Online extraction method for amino acid and acyl carnitine in inherited metabolic disease

Abstract

The invention discloses an on-line extraction method for amino acid and acyl carnitine in inherited metabolic diseases, and belongs to the technical field of clinical medicine detection.According to the method, simultaneous on-line extraction and real-time monitoring analysis of amino acid and acyl carnitine in inherited metabolic diseases can be achieved, a dry matrix spot form is used as an experimental sample, and the method is simple and convenient to operate. The online extraction process does not need complex offline pretreatment operation and a time-consuming chromatographic separation and detection device, is small in biological sample consumption, short in time consumption and high in extraction and detection efficiency, shows a satisfactory extraction effect, can improve the protonation effect of a target analyte in a positive ion mode by H < + > generated by ionized water, is high in sensitivity and good in universality, and is suitable for industrial production. And a novel method with potential is provided for early clinical screening of amino acid and acyl carnitine IMDs.

Description

Technical Field

[0001] The present application belongs to the field of clinical medical detection technology, and in particular relates to an online extraction method for amino acids and acylcarnitines in genetic metabolic diseases. Background Art

[0002] Inherited metabolic diseases (IMDs) refer to a class of diseases with defects in the body's metabolic function, which can cause a series of abnormal reactions and even death. Newborn screening (NBS) is the main effective measure for early detection and early treatment of IMDs. It has received widespread attention worldwide and has become a mandatory examination. There are many types of IMDs, and the single incidence rate is low, but the overall morbidity and mortality rate are high. From the perspective of different categories, IMDs mainly include amino acid metabolic diseases, organic acid metabolic diseases, and fatty acid oxidation disorder metabolic diseases. The metabolic small molecule indicators of these IMDs are mainly amino acids and acylcarnitines. With the rapid growth of clinical needs, there is an urgent need to develop IMDs detection technology to improve the quality of birth and social and economic benefits of the population.

[0003] The pretreatment for IMDs screening is mostly intermittent offline extraction, and its workflow mainly involves offline extraction (such as low-temperature precipitation, nitrogen blowing and re-dissolution, constant temperature oscillation, centrifugation) of metabolites in biological samples, and the obtained sample solution enters mass spectrometry detection. Currently, a variety of offline pretreatment technologies have been reported, which have improved the extraction efficiency of amino acids and acylcarnitine indicators, enriching the strength of IMDs screening development, but for large-scale NBS samples, the sample throughput and detection time of offline extraction limit high-throughput research. Therefore, it is urgent to establish a method that can simultaneously realize online extraction and real-time monitoring, so that the metabolites extracted from the samples can be analyzed more quickly, thereby improving the feasibility of large-scale sample research. Summary of the invention

[0004] The purpose of the present application is to provide an online extraction method for amino acids and acylcarnitines in genetic metabolic diseases, so as to solve the technical problems of the existing offline extraction methods of amino acids and acylcarnitines, such as complex pre-treatment, long time consumption and low detection efficiency.

[0005] To achieve the above purpose, the technical solution adopted in the present application is: to provide an online extraction method for amino acids and acylcarnitines in genetic metabolic diseases, which specifically comprises the following steps:

[0006] (i) transferring the biological matrix containing amino acids and acylcarnitines onto a filter paper sheet to prepare dry matrix spots;

[0007] (ii) The first injection needle and the second injection needle are connected to the capillary and the Nanotube in sequence; an extractant and a dry matrix spot are added to the first injection needle for online extraction to obtain an extraction sample solution; water is sucked into the second injection needle, and the extraction sample solution and water are mixed in the capillary to obtain a sample solution; a voltage is applied at the needle tip of the second injection needle and the tip of the Nanotube; the sample solution is passed through the Nanotube to form an electrospray at the tip of the Nanotube, and the electrospray is sprayed into a mass spectrometer for analysis.

[0008] In one embodiment,

[0009] The biological matrix is ​​one of serum, whole blood or artificial urine, and the amount of the biological matrix added is 3 μL.

[0010] In one embodiment,

[0011] Step (i) The diameter of the hole punched in the dry matrix spot is 5 mm.

[0012] In one embodiment,

[0013] The extracting agent in step (ii) is a mixture of methanol and isopropanol, a mixture of acetonitrile and trifluoroacetic acid, or one of methanol, acetonitrile or acetone mixed with formic acid. Preferably, the extracting agent is a mixture of methanol and isopropanol mixed with formic acid.

[0014] In one embodiment,

[0015] The volume ratio of methanol to isopropanol is 3:7-9:1, preferably, the volume ratio of methanol to isopropanol is 7:3; the content of trifluoroacetic acid accounts for 0.05% of acetonitrile, and the content of formic acid accounts for 0.1% of the extractant.

[0016] In one embodiment,

[0017] The amount of the extractant in step (ii) is 300-600 μL, preferably, 300 μL; the amount of water sucked into the second injection needle is 300 μL.

[0018] In one embodiment,

[0019] The flow rates of the first injection needle and the second injection needle were both 5 μL / min.

[0020] In one embodiment,

[0021] The outer diameter of the capillary is 164μm and the inner diameter is 100μm; the Nanotube is a borosilicate glass tube with an outer diameter of 2.0mm, an inner diameter of 1.56mm and a length of 10cm.

[0022] In one embodiment,

[0023] The voltage applied in step (ii) is 2.7 kV.

[0024] In one embodiment,

[0025] Step (ii) The distance between the tip of the nanotube and the entrance of the mass spectrometer is 4-6 mm.

[0026] The present application provides an online extraction method for amino acids and acylcarnitines in genetic metabolic diseases. The biological matrix is ​​used as an experimental sample in the form of dry matrix spots, and an injection needle is used as a carrying container to perform an online extraction process on the biological dry matrix spots. The formed dry matrix spots are not only highly stable, low in contamination risk, and easy to store, but also can greatly reduce sample consumption and detection time, thereby reducing the matrix effect; the amino acid and acylcarnitine IMDs are determined by combining the online extraction sample solution with externally doped ionized water, and the external doping of water is beneficial to reducing the matrix effect and improving the sensitivity; the H generated by the ionized water + , which can improve the protonation effect of the target analyte in the positive ion mode; the nano-electrospray sprayer formed by the micron-sized nanotube tip has the advantages of high sensitivity, small injection volume, and low residual effect, and is suitable for biological sample analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0028] Figure 1 Schematic diagram of extracting amino acid and acylcarnitine IMDs indicators from dry matrix spots;

[0029] Figure 2 Schematic diagram of extracting IMDs indicators for a single injection;

[0030] Figure 3 for Figure 1 Optimization result diagram of different voltage positions;

[0031] Figure 4 for Figure 1 Results of whether the extractant contains water;

[0032] Figure 5 Schematic diagram of extracting IMDs indicators for double injection;

[0033] Figure 6 for Figure 5 Results of different voltage position optimization and whether the extractant contains water;

[0034] Figure 7 Optimization results graph for different extractants;

[0035] Figure 8 This is the optimization result diagram of the volume ratio of methanol and isopropanol;

[0036] Fig. 9 is a comparison of mass spectra of Phe and C2 (10 μM) in dry serum spots;

[0037] Fig.10 It is a comparison chart of the signal intensity of 10 μM Phe, Leu, Val, Met, Cit, Arg, His, 50 μM Pro, Thr, 10 μM C2, 5 μM C3, 0.5 μM C5, C8, C10, C5DC and 1 μM C5OH mixed solution in dry serum spots;

[0038] Fig.11 This is a comparison chart of the signal intensities of Phe and C2 (10 μM) in the samples of bovine or sheep serum simulated dry serum spots, bovine or sheep whole blood simulated dry blood spots, and artificial synthetic dry urine spots. DETAILED DESCRIPTION

[0039] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clear, the present application is further described in detail. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0040] The amino acids and acylcarnitines contained in the dry matrix spots for detection specifically refer to the following acylcarnitines: phenylalanine (Phe), leucine (Leu), valine (Val), methionine (Met), citrulline (Cit), arginine (Arg), proline (Pro), histidine (His), threonine (Thr), acetylcarnitine (C2), propionylcarnitine (C3), isovalerylcarnitine (C5), octanoylcarnitine (C8), decanoylcarnitine (C10), hydroxyisovalerylcarnitine (C5OH) and glutarylcarnitine (C5DC).

[0041] Example 1

[0042] An on-line extraction method for amino acids and acylcarnitines in inherited metabolic diseases, e.g. Figure 2 As shown, the specific steps include:

[0043] (i) 3 μL of serum containing amino acids and acylcarnitines was transferred onto a filter paper to prepare a dry matrix spot, and the diameter of the hole punched in the dry matrix spot was 5 mm;

[0044] (ii) The first injection needle is connected to a capillary (the outer diameter of the capillary is 164 μm, and the inner diameter is 100 μm) and a nanotube (the nanotube is a high borosilicate glass tube, with an outer diameter of 2.0 mm, an inner diameter of 1.56 mm, and a length of 10 cm), 600 μL of acetonitrile (containing 0.05% trifluoroacetic acid) and 0.1% formic acid are mixed to prepare an extractant, and the extractant and dry matrix spots are added to the first injection needle for online extraction to prepare an extraction sample solution, and a voltage of 2.7 kV is applied to the needle tip of the first injection needle; the flow rate of the first injection needle is controlled by a syringe pump, and the flow rate is 5 μL / min; the extraction sample solution passes through the nanotube, and an electrospray is formed at the tip of the nanotube, and the electrospray is sprayed into a mass spectrometer for analysis, and the distance between the tip of the nanotube and the entrance of the mass spectrometer is 4-6 mm.

[0045] Example 2

[0046] This embodiment is different from Embodiment 1 in that no voltage is applied to the needle tip of the first injection needle, and a voltage of 2.7 kV is applied to the tip of the Nanotube, and the other steps are the same.

[0047] Example 3

[0048] This embodiment is different from Embodiment 1 in that a voltage of 2.7 kV is applied simultaneously to the needle tip of the first injection needle and the tip of the Nanotube, and the other steps are the same.

[0049] Example 4

[0050] The difference between this embodiment and embodiment 3 is that the extractant is composed of a mixture of 300 μL acetonitrile (containing 0.05% trifluoroacetic acid), 300 μL H 2 O and 0.1% formic acid, and the other steps are the same.

[0051] like Figure 3-4 As shown, by comparing Examples 1-3, when water is not directly added to the extractant, a higher signal intensity is obtained when voltage is applied simultaneously at the needle tip of the first injection needle and the tip of the Nanotube; by comparing Example 3 and Example 4, the signal intensity is better when water is not directly added to the extractant than when water is directly added.

[0052] Example 5

[0053] An on-line extraction method for amino acids and acylcarnitines in inherited metabolic diseases, e.g. Figure 5 As shown, the specific steps include:

[0054] (i) 3 μL of serum containing amino acids and acylcarnitines was transferred onto a filter paper to prepare a dry matrix spot, and the diameter of the hole punched in the dry matrix spot was 5 mm;

[0055] (ii) The first injection needle and the second injection needle are connected to a capillary (the outer diameter of the capillary is 164 μm, the inner diameter is 100 μm) and a Nanotube (the Nanotube is a high borosilicate glass tube, the outer diameter is 2.0 mm, the inner diameter is 1.56 mm, and the length is 10 cm) in sequence, 300 μL of acetonitrile (containing 0.05% trifluoroacetic acid) and 0.1% formic acid are mixed to prepare an extractant, and the extractant and the dry matrix spot are added to the first injection needle for online extraction to prepare an extraction sample solution; 300 μL of water is sucked into the second injection needle, and the extracted sample solution and water are mixed in the capillary to obtain a sample solution; the flow rate of the first injection needle and the second injection needle is controlled by a syringe pump, and the flow rate is 5 μL / min; a voltage of 2.7 kV is applied to the needle tip of the first injection needle, and the sample solution passes through the Nanotube to form an electrospray at the tip of the Nanotube, and the electrospray is sprayed into a mass spectrometer for analysis, and the distance between the tip of the Nanotube and the entrance of the mass spectrometer is 4-6 mm.

[0056] Example 6

[0057] The present embodiment is different from the embodiment 5 in that no voltage is applied to the needle tip of the first injection needle, and a voltage of 2.7 kV is applied to the needle tip of the second injection needle, and the other steps are the same.

[0058] Example 7

[0059] This embodiment is different from Embodiment 5 in that no voltage is applied to the needle tip of the first injection needle, and a voltage of 2.7 kV is applied to the tip of the Nanotube, and the other steps are the same.

[0060] Example 8

[0061] The present embodiment is different from the embodiment 5 in that a voltage of 2.7 kV is applied to the needle tips of the first injection needle and the second injection needle at the same time, and the other steps are the same.

[0062] Example 9

[0063] This embodiment is different from Embodiment 5 in that a voltage of 2.7 kV is applied simultaneously to the needle tip of the first injection needle and the tip of the Nanotube, and the other steps are the same.

[0064] Example 10

[0065] This embodiment is different from Embodiment 5 in that a voltage of 2.7 kV is applied simultaneously to the needle tip of the second injection needle and the tip of the Nanotube, and no voltage is applied to the needle tip of the first injection needle. The other steps are the same.

[0066] Depend on Figure 6 It can be seen that, compared with Example 3 and Examples 5-10, the signal strength is optimal when the second injection needle sucks water and voltage is applied simultaneously at the needle tip of the second injection needle and the nanotube tip sprayer.

[0067] Embodiment 11

[0068] The difference between this embodiment and embodiment 10 is that the extractant consists of 300 μL acetonitrile and 0.1% formic acid, and the other steps are the same.

[0069] Example 12

[0070] The difference between this embodiment and embodiment 10 is that the extractant consists of 300 μL methanol and 0.1% formic acid, and the other steps are the same.

[0071] Example 13

[0072] The difference between this embodiment and embodiment 10 is that the extractant consists of 300 μL acetone and 0.1% formic acid, and the other steps are the same.

[0073] Embodiment 14

[0074] The difference between this embodiment and embodiment 10 is that the extractant consists of 300 μL methanol / isopropanol and 0.1% formic acid, the volume ratio of methanol to isopropanol is 8:2, and the other steps are the same.

[0075] Comparative Examples 10-14, the composition of the extractant was optimized. Figure 7 In the above, Ⅰ is acetonitrile (0.1% formic acid), Ⅱ is methanol (0.1% formic acid), Ⅲ is acetone (0.1% formic acid), Ⅳ is acetonitrile (0.05% trifluoroacetic acid + 0.1% formic acid), Ⅴ is methanol, isopropanol and 0.1% formic acid, and the volume ratio of methanol to isopropanol is 8:2; Figure 7 It can be seen that the extraction efficiency is best when the extractant is methanol, isopropanol and 0.1% formic acid.

[0076] Embodiment 15

[0077] The difference between this embodiment and Embodiment 14 is that the volume ratio of methanol to isopropanol is 9:1, and the other steps are the same.

[0078] Example 16

[0079] The difference between this embodiment and Embodiment 14 is that the volume ratio of methanol to isopropanol is 7:3, and the other steps are the same.

[0080] Embodiment 17

[0081] The difference between this embodiment and Embodiment 14 is that the volume ratio of methanol to isopropanol is 6:4, and the other steps are the same.

[0082] Embodiment 18

[0083] The difference between this embodiment and Embodiment 14 is that the volume ratio of methanol to isopropanol is 5:5, and the other steps are the same.

[0084] Embodiment 19

[0085] The difference between this embodiment and Embodiment 14 is that the volume ratio of methanol to isopropanol is 3:7, and the other steps are the same.

[0086] Comparative Examples 14-19, by Figure 8 It can be seen that when the volume ratio of methanol to isopropanol is 7:3, the extraction efficiency is optimal.

[0087] Embodiment 20

[0088] This embodiment is different from Embodiment 16 in that the biological matrix is ​​whole blood, and the other steps are the same.

[0089] Embodiment 21

[0090] This embodiment is different from Embodiment 16 in that the biological matrix is ​​artificial urine, and the other steps are the same.

[0091] In order to prove the reliability of the effect of the present invention, the performance test of the extraction solution obtained under the optimal extraction conditions is carried out below.

[0092] like Fig. 9 As shown, the signal intensity of Phe and C2 in the dry serum spots obtained by mixing externally doped ionized water with the online extraction sample solution and combining with nano-electrospray was better than that of the conventional extraction method, with fewer impurities and comparable to the signal intensity of the standard solution.

[0093] like Fig.10 As shown, the signal intensity of the amino acid and acylcarnitine indicators obtained in the present application showed a high response, which was comparable to the signal intensity of the standard solution, verifying the feasibility of the present method for analyzing mixed amino acid and acylcarnitine IMDs in dry serum spots.

[0094] like Fig.11 As shown, the signal intensity of different biological dry matrix spots obtained in the present application is comparable to that of the standard solution, which verifies the universality of the present method for analyzing IMDs indicators in a variety of dry matrix spots.

[0095] The present application provides an online extraction method for amino acids and acylcarnitines in genetic metabolic diseases, which specifically comprises the following steps: a biological matrix containing amino acids and acylcarnitines is moved onto a filter paper sheet to prepare dry matrix spots; a first injection needle and a second injection needle are connected to a capillary and a Nanotube in sequence; an extractant and a dry matrix spot are added to the first injection needle for online extraction to obtain an extraction sample solution, water is sucked into the second injection needle, and the extraction sample solution and water are mixed in the capillary to obtain a sample solution; a voltage is applied at the needle tip of the second injection needle and the tip of the Nanotube, and the sample solution passes through the Nanotube and is injected into the capillary; a voltage is applied at the needle tip of the second injection needle and the tip of the Nanotube to inject the sample solution ... An electrospray is formed at the tip of the o tube, and the electrospray is sprayed into a mass spectrometer for analysis; the present application provides an analytical method for simultaneously realizing online extraction and real-time monitoring of amino acids and acylcarnitines in genetic metabolic diseases, in which dry matrix spots are used as experimental samples, and the online extraction process does not require complicated offline pretreatment operations and time-consuming chromatographic separation and detection devices, with low biological sample consumption, short time consumption, high extraction and detection efficiency, and satisfactory extraction effect. The target analyte has a good protonation response, high sensitivity, and good universality, providing a potential new method for early clinical screening of amino acids and acylcarnitine IMDs.

[0096] It should be noted that the raw materials, devices and instruments used in the present invention are conventional commercially available products unless otherwise specified.

[0097] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

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

Claims

1. An online extraction method for amino acids and acylcarnitines in genetic metabolic diseases, characterized in that: The specific steps include: (i) transferring the biological matrix containing amino acids and acylcarnitines onto a filter paper sheet to prepare dry matrix spots; (ii) the first injection needle and the second injection needle are connected to the capillary and the Nanotube in sequence, the extractant and the dry matrix spot are added to the first injection needle for online extraction to obtain an extraction sample solution, the second injection needle is sucked into water, the extraction sample solution and water are mixed in the capillary to obtain a sample solution; A voltage is applied at the needle tip of the second injection needle and the tip of the Nanotube; the sample solution is passed through the Nanotube to form an electrospray at the tip of the Nanotube, and the electrospray is sprayed into a mass spectrometer for analysis.

2. The online extraction method for amino acids and acylcarnitines in genetic metabolic diseases according to claim 1, characterized in that: In step (i), the biological matrix is ​​one of serum, whole blood or artificial urine, and the amount of the biological matrix added is 3 μL.

3. The online extraction method for amino acids and acylcarnitines in genetic metabolic diseases according to claim 1, characterized in that: The diameter of the holes punched in the dry matrix spots in step (i) is 5 mm.

4. The online extraction method for amino acids and acylcarnitines in genetic metabolic diseases according to claim 1, characterized in that: The extractant in step (ii) is a mixture of methanol and isopropanol, a mixture of acetonitrile and trifluoroacetic acid, or a mixture of methanol, acetonitrile or acetone and formic acid.

5. The online extraction method for amino acids and acylcarnitines in genetic metabolic diseases according to claim 4, characterized in that: The volume ratio of the methanol to the isopropanol is 3:7-9:1, the content of the trifluoroacetic acid accounts for 0.05% of the acetonitrile, and the content of the formic acid accounts for 0.1% of the extractant.

6. The online extraction method for amino acids and acylcarnitines in genetic metabolic diseases according to claim 1, characterized in that: The amount of the extractant in step (ii) is 300-600 μL; the amount of water sucked into the second injection needle is 300 μL.

7. The online extraction method for amino acids and acylcarnitines in genetic metabolic diseases according to claim 1, characterized in that: The flow rates of the first injection needle and the second injection needle are both 5 μL / min.

8. The online extraction method for amino acids and acylcarnitines in genetic metabolic diseases according to claim 1, characterized in that: The outer diameter of the capillary is 164 μm, and the inner diameter is 100 μm; the Nanotube is a borosilicate glass tube with an outer diameter of 2.0 mm, an inner diameter of 1.56 mm, and a length of 10 cm.

9. The online extraction method for amino acids and acylcarnitines in genetic metabolic diseases according to claim 1, characterized in that: The voltage applied in step (ii) is 2.7 kV.

10. The online extraction method for amino acids and acylcarnitines in genetic metabolic diseases according to claim 1, characterized in that: In step (ii), the distance between the tip of the Nanotube and the entrance of the mass spectrometer is 4-6 mm.

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