Metabolism analysis method for polymer nano material

Abstract

The invention discloses a method for analyzing metabolism of a polymer nano material. Reversed-phase liquid chromatography separation is combined with a data dependence acquisition mode in mass spectrometry, an animal sample purification solution is detected by utilizing an LC-HRMS technology, and when different monomers of the metabolite of the polymer nano material are relatively quantified, precursor ions are used as quantitative ions, and the concentration of the metabolite of the polymer nano material is determined. And taking the sum of signals of adduct ions in different charge states of each monomer as a response signal of the monomer. Compared with a traditional LC-MS metabolism analysis method needing to process mass ion information, the method provided by the invention has the advantages that the complexity of mass spectrum information is simplified, the identification process is optimized, the analysis efficiency is improved, and the full-molecular-weight profile analysis of the polymer metabolism behavior can be realized. The polymer omics analysis thought and strategy based on the structural similarity established by the method do not depend on a specific type of polymer, so that the method has good universality, and important technical support is provided for the metabolism research of the polymer.

Description

Technical Field

[0001] This invention relates to the field of analytical technology, and in particular to a method for metabolic analysis of polymer nanomaterials. Background Technology

[0002] Polymer nanomaterials have wide applications in pharmaceutical excipients, enabling covalent bonding with active pharmaceutical ingredients to significantly improve pharmacokinetic properties. For example, polyethylene glycol (PEG) is frequently used for this type of modification. Drug PEGylation refers to the process of covalently linking PEG to the active pharmaceutical ingredient. By increasing the drug's molecular weight, PEGylation can effectively reduce renal clearance and prolong the drug's half-life. Polymer nanomaterials are also components of various nanomedicine delivery systems (NDDS), serving as a crucial material basis for their carrier function. With the rapid development of NDDS, the in vivo processes of polymer nanomaterials are receiving increasing attention. Currently, most in vivo studies indicate that polymer nanomaterials are taken up by the reticuloendothelial system and tend to accumulate in organs over extended periods. Some polymers and their metabolites possess biological activity and may cause adverse reactions. For instance, acidic metabolites of PEG may disrupt the body's acid-base balance, leading to metabolic acidosis and hypercalcemia; some polymer nanomaterials metabolize in vivo to produce low-molecular-weight metabolites, which are excreted through the kidneys. These metabolites may accumulate in the kidneys, leading to kidney damage. Therefore, in-depth research into the in vivo metabolic behavior of polymers is of great significance for scientifically assessing their safety and promoting the safe design and development of related drugs.

[0003] Taking PEG as an example, studies have identified its monocarboxylated, dicarboxylic acid, and sulfated metabolites in serum, urine, and tissue samples from humans and animals. Other studies have shown that PEG can produce small amounts of oxalic acid during metabolism. However, a systematic and in-depth study of PEG's metabolic processes in vivo is still lacking. Key questions such as whether polymer chains break down and whether other metabolic pathways exist remain unclear. This situation is related to the limitations of current metabolic analysis techniques, hindering a systematic analysis of polymer metabolism and a comprehensive tracking of its metabolic trajectory in vivo.

[0004] The metabolic analysis of polymers presents significant technical challenges, and comprehensive and systematic studies on polymer metabolism are extremely rare. Taking PEG as an example, as a widely used polymer nanomaterial, its metabolic analysis faces even greater challenges compared to drug metabolism studies. The lack of characteristic chromophores in PEG limits the analytical capabilities of traditional high-performance liquid chromatography (HPLC) for it and its metabolites. Although liquid chromatography-tandem mass spectrometry (LC-MS) can provide detailed characterization information of the analyte, the inherent polydispersity of PEG's molecular weight and its tendency to form multiple charges during mass spectrometry ionization lead to a large number of precursor ions, complicating the mass spectrum, especially when analyzing PEG metabolites. Therefore, traditional LC-MS techniques are insufficient for comprehensive metabolic analysis of PEG. Previous studies have often employed radiolabeling to study polymer metabolism; however, radiolabels may be lost during polymer metabolism. Furthermore, when radiolabeling polymers, there is the problem of radiolabel dissociation due to chain shortening, and this problem becomes more pronounced as the polymer molecular weight increases.

[0005] Given that existing technologies are insufficient to fully and accurately analyze the metabolic behavior of polymers, there is a need to establish a metabolic analysis technique based on the full molecular weight profile in order to systematically elucidate the metabolic processes of polymers in vivo. Summary of the Invention

[0006] To overcome the limitations of existing technologies in comprehensively and accurately analyzing the metabolites of polymer nanomaterials in the human body, this invention proposes a "polymeromics" analysis strategy and provides a method for analyzing the metabolism of polymer nanomaterials. Based on the principle that polymers and their metabolites are a group of structurally similar compounds with a continuous molecular weight distribution, this method first identifies some representative metabolites, then systematically infers the composition and structure of the entire series of metabolites, and performs cluster analysis on the polymer metabolites to efficiently reveal metabolic behavior. This solves the problem of the time-consuming and laborious process of identifying the structure of each monomer individually.

[0007] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0008] A method for analyzing the metabolism of polymer nanomaterials combines reversed-phase liquid chromatography separation with data-dependent acquisition mode in mass spectrometry. LC-HRMS technology is used to detect purified animal sample solutions. When performing relative quantification of different monomers of polymer nanomaterial metabolites, precursor ions are used as quantitative ions, and the sum of signals from adduct ions of different charge states of each monomer is used as the response signal of that monomer.

[0009] The method for preparing the purified animal sample solution is to add a precipitant to the metabolite extract, mix well, centrifuge, and obtain the supernatant after protein precipitation.

[0010] The precipitant is selected from one of acetonitrile, methanol, ethanol, acetone, ammonium sulfate, and trichloroacetic acid.

[0011] The volume ratio of the precipitant to the metabolite extract is not less than 2:1.

[0012] The methods for obtaining the metabolite extract include:

[0013] (a) For blood samples: collect the supernatant after centrifugation;

[0014] (b) For fecal samples: after freeze-drying, grind thoroughly into powder, weigh the powder, add acetonitrile-water solution for ball milling, then extract by ultrasonication and centrifuge to obtain the supernatant;

[0015] (c) For tissue samples: rinse with physiological saline and homogenize, take the homogenate, add acetonitrile-water solution for ball milling, then sonicate and centrifuge, and take the supernatant;

[0016] (d) For urine samples: no extraction or processing is required; they can be used directly for subsequent analysis.

[0017] The volume ratio of the acetonitrile-water solution is 40 / 60, and the amount of the acetonitrile-water solution is 1.5-2.5 mL per 0.5 g sample; the sonication time is 15-25 min.

[0018] The reversed-phase liquid chromatography separation was performed using a PLRP-S column with 1 mM ammonium formate-water solution and acetonitrile / methanol solution as the mobile phase, with an acetonitrile / methanol volume ratio of 50 / 50, and gradient elution was used.

[0019] The precursor ions used for relative quantification are selected from [M+nH]. n+ [M+nNa] n+ [M+nK] n+ [M+nMg] 2n+ [M+nCa] 2n+ [M+nNH4] n+ [M-nH] n- [M+nCH3COO] n- [M+nHCOO] n- [M+nCl] n- One of them, where n is a positive integer ≥1 for all metabolites.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] Based on the "polymeromics" analysis strategy proposed in this invention, the structural similarity between polymer monomers can be utilized to systematically identify the structures of unknown metabolites, construct metabolite-metabolite networks, and thus elucidate the metabolic behavior of polymers in vivo and their metabolic characteristics. Compared to traditional LC-MS metabolic analysis methods that require processing massive amounts of ion information, the method proposed in this invention simplifies the complexity of mass spectrometry information. This not only optimizes the identification process and improves analytical efficiency but also helps to achieve full molecular weight profile resolution of polymer metabolic behavior. The "polymeromics" analysis approach and strategy based on structural similarity established in this invention are not dependent on specific types of polymers, thus possessing good universality and providing important technical support for polymer metabolic research. Attached Figure Description

[0022] Figure 1 The molecular weight distribution information of mPEG2000 metabolites identified in plasma samples is shown in Figure A, which is the molecular weight distribution information of PEG-CHO, and Figure B is the molecular weight distribution information of PEG-COOH.

[0023] Figure 2 The graphs show the molecular weight distribution of metabolites in rat fecal samples. A represents the molecular weight distribution of PEG-CHO, and B represents the molecular weight distribution of PEG-COOH.

[0024] Figure 3 The graph shows the molecular weight distribution of metabolites in rat urine samples. A is the molecular weight distribution graph of PEG-CHO, and B is the molecular weight distribution graph of PEG-COOH.

[0025] Figure 4 The graph shows the molecular weight distribution of PEG monocarboxylate identified in rat tissues. A is the molecular weight distribution of PEG monocarboxylate in heart tissue, B is the molecular weight distribution of PEG monocarboxylate in liver tissue, C is the molecular weight distribution of PEG monocarboxylate in spleen tissue, and D is the molecular weight distribution of PEG monocarboxylate in kidney tissue. Detailed Implementation

[0026] The embodiments described below are exemplary descriptions of key experimental evidence and are not intended to limit the core content and application scope of this invention due to the amount of evidence. It should be noted that all the accompanying drawings and corresponding descriptions merely illustrate the concept, principles, and representative experimental evidence of the disclosed embodiments of this invention. Where the chain of evidence is complete, it is unnecessary to show all the specific details and extended details of the various embodiments listed in this invention.

[0027] Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0028] All raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. mPEG2000 (monomethoxy polyethylene glycol 2000) was provided by Keykie Technologies (Beijing, China); LC-MS grade acetonitrile, methanol, and water were purchased from Fessell Technologies (Fairlawn, New Jersey, USA); and LC-MS grade ammonium formate was purchased from Sigma-Aldrich (Bellfont, Pennsylvania, USA).

[0029] Example 1

[0030] In this embodiment, blood samples from rats injected with mPEG2000 were first collected. After processing, the metabolites in the samples were analyzed using LC-HRMS technology.

[0031] The specific implementation method is as follows:

[0032] 1. Instruments

[0033] The Shimadzu LC-20AD high-performance liquid chromatograph and the Sciex Triple TOF 6600+ mass spectrometer are equipped with an electrospray ionization source.

[0034] 2. Sample preparation

[0035] Blood samples, approximately 400 μL each, were collected from rats injected with mPEG2000 (4.8 mg / kg) at different time points. Immediately after collection, the samples were centrifuged at 13300 rpm for 5 min at 4°C, and the supernatant (plasma) was separated and collected.

[0036] 3. Blood sample processing

[0037] Take a plasma sample, add acetonitrile to precipitate proteins (acetonitrile to plasma volume ratio of 2:1), vortex for 30 s, centrifuge at 13300 rpm for 5 min at 4°C, collect the supernatant, dilute appropriately with water, and perform LC-HRMS analysis.

[0038] 4. Detection Method

[0039] Mass spectrometry conditions: Electrospray ionization source in positive / negative ion scanning mode, spray voltage 5500 V, ion source temperature 550℃, nebulizing gas 50 psi, curtain gas 30 psi, desolvating gas 50 psi.

[0040] Chromatographic conditions: A PLRP-S column (2.1 × 150 mm, 3 μm, 300 Å, Agilent) was used. Mobile phase A was 1 mM ammonium formate-water solution, and mobile phase B was acetonitrile / methanol (50 / 50, v / v) solution. The elution gradient was: 0.01–2.00 min, 42% B; 2.00–45.00 min, 42–56% B; 45.00–65.00 min, 56–58% B; 65.00–70.00 min, 58–85% B; 70.00–77.00 min, 85% B; 77.00–77.20 min, 85–42% B; 77.20–81.60 min, 42% B. The flow rate was 0.3 mL / min, and the column temperature was 40°C.

[0041] By combining reversed-phase liquid chromatography (RPLC) separation with data-dependent acquisition (DDA) mode in mass spectrometry, the supernatant obtained was analyzed using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Due to the subtle differences in hydrophobicity between monomers of different molecular weights, RPLC can effectively separate individual monomers of the polymer, thereby enabling full molecular weight profile metabolic analysis of the polymer. DDA selectively transports precursor ions to the collision cell for cleavage only when preset conditions are met (the ions are the most abundant). This method can obtain high-quality MS / MS information of metabolites, improving the efficiency of metabolite identification.

[0042] In metabolic data analysis, because each monomer of PEG is a homologue and shares structural similarities, the metabolites produced after metabolism in vivo will also exhibit structural similarities. This will manifest as similarities in MS / MS spectra and regularities in retention times in the data acquired by RPLC-DDA. Utilizing this characteristic, some metabolites are initially identified by comparing the similarity between metabolic data and the MS / MS spectra of the PEG parent compound. Then, by comparing the identified metabolites with unknown metabolites using MS / MS spectra and retention times, the structure of unknown metabolites is further identified, increasing the number of metabolites discovered. When performing relative quantification of different monomers of metabolites, precursor ions are used as quantitative ions, and the sum of the signals from adduct ions of different charge states of each monomer is used as the response signal of that monomer. [M+nNH4] is used as the quantification ion. n+ As quantitative ions, [M+NH4] is included. + [M+2NH4] 2+ [M+3NH4] 3+ and [M+4NH4] 4+ .

[0043] 5. Analysis Results

[0044] The analytical method of this invention was used to detect metabolites in rat plasma samples, such as... Figure 1 As shown, cluster analysis reveals that these metabolites exhibit a bimodal distribution at the molecular weight level, namely, they are divided into two groups: high molecular weight and low molecular weight.

[0045] Example 2

[0046] In this embodiment, rat fecal samples were first collected after being injected with mPEG2000. After processing, the metabolites in the samples were analyzed using LC-HRMS technology.

[0047] The specific implementation method is as follows:

[0048] 1. Instruments

[0049] The Shimadzu LC-20AD high-performance liquid chromatograph and the Sciex Triple TOF 6600+ mass spectrometer are equipped with an electrospray ionization source.

[0050] 2. Sample preparation

[0051] Fecal samples were collected from rats at different time points after injection of mPEG2000 (4.8 mg / kg). The fecal samples were freeze-dried and thoroughly ground into a homogeneous powder. 0.5 g of the powder was weighed and added to 2.5 mL of acetonitrile / water (40 / 60, v / v) solution. The mixture was ball-milled at 1200 rpm for 4 min, followed by sonication for 25 min. The mixture was then centrifuged at 3500 rpm for 10 min, and the supernatant was collected.

[0052] 3. Fecal sample processing

[0053] Collect the fecal supernatant, add acetonitrile to precipitate proteins (acetonitrile to fecal supernatant volume ratio is 3:1), vortex for 30 s, centrifuge at 13300 rpm for 5 min at 4°C, collect the supernatant, dilute appropriately with water, and perform LC-HRMS analysis.

[0054] 4. Detection Method

[0055] Mass spectrometry conditions: Electrospray ionization source in positive / negative ion scanning mode, spray voltage 5500 V, ion source temperature 550℃, nebulizing gas 50 psi, curtain gas 30 psi, desolvating gas 50 psi.

[0056] Chromatographic conditions: A PLRP-S column (2.1 × 150 mm, 3 μm, 300 Å, Agilent) was used. Mobile phase A was 1 mM ammonium formate-water solution, and mobile phase B was acetonitrile / methanol (50 / 50, v / v) solution. The elution gradient was: 0.01–2.00 min, 42% B; 2.00–45.00 min, 42–56% B; 45.00–65.00 min, 56–58% B; 65.00–70.00 min, 58–85% B; 70.00–77.00 min, 85% B; 77.00–77.20 min, 85–42% B; 77.20–81.60 min, 42% B. The flow rate was 0.3 mL / min, and the column temperature was 40°C.

[0057] LC-HRMS technology specifically employs RPLC separation and DDA mass spectrometry scanning mode. During quantification, [M+nNH4] is used as the quantification factor. n+ As quantitative ions, [M+NH4] is included. + [M+2NH4] 2+ [M+3NH4] 3+ and [M+4NH4] 4+ .

[0058] 5. Analysis Results

[0059] The analytical method established in this invention was used to analyze metabolites in rat fecal samples. The results are as follows: Figure 2 As shown, metabolites of the polymer were detected in fecal samples, consistent with the types of metabolites identified in blood samples.

[0060] Example 3

[0061] In this embodiment, urine samples from rats injected with mPEG2000 were first collected. After processing, the metabolites in the samples were analyzed using LC-HRMS technology.

[0062] The specific implementation method is as follows:

[0063] 1. Instruments

[0064] The Shimadzu LC-20AD high-performance liquid chromatograph and the Sciex Triple TOF 6600+ mass spectrometer are equipped with an electrospray ionization source.

[0065] 2. Urine sample processing

[0066] Urine samples were collected from rats at different time points after injection of mPEG2000 (4.8 mg / kg). Acetonitrile was added to precipitate proteins (acetonitrile to urine volume ratio of 3:1), vortexed for 30 s, and centrifuged at 13300 rpm for 5 min at 4°C. The supernatant was collected, appropriately diluted with water, and analyzed by LC-HRMS.

[0067] 3. Detection Method

[0068] Mass spectrometry conditions: Electrospray ionization source in positive / negative ion scanning mode, spray voltage 5500 V, ion source temperature 550℃, nebulizing gas 50 psi, curtain gas 30 psi, desolvating gas 50 psi.

[0069] Chromatographic conditions: A PLRP-S column (2.1 × 150 mm, 3 μm, 300 Å, Agilent) was used. Mobile phase A was 1 mM ammonium formate-water solution, and mobile phase B was acetonitrile / methanol (50 / 50, v / v) solution. The elution gradient was: 0.01–2.00 min, 42% B; 2.00–45.00 min, 42–56% B; 45.00–65.00 min, 56–58% B; 65.00–70.00 min, 58–85% B; 70.00–77.00 min, 85% B; 77.00–77.20 min, 85–42% B; 77.20–81.60 min, 42% B. The flow rate was 0.3 mL / min, and the column temperature was 40°C.

[0070] LC-HRMS technology specifically employs RPLC separation and DDA mass spectrometry scanning mode. During quantification, [M+nNH4] is used as the quantification factor. n+ As quantitative ions, [M+NH4] is included. + [M+2NH4] 2+ [M+3NH4] 3+ and [M+4NH4] 4+ .

[0071] 4. Analysis Results

[0072] The analytical method established in this invention was used to analyze metabolites in rat urine samples. The results are as follows: Figure 3 As shown, metabolites of the polymer were detected in urine samples, consistent with those identified in blood samples.

[0073] Example 4

[0074] In this embodiment, rat tissue samples injected with mPEG2000 were first collected. After processing, the metabolites in the samples were analyzed using LC-HRMS technology.

[0075] The specific implementation method is as follows:

[0076] 1. Instruments

[0077] The Shimadzu LC-20AD high-performance liquid chromatograph and the Sciex Triple TOF 6600+ mass spectrometer are equipped with an electrospray ionization source.

[0078] 2. Sample preparation

[0079] Tissue samples were collected from rats at different time points after injection of mPEG2000 (4.8 mg / kg), rinsed with physiological saline, and homogenized. 0.5 g of tissue homogenate was taken, and 1.5 mL of acetonitrile / water (40 / 60, v / v) solution was added. The mixture was ball-milled at 1200 rpm for 4 min, followed by sonication for 15 min. The mixture was then centrifuged at 3500 rpm for 10 min, and the supernatant was collected.

[0080] 3. Tissue Sample Processing

[0081] Take the tissue supernatant, add acetonitrile to precipitate the protein (acetonitrile to tissue supernatant volume ratio is 3:1), vortex for 30 s, centrifuge at 13300 rpm for 5 min at 4°C, collect the supernatant, dilute it appropriately with water, and perform LC-HRMS analysis.

[0082] 4. Detection Method

[0083] Mass spectrometry conditions: Electrospray ionization source in positive / negative ion scanning mode, spray voltage 5500 V, ion source temperature 550℃, nebulizing gas 50 psi, curtain gas 30 psi, desolvating gas 50 psi.

[0084] Chromatographic conditions: A PLRP-S column (2.1 × 150 mm, 3 μm, 300 Å, Agilent) was used. Mobile phase A was 1 mM ammonium formate-water solution, and mobile phase B was acetonitrile / methanol (50 / 50, v / v) solution. The elution gradient was: 0.01–2.00 min, 42% B; 2.00–45.00 min, 42–56% B; 45.00–65.00 min, 56–58% B; 65.00–70.00 min, 58–85% B; 70.00–77.00 min, 85% B; 77.00–77.20 min, 85–42% B; 77.20–81.60 min, 42% B. The flow rate was 0.3 mL / min, and the column temperature was 40°C.

[0085] LC-HRMS technology specifically employs RPLC separation and DDA mass spectrometry scanning mode. During quantification, [M+nNH4] is used as the quantification factor. n+ As quantitative ions, [M+NH4] is included. + [M+2NH4] 2+ [M+3NH4] 3+ and [M+4NH4] 4+ .

[0086] 5. Analysis Results

[0087] The analytical method of this invention was used to detect metabolites in rat tissue samples. For example... Figure 4 As shown, the molecular weight distribution results of one of the metabolites, PEG monoformate, demonstrate that this method can resolve metabolites based on a complete molecular weight distribution profile.

[0088] In addition to acetonitrile, other protein precipitants added to the metabolite extract of animal sample purification solution can include methanol, ethanol, acetone, ammonium sulfate, or trichloroacetic acid. Experiments have shown that a volume ratio of precipitant to metabolite extract of at least 2:1 is sufficient for complete precipitation.

[0089] Alternatively, the precursor ion used for relative quantification is [M+nH]. n+ [M+nNa] n+ [M+nK] n+ [M+nMg] 2n+ [M+nCa] 2n+ [M+nNH4] n+ [M-nH] n- [M+nCH3COO] n- [M+nHCOO] n- [M+nCl] n-One of them. For all metabolites, n is a positive integer ≥ 1.

[0090] It should be understood that the above embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For those skilled in the art, various modifications or substitutions can be made without departing from the inventive concept, and all such modifications or substitutions should be considered within the scope of protection of this invention.

Claims

1. A method for metabolic analysis of polymer nanomaterials, characterized in that, By combining reversed-phase liquid chromatography separation with data-dependent acquisition mode in mass spectrometry, and using LC-HRMS technology to detect purified animal sample solutions, when performing relative quantification of different monomers of polymer nanomaterial metabolites, precursor ions are used as quantitative ions, and the sum of signals of adduct ions with different charge states of each monomer is used as the response signal of that monomer.

2. The method for metabolic analysis of polymer nanomaterials according to claim 1, characterized in that, The method for preparing the purified animal sample solution is to add a precipitant to the metabolite extract, mix well, centrifuge, and obtain the supernatant after protein precipitation.

3. The method for metabolic analysis of polymer nanomaterials according to claim 2, characterized in that, The precipitant is selected from one of acetonitrile, methanol, ethanol, acetone, ammonium sulfate, and trichloroacetic acid.

4. The method for metabolic analysis of polymer nanomaterials according to claim 2 or 3, characterized in that, The volume ratio of the precipitant to the metabolite extract is not less than 2:

1.

5. The method for metabolic analysis of polymer nanomaterials according to claim 2, characterized in that, The methods for obtaining the metabolite extract include: (a) For blood samples: collect the supernatant after centrifugation; (b) For fecal samples: after freeze-drying, grind thoroughly into powder, weigh the powder, add acetonitrile-water solution for ball milling, then extract by ultrasonication and centrifuge to obtain the supernatant; (c) For tissue samples: rinse with physiological saline and homogenize, take the homogenate, add acetonitrile-water solution for ball milling, then sonicate and centrifuge, and take the supernatant; (d) For urine samples: no extraction or processing is required; they can be used directly for subsequent analysis.

6. The method for metabolic analysis of polymer nanomaterials according to claim 5, characterized in that, The volume ratio of the acetonitrile-water solution is 40 / 60, and the amount of the acetonitrile-water solution is 1.5-2.5 mL per 0.5 g sample; the sonication time is 15-25 min.

7. The method for metabolic analysis of polymer nanomaterials according to claim 1, characterized in that, The reversed-phase liquid chromatography separation was performed using a PLRP-S column with 1 mM ammonium formate-water solution and acetonitrile / methanol solution as the mobile phase, with an acetonitrile / methanol volume ratio of 50 / 50, and gradient elution was used.

8. The method for metabolic analysis of polymer nanomaterials according to claim 1, characterized in that, The precursor ions used for relative quantification are selected from [M+nH]. n+ [M+nNa] n+ [M+nK] n+ [M+nMg] 2n+ [M+nCa] 2n+ [M+nNH4] n+ [M-nH] n- [M+nCH3COO] n- [M+nHCOO] n- [M+nCl] n- One of them, where n is a positive integer ≥1 for all metabolites.

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