Quinone compound online mass spectrometry method based on micro-droplet derivatization

Abstract

The invention belongs to the technical field of mass spectrometry, and provides a quinone compound online mass spectrometry method based on micro-droplet derivatization. The mass spectrometry method comprises the following steps: introducing a derivatization reagent solution into an ion source I to form micro-droplets I; introducing a quinone compound solution into an ion source II to form micro-droplets II; after the micro-droplet I and the micro-droplet II are fused, a derivatization reagent and a quinone compound are subjected to a conjugate addition reaction to obtain a zwitterionic product; and carrying out mass spectrometry on the zwitterionic product. According to the method, a derivatization reagent triphenylphosphine is adopted, rapid and online derivatization of quinone compounds is achieved through micro-droplet fusion, zwitterionic products are rapidly generated within the time scale of milliseconds, analysis steps are simplified, and derivatization time is shortened; the detection sensitivity of the quinone compounds is remarkably improved by 2-3 orders of magnitude, and a complicated pretreatment step is not needed; the method has universality and is also suitable for detection of low-concentration quinone compounds, and RSD is 3.3-7.3%.

Description

Technical Field

[0001] The invention relates to the technical field of mass spectrometry analysis, and in particular to an online mass spectrometry analysis method for quinone compounds based on microdroplet derivatization. Background Art

[0002] Quinone compounds are a class of aromatic compounds with conjugated unsaturated ketone properties. They are widely distributed in animals, plants and microorganisms and can participate in a variety of important metabolic activities and biological processes. For example, endogenous quinones can exist in the human body through the metabolism of catecholamines and estrogens, and some food-derived quinones can induce heme oxygenase HO-1 in aortic endothelial cells to protect blood vessels. However, the toxicity of some exogenous quinones has attracted the attention of researchers, especially polycyclic aromatic hydrocarbon quinones from environmental pollution. These quinones are mainly produced by incomplete combustion of fossil fuels and oxidative degradation of polycyclic aromatic hydrocarbons in the atmosphere. Studies have shown that polycyclic aromatic hydrocarbon quinones can produce reactive oxygen species (ROS). Excessive exposure to exogenous quinones caused by diet, medical treatment and air pollution poses a certain threat to human health and may even cause oxidative stress, DNA damage, lung damage, cytotoxicity, immunotoxicity or carcinogenesis. Therefore, the quantitative detection of quinone compounds in environmental and biological samples is of great significance.

[0003] Mass spectrometry has been widely used in the quantitative detection of quinone compounds due to its advantages of high sensitivity and high specificity. Quinone compounds have a conjugated structure and are not easy to ionize. Before entering mass spectrometry analysis, quinone compounds usually need to be chemically derivatized to improve the ionization efficiency of quinone compounds. Conventional derivatization methods require chromatographic separation and long-term pretreatment (3 to 11 hours) of quinone compounds, which is time-consuming and labor-intensive and not suitable for rapid detection of quinone compounds in complex matrices. Huang Guangming et al. studied the paper spray and toothpick spray detection of quinone compounds based on cysteamine derivatization, which has certain application potential. However, the cysteamine-based derivatization method still cannot meet the actual needs of high-sensitivity analysis. In addition, since the analytical performance of paper spray and toothpick spray is easily affected by multiple factors such as the physical structure of the carrier surface, the position of the carrier tip and the elution solvent, the signal fluctuation and repeatability of the results caused by each measurement and replacement of the carrier cannot be ignored. Even when calibrated with isotope internal standards, the relative standard deviation (RSD) of three measurements at low concentrations is still as high as 20 to 30%.

[0004] In recent years, the unique acceleration characteristics of micro-droplet chemical reactions have been gradually applied to the field of chemical analysis, including the highly sensitive derivatization detection of compounds such as sugars, volatile organic small molecules, and lipids. By fusing two coaxial or cross micro-droplets containing the compound to be detected and the derivatization reagent respectively in the gas phase, chemical reactions can be quickly achieved during the flight of the micro-droplets towards the mass spectrometry inlet, thereby introducing more reactive ionization groups into the target molecules, realizing the efficient ionization of the compounds to be detected, and improving the detection sensitivity. In addition, the derivatization method based on micro-droplets also exhibits certain matrix tolerance and good linearity, showing certain application potential in the quantitative analysis of actual samples (such as serum, urine, beer, etc.).

[0005] Therefore, there is still a great demand for developing an on-line derivatization mass spectrometry analysis method for quinone compounds in complex matrices. Summary of the Invention

[0006] The purpose of the present invention is to provide an on-line mass spectrometry analysis method for quinone compounds based on micro-droplet derivatization in view of the deficiencies of the prior art.

[0007] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0008] The present invention provides an on-line mass spectrometry analysis method for quinone compounds based on micro-droplet derivatization, comprising the following steps:

[0009] 1) Introduce the derivatization reagent solution into ion source I to form micro-droplet I; introduce the quinone compound solution into ion source II to form micro-droplet II;

[0010] After the fusion of micro-droplet I and micro-droplet II, the derivatization reagent and the quinone compound undergo a conjugate addition reaction to obtain an amphoteric ion product;

[0011] 2) Perform mass spectrometry analysis on the amphoteric ion product.

[0012] Preferably, in step 1), the derivatization reagent solution contains a derivatization reagent and acetonitrile, and the derivatization reagent is triphenylphosphine.

[0013] Preferably, in step 1), the concentration of the derivatization reagent in the derivatization reagent solution is 1 - 10 mmol / L.

[0014] Preferably, in step 1), the quinone compound solution contains a quinone compound and an acetonitrile aqueous solution.

[0015] Preferably, the concentration of the quinone compound in the quinone compound solution is 0.1 - 2 μmol / L; the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 2 - 4:6 - 8.

[0016] Preferably, the quinone compound comprises one or more of p-benzoquinone compounds, naphthoquinone compounds and anthraquinone compounds.

[0017] Preferably, the structural formula of the p-benzoquinone compound is Wherein, R is hydrogen, methyl, amino, hydroxyl, alkyl, alkoxy, halogen or benzene ring.

[0018] Preferably, the ion source I and the ion source II are independently an electrospray ion source, an electrospray extraction ion source, a desorption electrospray ion source, a sonic spray ion source or a dielectric barrier discharge ion source.

[0019] Preferably, the temperature of the ion source I and the ion source II are independently 420-480° C., and the atomizing gas pressure of the ion source I and the ion source II are independently 70-80 psi.

[0020] Preferably, the mass spectrometry analysis in step 2) is performed in electrospray mass spectrometry positive ion mode.

[0021] The beneficial effects of the present invention include the following:

[0022] 1) The present invention adopts a new derivatization reagent triphenylphosphine, and realizes rapid, online derivatization of quinone compounds by microdroplet fusion; at the gas-liquid interface of the microdroplets, the derivatization reaction of the derivatization reagent triphenylphosphine and the quinone compounds is accelerated, and zwitterion products can be quickly generated within a millisecond time scale, which are further used for mass spectrometry analysis; the existing off-line derivatization analysis method requires 3 to 11 hours to complete the derivatization reaction, and the method of the present invention greatly simplifies the analysis steps and shortens the derivatization time.

[0023] 2) Compared with the existing online derivatization analysis methods, the online mass spectrometry analysis method of quinone compounds based on microdroplet derivatization of the present invention significantly improves the detection sensitivity of quinone compounds by 2 to 3 orders of magnitude, and is simple and fast to operate without complicated pre-treatment steps.

[0024] 3) The mass spectrometry analysis method of the present invention is universal for quinone compounds and is also suitable for the detection of low-concentration quinone compounds. The RSD of repeated measurements is only 3.3-7.3%, while the RSD of existing analysis methods is as high as 20-30%. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of forming microdroplets for ion source;

[0026] Figure 2 The results of the derivatization of quinone compounds in Example 1, wherein (a) is BQ, (b) is MBQ, (c) is NQ, and (d) is AQ;

[0027] Figure 3 is the collision induced dissociation (CID) spectrum of Example 1, wherein (a) is BQ, (b) is MBQ, (c) is NQ, and (d) is AQ;

[0028] Figure 4 The signal-to-noise ratio of mass spectrometry analysis for Example 1 and Comparative Examples 1 to 3;

[0029] Figure 5 is the relative signal intensity of the quinone compound derivatization products in Examples 1 to 2 and Comparative Examples 4 to 9;

[0030] Figure 6 is the relative signal intensity of the quinone compound derivatization product in Example 1 and Comparative Examples 10 to 14;

[0031] Figure 7 is the relative signal intensity of the quinone compound derivatization products in Example 1, Example 3, and Comparative Examples 15 to 16;

[0032] Figure 8 is the relative signal intensity of the quinone compound derivatization products in Example 1 and Comparative Examples 17 to 20;

[0033] Figure 9 are the relative signal intensities of the quinone compound derivatization products in Example 1 and Comparative Examples 21-22, wherein (a) is Comparative Example 21, (b) is Comparative Example 22, and (c) is Example 1;

[0034] Figure 10 The standard curve for quantitative analysis of quinone compounds, where (a) is BQ, (b) is MBQ, (c) is NQ, and (d) is AQ;

[0035] Figure 11 These are the detection results of the mass spectrometry analysis method of Example 1 in a complex matrix, wherein (a) is a human serum sample and (b) is a human urine sample. DETAILED DESCRIPTION

[0036] The present invention provides an online mass spectrometry analysis method for quinone compounds based on microdroplet derivatization, comprising the following steps:

[0037] 1) introducing a derivatization reagent solution into an ion source I to form microdroplets I; introducing a quinone compound solution into an ion source II to form microdroplets II;

[0038] After the fusion of microdroplets I and II, the derivatization reagent and the quinone compound undergo a conjugate addition reaction to obtain a zwitterionic product;

[0039] 2) The zwitterion products are subjected to mass spectrometry analysis.

[0040] In the present invention, the derivatization reagent solution in step 1) preferably comprises a derivatization reagent and acetonitrile, and the derivatization reagent is preferably triphenylphosphine.

[0041] In the present invention, the concentration of the derivatization reagent in the derivatization reagent solution in step 1) is preferably 1-10 mmol / L, more preferably 1.5-8 mmol / L, and even more preferably 2 mmol / L.

[0042] In the present invention, the quinone compound solution in step 1) preferably comprises a quinone compound and an acetonitrile aqueous solution.

[0043] In the present invention, the concentration of quinone compounds in the quinone compound solution is preferably 0.1-2 μmol / L, more preferably 0.5-1.5 μmol / L, and more preferably 1 μmol / L; the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is preferably 2-4:6-8, more preferably 2.5-3.5:6.5-7.5, and more preferably 3:7.

[0044] In the present invention, the quinone compound preferably comprises one or more of p-benzoquinone compounds, naphthoquinone compounds and anthraquinone compounds.

[0045] In the present invention, the structural formula of the p-benzoquinone compound is Among them, R is preferably hydrogen, methyl, amino, hydroxy, alkyl, alkoxy, halogen or benzene ring.

[0046] In the present invention, the ion source I and the ion source II are preferably independently an electrospray ion source, an electrospray extraction ion source, a desorption electrospray ion source, a sonic spray ion source or a dielectric barrier discharge ion source.

[0047] In the present invention, the temperature of the ion source I and the ion source II is independently preferably 420-480°C, further preferably 430-470°C, and more preferably 450°C; the atomizing gas pressure of the ion source I and the ion source II is independently preferably 70-80psi, further preferably 72.5-78psi, and more preferably 75psi.

[0048] In the present invention, the conjugate addition reaction in step 1) forms a CP bond at the 2-substitution position of the quinone compound, and then obtains a zwitterionic product by proton transfer; the reaction principle is as follows:

[0049]

[0050] In the present invention, the derivatization reagent solution in step 1) is preferably introduced into the ion source I via a syringe pump, and the flow rate of the derivatization reagent solution is preferably 10 to 50 μL / min, more preferably 20 to 40 μL / min.

[0051] In the present invention, in step 1), the microdroplets I and II collide and mix in the gas phase, and the derivatization reaction of the derivatization reagent and the quinone compound is accelerated at the gas-liquid interface of the microdroplets.

[0052] In the present invention, the mass spectrometry analysis in step 2) is preferably performed in electrospray mass spectrometry positive ion mode.

[0053] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] In the embodiments and comparative examples of the present invention, the specific conditions for performing mass spectrometry analysis in the electrospray mass spectrometry positive ion mode are: ion transfer tube temperature: 450°C; nebulizer gas pressure: 72.5psi; injection pump flow rate: 10μL / min; S-lens RF level: 60%; spray voltage: +4.5kV; maximum injection time: 100ms; and number of microscans: 1.

[0055] Example 1

[0056] The derivatization reagent triphenylphosphine and acetonitrile were mixed to obtain a derivatization reagent solution with a concentration of 1 mmol / L. 1,4-p-benzoquinone (BQ) and an acetonitrile aqueous solution (the volume ratio of acetonitrile to water in the acetonitrile aqueous solution was 3:7) were mixed to obtain a BQ solution with a BQ concentration of 1 μmol / L.

[0057] The derivatization reagent solution was injected into the electrospray ion source I at a flow rate of 10 μL / min through a syringe pump, and the electrospray ion source I formed microdroplets I at a temperature of 450°C and a nebulizer gas pressure of 72.5 psi. The BQ solution was injected into the electrospray ion source II, and the electrospray ion source II formed microdroplets II at a temperature of 450°C and a nebulizer gas pressure of 72.5 psi. After the fusion of microdroplets I and II, the derivatization reagent and BQ underwent a conjugate addition reaction, and then a zwitterion product was generated by proton transfer. Subsequently, mass spectrometry analysis was performed in the positive ion mode of electrospray mass spectrometry, and the zwitterion product obtained a proton to generate a phosphonium ion, which can be detected by mass spectrometry.

[0058] The above steps were repeated with BQ replaced by methyl-p-benzoquinone (MBQ), 1,4-naphthoquinone (NQ) and anthraquinone (AQ) respectively.

[0059] Figure 2 These are the derivatization test results of the quinone compounds of Example 1, wherein (a) is BQ, (b) is MBQ, (c) is NQ, and (d) is AQ.

[0060] Figure 31 is the collision induced dissociation (CID) spectrum of Example 1, wherein (a) is BQ, (b) is MBQ, (c) is NQ, and (d) is AQ.

[0061] Depend on Figure 2 (a) A peak with a mass-to-nuclear ratio (m / z) of 371 can be observed, which is the protonated ion [BQ+PPh3+H] + .Depend on Figure 3 In (a), m / z 293 and m / z 215 correspond to the loss of two consecutive neutral benzene molecules, respectively, and m / z 262 corresponds to the PPh3 positive ion generated by the CP bond break. This result confirms that Example 1 successfully achieved the online derivatization of BQ, and the obtained zwitterionic product combined with H in the positive ion mode of electrospray mass spectrometry. + It is shown that the present invention can realize the online derivatization and mass spectrometry detection of quinone compounds by using triphenylphosphine as a derivatization reagent.

[0062] Depend on Figure 2 (b) A peak with m / z of 385 can be observed, which is the protonated ion [MBQ+PPh3+H] + .Depend on Figure 3 In (b), it can be observed that m / z 307 and m / z 229 correspond to the loss of two consecutive neutral benzene molecules, respectively, and m / z 262 corresponds to the PPh3 positive ion generated by the cleavage of the CP bond.

[0063] Depend on Figure 2 (c) A peak with m / z of 421 can be observed, which is the protonated ion

[0064] [NQ+PPh3+H] + .Depend on Figure 3 In (c), it can be observed that m / z 343 and m / z 265 correspond to the loss of two consecutive neutral benzene molecules, respectively, and m / z 263 corresponds to the PPh3 positive ion generated by the cleavage of the CP bond.

[0065] Depend on Figure 2 In (d), a peak with m / z of 471 can be observed, which is the protonated ion

[0066] [AQ+PPh3+H] + .Depend on Figure 3 In (d), it can be observed that m / z 393 and m / z 315 correspond to the loss of two consecutive neutral benzene molecules, respectively, and m / z 263 corresponds to the PPh3 positive ion generated by the cleavage of the CP bond.

[0067] Example 2

[0068] The nebulizing gas pressure of the electrospray ion source I and the electrospray ion source II in Example 1 was modified to 80 psi, and the rest was the same as in Example 1.

[0069] Example 3

[0070] The concentration of the derivatization reagent solution in Example 1 was modified to 2 mmol / L, and the rest was the same as in Example 1.

[0071] Comparative Example 1

[0072] The derivatization reagent solution in Example 1 was replaced with a mixture of methanol and water, wherein the volume ratio of methanol to water was 1:1. Others were the same as in Example 1.

[0073] Comparative Example 2

[0074] The triphenylphosphine in Example 1 was replaced by cysteamine, and the rest was the same as Example 1.

[0075] Comparative Example 3

[0076] The triphenylphosphine in Example 1 was replaced by aniline, and the rest was the same as in Example 1.

[0077] Figure 4 is the signal-to-noise ratio of mass spectrometry analysis of Example 1 and Comparative Examples 1 to 3. Figure 4 It can be seen that the microdroplet derivatization mass spectrometry analysis method using triphenylphosphine as the derivatization reagent of the present invention obtains the highest signal-to-noise ratio. The signal-to-noise ratio of BQ under triphenylphosphine derivatization conditions is 5.7 times that under cysteamine derivatization conditions, the signal-to-noise ratio of MBQ under triphenylphosphine derivatization conditions is 5.2 times that under cysteamine derivatization conditions, the signal-to-noise ratio of NQ under triphenylphosphine derivatization conditions is 13.5 times that under cysteamine derivatization conditions, and the signal-to-noise ratio of AQ under triphenylphosphine derivatization conditions is 34.9 times that under cysteamine derivatization conditions; while no reaction product was observed under the conditions where methanol and aniline were used as derivatization reagents.

[0078] Comparative Example 4

[0079] The nebulizing gas pressure of the electrospray ion source I and the electrospray ion source II in Example 1 was modified to 29 psi, and the rest was the same as in Example 1.

[0080] Comparative Example 5

[0081] The nebulizing gas pressure of the electrospray ion source I and the electrospray ion source II in Example 1 was modified to 36.5 psi, and the rest was the same as in Example 1.

[0082] Comparative Example 6

[0083] The nebulizing gas pressure of the electrospray ion source I and the electrospray ion source II in Example 1 was modified to 43.5 psi, and the rest was the same as in Example 1.

[0084] Comparative Example 7

[0085] The nebulizing gas pressure of the electrospray ion source I and the electrospray ion source II in Example 1 was modified to 51 psi, and the rest was the same as in Example 1.

[0086] Comparative Example 8

[0087] The nebulizing gas pressure of the electrospray ion source I and the electrospray ion source II in Example 1 was modified to 58 psi, and the rest was the same as in Example 1.

[0088] Comparative Example 9

[0089] The nebulizing gas pressure of the electrospray ion source I and the electrospray ion source II in Example 1 was modified to 65 psi, and the rest was the same as in Example 1.

[0090] Figure 5 is the relative signal intensity of the quinone compound derivatization product in Examples 1 to 2 and Comparative Examples 4 to 9. Figure 5 It can be seen that as the nebulizer gas pressure of the ion source increases from 29psi to 72.5psi, the relative signal intensity of the derivatization products of quinone compounds gradually increases, reaching the highest value when the nebulizer gas pressure is 72.5psi; the relative signal intensity no longer increases as the nebulizer gas pressure continues to increase. This result is related to the gradual decrease in the size of the microdroplets during the increase in the nebulizer gas pressure. The smaller the size of the microdroplets, the larger the specific surface area, which is conducive to accelerating the derivatization reaction and increasing the relative signal intensity of the reaction products.

[0091] Comparative Example 10

[0092] The temperature of the electrospray ion source I and the electrospray ion source II in Example 1 was modified to 200° C., and the rest was the same as in Example 1.

[0093] Comparative Example 11

[0094] The temperature of the electrospray ion source I and the electrospray ion source II in Example 1 was modified to 250° C., and the rest was the same as in Example 1.

[0095] Comparative Example 12

[0096] The temperature of the electrospray ion source I and the electrospray ion source II in Example 1 was modified to 300° C., and the rest was the same as in Example 1.

[0097] Comparative Example 13

[0098] The temperature of the electrospray ion source I and the electrospray ion source II in Example 1 was modified to 350° C., and the rest was the same as in Example 1.

[0099] Comparative Example 14

[0100] The temperature of the electrospray ion source I and the electrospray ion source II in Example 1 was modified to 400° C., and the rest was the same as in Example 1.

[0101] Figure 6 is the relative signal intensity of the quinone compound derivatization products in Example 1 and Comparative Examples 10 to 14. Figure 6 It can be seen that as the temperature of the ion source increases from 200°C to 450°C, the relative signal intensity of the derivatization products of quinone compounds gradually increases. This result shows that the increase in the ion source temperature is conducive to the evaporation of the microdroplet solvent, thereby reducing the size of the microdroplets and promoting the derivatization reaction.

[0102] Comparative Example 15

[0103] The concentration of the derivatization reagent solution in Example 1 was modified to 0.1 mmol / L, and the rest was the same as in Example 1.

[0104] Comparative Example 16

[0105] The concentration of the derivatization reagent solution in Example 1 was modified to 0.5 mmol / L, and the rest was the same as in Example 1.

[0106] Figure 7 is the relative signal intensity of the quinone compound derivatization products in Example 1, Example 3, and Comparative Examples 15-16. Figure 7 It can be seen that as the concentration of the derivatization reagent solution increases from 0.1 mmol / L to 1 mmol / L, the relative signal intensity of the derivatization product of quinone compounds first gradually increases and then tends to stabilize; when the concentration of the derivatization reagent solution exceeds 1 mmol / L, the relative signal intensity basically no longer increases.

[0107] Comparative Example 17

[0108] The volume ratio of acetonitrile to water in the acetonitrile aqueous solution in the quinone compound solution of Example 1 was modified to 100:0, and the rest was the same as Example 1.

[0109] Comparative Example 18

[0110] The volume ratio of acetonitrile to water in the acetonitrile aqueous solution in the quinone compound solution of Example 1 was modified to 7:3, and the rest was the same as Example 1.

[0111] Comparative Example 19

[0112] The volume ratio of acetonitrile to water in the acetonitrile aqueous solution in the quinone compound solution of Example 1 was modified to 1:1, and the rest was the same as Example 1.

[0113] Comparative Example 20

[0114] The volume ratio of acetonitrile to water in the acetonitrile aqueous solution in the quinone compound solution of Example 1 was changed to 0:100, and the rest was the same as Example 1.

[0115] Figure 8 is the relative signal intensity of the quinone compound derivatization products in Example 1 and Comparative Examples 17 to 20. Figure 8 It can be seen that as the volume of water in the acetonitrile aqueous solution increases, the relative signal intensity of the derivatization product of quinone compounds gradually increases, reaching the highest value when the volume ratio of acetonitrile to water is 3:7. This result shows that compared with acetonitrile, water is conducive to maintaining the state of microdroplets for a longer time and prolonging the time of accelerated reaction in microdroplets; the ionization and proton aggregation of water on the surface of microdroplets provide an acidic environment for the derivatization reaction, promoting the derivatization reaction.

[0116] Comparative Example 21

[0117] The derivatization reagent solution in Example 1 was omitted, and the rest was the same as in Example 1.

[0118] Comparative Example 22

[0119] The electrospray mass spectrometry positive ion mode of Comparative Example 21 was changed to electrospray mass spectrometry negative ion mode, and the rest was the same as Comparative Example 21.

[0120] Figure 9 is the relative signal intensity of the quinone compound derivatization products in Example 1 and Comparative Examples 21-22, wherein (a) is Comparative Example 21, (b) is Comparative Example 22, and (c) is Example 1. Figure 9 It can be seen that without the derivatization reaction of the derivatization reagent, the positive ion mode ( Figure 9 a) No ionization signals of quinone compounds were observed. Figure 9 b), three ionization signals of BQ, MBQ and NQ can be observed, but no signal of AQ ionization is observed; among them, the mass spectrum result of BQ ionization is 108.0214, which has an error of -2.8ppm compared with the theoretical value (108.0217); the mass spectrum result of MBQ ionization is 122.0370, which has an error of -2.5ppm compared with the theoretical value (122.0373); the mass spectrum result of NQ ionization is 158.0371, which has an error of -1.3ppm compared with the theoretical value (158.0373). When the positive ion mode ( Figure 9 c), the ionization signals of BQ, MBQ, NQ, and AQ can all be observed. From the detection signal-to-noise ratio, the detection signal-to-noise ratio of BQ increased by 14.4 times, the detection signal-to-noise ratio of MBQ increased by 7.4 times, and the detection signal-to-noise ratio of NQ increased by 15.2 times.

[0121] Characterization of the analytical performance of the mass spectrometry method: According to the parameter settings of Example 1, the internal standard method was adopted, BQ-D3 reagent was used as the internal standard reagent for BQ and MBQ, NQ-D5 reagent was used as the internal standard reagent for NQ and AQ, and the concentration of the internal standard reagent was 100nmol / L. The quantitative analysis of quinone compounds was performed by the selected reaction detection mode (SRM) to characterize the analytical performance of the mass spectrometry method of the present invention. The analytical performance results are shown in Table 1. Among them, the analysis results of RSD are expressed as the ratio of the signal intensity of quinone compounds and internal standard reagent (IS) (I 醌类化合物 / I IS ) for reference.

[0122] Figure 10 The standard curves for quantitative analysis of quinone compounds, where (a) is BQ, (b) is MBQ, (c) is NQ, and (d) is AQ.

[0123] Table 1 Analysis performance results of the mass spectrometry method of the present invention

[0124]

[0125] As can be seen from Table 1, the LOD of the mass spectrometry analysis method of the present invention for the four quinone compounds is as low as nmol / L level, with high sensitivity, which is close to the results of the existing HPLC-ESI method and the methanol solution derivatization method; however, the existing HPLC-ESI method and the methanol solution derivatization method both require a pre-treatment time of up to 59 hours, which does not meet the needs of rapid detection. Compared with the in-situ or online derivatization method based on paper spray and toothpick spray, the sensitivity of the mass spectrometry analysis method of the present invention is improved by 2 to 3 orders of magnitude. The mass spectrometry analysis method of the present invention obtains good linearity for quinone compounds in a wide concentration range, and the recovery rate reaches 90.2 to 110.2%, indicating that the mass spectrometry analysis method of the present invention can realize the detection of quinone compounds in a wide linear range, and still has good accuracy at lower concentrations. The mass spectrometry analysis method of the present invention has high repeatability and accuracy, with an RSD of 3.3 to 7.3%, which is significantly better than paper spray (RSD of 3 to 20%) and toothpick spray (30%).

[0126] The mass spectrometry analysis method of the present invention is also applicable to the mass spectrometry analysis of quinone compounds in complex matrices. Human serum and human urine were used as research objects, 100 nmol / L quinone compounds were added to obtain spiked samples, and mass spectrometry analysis was performed according to the parameter settings of Example 1. The LODs of BQ, MBQ, NQ, and AQ in serum spiked samples were 26.2 nmol / L, 13.6 nmol / L, 15.0 nmol / L, and 23.1 nmol / L, respectively, and the recoveries were 90.7%, 85.4%, 95.3%, and 92.9%, respectively. Figure 11These are the detection results of the mass spectrometry analysis method of Example 1 in a complex matrix, wherein (a) is a human serum sample and (b) is a human urine sample.

[0127] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An online mass spectrometry analysis method for quinone compounds based on microdroplet derivatization, characterized in that: The following steps are included: 1) introducing a derivatization reagent solution into an ion source I to form microdroplets I; introducing a quinone compound solution into an ion source II to form microdroplets II; After the fusion of microdroplets I and II, the derivatization reagent and the quinone compound undergo a conjugate addition reaction to obtain a zwitterionic product; 2) The zwitterion products are subjected to mass spectrometry analysis.

2. The method for online mass spectrometry analysis of quinone compounds based on microdroplet derivatization according to claim 1, characterized in that: Step 1) The derivatization reagent solution comprises a derivatization reagent and acetonitrile, and the derivatization reagent is triphenylphosphine.

3. The method for online mass spectrometry analysis of quinone compounds based on microdroplet derivatization according to claim 2, characterized in that: Step 1) The concentration of the derivatization reagent in the derivatization reagent solution is 1-10 mmol / L.

4. The method for online mass spectrometry analysis of quinone compounds based on microdroplet derivatization according to any one of claims 1 to 3, characterized in that: Step 1) The quinone compound solution comprises a quinone compound and an acetonitrile aqueous solution.

5. The method for online mass spectrometry analysis of quinone compounds based on microdroplet derivatization according to claim 4, characterized in that: The concentration of the quinone compound in the quinone compound solution is 0.1-2 μmol / L; the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 2-4:6-8.

6. The method for online mass spectrometry analysis of quinone compounds based on microdroplet derivatization according to claim 5, characterized in that: The quinone compounds include one or more of p-benzoquinone compounds, naphthoquinone compounds and anthraquinone compounds.

7. The method for online mass spectrometry analysis of quinone compounds based on microdroplet derivatization according to claim 6, characterized in that: The structural formula of the p-benzoquinone compound is Wherein, R is hydrogen, methyl, amino, hydroxyl, alkyl, alkoxy, halogen or benzene ring.

8. The method for online mass spectrometry analysis of quinone compounds based on microdroplet derivatization according to claim 1, characterized in that: The ion source I and the ion source II are independently an electrospray ion source, an electrospray extraction ion source, a desorption electrospray ion source, a sonic spray ion source or a dielectric barrier discharge ion source.

9. The method for online mass spectrometry analysis of quinone compounds based on microdroplet derivatization according to claim 8, characterized in that: The temperatures of the ion source I and the ion source II are independently 420-480° C., and the atomizing gas pressures of the ion source I and the ion source II are independently 70-80 psi.

10. The method for online mass spectrometry analysis of quinone compounds based on microdroplet derivatization according to claim 9, characterized in that: Step 2) The mass spectrometry analysis is performed using electrospray mass spectrometry positive ion mode.

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