number after protonation. Then, through mass difference identification, search for the corresponding isotope-labeled molecular formula and calculate their signal intensity ratio. Here, taking the calculation of the 2 H / 1 H signal intensity ratio as an example:

[0031] 1 H = 1.0078

[0032] 2 H = 2.0141

[0033] Δ = 2 H - 1 H = 1.0063

[0034] Assume that the mass number after protonation of the reference is M. By calculating M + n×1.0063 u (±0.0005 u), (where n represents the number of deuterium atoms, n = 1, 2,... 10; u represents the atomic mass unit, u = 1.67*10 -27 kg) to find the 2 H / 1 H isotope pair mass spectrometry characteristics and calculate the 2 H and 1 H signal intensity ratio in the gas sample.

[0035] The steps of obtaining the gas sample can be repeated at different times after the subject ingests deuterated water, and deuterium-resolved mass spectra of these gas samples can be obtained to obtain a time series of the amount of deuterium-labeled metabolites, particularly the aforementioned isotope ratio.

[0036] On the other hand, the present invention provides a mass spectrometry system configured to perform the method of the present invention. The mass spectrometry system includes:

[0037] A mass spectrometer configured to receive a gas sample containing volatile substances exhaled by a subject after ingesting deuterated water, the mass spectrometer including an ion source for ionizing at least part of the volatile metabolites in the gas sample and a mass analyzer for obtaining a deuterium-resolved mass spectrum of the ionized gas sample; and a data processing system configured to process the deuterium-resolved mass spectrum to identify and / or quantify at least one mass spectrometry characteristic related to at least one deuterium-labeled metabolite in the gas sample.

[0038] In advantageous embodiments, the ion source is a SESI source and the mass analyzer is a mass analyzer of the Orbitrap type. However, other types of ion sources and high-resolution mass analyzers can be used.

[0039] In some embodiments, the mass spectrometry system can be specifically configured to process exhaled breath samples in real time. To this end, the system can include an interface for connecting a breathing mask or an exhalation mouthpiece to the mass spectrometry system. In this way, the gas exhaled by the subject can be sampled, the exhaled breath forms a gas sample, and the gas sample is transmitted to the ion source in real time. The mass spectrometry system can also include the above-mentioned breathing mask or exhalation mouthpiece. In other embodiments, the gas sample can be stored before determining the mass spectrum of the gas sample, i.e., the gas sample can be analyzed offline. For example, the gas sample can be collected by having the subject exhale into a bag, and the gas sample can be stored in the bag for subsequent offline analysis, which is common in the urea breath test for identifying Helicobacter pylori infection. The mass spectrometry system can accordingly include an exhalation mouthpiece for connecting to the breath test bag.

[0040] The data processing system of the mass spectrometry system can be configured to execute any of the above data processing procedures. To this end, the data processing system can include a computer processor and a program memory storing program data that causes the computer processor to execute the data processing procedures. The data processing system can in particular be a suitably programmed general-purpose computer. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The following is a brief description of the invention, and the drawings are used to illustrate the preferred embodiments of the invention, for illustration only and not for limitation.

[0042] Figure 1 A schematic diagram of an experimental setup for real-time in vivo monitoring of 2 H-labeled metabolites excreted by mice is shown.

[0043] Figures 2A - 2C Mass spectra of metabolites are shown, taking three metabolites as examples.

[0044] Figures 2D - 2F The 2 H / 1 H isotope ratios of these metabolites are shown as a time series.

[0045] Figure 3 A schematic diagram of an experimental setup for real-time in vivo monitoring of 2 H-labeled metabolites exhaled by human subjects is shown.

[0046] Figure 4 A flow chart of mass spectrometry is shown.

[0047] Figures 5A - 5C Shows the time series of the isotope ratios of selected metabolites in the exhaled breath of a human subject. Detailed implementation

[0048] Definition:

[0049] Isotopologue: An isotopologue is a molecule that differs only in its isotopic composition. They have the same chemical formula and atomic bonding arrangement, but at least one atom has a different number of neutrons from the parent.

[0050] Deuterated water: Refers to water that includes heavy water ( 2 H 2 O, with two deuterium isotopes per molecule) and semi - heavy water ( 2 H 1 HO, with one deuterium isotope per molecule), and its content is higher than natural abundance.

[0051] Metabolite: The definition of metabolite is the same as the usual definition, referring to any intermediate or end product in a metabolic process. Water is not included.

[0052] Deuterated metabolite: A metabolite in which at least one proton is replaced by a deuteron. Deuterated water is not considered a deuterated metabolite.

[0053] Mass spectrometer: A mass spectrometer includes an ion source (ionizer) and a mass analyzer. The ion source converts a portion of the sample into ions. The extraction system removes the ions from the sample and then directs them to the mass analyzer.

[0054] SESI: Secondary electrospray ionization (SESI) is a spray - type ambient ionization method in which ions are generated by electrospray and then charged by collision with vapor molecules in the gas phase. When combined with high - resolution mass spectrometry (SESI - HRMS), it can capture fine isotopic structures.

[0055] Orbitrap mass analyzer: Orbitrap is an ion trap mass analyzer composed of an external barrel - shaped electrode and a coaxial internal conical electrode. It traps ions in an orbit around the conical electrode. By detecting the image current of the trapped ions and using Fourier transform of the frequency signal, it is converted into a mass spectrum.

[0056] Deuterium - resolved mass spectrum: The obtained mass spectrum has sufficient resolution to distinguish between a deuterium - labeled molecule and its isotopologues. Although their total mass integer (i.e., the molecular mass rounded to the nearest Dalton) is the same, the isotopologues contain a smaller number of deuteriums and contain heavy isotopes of carbon, nitrogen, or oxygen (e.g., 13C, 15 N, 17 O or 18 O). These heavy isotopes are usually present in low natural abundances in organic molecules. Deuterium-resolved mass spectra can be obtained by using a mass spectrometer with a sufficiently high resolution. The best resolution should reach 120,000 at m / z 200. This high resolution can be achieved by commercially available Orbitrap-type mass spectrometers, enabling clear separation of the mass spectral lines of different isotopes. It should be noted that the resolution of the Orbitrap-type mass spectrometer scales as 1 / sqrt(m / z) with increasing m / z. Therefore, for lighter molecules, the characteristic separation of different isotopes with the same integer total mass is easier, while it is more difficult for heavier molecules.

[0057] Isotope ratio: The term "isotope ratio" or "isotopic ratio" is understood to indicate the ratio of the concentration of a specific isotopologue in a sample to the concentration of another isotopologue of the same metabolite in the same sample. The isotopic ratio can be well approximated by the intensity ratio of the relevant mass spectral features ("peaks") in the mass spectrum and can thus be equated with this ratio.

[0058] Respiratory mask: A respiratory mask is a mask that covers the mouth and usually also other facial or head areas, designed to direct the wearer's breath to a specific device.

[0059] Example 1: Analysis of exhaled gases from mice

[0060] Method: Two healthy specific pathogen-free (SPF) female C57BL / 6 mice, weighing approximately 18 - 22 grams, were selected for this experiment. The mice were purchased from the Guangdong Provincial Medical Laboratory Animal Center (Foshan, China), housed under SPF conditions, with an environmental temperature of 22 ± 2 °C, a 12-hour light / dark cycle, and provided with standard mouse feed and free access to water. The mice underwent a one-week acclimation period before the experiment began. This animal study has been approved by the Experimental Animal Ethics Committee of Jinan University and was conducted in accordance with the national guidelines for the management and use of experimental animals. Heavy water ( 2 H 2 O, 99.9 atom %) 2 H) was purchased from Merck KGaA (Darmstadt, Germany).

[0061] SESI-HRMS analysis: On day zero (D0, i.e., the day before starting the 2 H 2 O treatment), both mice drank standard drinking water (H 2 O) to obtain baseline measurement data. Subsequently, the mice were randomly divided into two groups. The treatment group drank 80% (v / v) 2 H 2 O; the control group drank H2 O. Volatiles emitted by mice were measured by SESI-HRMS on days 1, 2, 3, 10, 12, and 13, with each measurement lasting 20 minutes. The volatile components of the mice were released through the skin and / or respiration, and SESI-HRMS analysis was performed according to the method reported previously.

[0062] Figure 1 Shows the experimental setup for real-time monitoring of 2 H-labeled compounds in the exhaled air of mice. Mice were given 2 H 2 O or H 2 O and placed inside a polypropylene tube. Medical-grade air at a flow rate of 1 liter per minute generated by a zero-air generator (Beijing Anjiehua Co., Ltd., Beijing, China) was controlled by a flow meter. Volatiles released by the mice through the skin and / or respiration were transported to a self-made SESI source 11 in the laboratory. The samples were ionized in the SESI source and directly analyzed by a high-resolution mass spectrometer (HRMS, Thermo Fisher Q-Exactive Orbitrap MS, Waltham, Massachusetts, USA) without sample pretreatment and chromatographic separation. Mass spectrometry measurements were performed in polarity-switching mode with a spray voltage of 2.5 kV; the temperature of the ion transfer capillary was 150 °C. The mass range was m / z 50 - 750, and the resolution was set to 70,000 (at m / z 200). The S-lens RF level was 50, and the microscan was set to 1.

[0063] Data analysis: Pretreatment: Data analysis was performed using MATLAB software (version 2020b, MathWorks Inc., USA). The original mass spectrometry data files were converted to the mzXML format using msConvert (Proteowizard). Positive-ion scans and negative-ion scans in each file were filtered. Subsequently, we calculated the average mass spectrum of 100 scans in each file and recalibrated these mass spectra using polynomial fitting. After calibration, the mass accuracy across the entire mass range was within 2 ppm for both polarities. Then, the mass spectra were centered and binned using the kernel density function, and the bandwidth matching the instrument resolution at each m / z was selected. The final feature list was obtained by centering the generated kernel density function. The signal intensity of each feature was calculated as the sum of the peak intensities within the full width at half maximum. Thus, a data matrix was obtained, including 14 samples (seven time points after the mice drank H 2 O and seven time points after the mice drank 2 H 2 O) × 3476 features in the positive scan mode × 1407 features in the negative scan mode.

[0064] Post - processing: First, in a further filtering step, we only considered cases where the signal intensity was greater than zero for at least four out of the seven data points of 2 H 2 O, thus further reducing the number of features. Based on the measured exact mass, in both positive and negative modes, the molecular formulas were determined for the remaining 1350 positive - mode features and 1131 negative - mode features with a tolerance of 2 ppm. For positive - mode features, protonated species containing only C, H, N, and O elements and conforming to the "seven - golden rules" were considered; for negative - mode features, de - protonated species containing only C, H, N, and O elements and conforming to the "seven - golden rules" were considered. By identifying mass differences Δ = n * 1.0063 u (±0.0005 u), where n = 1, 2, … 10, the mass - spectral features of 2 H / 1 H isotope pairs were searched. The 2 H / 1 H signal - intensity ratios for the mice at all time points were calculated. From the results, only those showing an increasing trend of 2 H 2 O relative to control mice were selected for the 2 H / 1 H time - trace (i.e., 60 isotope pairs). In addition, to explore the similarities between the different kinetics of these ratio time - traces, we performed hierarchical - clustering analysis. Then, using the metabolite set of "major chemical classes of lipids and non - lipids" in Metabo Analyst R software (version 3.0.3), the potential compound candidates were associated with the metabolites undergoing 2 H enrichment. In addition, using the "cross - reference" function of Metabo Analyst R software, the compound results were cross - referenced with the HMDB and KEGG databases.

[0065] Results: We identified 60 2 H / 1 H isotope pairs that met the two criteria of mass difference and increasing intensity (among them, 26 were in positive - ion mode and 34 were in negative - ion mode). Figure 2A Taking [C 3 H 3 O 3 - as an example, the deuterium - resolved mass - spectrum of its M + 1 isotope is shown. It shows the mass - spectra of the experiments where mice drank H 2 O (dashed line) and 2 H 2 O (solid line), and the mass - spectrum at m / z 88 (actual resolution of 130,000), which is consistent with the actual mass - spectral resolution, of [C 3 H 4 O​3 -H] - The simulated mass spectrum (circles) of this ion. This ion has been identified as pyruvate in previous studies. Figure 2B and Figure 2C show two other examples at m / z 148 in the positive ion mode and at m / z 88 in the negative ion mode, respectively.

[0066] After we identified the species with significant 2 H enrichment, we further studied their evolution over time during the experiment. Figures 2D through 2F shows the three representative 2 H 2 O and 2 H 2 H / 1 H ratio time traces of two mice (i.e., the mice drinking 2 H / 1 H ratio) over 13 days. In most cases, the maximum value of the 2 H 2 H ratio appears on the 12th day after starting to drink Figures 2D through 2F ). Although most of the traces peak on the 12th day, significant differences in kinetic characteristics can be observed simply by eye (see

[0067] Discussion: This example demonstrates the applicability of SESI-HRMS in real-time detection of in vivo metabolic 2 H enrichment. This technique has the required sensitivity (i.e., in the range of parts per trillion) to detect the emission of species under physiological conditions without any sample preconcentration, while providing the required mass resolution to separate fine isotope structures. Figure 2A This is well illustrated by the M+1 mass spectrum of deprotonated pyruvate, which shows the control mice (dashed line), the mice drinking 2 H 2 O, and the simulated mass spectrum (circles) (resolution of approximately 122,000), and this resolution is comparable to the actual mass resolution at this specific mass. First, it shows a perfect match between the simulated mass spectrum and the mass spectrum obtained from the experiment of the mice drinking H 2 O, which indicates the reliability of the calibration process (within 2 ppm) and the possibility of resolving 13 C and 2 H isotope peaks, although they have the same nominal mass number. The perfect match between the experimental mass spectrum and the simulated mass spectrum in terms of m / z and relative intensity further strengthens the credibility of all the determined ion formulas. Another example is shown in Figure 2BThe most likely molecular formula at m / z 147.11268 is calculated to be [C 6 H 14 O 2 N 2 +H] + , which has been identified as lysine in human breath. 15 N and 13 Simulated mass spectra and experimental mass spectra of C isotopologues (for drinking H 2 O mice) perfectly match in terms of m / z position and (relative) signal intensity, and the natural abundance of the latter 2 The H isotopologues were also partially resolved (resolution of about 94,000), so the molecular formula determined by this method has a high degree of confidence. Figure 2C An example is shown for the short chain fatty acid butyrate. More M+1 mass spectra have been resolved sufficiently to confirm the molecular formula.

[0068] exist Figures 2A - 2C In all cases shown in the figure, the mice that drank heavy water 2 H / peak increased significantly, indicating that long-term drinking 2 H 2 O, non-invasive, in vivo monitoring of metabolites 2 The enrichment of H / is feasible. Figures 2D - 2F As explained in the figure, these figures show Figures 2A - 2C The species shown in 2 H / 1 The H ratio time trace. By simple visual inspection, it is obvious that, as expected, the ratio of the control mice drinking water remained stable near zero over several days. In contrast, for the 2 H 2 O mice had a match on day 0 (i.e., baseline), but over the following days, the ratio increased significantly and deviated from that of control mice. 2 H / 1 H tracks dynamics. For example, pyruvate peaked at nearly 4% on day 3 and then remained in this range until the end of the experiment ( Figure 2D ). Lysine increased steadily, peaking at about 1% on day 3 and then approaching 1.5% on day 12 ( Figure 2E In contrast, butyrate remained at natural abundance levels on the first day, then rose to a peak of nearly 30% on the second and third days. It then dropped to about 13% on the following days ( Figure 2F ). Interestingly, this is one of four short-chain fatty acids we found with detectable examples of two hydrogen atom exchanges in its structure. 2 H / 1The H kinetic profiles are very similar, but their maximum ratios are approximately ten times lower (i.e., about 3% on the third day instead of 30%). We found that all short-chain fatty acids as well as lactic acid exhibited similar behavior. However, for the latter, the ratio tended to increase significantly over time (i.e., the accumulation effect).

[0069] These 2 H / 1 The range and kinetic differences in the H / H ratios suggest that the final enrichment mechanisms of the detected metabolites 2 of H are not the same. For example, it was found that the maximum 4 H 2 2 H 4 O 2 / C 4 H 6 O 2 of 2 H / 1 H ratio was close to 1, but this was an outlier as most of the identified 2 H n / H ratios corresponded to n = 1, with a median ratio of approximately 3% (median = 0.0296; interquartile range = 0.0631).

[0070] There are two main mechanisms to explain the 2 H / H 1 exchange that occurs: enzyme-catalyzed irreversible exchange or non-enzyme-catalyzed reversible exchange. In the latter, 2 H 2 O forms N- 2 H, O- 2 H, and S- 2 H bonds, and this exchange occurs faster and more frequently because when hydrogen is bonded to a heteroatom, due to the inherent electronegativity provided by the lone pair of electrons, 2 H / H 1 exchange is more prominent; while in the enzyme-catalyzed exchange, 2 H 2 O forms C- 2 H bonds, and this process is slower and depends on the amount of enzymes available and produced in the organism. Although all the molecular formulas identified in this study contain O and / or N atoms, it is not yet clear whether the observed 2 H enrichment corresponds to enzyme-catalyzed, non-enzyme-catalyzed, or both reaction types present.

[0071] Cluster analysis revealed three main patterns in these ratio time series. The first group included pyruvate and lactate. The similarity in their increasing trend over time can be explained by anaerobic glycolysis in red blood cells, muscle cells, or the gut microbiota, where glucose produces pyruvate, which is then converted to lactate when oxygen supply is limited. The second group included lysine, interestingly, which clustered with m / z 161.1284, corresponding to the molecular formula of methyl lysine. In this group and the third group, we found three short-chain fatty acids (i.e., acetic, propionic, and butyric acids). These are the main products of gut microbial activity, and their importance in regulating other metabolic, endocrine, and immune functions is becoming increasingly evident.

[0072] Example Two

[0073] Human Breath Analysis: Figure 3 Schematically shows an experimental setup for real-time monitoring of deuterium ([ 2 H) uptake in metabolites in the human body. Similar to Example 1, a mass spectrometer 10 including a SESI source 11 and an Orbitrap high-resolution mass analyzer 12 is used. The SESI source 11 is coupled to an exhalation nozzle 14 through a pipe 15, providing an interface for real-time analysis of the exhaled gas of a human subject 60. The raw mass spectra obtained from the mass spectrometer 10 can be analyzed by a suitably programmed general-purpose computer data processing system 13.

[0074] The method of monitoring in vivo 2 H uptake using this setup is schematically shown in the Figure 4 flowchart. In step 21, the subject orally ingests deuterated water. The recommended dose is 1 to 20 grams of 70% 2 H 2 O per kilogram of body water (where body water in males is estimated to be 0.6 times body weight and in females is 0.5 times body weight), or approximately 0.5 to 10 grams of 70% 2 H 2 O per kilogram of body weight. At these doses, deuterated water is known to have no toxic effects on the human body. For example, a 70-kg subject orally ingested 50 ml of 2 H 2 O (99.9%, v / v) in one minute, corresponding to approximately 1 gram of 70% 2 H 2 O per kilogram of body weight. The subject exhales into the exhalation nozzle at multiple different time points after ingestion to obtain multiple exhaled samples (step 22), and the deuterium-resolved mass spectra of these exhaled samples are determined in real time (step 23). Then, these mass spectra are analyzed to determine the presence of deuterium-labeled metabolites in the exhaled samples (step 24), and the isotope ratios of deuterium and protonated isotopes are determined for multiple metabolites (step 25).

[0075] The data processing procedure is very similar to Example 1 above. Specifically, the data processing involves separating mass spectrometry features related to deuterated metabolites and isotopic components with the same nominal mass number but different deuterium numbers. To this end, as in Example 1, classification is performed by using a Kernel density function with a bandwidth matching the instrument resolution at each m / z.

[0076] Dozens of deuterated metabolites have been identified and quantified by determining the isotope ratios relative to their fully protonated isotopes. Figures 5A - 5C Selected time series of the measured isotope ratios are shown. Various different patterns have been observed in these time series. For example, for some metabolites, as Figure 5A shown, a monotonic increase in the isotope ratio was observed within six hours after ingestion, while other metabolites exhibited more complex patterns, as Figure 5B and 5C shown. The underlying mechanisms leading to these time series are the subject of ongoing research.

[0077] Conclusion: The SESI-HRMS technique allows for non-invasive real-time monitoring of the deuterium labeling of metabolites in animals and humans by collecting volatile compounds excreted through the skin or breath after oral ingestion of deuterated water. The results obtained indicate that the currently proposed method provides new opportunities for expanding current metabolic research using deuterium tracer techniques, particularly those focusing on anaerobic glycolysis, lysine methylation, and the gut microbiome, by monitoring changes in short-chain fatty acids.

[0078] Improvement: In this study, secondary electrospray ionization was combined with high-resolution mass spectrometry (SESI-HRMS), and an Orbitrap-type mass spectrometer was used. Although this combination has proven to be particularly suitable for the current purpose, the currently proposed method is not limited to a specific ionization method and other types of ion sources, such as plasma ionization or atmospheric pressure chemical ionization, can also be employed.

[0079] The currently proposed method is not limited to a specific deuterated water administration protocol (e.g., single dose, multiple doses, or continuous administration over a certain period of time) or a specific protocol regarding the relationship between collecting gas samples and deuterated water administration time. This is demonstrated in Examples 1 and 2 above: In Example 1, the subject continuously ingested deuterated water over a long period of time, and gas samples were collected during this period. In Example 2, the subject ingested deuterated water only once within a very short period of time, and gas samples were collected after stopping the ingestion of deuterated water. There are many other feasible protocols, including those in which the ingestion of deuterated water follows a more complex pattern.

[0080] Although mass spectrometry was performed in real time in the above examples, gas samples can also be stored in one or more gas bags and analyzed offline.

[0081] References

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Claims

1. A mass spectrometry method, characterized in that, it includes: obtaining a gas sample containing volatile metabolites released after a human or animal subject ingests deuterium-labeled water; determining a mass spectrum of the deuterium-resolved mass spectrometry of the gas sample, wherein the deuterium-resolved mass spectrometry is a mass spectrometry technique capable of resolving deuterium-labeled volatile metabolites; the determining the mass spectrum of the deuterium-resolved mass spectrometry of the gas sample specifically includes: immediately and in real time determining the deuterium-resolved mass spectrometry after obtaining the gas sample from the subject; the gas sample includes the gas exhaled when the subject breathes; and processing the mass spectrum of the deuterium-resolved mass spectrometry to determine the presence or content of at least one deuterium-labeled metabolite in the gas sample; the processing the mass spectrum of the deuterium-resolved mass spectrometry specifically includes: performing a mass spectrometry separation step, and using a kernel density function to separate the mass spectrometry features related to deuterium-labeled metabolites from the mass spectrometry features of metabolite isotopes having the same mass number but containing no deuterium or less deuterium; the kernel density function is specifically: Among them, represents the density estimate value at the position ; represents the total number of data points; represents the bandwidth; represents the th m / z measurement value; represents the standard m / z numerical value; the kernel function adopts a Gaussian kernel function, specifically: The kernel density function calculates the corresponding bandwidth using the following formula :[[]]END]] wherein, Among them, represents the standard m / z value of the substance to be measured, and is the resolution of the mass spectrometer; Among them, is the standard deviation of all measured m / z , and n is the number of samples.

2. A mass spectrometry method according to claim 1, characterized in that, it further includes: before obtaining the gas sample from the subject, the subject ingests deuterium-labeled water.

3. A mass spectrometry method according to claim 2, characterized in that, the processing the mass spectrum of the deuterium-resolved mass spectrometry specifically includes: determining the isotope ratio between a deuterium-labeled metabolite in the gas sample and a certain isotope of the metabolite as a reference.

4. A mass spectrometry method according to claim 3, characterized in that, the processing the mass spectrum of the deuterium-resolved mass spectrometry further includes: after the subject ingests deuterium-labeled water, obtaining a plurality of gas samples from the subject at a plurality of different times, and determining the sequence of the content change of the deuterium-labeled metabolite over time according to the mass spectrum of the resolvable deuterium-labeled volatile metabolite.

5. A mass spectrometer (10), characterized in that, configured to receive a gas sample containing volatile substances excreted after a subject ingests deuterium-labeled water, the mass spectrometer (10) includes: an ion source (11) for ionizing at least a part of the volatile substances in the gas sample, and a mass analyzer (12) for determining the mass spectrum of the resolvable deuterium-labeled volatile metabolites in the ionized gas sample; the gas sample is specifically: a gas sample containing volatile metabolites released after a human or animal subject ingests deuterium-labeled water; the mass analyzer (12) is further configured to immediately and in real time determine the deuterium-resolved mass spectrometry after obtaining the gas sample from the subject; the gas sample includes the gas exhaled when the subject breathes; a data processing system (13) configured to process the mass spectrum of the deuterium-resolved mass spectrometry to identify and / or quantify at least one mass spectrometry feature associated with the presence of at least one deuterium-labeled metabolite in the gas sample; the data processing system (13) of the mass spectrometry system is configured to perform a mass spectrometry separation step to separate the mass spectrometry features of metabolite isotopes having the same mass integer but containing no deuterium or containing less deuterium; Processing the mass spectrometry diagram of the deuterium-resolved mass spectrometry specifically includes: performing a mass spectrometry separation step, and using a kernel density function to separate the mass spectrometry features related to deuterium-labeled metabolites from the mass spectrometry features of isotopic metabolites with the same mass number but without deuterium or with less deuterium. The specific form of the kernel density function is: Among them, represents the density estimate value at the position ; represents the total number of data points; represents the bandwidth; represents the th m / z measurement value; represents the standard m / z numerical value; The kernel function adopts a Gaussian kernel function, specifically: The kernel density function calculates the corresponding bandwidth using the following formula :[[]] where Among them, represents the standard m / z value of the substance to be measured, which is the resolution of the mass spectrometer; where, is the standard deviation of all measured m / z , and n is the number of samples.

6. A mass spectrometer according to claim 5, characterized in that where the ion source (11) is a secondary electrospray ionization source, and / or the mass analyzer (12) is an Orbitrap type mass spectrometer.

7. A mass spectrometer according to claim 6, comprising a breathing mask or an exhalation nozzle for transmitting the air exhaled by the subject to the ion source (11) in real time.

8. A mass spectrometer according to claim 7, characterized in that the data processing system (13) is configured to determine the isotope ratio between the deuterium-labeled metabolite and a certain isotope of the metabolite as a reference in the gas sample.

9. A mass spectrometer according to claim 8, characterized in that the data processing system (13) is configured to determine the time series amount of the deuterium-labeled metabolite according to the mass spectrometry diagrams of multiple deuterium-resolved mass spectra of samples obtained after the subject ingests deuterium-labeled water at different time points.

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