Rapid detection method for serum 25-hydroxy vitamin D based on nano electrospray three-stage tandem mass spectrometry

Abstract

The invention discloses a rapid detection method of serum 25-hydroxy vitamin D based on nano electrospray three-stage tandem mass spectrometry, and belongs to the technical field of detection of 25-hydroxy vitamin D. In order to avoid the loss of detection flux and guarantee the avoidance of sensitivity and specificity, the detection method comprises the steps of sample pretreatment, liquid-liquid extraction treatment, derivatization reaction, redissolution, analysis and the like, a DI-nanoESI-MRM3 enhanced detection method is used for qualitatively and quantitatively analyzing 25 (OH) D in serum, a traditional LC-MS / MS detection method is replaced, and the detection efficiency is improved. After an analysis solvent is prepared, an analyte is directly introduced into an MS system, a separation step is skipped, and the analyte is used for response instead of chromatographic separation for quantification; and meanwhile, the selectivity of the MRM3 on the target conversion provides an additional recognition dimension to replace the deletion of retention time information, and the method can provide high-throughput screening, so that the solvent consumption is remarkably reduced without influencing the sensitivity and the selectivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of 25-hydroxyvitamin D detection, and more specifically, relates to a rapid detection method for serum 25-hydroxyvitamin D based on nanoelectrospray triple quadrupole mass spectrometry. Background Art

[0002] Vitamin D is a fat-soluble vitamin that is crucial for maintaining the dynamic balance of calcium in the human body and is closely related to aspects such as bone health, autoimmunity, and cardiovascular health. Vitamin D deficiency, abnormal metabolism, or overdose can all have a significant impact on health. Therefore, accurate detection results are crucial for clinical assessment of the body's basal level, formulation of intervention plans, and other aspects.

[0003] Clinical serum vitamin D detection methods mainly include chemiluminescence immunoassay (CLIA) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). When dealing with complex matrices such as serum, conventional MRM quantification may lack sufficient sensitivity or accuracy because a large number of interfering compounds are co-extracted from the matrix. Interfering compounds can be effectively separated by LC before detection, but this greatly reduces the detection throughput.

[0004] Therefore, how to effectively avoid the loss of detection throughput while ensuring sensitivity and specificity has become the focus of research on sample detection methods in the field. Summary of the Invention

[0005] The present invention aims at the technical problems existing in the prior art and provides a rapid detection method for serum 25-hydroxyvitamin D based on nanoelectrospray triple quadrupole mass spectrometry.

[0006] To solve the above technical problems, the technical solution provided by the present invention is as follows: A rapid detection method for serum 25-hydroxyvitamin D based on nanoelectrospray triple quadrupole mass spectrometry, comprising the following steps:

[0007] S1. Sample pretreatment: Take 50 - 100 μL of serum and add it to a centrifuge tube. Then add 50 - 100 μL of internal standard working solution or quality control solution and 250 - 1000 μL of acetonitrile. Then vortex for 3 - 8 minutes and centrifuge at 8000 - 12000 rpm for 3 - 8 minutes at room temperature;

[0008] S2. Liquid-liquid extraction treatment: Collect the supernatant prepared in S1 into a second centrifuge tube. Then add 300 - 600 μL of n-hexane and 200 - 400 μL of water, vortex for 3 - 8 minutes, and then perform liquid-liquid extraction;

[0009] S3, Derivatization reaction: After centrifuging the second centrifuge tube at 8000 - 12000 rpm for 3 - 8 minutes, transfer the upper organic layer in the second centrifuge tube to the third centrifuge tube, dry it, and then dissolve it in a mixture of 200 - 400 μL of ethyl acetate and dichloromethane. Add 60 - 120 μL of isonicotinyl chloride derivative solution;

[0010] S4, Reconstitution: Dry the mixture in the third centrifuge tube and reconstitute it with 0.5 - 2 mL of methanol. Analyze the final mixture by MS 3 analysis.

[0011] Preferably, the preparation method of the internal standard working solution or quality control solution is as follows:

[0012] Dilute the 25(OH)D stock solution with acetonitrile to concentrations of 10, 20, 50, 100, 200, 400, 600, and 1000 ng / mL to prepare the working solution; or prepare quality control solutions with 4 different concentrations of 30, 75, 150, and 500 ng / mL.

[0013] Preferably, the volume ratio of each component in the mixture solution of ethyl acetate and dichloromethane in S3 is: ethyl acetate:dichloromethane is 1:3.

[0014] Preferably, the preparation method of the isonicotinyl chloride derivative solution in S3 is as follows: Disperse 10 mg of INC in 2 ml of dichloromethane to prepare an INC suspension.

[0015] Preferably, the usage method of the isonicotinyl chloride derivative solution in S3 is as follows: Shake the suspension for 1 minute and then use it for the derivatization of 25(OH)D.

[0016] Preferably, pass nitrogen at 15 - 60 °C through the mixture in the third centrifuge tube until the mixture is dry.

[0017] Preferably, it further includes the following steps:

[0018] Inject the reconstituted mixture in the third centrifuge tube into a glass capillary for MS 3 analysis.

[0019] Preferably, for MS 3 The analysis ion pairs are: For 25(OH)D3, the MRM 3 ion pair is m / z 506.3→488.3→365.3; for 25(OH)D2, the MRM 3 ion pair is m / z 518.3→500.3→377.3.

[0020] Preferably, the specifications of the glass capillary are: inner diameter 1.56 mm, outer diameter 2 mm; the volume of the detection solution extracted from the glass capillary is 40 - 120 μL.

[0021] Preferably, acetonitrile is used as the protein precipitation reagent in S1, and methanol can also be used as the protein precipitation reagent; n-hexane is used as the extraction solution reagent in S2, and the extraction solution reagent can be replaced with ethyl acetate, dichloromethane or methyl tert-butyl ether.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention uses DI-nanoESI-MRM 3 Enhanced detection method for qualitative and quantitative analysis of 25(OH)D in serum to replace the traditional LC-MS / MS detection method. After the analytical solvent is prepared, the analyte is directly introduced into the MS system, skipping the separation step, and using the analyte response instead of chromatographic separation for quantification; at the same time, MRM 3 The selectivity for the target transition provides an additional dimension of identification to replace the lack of retention time information. This method can provide high-throughput screening, thus significantly reducing solvent consumption without affecting sensitivity and selectivity. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 It is the typical secondary mass spectrum of 25(OH)D2 after derivation in the embodiment of the present invention;

[0026] Figure 2 It is the typical tertiary mass spectrum of 25(OH)D2 after derivation in the embodiment of the present invention;

[0027] Figure 3 It is the typical secondary mass spectrum of 25(OH)D3 after derivation in the embodiment of the present invention;

[0028] Figure 4 It is the typical tertiary mass spectrum of 25(OH)D3 after derivation in the embodiment of the present invention;

[0029] Figure 5 It is the mass spectrum response diagram of different protein precipitation reagents in the embodiment of the present invention;

[0030] Figure 6 It is the mass spectrum response diagram of different extraction reagents in the embodiment of the present invention;

[0031] Figure 7It is a linear schematic diagram of 25(OH)D2 within the concentration range of 10 - 1000 ng / mL in the embodiment of the present invention;

[0032] Figure 8 It is a linear schematic diagram of 25(OH)D3 within the concentration range of 10 - 1000 ng / mL in the embodiment of the present invention. Specific embodiments

[0033] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application clearer, the following further details this application in conjunction with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0034] Embodiment 1

[0035] The embodiment of the present invention provides a rapid detection method for serum 25-hydroxyvitamin D based on nanoelectrospray triple quadrupole mass spectrometry, including the following steps:

[0036] S1. Sample pretreatment: Take 50 - 100 μL of serum and add it to a centrifuge tube, add 50 - 100 μL of internal standard working solution or quality control solution, and 250 - 1000 μL of acetonitrile thereto, then vortex for 3 - 8 minutes and centrifuge at 8000 - 12000 rpm for 3 - 8 minutes at room temperature;

[0037] S2. Liquid - liquid extraction treatment: Collect the supernatant prepared in S1 into a second centrifuge tube, then add 300 - 600 μL of n - hexane and 200 - 400 μL of water, vortex for 3 - 8 minutes and then perform liquid - liquid extraction;

[0038] S3. Derivatization reaction: After centrifuging the second centrifuge tube at 8000 - 12000 rpm for 3 - 8 minutes, transfer the upper organic layer in the second centrifuge tube to a third centrifuge tube, dry it, and then dissolve it in a mixture of 200 - 400 μL of ethyl acetate and dichloromethane, and add 60 - 120 μL of isonicotinyl chloride derivative solution;

[0039] S4. Reconstitution: Dry the mixture in the third centrifuge tube and reconstitute it with 0.5 - 2 mL of methanol, and perform MS 3 analysis.

[0040] The present invention adopts DI - nanoESI - MRM 3 enhanced detection method for qualitative and quantitative analysis of 25(OH)D in serum, to replace the traditional LC - MS / MS detection method. After preparing the analytical solvent, the analyte is directly introduced into the MS system, skipping the separation step, and using the analyte response instead of chromatographic separation for quantification; at the same time, MRM 3The selectivity for target transformation provides an additional identification dimension to replace the lack of retention time information. This method can provide high-throughput screening, thus significantly reducing solvent consumption without affecting sensitivity and selectivity.

[0041] Example 2

[0042] 25(OH)D is 25-hydroxyvitamin D.

[0043] I. Solution preparation

[0044] The preparation method of the internal standard working solution or quality control solution is as follows:

[0045] Dilute the 25(OH)D stock solution with acetonitrile to concentrations of 10, 20, 50, 100, 200, 400, 600, and 1000 ng / mL to prepare working solutions; four different concentrations (30, 75, 150, and 500 ng / mL) of quality control solutions are independently prepared in the same or similar manner.

[0046] The preparation method of the isonicotinoyl chloride derivative solution is as follows:

[0047] Disperse 10 mg of INC (isonicotinoyl chloride solution) in 2 mL of dichloromethane to prepare an INC suspension.

[0048] The usage method of the isonicotinoyl chloride derivative solution is as follows:

[0049] Shake the prepared suspension for 1 minute, and then use it for the derivatization of 25(OH)D.

[0050] II. The specific operation steps are as follows:

[0051] S1. Sample protein precipitation treatment: Place the frozen calf serum sample at room temperature to thaw. After gently mixing it upside down and evenly, take 50 - 100 μL of serum and add it to a centrifuge tube. Subsequently, add 50 - 100 μL of the internal standard working solution or quality control solution and 250 - 1000 μL of acetonitrile to the calf serum, then vortex for 3 - 8 minutes and centrifuge at 8000 - 12000 rpm for 3 - 8 minutes at room temperature;

[0052] S2. Liquid-liquid extraction treatment: Collect the supernatant prepared in S1 into a second centrifuge tube, then add 300 - 600 μL of n-hexane and 200 - 400 μL of water, vortex for 3 - 8 minutes, and then perform liquid-liquid extraction;

[0053] S3. Derivatization reaction: After centrifuging the second centrifuge tube at 8000 - 12000 rpm for 3 - 8 minutes, transfer the upper organic layer in the second centrifuge tube to the third centrifuge tube, dry it under a nitrogen atmosphere, then redissolve it in a 200 - 400 μL mixture of ethyl acetate and dichloromethane. Finally, add 60 - 120 μL of the pre-prepared isonicotinyl chloride derivative solution.

[0054] S4. Reconstitution: Dry the mixture in the third centrifuge tube under a nitrogen atmosphere and reconstitute it with 0.5 - 2 mL of methanol.

[0055] S5. Detection and analysis: Inject the reconstituted mixture in the third centrifuge tube into a glass capillary for MS 3 analysis, and the volume of the detection solution extracted from the glass capillary is 40 - 120 μL.

[0056] Furthermore, the centrifuge tube used in this example is a polypropylene conical centrifuge tube, and the specifications of the glass capillary used in step S5 are: inner diameter 1.56 mm, outer diameter 2 mm.

[0057] In step S3, the volume ratio of each component in the mixture solution of ethyl acetate and dichloromethane is: ethyl acetate:dichloromethane is 1:3.

[0058] In step S4, the drying condition of the mixture is: Pass the mixture in the third centrifuge tube through a nitrogen atmosphere at 15 - 60 °C until the mixture is dry.

[0059] In step S5, the MS 3 analysis ion pairs are: For 25(OH)D3, the MRM 3 ion pair is m / z 506.3→488.3→365.3; For 25(OH)D2, the MRM 3 ion pair is m / z 518.3→500.3→377.3.

[0060] Example 3

[0061] S1. Place the frozen calf serum sample at room temperature to thaw. After inverting and gently mixing evenly, take 50 μL of serum and transfer it to a centrifuge tube. Subsequently, add 50 μL of the internal standard working solution or quality control solution and 250 μL of acetonitrile to the calf serum, then vortex for 5 minutes and centrifuge at 10000 rpm for 5 minutes at room temperature.

[0062] S2. Collect the supernatant prepared in S1 into the second centrifuge tube, then add 500 μL of n-hexane and 200 μL of water, and perform liquid-liquid extraction after vortexing for 5 minutes.

[0063] S3. After centrifuging the second centrifuge tube at 10,000 rpm for 5 minutes, transfer the upper organic layer in the second centrifuge tube to the third centrifuge tube, dry it under a nitrogen environment, and then redissolve it in a 300-μL mixture of ethyl acetate and dichloromethane (1 / 3, v / v). Finally, add 80 μL of the pre-prepared isonicotinyl chloride derivative solution.

[0064] S4. Dry the mixture in the third centrifuge tube under a nitrogen environment at 40 °C and reconstitute it with 1 mL of methanol.

[0065] S5. Inject 80 μL of the reconstituted mixture detection solution from the third centrifuge tube into a glass capillary for MS 3 analysis.

[0066] In this example, for 25(OH)D3, the MRM 3 ion pairs are m / z 506.3→488.3→365.3; for 25(OH)D2, the MRM 3 ion pairs are m / z 518.3→500.3→377.3.

[0067] In this example, the typical fragmentation pattern of the ions is as Figures 1 - 4 shown.

[0068] Furthermore, as a preferred embodiment of the present invention, in step S1, acetonitrile is used as the protein precipitation reagent, and methanol can also be selected as the protein precipitation reagent. The mass spectrometry response is as Figure 5 shown.

[0069] In step S2, n-hexane is used as the extraction solution reagent, and the extraction solution reagent can be replaced with ethyl acetate, dichloromethane or methyl tert-butyl ether. The mass spectrometry response is as Figure 6 shown.

[0070] The mass spectrometry conditions are shown in Table 1:

[0071]

[0072] The standard curve results are as Figure 7 , Figure 8 and Table 2.

[0073] In the linear range of 10–1000 ng / mL, the linear correlations of 25(OH)D2 and 25(OH)D3 are good, and the correlation coefficients R 2 are 0.9956 and 0.9948 respectively, and the accuracy ranges are 94.02-110.66% and 95.39-109.06%.

[0074] Table 2: Standard curve results

[0075]

[0076] Table 3: DI-nanoESI-MRM proposed in the embodiments of the present invention 3 Comparison of the method with the methods for determining 25(OH)D in different matrices reported previously

[0077]

[0078] In Table 3, [1] represents Comparative Example 1 and [2] represents Comparative Example 2.

[0079] In summary, compared with previous studies, the embodiments of the present invention have higher sensitivity and shorter sample pretreatment time compared with Comparative Example 1 and Comparative Example 2, and can meet the needs of detecting a large number of clinical samples.

[0080] The present invention adopts DI-nanoESI-MRM 3 enhanced detection method for qualitative and quantitative analysis of 25(OH)D in serum to replace the traditional LC-MS / MS detection method. After the analytical solvent is prepared, the analyte is directly introduced into the MS system, skipping the separation step, and quantitative analysis is performed using the analyte response instead of chromatographic separation; at the same time, MRM 3 The selectivity for the target transition provides an additional dimension of identification to replace the lack of retention time information. This method can provide high-throughput screening, thus significantly reducing solvent consumption without affecting sensitivity and selectivity.

[0081] In the description of the present invention, it should be understood that the orientation or positional relationship indicated by terms such as "length", "width", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus cannot be construed as a limitation to the present application.

[0082] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present application, "a plurality" means two or more unless otherwise specifically defined.

[0083] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A method for rapid detection of serum 25-hydroxyvitamin D based on nano-electrospray tandem mass spectrometry, characterized in that: The following steps are involved: S1. Sample pretreatment: 50-100 μL of serum was added to a centrifuge tube, and 50-100 μL of internal standard working solution or quality control solution and 250-1000 μL of acetonitrile were added thereto, followed by vortexing for 3-8 minutes and centrifuging at 8000-12000 rpm for 3-8 minutes at room temperature; S2, liquid-liquid extraction treatment: collect the supernatant prepared in S1 into a second centrifuge tube, then add 300-600 μL of n-hexane and 200-400 μL of water, vortex for 3-8 minutes and then perform liquid-liquid extraction; S3, derivatization reaction: after centrifuging the second centrifuge tube at 8000-12000 rpm for 3-8 minutes, the upper organic layer in the second centrifuge tube is transferred to a third centrifuge tube, dried, and then dissolved in a mixture of 200-400 μL of ethyl acetate and dichloromethane, and 60-120 μL of isonicotinoyl chloride derivatization solution is added; S4. Redissolution: Dry the mixture in the third centrifuge tube and reconstitute it with 0.5-2 mL of methanol. 3 analyze.

2. The method for rapid detection of serum 25-hydroxyvitamin D based on nano-electrospray ionization triple-tandem mass spectrometry according to claim 1, characterized in that: The preparation method of the internal standard working solution or the quality control solution is as follows: The 25(OH)D stock solution was diluted with acetonitrile to concentrations of 10, 20, 50, 100, 200, 400, 600, and 1000 ng / mL to prepare working solutions; or quality control solutions of 30, 75, 150, and 500 ng / mL were prepared.

3. The method for rapid detection of serum 25-hydroxyvitamin D based on nano-electrospray ionization triple-tandem mass spectrometry according to claim 1, characterized in that: The volume ratio of each component in the mixture solution of ethyl acetate and dichloromethane in S3 is: ethyl acetate: dichloromethane is 1:

3.

4. The method for rapid detection of serum 25-hydroxyvitamin D based on nano-electrospray ionization triple-tandem mass spectrometry according to claim 1, characterized in that: The preparation method of the isonicotinoyl chloride derivative solution in S3 is as follows: 10 mg of INC is dispersed in 2 ml of dichloromethane to prepare an INC suspension.

5. The method for rapid detection of serum 25-hydroxyvitamin D based on nano-electrospray ionization triple-tandem mass spectrometry according to claim 1, characterized in that: The method of using the isonicotinoyl chloride derivatization solution in S3 is as follows: the suspension is shaken for 1 minute and then used for the derivatization of 25(OH)D.

6. The method for rapid detection of serum 25-hydroxyvitamin D based on nano-electrospray ionization triple-tandem mass spectrometry according to claim 1, characterized in that: The mixture in the third centrifuge tube is passed through nitrogen gas at 15-60° C. until the mixture is dry.

7. The method for rapid detection of serum 25-hydroxyvitamin D based on nano-electrospray ionization triple-tandem mass spectrometry according to claim 1, characterized in that: The following steps are also included: The recombinant mixture in the third centrifuge tube was injected into a glass capillary tube for MS. 3 analyze.

8. The method for rapid detection of serum 25-hydroxyvitamin D based on nano-electrospray ionization triple-tandem mass spectrometry according to claim 1, characterized in that: The MS 3 The analytical ion pairs are: for 25(OH)D3, MRM 3 The ion pairs are m / z 506.3→488.3→365.3; for 25(OH)D2, MRM 3 The ion pair was m / z 518.3→500.3→377.

3.

9. The method for rapid detection of serum 25-hydroxyvitamin D based on nano-electrospray tandem mass spectrometry according to claim 7, characterized in that: The specifications of the glass capillary are: inner diameter 1.56 mm, outer diameter 2 mm; the volume of the detection solution extracted in the glass capillary is 40-120 μL.

10. The method for rapid detection of serum 25-hydroxyvitamin D based on nano-electrospray ionization triple-tandem mass spectrometry according to claim 1, characterized in that: In the S1, acetonitrile is a protein precipitation reagent, and methanol can also be used as a protein precipitation reagent; in the S2, n-hexane is an extraction solution reagent, and the extraction solution reagent can be replaced by ethyl acetate, dichloromethane or methyl tert-butyl ether.

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