Method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in-situ labeling

Abstract

The invention relates to the field of biological detection and analysis, and particularly discloses a method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dried blood sampling and in-situ labeling. According to the method, through synchronous dry blood sampling and in-situ labeling reaction, chemical derivatization of a target compound is completed in the sampling process, the target compound is converted into a non-volatile and chemically stable product, and the loss of the target compound is effectively avoided. Meanwhile, the adopted derivatization reagent can introduce chromophores or easy-to-ionize groups on target molecules, so that the chromatographic retention capability and the detection sensitivity of the target molecules are remarkably enhanced. According to the method, additional sample pretreatment is not needed, sampling and marking are performed synchronously, experimental errors are reduced, and sample stability and detection precision are improved. The method can be widely applied to clinical diagnosis, metabonomics research and biomedical analysis, and a novel and convenient technical scheme is provided for efficient analysis of volatile metabolites in trace blood samples.

Description

Technical Field

[0001] The invention belongs to the field of biological analysis and chromatography-mass spectrometry detection, and specifically relates to a method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dried blood spot or dried blood tip sampling and in-situ labeling. Background Art

[0002] Blood is an important carrier of human metabolic activities, and the metabolites in it can reflect the physiological state and the development of diseases. Therefore, the analysis of blood metabolites is of great significance in clinical diagnosis, metabolomics research, disease screening and personalized medicine. Volatile fatty acids and fatty aldehydes are key endogenous metabolites in the blood, and play an important role in physiological and pathological processes such as inflammatory response, energy metabolism, intestinal flora balance and nervous system diseases. For example, volatile fatty acids are involved in short-chain fatty acid metabolism and are closely related to diabetes, cardiovascular disease and intestinal flora imbalance, while volatile fatty aldehydes can be used as biomarkers of lipid peroxidation and oxidative stress.

[0003] Blood collection is important for analyzing volatile fatty acids and fatty aldehyde metabolites in the blood. In terms of blood collection methods, there are two methods: venous sampling and peripheral blood sampling. Conventional testing usually relies on venous blood sampling, but this method requires professional medical staff to operate, the blood collection volume is large, and the storage and transportation conditions are high. It is not suitable for large-scale screening, children and elderly patients, or resource-constrained areas. In contrast, peripheral blood sampling (such as fingertip blood, heel blood) is a more convenient, trace, and autonomous method that only requires a slightly upgraded blood sample to complete the test. Therefore, it has attracted much attention in applications such as neonatal disease screening, chronic disease monitoring, and personalized health management.

[0004] Dried blood spot (DBS) technology and dried blood tip technology (collectively referred to as dried blood sampling technology or dried blood technology) are micro-blood collection technologies based on peripheral blood. Their core advantages are: (1) the required blood sample volume is extremely small (μL level), which is suitable for low sample volume analysis; (2) no cold chain storage is required, the sample stability is high, and it is convenient for long-term storage; (3) the operation is simple, and it is easy to collect and mail remotely; (4) it reduces the risk of cross infection and is suitable for large-scale epidemiological studies. Due to these advantages, dried blood sampling technology has been widely used in newborn screening, drug monitoring, environmental exposure assessment and metabolomics analysis.

[0005] Although dry blood sampling technology has many advantages in the analysis of blood metabolites, it still has obvious shortcomings in the detection of volatile fatty acids and fatty aldehydes. First, due to the high volatility of volatile fatty acids and fatty aldehydes, gas phase losses are prone to occur during the sampling and drying process of dry blood, resulting in deviations in the test results. Secondly, the chemical stability of these compounds is poor and they are easily affected by environmental factors (such as temperature and humidity). Oxidation, degradation or transformation may occur during storage. In addition, the lack of chromophores and easily ionized groups in the molecular structure of volatile fatty acids and fatty aldehydes leads to weak chromatographic responses during high-performance liquid chromatography (HPLC) or gas chromatography (GC) analysis, low ionization efficiency in mass spectrometry detection, limited sensitivity and selectivity of direct detection, and difficult to accurately measure. Summary of the invention

[0006] In view of the defects and problems existing in the detection of volatile fatty acids and fatty aldehydes based on the current dry blood sampling technology, the present invention provides a method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling.

[0007] The present invention provides a method for detecting volatile fatty acids and fatty aldehydes in trace peripheral blood based on dried blood sampling and in situ labeling, comprising the following steps: S1, collecting a trace amount of peripheral blood using a dried blood spot or a dried blood tip, adding a derivatization reagent for in situ labeling, and then drying to obtain a dried sample;

[0008] S2, adding a desorption solvent to the dried sample for desorption to obtain a desorption solution;

[0009] S3. The desorption liquid is subjected to instrumental analysis to achieve quantitative detection of volatile fatty acids or volatile fatty aldehydes.

[0010] In the above-mentioned method for detecting volatile fatty acids and fatty aldehydes in trace peripheral blood based on dry blood sampling and in situ labeling, in step S1, for the detection of volatile fatty acids, any one of Cholamine, acyl chlorides, esterification reagents, amines, and hydroxylamines is selected as a derivatization reagent; for the detection of volatile fatty aldehydes, any one of Girard's P, hydroxylamine, hydrazines, aromatic amines, phenylhydrazones, and dansyl chloride is selected as a derivatization reagent.

[0011] The above-mentioned method for detecting volatile fatty acids and fatty aldehydes in trace peripheral blood based on dried blood sampling and in situ labeling adopts the dried blood spot technology to collect trace peripheral blood. A blood collection needle is used to puncture the peripheral part, and 20-30 μL of blood is dripped onto a dried blood spot card or filter paper and the blood is evenly spread in the blood collection area. A derivatization reagent solution is added when the blood sample is not completely dry, and then the blood is dried to obtain a dried derivatized blood spot sample.

[0012] In the above-mentioned method for detecting volatile fatty acids and fatty aldehydes in trace peripheral blood based on dry blood sampling and in situ labeling, in step S2, a derivatized blood spot sample after drying is taken, a desorption solvent is added, and the target analyte is released by shaking or ultrasonic treatment to obtain a desorption liquid, the desorption liquid is centrifuged to obtain a supernatant, and the supernatant is subjected to quantitative detection of volatile fatty acids or volatile fatty aldehydes.

[0013] The above-mentioned method for detecting volatile fatty acids and fatty aldehydes in trace peripheral blood based on dry blood sampling and in situ labeling, when using a dry blood pipette tip to collect trace peripheral blood, a hydrophilic fiber material is loaded in the pipette tip, and a micropipette is used to quantitatively absorb 20-30 μL of peripheral blood, and the blood is moved up and adsorbed to the fiber material by negative pressure; the pipette tip is placed in a drying device, and the reverse drainage technology is used to make the blood sample evenly adhere and dry and fix under constant temperature conditions.

[0014] In the above-mentioned method for detecting volatile fatty acids and fatty aldehydes in trace peripheral blood based on dry blood sampling and in situ labeling, in step S2, a desorption solvent is added to the dried dry blood tip and the target metabolites are released from the fiber matrix by repeated blowing or slight vortexing to obtain an extract, and the extract is subjected to quantitative detection of volatile fatty acids or volatile fatty aldehydes.

[0015] In the above-mentioned method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling, the desorption solvent is selected from any one of the following: (a) a single solvent among methanol, acetonitrile or acetone; (b) a mixed solution of at least one solvent among methanol, acetonitrile, acetone and water; (c) a mixed solution of two or more solvents among methanol, acetonitrile and acetone.

[0016] The above-mentioned method for detecting volatile fatty acids and fatty aldehydes in trace peripheral blood based on dry blood sampling and in situ labeling adopts high performance liquid chromatography-fluorescence detection, high performance liquid chromatography-mass spectrometry, liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry (GC-MS) to analyze the target derivatization products for quantitative detection of volatile fatty acids or volatile fatty aldehydes.

[0017] In the above-mentioned method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling, when performing in situ labeling in step S1, the in situ labeling reaction of volatile fatty acids can also be promoted by adding a buffer system or a catalyst; the in situ labeling reaction of volatile fatty aldehydes can also be promoted by adding a buffer system or a catalyst or a reaction medium.

[0018] In the above-mentioned method for detecting volatile fatty acids and fatty aldehydes in trace peripheral blood based on dry blood sampling and in situ labeling, the buffer system is optimized according to the properties of the detection target, wherein: the derivatization reaction of volatile fatty acids adopts pH 6.0-8.5 phosphate buffer; the derivatization reaction of volatile fatty aldehydes uses a weak acid buffer, such as ammonium acetate buffer or phosphate buffer; the selection principle of the catalyst is: the labeling of volatile fatty acids adopts EDC / HOBt condensation agent; 0.05% EDTA is added to the labeling system of volatile fatty aldehydes; the reaction medium is methanol, acetonitrile or a mixed solvent thereof with water.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling proposed in the present invention completes in situ derivatization during the sampling process, converts volatile fatty acids and fatty aldehydes into non-volatile, stable derivatization products, which can effectively avoid analyte loss and avoid detection deviation caused by volatility loss.

[0021] 2. The detection method of trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling of the present invention can introduce chromophores or easily ionized groups, improve reaction activity and conversion rate, significantly improve the chromatographic separation ability and mass spectrometry detection signal of the target analyte, thereby improving analytical sensitivity and quantitative accuracy. 3. The detection method of trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling of the present invention can simplify the pretreatment process, complete the derivatization simultaneously during sampling, eliminate the additional derivatization step, simplify the experimental operation, and improve the detection efficiency.

[0022] 4. The method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling of the present invention is applicable to a variety of analytical platforms, and the obtained derivatization products can be directly applied to HPLC-MS or GC-MS detection without the need for additional derivatization steps, thereby improving the operability and applicability of the experiment.

[0023] 5. The method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling of the present invention combines dry blood spot sampling with in situ labeling, so that samples can be stored for a long time at room temperature, reducing the need for cold chain transportation and facilitating remote sample collection and centralized testing.

[0024] 6. The method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling of the present invention is suitable for clinical disease screening, metabolomics research, drug metabolism analysis and biomedical monitoring, and can be widely used in the fields of early disease diagnosis, nutritional metabolism research and personalized medicine.

[0025] 7. The present invention optimizes the derivatization reaction system and adopts the following technical means to achieve efficient labeling reaction: (1) Buffer system: The derivatization reaction of volatile fatty acids adopts pH 6.0-8.5 phosphate buffer to stabilize the carboxylic acid condensation reaction; the derivatization reaction of volatile fatty acids uses a weak acid buffer, including ammonium acetate buffer or phosphate buffer (pH <7); (2) Catalyst: EDC / HOBt condensation agent is used for carboxylic acid activation in volatile fatty acid labeling; 0.05% EDTA is added to volatile fatty aldehyde labeling to inhibit metal ion catalytic side reactions; (3) Reaction medium: methanol, acetonitrile or a mixed solvent thereof with water is selected to balance the solubility of the reagent, the reaction environment and the stability of the metabolite. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The present invention is a flow chart of the overall process of detecting volatile fatty acids and fatty aldehydes in fingertip blood samples by dry blood sampling and in situ labeling.

[0027] Figure 2 Schematic diagram of the process for the detection of volatile fatty acids and fatty aldehydes in fingertip blood samples using dried blood spot sampling combined with in situ labeling.

[0028] Figure 3 Schematic diagram of the process of detecting volatile fatty acids and fatty aldehydes in peripheral blood samples using dried blood tip sampling combined with in situ labeling technology.

[0029] Figure 4 Results of volatile fatty acids detection in fingertip blood samples using dried blood tip sampling and in situ labeling combined with LC-MS / MS.

[0030] Figure 5 Detection results of volatile fatty aldehydes in fingertip blood samples using dried blood tip sampling and in situ labeling combined with LC-MS / MS. DETAILED DESCRIPTION

[0031] Based on the existing dry blood sampling technology, the present invention innovatively introduces the synchronous sampling and in situ labeling strategy, such as Figure 1 As shown, the storage stability and detection sensitivity of volatile fatty acids and fatty aldehydes are significantly improved. The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0032] Example 1: This example provides a method for synchronous detection of trace peripheral blood volatile fatty acids and fatty aldehydes sampling markers. The method is based on dried blood spot sampling technology (DBS). The collection and detection process is as follows: Figure 2 As shown, it specifically includes the following contents.

[0033] (1) Peripheral blood sampling and dried blood spot preparation: Use a blood collection needle to puncture the fingertip, collect 20-30 μL of fingertip blood, and drip it onto the dried blood spot card to ensure that the blood penetrates and evenly diffuses to the sample area; before the blood is dry, add 20 μL of derivatization reagent solution to allow it to fully contact with the target compound for derivatization reaction, wherein a solution containing 100 mM Cholamine and 1 mM formic acid is used as a derivatization reagent for the derivatization of volatile fatty acids, and 100 mM Girard's P is used as a derivatization reagent for the derivatization of volatile fatty aldehydes;

[0034] The derivatized dried blood spots were placed in a ventilated environment to dry naturally, or dried at 37°C for 1-2 hours, and then sealed and stored to prevent moisture from affecting sample stability.

[0035] (2) Target extraction: Use a hole puncher or scissors to cut a dry blood spot and place it in a 1.5 mL centrifuge tube. Add 200 μL of 50% methanol solution as a desorption solvent to the centrifuge tube to fully release the target product. Vortex gently at room temperature for 5 min to ensure that the sample is completely infiltrated, then centrifuge at 12,000 rpm for 5 min, and take the supernatant for LC-MS / MS analysis.

[0036] (3) Liquid chromatography-mass spectrometry (LC-MS / MS) analysis:

[0037] Liquid chromatography (LC) conditions:

[0038] Chromatographic column: ACQUITY UPLC BEH C18 (1.7 μm, 2.1 mm × 50 mm)

[0039] Mobile phase A: 0.1% formic acid in water

[0040] Mobile phase B: 0.1% formic acid in acetonitrile

[0041] Elution method: gradient elution

[0042] Flow rate: 0.2mL / min

[0043] Column temperature: 30°C

[0044] Injection volume: 2~5μL.

[0045] Mass spectrometry (MS) detection conditions:

[0046] Mass spectrometer: Triple quadrupole mass spectrometer (QqQ-MS)

[0047] Ion source mode: electrospray ionization (ESI + )

[0048] Ion source voltage: 2.8-3.0kV

[0049] Capillary temperature: 300-400℃

[0050] Data acquisition mode: reaction monitoring mode (MRM).

[0051] Example 2: This example provides a method for synchronous detection of trace peripheral blood volatile fatty acids and fatty aldehydes by sampling and labeling. The difference from Example 1 is that this example uses a method of pre-coating a derivatization reagent for in situ labeling, which specifically includes the following contents:

[0052] (1) Pretreatment of dried blood spot card and application of derivatization reagent:

[0053] Derivatization reagents were pre-coated on the sample area of ​​the dried blood spot card, wherein the volatile fatty acid labeling area was coated with 20 μL of a solution containing 100 mM Cholamine and 1 mM formic acid, and the volatile fatty aldehyde labeling area was coated with 20 μL of a 100 mM Girard's P solution;

[0054] The coated dried blood spot card was placed in a 37°C oven to dry for 30 minutes to ensure that the reagent was evenly distributed and a stable coating was formed.

[0055] (2) Peripheral blood sampling and dried blood spot preparation:

[0056] Use a blood collection needle to puncture the fingertip, collect 20-30 μL of fingertip blood, and drip it onto the pre-treated dry blood spot card to ensure that the blood is fully in contact with the derivatization reagent;

[0057] Place the dried blood spot card in a ventilated environment to dry naturally, or dry it at 37°C for 1-2 hours, and then seal it for storage.

[0058] (3) Target desorption:

[0059] Use a hole punch or scissors to cut out dried blood spots and place them in a 1.5 mL centrifuge tube;

[0060] Add 200 μL of 50% methanol solution as desorption solvent to the centrifuge tube and vortex gently for 5 min at room temperature to ensure sufficient release of the target product;

[0061] The mixture was centrifuged at 12,000 rpm for 5 minutes and the supernatant was used for LC-MS / MS analysis.

[0062] (4) Liquid chromatography-mass spectrometry (LC-MS / MS) analysis: Same as Example 1

[0063] This embodiment simplifies the operation steps and improves the labeling efficiency by pre-coating the derivatization reagent, while avoiding the problem of uneven mixing of the derivatization reagent and blood, and is suitable for high-throughput sample analysis.

[0064] Example 3: This example provides a method for synchronous detection of trace peripheral blood volatile fatty acids and fatty aldehydes by sampling and labeling. The method is based on the dry blood tip sampling technology, specifically using the quantitative blood sampling device disclosed in the patent publication number CN115200943A for sampling and derivatization in situ labeling. The collection and detection process is as follows: Figure 3 As shown, it specifically includes the following contents.

[0065] (1) Peripheral blood sampling: A micropipette tip loaded with hydrophilic fibers is used as a blood collection device. When used for blood collection, a fixed volume of fingertip blood sample is collected using a micropipette. By rotating the pipette knob, negative pressure is used to move the blood up along the inner wall of the tip and it is absorbed by the hydrophilic fiber material to ensure stable sample collection. This technology avoids the problem of uneven blood spot diffusion in conventional DBS sampling and provides more accurate sample volume control.

[0066] (2) In situ labeling: Use a micropipette to collect a 20 μL fingertip blood sample, allowing the blood to move up along the inner wall of the tip and be absorbed by the hydrophilic fiber material; rotate the pipette knob to draw up another 20 μL of the derivatization reagent solution to ensure that the reagent is in full contact with the sample and complete the in situ labeling reaction; place the sampled and labeled dry blood tip in a tip box containing a desiccant for subsequent storage and analysis.

[0067] For the detection of volatile fatty acids, this embodiment selects a solution containing 100mM Cholamine and 1mM formic acid as a derivatization labeling reagent to improve the chromatographic separation effect and mass spectrometry detection sensitivity. In addition to Cholamine, any of acyl chlorides, esterification agents, amines, and hydroxylamines can also be selected.

[0068] For the detection of volatile fatty aldehydes, this example selects a solution of 100 mM Girard's P and 1 mM formic acid as a derivatization labeling reagent to enhance the mass spectrometry response signal of the target analyte. In addition, any one of hydroxylamine, hydrazines, aromatic amines, phenylhydrazones, and dansyl chloride can also be selected.

[0069] (3) Sample extraction and detection: Add 200 μL of 50% methanol solution as a desorption solvent to the dry blood tip to fully wet the fiber material so that the target product is released from the matrix; after appropriate vortexing or standing (e.g., 5 min), the obtained desorption solution is directly used for LC-MS / MS detection.

[0070] In terms of liquid chromatography (LC) separation conditions, an ACQUITY UPLC BEH C18 column (particle size 1.7 μm, column size 2.1 mm × 50 mm) was used for separation. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. A gradient elution program was used for separation to optimize the retention time and peak shape of the target compound. The chromatographic flow rate was set to 0.2 mL / min, the column temperature was 30 ° C, and the injection volume was set between 2 and 5 μL to ensure good separation effect and stable signal response.

[0071] In terms of mass spectrometry (MS) detection, triple quadrupole mass spectrometry (QqQ-MS) was used for high-sensitivity quantitative analysis of target compounds. The electrospray ionization source (ESI) working mode was set to positive ion mode (ESI + ) to improve the ionization efficiency of the target compound. The ion source voltage was adjusted to 2.8-3.0 kV and the capillary temperature was controlled at 300-400 °C to ensure efficient ionization. Data acquisition adopted the reaction monitoring mode (MRM) to achieve highly selective and sensitive quantitative detection.

[0072] Example 4: This example verifies the application of the method of Example 3 in the detection of volatile fatty acids and fatty aldehydes in fingertip blood samples.

[0073] In this example, the pipette tips used were from Thermo Scientific TM Labtip 200μL polypropylene pipette tip was combined with 5mg cotton wool fiber to prepare hydrophilic fiber loaded pipette tip for quantitative blood collection operation. The performance of this method in the analysis of volatile metabolites (volatile fatty acids and volatile fatty aldehydes) was evaluated using spiked whole blood samples as the test matrix.

[0074] (1) Detection of volatile fatty acids

[0075] Use a cotton fiber-loaded pipette tip to draw 20 μL of a whole blood sample spiked with a 1 μM volatile fatty acid mixed standard solution (including acetic acid, propionic acid, butyric acid, and valeric acid). Subsequently, draw 20 μL of a derivatization reagent solution (containing 100 mM Cholamine, 20 mM HOBT, 20 mM HATU, and 20 mM TEA), and rotate the knob of the micropipette to allow the derivatization reagent solution to move up along the inner wall of the pipette tip and be fully absorbed by the cotton fiber. Place the pipette tip in a pipette tip box containing a desiccant and let it stand at room temperature for 48 hours to ensure that the target metabolites are completely derivatized. Finally, use 200 μL of a 50% methanol solution for desorption, centrifuge the resulting desorbed solution at 12,000 rpm for 5 minutes, and take the supernatant for LC-MS / MS analysis. The results are as follows Figure 4 shown.

[0076] The experimental results showed that the labeled products of the four volatile fatty acids could be successfully detected, and no interference signals were found in the blank control. This experimental result further verified the feasibility of the dried blood tip / in situ labeling strategy in the analysis of volatile fatty acids.

[0077] (2) Detection of volatile fatty aldehydes

[0078] First, use a cotton fiber-loaded pipette tip to draw 20 μL of a 1 μM volatile fatty aldehyde mixed standard solution (including valeraldehyde, hexanal, heptanal, and octanal) spiked whole blood sample. Subsequently, draw 20 μL of a derivatization reagent solution (containing 100 mM Girard's P and 1 mM formic acid), and rotate the micropipette knob to allow the derivatization reagent solution to move up along the inner wall of the pipette tip and be fully absorbed by the cotton fiber. Subsequently, place the pipette tip in a pipette tip box containing a desiccant and let it stand at room temperature for 48 hours to ensure that the derivatization reaction is fully completed. Finally, use 200 μL of 50% methanol solution for desorption, and the resulting desorbed solution is directly used for LC-MS / MS detection. The results are shown in the figure. Figure 5 shown.

[0079] The experimental results showed that the labeled products of the four volatile fatty aldehydes could be successfully detected, and no interference signals appeared in the blank control, which verified the effectiveness and feasibility of the dried blood tip / in situ labeling strategy in the analysis of volatile fatty aldehydes.

[0080] Comparative Example 1: In order to verify the labeling effect of different derivatization reagents, this comparative example is based on Example 4. The experiment uses the same spiked whole blood sample as the test matrix. The difference is that 20 μL, 100 mM Cholamine reagent is used for derivatization labeling, specifically:

[0081] A 20 μL, 1 μM volatile fatty acid mixed standard solution (including acetic acid, propionic acid, butyric acid and valeric acid) spiked whole blood sample was taken using a cotton fiber loaded pipette tip. Subsequently, 20 μL of 100 mM Cholamine derivatization reagent was taken, and the derivatization reagent solution was moved up along the inner wall of the pipette tip by rotating the micropipette knob and fully absorbed by the cotton fiber. The pipette tip was placed in a pipette tip box containing a desiccant and allowed to stand at room temperature for 48 hours to ensure complete derivatization of the target metabolites. Finally, 200 μL of 50% methanol solution was used for desorption, and the resulting desorbed solution was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was taken for LC-MS / MS analysis.

[0082] The experimental results showed that the derivatized products of the four volatile fatty acids could not be detected, indicating that Cholamine alone could not directly react with volatile fatty acids.

[0083] Comparative Example 2: This comparative experiment is used to verify the detection of target volatile metabolites (volatile fatty acids and volatile fatty aldehydes) when derivatization is not performed synchronously during the sampling stage.

[0084] (1) Detection of volatile fatty acids

[0085] The same spiked whole blood samples were used as the test matrix in the experiment, except that in the comparative experiment, derivatization was not performed simultaneously during the sampling stage, but during the subsequent desorption stage.

[0086] Use a loaded cotton fiber pipette tip to absorb 20 μL of a whole blood sample spiked with a 1 μM volatile fatty acid mixed standard solution (including acetic acid, propionic acid, butyric acid, and valeric acid). Place the pipette tip in a pipette tip box containing a desiccant and leave it at room temperature for 48 hours to dry the sample.

[0087] Desorption was performed by 200 μL of 50% methanol solution, and the desorbed solution was mixed with 20 μL of derivatization reagent solution (containing 100 mM Cholamine, 20 mM HOBT, 20 mM HATU and 20 mM TEA). The reaction was carried out at room temperature for 2 h to complete the derivatization. After the reaction was completed, it was used for LC-MS / MS analysis.

[0088] Experimental results: No signal peaks of the derivatized products of the target volatile fatty acids were observed in the LC-MS / MS detection. This indicates that when the derivatization treatment was not performed synchronously during the sampling stage, the volatile fatty acids had been volatilized and lost during the drying process, resulting in the inability to detect their derivatized products.

[0089] (2) Detection of volatile fatty aldehydes

[0090] Use a cotton fiber loaded ordinary pipette tip to absorb 20 μL of 1 μM volatile fatty aldehyde mixed standard solution (including valeraldehyde, hexanal, heptanal and octanal) spiked whole blood sample. Place the pipette tip in a pipette tip box containing desiccant and let it stand at room temperature for 48 hours to dry the sample.

[0091] Desorption was performed by 200 μL of 50% methanol solution, and the desorbed solution was mixed with 20 μL of derivatization reagent solution (containing 100 mM Girard's P and 1 mM formic acid). The reaction was allowed to proceed for 2 h at room temperature to complete the derivatization. After the reaction, the sample was directly used for LC-MS / MS analysis.

[0092] Experimental results: No signal peak of the derivatization product of the target volatile fatty aldehyde was observed by LC-MS / MS detection. This indicates that when the derivatization treatment was not carried out synchronously during the sampling stage, the volatile fatty aldehyde had been volatilized and lost during the drying process, resulting in the inability to detect its derivatization product in the end.

[0093] The experimental results of this comparative example show that if derivatization is not performed simultaneously during the sampling stage, the target volatile metabolites (volatile fatty acids and volatile fatty aldehydes) will be lost during the drying process, resulting in the inability to detect the target in the subsequent LC-MS / MS analysis. This result is in sharp contrast to the detection results of Example 4, further proving that the present method improves the capture efficiency of volatile metabolites through the dry blood suction tip / in situ labeling strategy and ensures the accuracy and sensitivity of subsequent LC-MS / MS analysis. The method of the present invention can be used in a variety of applications, such as:

[0094] (1) Clinical diagnosis: used to detect volatile metabolites in peripheral blood to assist in disease screening and monitoring;

[0095] (2) Metabolomics research: used to analyze metabolites in peripheral blood and explore disease biomarkers;

[0096] (3) Biomedical analysis: used for biological monitoring related to drug metabolism, physiological metabolic abnormalities and environmental exposure;

[0097] (4) Forensic medicine: used for the storage, detection and identification of trace blood samples under special circumstances.

[0098] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling, characterized in that: The following steps are involved: S1. Collect a small amount of peripheral blood using a dried blood spot or a dried blood tip, add a derivatization reagent for in-situ labeling, and then dry the sample to obtain a dried sample; S2, adding a desorption solvent to the dried sample for desorption to obtain a desorption solution; S3. The desorption liquid is subjected to instrumental analysis to achieve quantitative detection of volatile fatty acids or volatile fatty aldehydes.

2. The method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling according to claim 1, characterized in that: In step S1, for the detection of volatile fatty acids, any one of Cholamine, acyl chloride, esterification reagent, amines, and hydroxylamines is selected in combination with the reaction medium as a derivatization reagent; for the detection of volatile fatty aldehydes, any one of Girard's P, hydroxylamine, hydrazines, aromatic amines, phenylhydrazones, and dansyl chloride is selected as a derivatization reagent.

3. The method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling according to claim 1, characterized in that: When using the dried blood spot technology to collect a trace amount of peripheral blood, use a blood collection needle to puncture the peripheral part, drip 20-30μL of blood onto the dried blood spot card or filter paper and allow the blood to spread evenly in the blood collection area. When the blood sample is not completely dry, add the derivatization reagent solution, and then dry it to obtain a dried derivatized blood spot sample.

4. The method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dried blood spot sampling and in situ labeling according to claim 3, characterized in that: In step S2, the dried derivatized blood spot sample is added with a desorption solvent and subjected to oscillation or ultrasonic treatment to release the target analyte to obtain a desorption solution, the desorption solution is centrifuged to obtain a supernatant, and the supernatant is subjected to quantitative detection of volatile fatty acids or volatile fatty aldehydes.

5. The method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling according to claim 1, characterized in that: When using a dry blood tip to collect a trace amount of peripheral blood, load the hydrophilic fiber material in the pipette tip, use a micropipette to quantitatively absorb 20-30μL of peripheral blood, and use negative pressure to move the blood upward and adsorb to the fiber material; place the pipette tip in a drying device, use reverse drainage technology to make the blood sample evenly adhere and dry and fix under constant temperature conditions.

6. The method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling according to claim 5, characterized in that: In step S2, a desorption solvent is added to the dried blood suction tip and the target metabolites are released from the fiber matrix by repeated blowing or slight vortexing to obtain a desorption solution, and the desorption solution is subjected to quantitative detection of volatile fatty acids or volatile fatty aldehydes.

7. The method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling according to claim 1, characterized in that: The desorption solvent is selected from any of the following forms: (a) a single solvent among methanol, acetonitrile or acetone; (b) a mixed solution of at least one solvent among methanol, acetonitrile and acetone and water; (c) a mixed solution of two or more solvents among methanol, acetonitrile and acetone.

8. The method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling according to claim 1, characterized in that: The target derivatization products are analyzed by high performance liquid chromatography-fluorescence detection, high performance liquid chromatography-mass spectrometry, liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry for quantitative detection of volatile fatty acids or volatile fatty aldehydes.

9. The method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling according to claim 2, characterized in that: When performing in situ labeling in step S1, for volatile fatty acids, the in situ labeling reaction can be promoted by adding a buffer system or a catalyst to the derivatization reagent; for volatile fatty aldehydes, the in situ labeling reaction can be promoted by adding a buffer system or a catalyst or a reaction medium to the derivatization reagent.

10. The method for detecting trace peripheral blood volatile fatty acids and fatty aldehydes based on dry blood sampling and in situ labeling according to claim 9, characterized in that: The buffer system is optimized according to the properties of the detection target, wherein: the derivatization reaction of volatile fatty acids uses a pH 6.0-8.5 phosphate buffer; the derivatization reaction of volatile fatty aldehydes uses a weak acid buffer, such as ammonium acetate buffer or phosphate buffer; the selection principle of the catalyst is: the labeling of volatile fatty acids uses an EDC / HOBt condensation agent; 0.05% EDTA is added to the labeling system of volatile fatty aldehydes; the reaction medium is methanol, acetonitrile or a mixed solvent thereof with water.

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