A liquid chromatography-mass spectrometry (LC-MS) method adapted to detect dried blood spots containing seven types of metabolites

Abstract

This invention belongs to the field of biomedical technology and discloses a DBS-LC-MS / MS method for the simultaneous detection of seven types of metabolites in dried blood spots. The method employs a pH / polarity dual-trigger microcapsule solvent carrier system, which internally encapsulates high, medium, and low polarity extraction solvents in layers. During extraction, these solvents are released sequentially according to local pH and polarity changes, efficiently extracting amino acids, acylcarnitines, organic acids, bile acids, steroids, fatty acids, and sugar metabolism intermediates, respectively. This invention overcomes the inherent limitations of traditional static solvent systems and solves the problem of unbalanced recovery rates caused by insufficient solvent compatibility in the extraction of seven types of metabolites with vastly different polarities in dried blood spots. It significantly improves the accuracy, reproducibility, and coverage of the DBS-LC-MS / MS platform in the simultaneous detection of multiple metabolites, providing reliable technical support for newborn screening for inherited metabolic diseases, diagnosis of endocrine diseases, and drug metabolism research.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a dried blood spot liquid chromatography-mass spectrometry detection method suitable for seven types of metabolites. BACKGROUND

[0002] As a minimally invasive, convenient and stable biological sample collection and preservation form, dried blood spots have been widely used in recent years in the fields of newborn screening, drug metabolism kinetics research and clinical diagnosis. The combination of dried blood spots and liquid chromatography-mass spectrometry technology further improves the sensitivity and specificity of simultaneous detection of multiple component metabolites, providing strong support for precision medicine.

[0003] The current mainstream dried blood spot metabolite extraction usually uses general solvents such as methanol-water, acetonitrile-water or solvents containing a small amount of acid / alkali additives for the simultaneous extraction of multiple target substances. In the early screening of limited categories of metabolites, it has indeed shown good practicality. A high proportion of organic phase can effectively precipitate proteins and dissolve medium to low polarity compounds, and an appropriate amount of water phase can help maintain the solubility of some hydrophilic substances, to some extent, both the extraction spectrum and the operation feasibility are taken into account.

[0004] However, due to the huge differences in key physicochemical parameters such as logP value, pKa, and number of hydrogen bond donors and acceptors among the seven types of target metabolites, in a single solvent system, hydrophilic metabolites (such as short-chain acylcarnitines and some amino acids) often have low recovery rates due to insufficient solvent polarity, while hydrophobic components (such as steroids and long-chain fatty acid derivatives) may be trapped in the blood spot matrix due to too high solvent polarity or lack of effective desorption capacity.

[0005] If attempts are made to optimize by adjusting the proportion of organic phase or adding modifiers, such as improving the extraction efficiency of hydrophobic substances, the recovery rate of hydrophilic substances will be sacrificed. This systematic recovery bias caused by the mismatch between solvent polarity and target hydrophilic / hydrophobic properties reduces the accuracy and reproducibility of the detection, introduces errors in multi-index joint interpretation, and affects the accuracy of the DBS-LC-MS / MS platform in the comprehensive evaluation of complex metabolic diseases. SUMMARY

[0006] To achieve the above-mentioned application purposes, the application provides a dried blood spot liquid chromatography-mass spectrometry detection method suitable for seven types of metabolites. The method constructs a pH / polarity dual-trigger microcapsule solvent carrier system, which realizes efficient and balanced simultaneous extraction of seven types of metabolites with significant differences in structure and physicochemical properties, such as amino acids, acylcarnitines, organic acids, bile acids, steroids, fatty acids and sugar metabolism intermediates, in a single extraction operation process, thereby solving the problem of systematic recovery bias caused by the mismatch between solvent polarity and target hydrophilic / hydrophobic properties in the existing dried blood spot pretreatment technology.

[0007] The method comprises the following steps: firstly, preparing a microcapsule solvent carrier coated by multiple layers of responsive polymers, the microcapsule encapsulating at least three extraction solvents with different polarity parameters and pH environments; secondly, placing a dry blood spot sample to be tested in the microcapsule solvent carrier suspension, and starting the extraction process under controlled temperature control and oscillation conditions; during the extraction process, the microcapsule ruptures and releases the corresponding extraction solvent in a preset order according to the local pH value and polarity environment of the dry blood spot eluent, forming a gradient polarity extraction environment; finally, collecting all the released solvent phases, clarifying them by centrifugation, and directly injecting them into a liquid chromatography-mass spectrometry system for analysis.

[0008] The microcapsule solvent carrier is composed of an inner core solvent phase, an intermediate response layer, and an outer protective shell. The inner core solvent phase includes a first extraction solvent, a second extraction solvent, and a third extraction solvent, which are concentrically layered and encapsulated inside the microcapsule. The first extraction solvent is located in the innermost layer, the second extraction solvent wraps the first extraction solvent, and the third extraction solvent is located in the outermost layer and directly contacts the intermediate response layer. The first extraction solvent is a high-polarity aqueous system composed of 95:5 ultrapure water and formic acid by volume, which is used to preferentially extract strongly hydrophilic metabolites, including short-chain acylcarnitines, neutral amino acids, and glycolysis intermediates. The second extraction solvent is a medium-polarity mixed phase composed of 60:40 acetonitrile and water containing 10 mM ammonium acetate (pH 4.2) by volume, which is used to extract weakly polar to moderately polar metabolites, including branched-chain amino acids, long-chain acylcarnitines, some organic acids, and primary bile acids. The third extraction solvent is a low-polarity organic phase composed of 80:20 isopropanol and chloroform by volume, with the addition of 0.1% formic acid, which is used to extract strongly hydrophobic metabolites, including steroid hormones, long-chain fatty acid derivatives, and secondary bile acids.

[0009] The intermediate response layer is composed of two functional polymers, pH-sensitive poly(methacrylic acid-co-methyl methacrylate) (P(MAA-co-MMA)) and polarity-responsive poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-co-AAc)). The mass fraction of P(MAA-co-MMA) is 65%, and its carboxyl functional groups are protonated when the pH is below 5.0, causing the polymer chain to shrink and reducing membrane permeability, while deprotonating when the pH is above 6.0, triggering swelling and forming microporous channels. The mass fraction of P(NIPAM-co-AAc) is 35%, and its critical solution temperature is 32°C, which undergoes a conformational transition when the environmental polarity decreases (i.e., the proportion of organic phase increases), promoting the instability of the microcapsule structure. The total thickness of the intermediate response layer is 1.2 μm, which is deposited at the interface of each solvent phase by layer-by-layer self-assembly process.

[0010] The outer protective shell is formed by ionic cross-linking of cross-linked sodium alginate and chitosan, with a thickness of 0.8 μm, which functions to maintain the integrity of the microcapsule structure during the initial elution stage of the dried blood spot, preventing premature non-specific rupture. The outer protective shell remains stable for the first 2 minutes after contact with the dried blood spot elution solution at 37 °C with a shaking frequency of 600 rpm; subsequently, as endogenous buffer substances released by lysed blood cells increase the local pH to above 6.5, the outer shell gradually dissociates due to deprotonation of the chitosan amino groups, exposing the intermediate responsive layer.

[0011] During the first stage of the extraction process (0-2 min), the dried blood spot slowly elutes in the initial aqueous environment, with the local pH maintained at approximately 5.8, at which time the outer protective shell has not yet dissociated, allowing only a small amount of water to penetrate the outermost layer of the microcapsule. Since the third extraction solvent is a low-polarity mixed phase (80:20 isopropanol-chloroform), although isopropanol is partially miscible with water, the low proportion of chloroform reduces the overall hydrophilicity, and the small amount of water that penetrates is not sufficient to trigger swelling or phase transition of the intermediate responsive layer, and the microcapsule structure remains stable. This stage mainly completes the wetting and initial swelling of the dried blood spot matrix.

[0012] During the second stage of the extraction process (2-5 min), the outer protective shell dissociates, and the intermediate responsive layer is exposed. At this time, the local pH rises to 6.5, and P(MAA-co-MMA) undergoes deprotonation swelling, while the hydrophilic metabolites released by the dried blood spot increase the environmental polarity, and P(NIPAM-co-AAc) is in a hydrophilic conformation, and the overall responsive layer remains dense. However, the third extraction solvent is incompatible with the high-polarity environment, and the interfacial energy rises sharply, causing the outermost layer of the microcapsule structure to become unstable, and the third extraction solvent is preferentially released. The low-polarity solvent released quickly infiltrates the hydrophobic regions of the dried blood spot, dissolving steroids, long-chain fatty acid derivatives, and secondary bile acids.

[0013] During the third stage of the extraction process (5-10 min), as the third extraction solvent is released, the overall polarity of the system decreases, and P(NIPAM-co-AAc) undergoes a hydrophobic phase transition, triggering the expansion of the micropores in the intermediate responsive layer. At the same time, the microenvironment pH in which the second extraction solvent is located is still above 6.0, and P(MAA-co-MMA) continues to swell, which together causes the interface encapsulating the second extraction solvent to rupture. The second extraction solvent is then released, and its 60% acetonitrile content effectively precipitates residual proteins, while the 10 mM ammonium acetate buffer system maintains a pH of 4.2, optimizing the ionization state of organic acids and bile acids, and improving their extraction efficiency.

[0014] In the fourth stage (10-15 min) of the extraction process, the acetonitrile proportion in the system is further diluted due to the release of the second extraction solvent, the polarity rises, and P(NIPAM-co-AAc) restores hydrophilicity, but at this time the microenvironment of the inner first extraction solvent presents high ionic strength and high water activity due to the dissolution of the previous metabolites. This environment triggers the excessive swelling of P(MAA-co-MMA) at the inner interface, causing the innermost membrane structure to break and release the first extraction solvent. The high water phase environment ensures that strong hydrophilic metabolites such as short-chain acylcarnitines, glycine, and serine are completely dissolved, avoiding their loss by precipitation in the organic phase.

[0015] The average particle size of the microcapsules is 15 μm, and the standard deviation of the particle size distribution is less than 2 μm, which is prepared by microfluidic droplet generation technology combined with ultraviolet light-induced cross-linking process. The drug loading capacity of the microcapsules is 0.35 microliters of the first extraction solvent, 0.40 microliters of the second extraction solvent, and 0.25 microliters of the third extraction solvent per milligram of microcapsules, and the total solvent release efficiency reaches more than 98% within 15 minutes.

[0016] The dry blood spot sample has a diameter of 3.2 mm and is collected on Whatman 903 filter paper. The blood spot drying time is 2 hours, and the storage condition is room temperature and light protection. When extracting, a single dry blood spot sample is mixed with 500 microliters of microcapsule suspension (concentration of 20 mg / mL) in a 1.5 milliliter polypropylene centrifuge tube, and placed in a 37°C constant temperature oscillator at 600 rpm for 15 minutes.

[0017] After the extraction is completed, the mixture is centrifuged at 14000 x g for 10 min, 200 microliters of supernatant is taken, filtered through a 0.22 μm polytetrafluoroethylene filter membrane, and then injected into a liquid chromatography-mass spectrometry system. The liquid chromatography uses a C18 reversed-phase chromatographic column (100 mm x 2.1 mm, 1.7 μm particle size), the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile containing 0.1% formic acid, and the gradient elution program is: 0-1 min, 5% B; 1-8 min, 5%-95% B; 8-10 min, 95% B; 10-10.1 min, 95%-5% B; 10.1-12 min, 5% B.

[0018] As a preferred embodiment of the present application, the formic acid concentration in the first extraction solvent can be adjusted to 0.5%-2.0% to optimize the protonation efficiency of amino acids; the ammonium acetate concentration in the second extraction solvent can be adjusted in the range of 5-20 mM to match the pKa values of different organic acids; the proportion of chloroform in the third extraction solvent can be varied between 10%-30% to enhance the solubility of lipid-soluble steroids. All adjustments need to be synchronized to calibrate the polymer composition ratio of the intermediate response layer to ensure the consistency of the trigger threshold and the solvent release timing.

[0019] As another preferred embodiment of the present application, the microcapsules can integrate magnetic nanoparticles in the outer protective shell, facilitating the rapid separation of microcapsule residues by an external magnetic field after extraction, simplifying the supernatant acquisition step, and being suitable for high-throughput automated platforms.

[0020] In addition, the method of the present application also has advantages in terms of dry blood spot sample stability. Since the microcapsules maintain a closed state at the beginning of extraction, they effectively isolate light-sensitive metabolites (such as vitamin D3 derivatives and certain unsaturated fatty acids) from the external environment.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] The present application achieves the technical effect of releasing optimized extraction solutions in stages according to the categories of metabolites in a single operation process by constructing a pH / polarity dual-trigger microcapsule solvent carrier. This method breaks through the limitations of traditional static solvent systems, solves the problem of unbalanced recovery rate of seven types of metabolites with large polarity span in dry blood spot extraction due to insufficient solvent adaptability, and significantly improves the accuracy, reproducibility, and coverage of the DBS-LC-MS / MS platform in the simultaneous detection of multiple-component metabolites. DETAILED DESCRIPTION

[0023] The present application provides a dry blood spot liquid chromatography-mass spectrometry detection method suitable for seven types of metabolites. The core of the method is to construct a pH / polarity dual-trigger microcapsule solvent carrier system, release extraction solvents with targeted polarity and pH environment in stages according to the physicochemical properties of target metabolites in a single extraction operation process, and achieve efficient and balanced simultaneous extraction of seven types of metabolites with large structural and polarity span, such as amino acids, acylcamitines, organic acids, bile acids, steroids, fatty acids, and sugar metabolism intermediates. This method completely avoids the systematic recovery bias problem caused by the inability to consider both hydrophilic and hydrophobic components in traditional single solvent systems.

[0024] The technical solutions of the present application will be described in detail below in conjunction with specific examples and comparative examples to ensure that those skilled in the art can fully understand and implement the present application.

[0025] Example 1: The average particle size of the microcapsules is 15 μm, the intermediate response layer is P(MAA-co-MMA) 65% + P(NIPAM-co-AAc) 35%, the first solvent is ultrapure water: formic acid = 95:5, the second solvent is acetonitrile: ammonium acetate aqueous solution = 60:40, the third solvent is isopropanol: chloroform = 80:20 with the addition of 0.1% formic acid, the extraction is performed at 37℃ with 600 rpm oscillation for 15 min, and the liquid chromatography-mass spectrometry gradient elution is performed.

[0026] Preparation process: layered microcapsules were prepared by microfluidic technology; dry blood spot mixed with microcapsule suspension for extraction; centrifugal filtration followed by LC-MS analysis.

[0027] Example 2: microcapsule average particle size 13 μm, the rest of the formulation and process are the same as example 1;

[0028] Preparation process: the same as example 1.

[0029] Example 3: microcapsule average particle size 17 μm, the rest of the formulation and process are the same as example 1;

[0030] Preparation process: the same as example 1.

[0031] Example 4: the second solvent acetonitrile: ammonium acetate aqueous solution = 50:50, the rest of the formulation and process are the same as example 1;

[0032] Preparation process: the same as example 1.

[0033] Example 5: the second solvent acetonitrile: ammonium acetate aqueous solution = 70:30, the rest of the formulation and process are the same as example 1;

[0034] Preparation process: the same as example 1.

[0035] Example 6: extraction time 12 min, the rest of the formulation and process are the same as example 1;

[0036] Preparation process: the same as example 1.

[0037] Example 7: extraction temperature 40℃, the rest of the formulation and process are the same as example 1;

[0038] Preparation process: the same as example 1.

[0039] Example 8: the outer shell of the microcapsule contains 0.5% Fe3O4@SiO2 magnetic nanoparticles, the rest of the formulation and process are the same as example 1;

[0040] Preparation process: the same as example 1 (additional magnetic field separation of microcapsule residues after extraction).

[0041] Comparative example 1: no microcapsule, using methanol: water = 80:20 single solvent extraction; the rest of the detection conditions are the same as example 1;

[0042] Preparation process: dry blood spot mixed with single solvent and oscillated for 15 min, centrifugal filtration followed by LC-MS analysis.

[0043] Comparative example 2: microcapsule without pH / polarity responsive layer, three-layer solvent is released at the same time; the rest of the formulation and process are the same as example 1;

[0044] Preparation process: the same as example 1 (microcapsule without responsive layer design).

[0045] Test method:

[0046] Extraction performance test: The recovery rate of 7 types of metabolites was determined by standard addition recovery experiment; the relative standard deviation RSD was calculated; the extraction efficiency of hydrophilic, hydrophobic and moderately polar metabolites was respectively calculated.

[0047] Detection accuracy test: The characteristic ion pairs were detected by liquid chromatography-mass spectrometry in multiple reaction monitoring mode; the reproducibility of retention time and peak area was verified; the degradation rate of photosensitive metabolites was evaluated.

[0048] Process adaptability test: The microcapsule solvent release timing was observed; the post-extraction processing efficiency was tested; the dispersion uniformity of microcapsules of different particle sizes was verified.

[0049] The test data are shown in Tables 1 and 2.

[0050] Table 1 Comparison table of average recovery rate, RSD and recovery rate of hydrophilic metabolites

[0051] Test Item Average Recovery (%) RSD (%) Recovery of Hydrophilic Metabolite (%) Example 1 94 6.2 92 Example 2 92 6.8 90 Example 3 93 6.5 91 Example 4 91 7 89 Example 5 95 5.8 93 Example 6 90 7.2 88 Example 7 93 6.3 91 Example 8 94 6 92 Comparative Example 1 75 15.8 68 Comparative Example 2 82 10.5 78

[0052] Table 2 Comparison table of recovery rate of hydrophobic metabolites and degradation rate of photosensitive metabolites

[0053]

[0054] Examples 1-8 have a gradient release adaptability solvent, the average recovery rate is ≥90%, and the RSD is ≤7.2%, which is much better than the comparative examples; the single solvent of Comparative Example 1 cannot consider both hydrophilic and hydrophobic metabolites, and Comparative Example 2 has no responsive release, resulting in unbalanced recovery rate, which confirms that the double-trigger microcapsule is the key to balanced extraction.

[0055] The microcapsule particle size is in the range of 13-17 μm, and the extraction performance is stable; the proportion of the second solvent acetonitrile increases (Example 4→1→5), and the recovery rate of moderately polar metabolites increases; the extraction time and temperature are in the optimized range, and the performance is less affected by shortening the time or increasing the temperature.

[0056] The RSD of Examples 1-8 is ≤7.2%, and the degradation rate of photosensitive metabolites is ≤4%, which is significantly better than the comparative examples due to the closed protection and precise release of the microcapsule.

[0057] Examples 1-8 cover 7 types of metabolites, have balanced recovery rate and good reproducibility, are suitable for neonatal screening, clinical diagnosis and other scenes; the integrated magnetic particles (Example 8) improve the processing efficiency and are suitable for high-throughput detection.

[0058] The method described in the application solves the problem of unbalanced recovery rate of traditional single solvent extraction by gradient solvent release of pH / polarity double-trigger microcapsules, different parameter combinations can achieve efficient and accurate detection, and is suitable for synchronous analysis of multiple types of metabolites.

[0059] The above merely provides the preferred embodiments of the application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall fall in the protection scope of the present application.

Claims

1. A method for detecting 7-class metabolites from dried blood spot by liquid chromatography-mass spectrometry, characterized in that, The method comprises the following steps: Preparation of a microcapsule solvent carrier composed of an inner core solvent phase, an intermediate response layer and an outer protective shell, wherein the inner core solvent phase comprises a first extraction solvent, a second extraction solvent and a third extraction solvent encapsulated in a concentric spherical layered structure, and the intermediate response layer is composed of pH-sensitive poly(methacrylic acid-co-methyl methacrylate) and polarity-responsive poly(N-isopropyl acrylamide-co-acrylic acid); Placing the dry blood spot sample to be tested in the suspension of the microcapsule solvent carrier and oscillating extraction; During the extraction process, the microcapsule solvent carrier ruptures and releases the corresponding extraction solvent in a preset order of the third extraction solvent, the second extraction solvent and the first extraction solvent according to the changes in the local pH value and the polarity environment of the dry blood spot eluent, forming a gradient polarity extraction environment, wherein the first extraction solvent is ultrapure water and formic acid at a volume ratio of 95:5, the second extraction solvent is acetonitrile and an aqueous solution containing 10 mM ammonium acetate at a volume ratio of 60:40, and the third extraction solvent is isopropanol and chloroform with the addition of 0.1% formic acid at a volume ratio of 80:20; After the extraction is completed, the supernatant is collected by centrifugation, filtered through a filter membrane and directly injected into a liquid chromatography-mass spectrometry system for analysis.

2. The method for the detection of 7-class metabolites adapted for dried blood spot liquid chromatography-mass spectrometry according to claim 1, characterized in that, The microcapsule solvent carrier encapsulates at least three extraction solvents with different polarity parameters and pH environments.

3. The method of claim 2, wherein the 7-type metabolite adapted dried blood spot LC-MS / MS method is characterized by, The microcapsule solvent carrier is composed of an inner core solvent phase, an intermediate response layer and an outer protective shell.

4. The method of claim 1, wherein the 7-type metabolite adapted dried blood spot LC-MS / MS method is characterized by, The poly(methacrylic acid-co-methyl methacrylate) is deprotonated to induce swelling when the pH is higher than 6.0, and the poly(N-isopropyl acrylamide-co-acrylic acid) has a critical solution temperature of 32℃ and undergoes a hydrophobic phase transition when the polarity of the system decreases.

5. The method of claim 3, wherein the 7-type metabolite adapted dried blood spot LC-MS / MS method is characterized by, The outer protective shell is formed by ionic crosslinking of crosslinked sodium alginate and chitosan, which remains structurally stable after contacting the dry blood spot eluent, and then dissociates due to the local increase in pH, and after dissociation, the release of the solvent is initiated.

6. The method of claim 1, wherein the 7-class metabolite adapted dried blood spot LC-MS / MS method is characterized by, The third extraction solvent is preferentially released in the early stage of the extraction process and is used to extract steroid hormones, long-chain fatty acid derivatives and secondary bile acids.

7. The method of claim 1, wherein the 7-class metabolite adapted dried blood spot LC-MS / MS method is characterized by, The second extraction solvent is released in the middle stage of the extraction process, and its acetonitrile component is used to precipitate residual proteins, and the ammonium acetate buffer system maintains a local pH of 4.2 to optimize the extraction efficiency of organic acids and bile acids.

8. The method of claim 1, wherein the 7-class metabolite adapted dried blood spot LC-MS / MS method is characterized by, The first extraction solvent is released in the late stage of the extraction process, and its high water phase environment ensures the complete dissolution of short-chain acylcarnitines, neutral amino acids and glycolytic intermediates.

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