CN116818945A - Analysis and detection method of naphthoquinone compounds based on mercaptoethanol derivatization strategy - Google Patents
Analysis and detection method of naphthoquinone compounds based on mercaptoethanol derivatization strategy Download PDFInfo
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Abstract
The invention discloses an analysis and detection method of naphthoquinone compounds based on a mercaptoethanol derivatization strategy, and belongs to the technical field of medicine analysis. Firstly, introducing mercaptoethanol which is an ionizable group into a juglone structure which is difficult to ionize through Michael addition reaction of mercaptoethanol and naphthoquinone compounds, then separating a juglone to be detected after being derivatized with mercaptoethanol through liquid chromatography, converting the juglone to charged gas phase ions in an ion source, analyzing and detecting the charged gas phase ions through triple quadrupole-tandem mass spectrometry, and quantitatively analyzing the to-be-detected substances through specific reaction ion pairs formed by parent ions and ion pairs. The invention establishes a high-efficiency and accurate mass spectrometry analysis and detection method for the derivatization of the juglone-mercaptoethanol, the stability of the derivatization product is better, the mass spectrometry detection sensitivity is higher, and an analysis and detection strategy for trace naphthoquinone compounds is provided.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to an analysis and detection method of naphthoquinone compounds based on a mercaptoethanol derivatization strategy.
Background
Naphthoquinone compounds are small molecular compounds widely existing in the nature, and the compounds play an important role in the treatment process of various diseases such as cancer resistance, inflammation resistance, oxidation resistance and virus resistance due to the unique structure and biological activity of the compounds, so that the naphthoquinone compounds have wide application prospect. For example, vitamin K3, also known as menaquinone, has procoagulant function and can be used to treat hemorrhagic diseases caused by vitamin K deficiency, including neonatal hemorrhage, hypoprothrombinemia, etc.; juglone has hemostatic and antibacterial activities, and can be used for treating eczema, cow leather and tinea; 1,4 naphthoquinone is mainly used as a synthetic pesticide bactericide and a dye intermediate; benzoquinone can be used as leather tanning agent, photographic developer and raw material for making dye, medicine and cosmetics. In recent years, the rapid development of liquid chromatography tandem mass spectrometry (LC-MS/MS) provides a possible solution for quantitative analysis of naphthoquinone compounds. Compared with the traditional quantitative methods of naphthoquinone compounds, such as ultraviolet-visible spectrophotometry, high performance liquid chromatography, gas chromatography-mass spectrometry, and the like, the liquid chromatography-tandem mass spectrometry has great advantages in all aspects of accuracy, precision, selectivity, sensitivity, and the like. At present, no liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring naphthoquinone compounds in biological samples with high sensitivity and good selectivity is reported in the existing research. Therefore, establishing an analytical method capable of accurately and sensitively quantifying naphthoquinone compounds is a problem to be solved.
The main reason that naphthoquinone compounds are difficult to quantify is that the naphthoquinone compounds have unsaturated structures, only C, H, O in molecular combination are not easy to ionize in a mass spectrum ESI ion source, and are not easy to break up in a secondary mass spectrum, so that stable fragments are generated. On the other hand, naphthoquinone compounds are stable in organic solvents, but are highly unstable in biological samples, and can bind to cysteine and glutathione of proteins, resulting in extremely low concentration levels, and thus quantitative analysis cannot be accomplished. Therefore, the sensitivity of directly measuring naphthoquinone compounds by using a liquid chromatography-mass spectrometry technology is low, and the quantitative requirement of the naphthoquinone compounds in biological samples cannot be met. Sreelatha, T and the like are combined with thiol and amide to synthesize substituted 1, 4-naphthoquinone, silver oxide is used as a catalyst to obtain an intermediate product, the operation steps are relatively complex, and the cost is high. In addition, ryu, C.K, etc., introduce thiols into quinone compounds by bromination reaction to form intermediates, but this method requires the addition of a palladium catalyst to form the target compound. As one of representative drugs among naphthoquinone compounds, juglone has a problem of low sensitivity and instability in quantitative analysis.
Disclosure of Invention
In order to solve the technical problems in the background art, the invention aims to establish an analysis and detection method of naphthoquinone compounds based on a mercaptoethanol derivatization strategy, wherein the method uses juglone as a representative compound, and uses a method based on a 1, 4-Michael addition reaction principle and through pre-column derivatization to introduce an ionizable group mercaptoethanol into a juglone structure which is difficult to ionize, so that the strategy of mass spectrum detection specificity and sensitivity is improved, and a specific reaction ion pair obtained through multiple reaction monitoring is used for quantitatively analyzing a substance to be detected, the pre-column derivatization method enables the response value of the quinone compounds in mass spectrum to be obviously improved, and the mass spectrum detection sensitivity to be obviously improved.
The invention aims at realizing the following technical scheme:
the analysis and detection method of naphthoquinone compounds based on mercaptoethanol derivatization strategy mainly comprises the following steps:
(1) Carrying out derivatization reaction on mercaptoethanol and naphthoquinone compounds;
(2) Separating by liquid chromatography to obtain derivatized naphthoquinone compounds;
(3) And converting the derivatized naphthoquinone compounds into charged gas-phase ions in an ion source, and analyzing and detecting by triple quadrupole-tandem mass spectrometry.
Based on the technical scheme, further, the naphthoquinone compound comprises juglone.
Based on the above technical scheme, further, the derivatization reaction in the step (1) specifically includes: dissolving naphthoquinone compounds, internal standard and mercaptoethanol in the reaction solution, adjusting the pH of the reaction solution to 4-10, and reacting for 0.5-8 hours at 25-80 ℃.
Based on the technical scheme, the reaction solution is water-acetonitrile solution with the volume ratio of 1:10-10:1; the internal standard is menadione, and the concentration ratio of the internal standard to the naphthoquinone compound is 1:1; the concentration of naphthoquinone compounds is 1-100 mug/mL, and the concentration of mercaptoethanol is 0.1-100 mg/mL.
Based on the technical scheme, further, the liquid chromatography separation conditions in the step (2) are as follows: chromatographic column: water meters Acquity UPLC BEH C 18 Column, 2.1mm x 50mm i.d.,1.7 μm particle size; mobile phase: phase A: water containing 0.1% formic acid by volume, phase B: acetonitrile; gradient elution; column temperature: 40 ℃; flow rate: 0.4mL/min; sample injection amount: 10 mu L.
Based on the above technical scheme, further, the gradient elution procedure is as follows: 0.1min 80% A,1.5min 80% A,3.0min10% A,3.5min 10% A,3.6min 80% A,5.0min 80% A.
Based on the technical scheme, further, the specific process of the step (3) is as follows: converting the derivatized naphthoquinone compound into charged gas-phase ions in an ion source, entering a mass spectrum first heavy mass analyzer Q1, screening by an electric field, enabling ions with specific mass-to-charge ratio to enter a second heavy mass analyzer Q2, performing collision induction cracking to obtain a series of fragment ions, enabling the fragment ions to enter a third heavy mass analyzer rod Q3 or an ion trap, selecting the ions with stable high signal response again under the action of the specific electric field as sub-ions to enter a detector for detection, and performing quantitative analysis detection on an object to be detected based on an analysis method for establishing multiple reaction monitoring by using ion pairs formed by parent ions and the sub-ions.
Based on the technical scheme, the collision energy is 5-15 eV.
Based on the technical scheme, further, the parent ion of the naphthoquinone compound is m/z174.15; the secondary fragment ions are m/z 88.9, 116.8, 117.4 and 144.8; specific parent ions of naphthoquinone compounds after derivatization with mercaptoethanol are m/z251.0, and fragment ions are m/z190.9 and 121.0.
Based on the technical scheme, further, the specific ion pair of the naphthoquinone compound after the derivatization of the mercaptoethanol is m/z251.0/190.9; the ion pair of the naphthoquinone compound is m/z 174.15/144.8.
Based on the technical scheme, further, the mass spectrum conditions are as follows: a Q-trap 6500 tandem mass spectrometer equipped with an ESI ionization source and analysis data processing software; detecting juglone in a negative ion mode; detecting the naphthoquinone compound after the derivatization of mercaptoethanol in a positive ion mode; ion ejection voltage 5000V; the temperature is 50 ℃; source internal gas 1: nitrogen pressure 40psi; gas 2: nitrogen pressure 40psi; air curtain gas: nitrogen pressure 35psi; MRM scan mode, declustering voltage: 60V.
Based on the technical scheme, the method further comprises the steps of sample pretreatment and standard curve preparation before measuring the juglone and the juglone derivatized with mercaptoethanol in the biological sample.
The invention provides a quantitative analysis and detection method of naphthoquinone compounds based on a mercaptoethanol derivatization strategy for the first time, and establishes a Multiple Reaction Monitoring (MRM) analysis method by utilizing high resolution and high mass accuracy of liquid chromatography-tandem mass spectrometry (LC-MS/MS); introducing an ionizable functional group onto the naphthoquinone compound with an unsaturated structure by a pre-column derivatization method, so that the response and detection sensitivity of the naphthoquinone compound in mass spectrum are obviously improved, and quantitative analysis and detection of the quinone compound in a complex sample are realized; the corresponding collision energy in the Mass Spectrum (MS) parameters was 13eV; the method comprises the steps of converting an object to be detected into gas-phase ions in an ion source, enabling the gas-phase ions to enter a mass spectrum, enabling the first-stage mass spectrum in a triple quaternary rod to scan ions with specific mass-to-charge ratio, enabling the ions to enter a collision chamber, enabling excimer ions to collide in the collision chamber to induce cracking, enabling the ions to enter a third-stage mass spectrum, enabling the third-stage mass spectrum to scan the ions with specific mass-to-charge ratio, enabling the analytes to collide through energy to generate the ions, and enabling the ions to be scanned and analyzed to establish a stable and reliable LC-MS/MS analysis method. In conclusion, the quantitative analysis of juglone and juglone compounds after derivatization with mercaptoethanol is completed by using a Multiple Reaction Monitoring (MRM) analysis method. The parent ion of the selected juglone is m/z174.15; the secondary fragment ions are m/z 88.9, 116.8, 117.4 and 144.8; specific parent ion of juglone-mercaptoethanol derivative after derivatization with mercaptoethanol is m/z251.0, and fragment ion is m/z190.9, 121.0.
The invention has the beneficial effects that:
1) The invention adopts a pre-column derivatization method to introduce an ionizable group into the structure of a naphthoquinone compound which is difficult to ionize, adopts mercaptoethanol as a derivatization reagent to carry out Michael addition with juglone, and has active chemical property because the mercaptoethanol is stable in an organic reagent; under the condition of room temperature, sulfhydryl groups are easy to carry out addition reaction with unsaturated double bonds, and the reaction efficiency is high; the product after derivatization reaction with mercaptoethanol contains sulfur atoms, so that the product is more easily charged in a mass spectrum, the detection sensitivity of the mass spectrum can be improved, the problem that the response of an analyte to be detected is very low due to the fact that no ionizable functional group exists in the unsaturated chemical structure of juglone is solved, the response value is improved by 750 times, and the detection sensitivity is improved to a large extent.
2) The invention provides a quantitative analysis and detection method of naphthoquinone compounds based on a mercaptoethanol derivatization strategy for the first time, and establishes a Multiple Reaction Monitoring (MRM) analysis method by utilizing high resolution and high mass accuracy of liquid chromatography-tandem mass spectrometry (LC-MS/MS), wherein the method has good reproducibility and high accuracy, and the pre-column derivatization method can obviously improve the mass spectrum detection response and sensitivity, so that the method is suitable for quantitative analysis and detection of trace quinone compounds in complex biological samples.
Drawings
FIG. 1 is a secondary mass spectrum and chromatogram of fragments of the reaction product of the derivatization of juglone with mercaptoethanol by tandem mass spectrometry obtained in example 1 and example 10.
FIG. 2 is a scanning mass spectrum of juglone compound obtained in example 1 after derivatization with mercaptoethanol.
FIG. 3 is a graph showing the results of optimized derivatization reaction time conditions for juglone compounds based on mercaptoethanol derivatization strategy obtained in examples 1-3.
FIG. 4 is a graph showing the results of optimized derivatization reaction temperature conditions of juglone compounds based on mercaptoethanol derivatization strategy obtained in examples 4-6.
FIG. 5 is a graph showing the results of optimized derivatization reaction pH conditions of juglone compounds based on mercaptoethanol derivatization strategy obtained in examples 7-9.
FIG. 6 is a typical chromatogram of a juglone compound obtained in example 10 after derivatization of juglone.
FIG. 7 is a graph showing the calibration of juglone compounds obtained in example 10 after derivatization with mercaptoethanol.
FIG. 8 is a graph comparing the mass spectral response of the underivatized and derivatized juglone compounds obtained in comparative examples 1-2.
Detailed Description
The invention is further illustrated below in connection with specific embodiments, but the invention is not limited to these specific embodiments.
The technical scheme of the invention is that an ionizable functional group is introduced onto a naphthoquinone compound without an ionizable structure by adopting a pre-column derivatization method, and the naphthoquinone compound is quantitatively analyzed by utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). The object to be detected is converted into gas-phase ions in an ion source and enters a mass spectrum, the first-stage mass spectrum in a triple quaternary rod scans ions with specific mass-to-charge ratio and enters a collision chamber, the excimer ions in the collision chamber undergo collision induced fragmentation to form ion ions and enter a third-stage mass spectrum, and the third-stage mass spectrum scans the ion ions with specific mass-to-charge ratio and enters a detector. Quantitative analysis of the analyte is performed based on the ion reaction formed by the parent and child ions in an analytical method for establishing Multiple Reaction Monitoring (MRM). The invention can be used for the analysis and detection of naphthoquinone compounds in complex samples, is very suitable for the quantitative analysis and detection of trace quinone compounds in complex biological samples,
the conditions for the mass spectrometry absolute quantification method of juglone compounds based on the mercaptoethanol derivatization strategy in the examples are as follows:
the chromatographic conditions are as follows: a high performance liquid chromatography system; chromatographic column: waters Acquity UPLC BEH C 18 Column, 2.1mm x 50mm i.d.,1.7 μm particle size; mobile phase: water containing 0.1% formic acid by volume fraction and acetonitrile, gradient elution; column temperature: 40 ℃; flow rate: 0.4mL/min; sample injection amount: 10. Mu.L;
the gradient elution procedure in the chromatographic conditions is shown in Table 1.
TABLE 1 gradient elution procedure
Wherein A is water containing 0.1% formic acid by volume and B is acetonitrile.
The mass spectrum conditions are as follows: a Q-trap 6500 tandem mass spectrometer equipped with an ESI ionization source and analysis data processing software; ion source: an ESI ionization source; detecting juglone as negative ions; detecting the product of derivatization of mercaptoethanol and juglone in a positive ion mode; ion ejection voltage 5000V; the temperature is 500 ℃; source internal gas 1: nitrogen pressure 40psi; gas 2: nitrogen pressure 40psi; air curtain gas: nitrogen pressure 35psi; MRM scan mode, declustering voltage: 60V; collision energy: 13eV.
The following examples optimize the conditions for derivatization of juglone with mercaptoethanol:
example 1:
1mg of juglone was dissolved with 1mL of DMSO solution and diluted to 1 μg/mL with acetonitrile-water (v/v=1/1) solution; mercaptoethanol was formulated with acetonitrile-water (v/v=1/1) solution to 1mg/mL; 1mg of internal standard menaquinone was weighed out, dissolved with acetonitrile-water (v/v=1/1) solution and diluted to 1 μg/mL;
taking 300 mu L of an object solution to be detected, 300 mu L of an internal standard solution and 600 mu L of a mercaptoethanol solution, and carrying out derivatization reaction for 0.5h at the temperature of 50 ℃; two groups, 300. Mu.L each, 200. Mu.L water in the control group and 30. Mu.L 3%H in the experimental group were taken 2 O 2 Incubating the solution at 25 ℃ for 10min; after the experimental group was complemented to 500. Mu.L with 170. Mu.L of water, 200. Mu.L of each was analyzed by liquid chromatography-tandem mass spectrometry, and the chromatogram and the mass spectrum response were recorded.
Example 2:
1mg of juglone was dissolved with 1mL of DMSO solution and diluted to 1 μg/mL with acetonitrile-water (v/v=1/1) solution; mercaptoethanol was formulated with acetonitrile-water (v/v=1/1) solution to 1mg/mL; 1mg of internal standard menaquinone was weighed out, dissolved with acetonitrile-water (v/v=1/1) solution and diluted to 1 μg/mL;
taking 300 mu L of an object solution to be detected, 300 mu L of an internal standard solution and 600 mu L of a mercaptoethanol solution, and carrying out derivatization reaction for 4 hours at 50 ℃; two groups, 300. Mu.L each, 200. Mu.L water in the control group and 30. Mu.L 3%H in the experimental group were taken 2 O 2 Incubating the solution at 25 ℃ for 10min; after the experimental group was complemented to 500. Mu.L with 170. Mu.L of water, 200. Mu.L of each was analyzed by liquid chromatography-tandem mass spectrometry, and the chromatogram and the mass spectrum response were recorded.
Example 3:
1mg of juglone was dissolved with 1mL of DMSO solution and diluted to 1 μg/mL with acetonitrile-water (v/v=1/1) solution; mercaptoethanol was formulated with acetonitrile-water (v/v=1/1) solution to 1mg/mL; 1mg of internal standard menaquinone was weighed out, dissolved with acetonitrile-water (v/v=1/1) solution and diluted to 1 μg/mL;
taking 300 mu L of an object solution to be detected, 300 mu L of an internal standard solution and 600 mu L of a mercaptoethanol solution, and carrying out derivatization reaction for 8 hours at 50 ℃; two groups, 300. Mu.L each, 200. Mu.L water in the control group and 30. Mu.L 3%H in the experimental group were taken 2 O 2 Incubating the solution at 25 ℃ for 10min; after the experimental group was complemented to 500. Mu.L with 170. Mu.L of water, 200. Mu.L of each was analyzed by liquid chromatography-tandem mass spectrometry, and the chromatogram and the mass spectrum response were recorded.
Example 4:
1mg of the weighed juglone was dissolved with 1mL of DMSO solution and diluted to 1. Mu.g/mL with acetonitrile-water (v/v=1/1) solution; mercaptoethanol was formulated with acetonitrile-water (v/v=1/1) solution to 1mg/mL; 1mg of internal standard menaquinone was weighed out, dissolved with acetonitrile-water (v/v=1/1) solution and diluted to 1 μg/mL;
taking 300 mu L of an object solution to be detected, 300 mu L of an internal standard solution and 600 mu L of a mercaptoethanol solution, and carrying out derivatization reaction at 25 ℃ for 1h respectively; two groups of 300. Mu.L of each were added with 30. Mu.L of 3%H 2 O 2 Solution and 30. Mu.L of 30% H 2 O 2 Adding 30 mu L of water into each group, and incubating for 10min at 25 ℃; after each group was accumulated with 140. Mu.L of water to 500. Mu.L, 200. Mu.L of each was subjected to liquid chromatography-tandem mass spectrometry analysis, and the chromatogram and the mass spectrum response were recorded.
Example 5:
1mg of the weighed juglone was dissolved with 1mL of DMSO solution and diluted to 1. Mu.g/mL with acetonitrile-water (v/v=1/1) solution; mercaptoethanol was formulated with acetonitrile-water (v/v=1/1) solution to 1mg/mL; 1mg of internal standard menaquinone was weighed out, dissolved with acetonitrile-water (v/v=1/1) solution and diluted to 1 μg/mL;
taking 300 mu L of an object solution to be detected, 300 mu L of an internal standard solution and 600 mu L of a mercaptoethanol solution, and carrying out derivatization reaction at 80 ℃ for 1h respectively; two groups of 300. Mu.L of each were added with 30. Mu.L of 3%H 2 O 2 Solution and 30. Mu.L of 30% H 2 O 2 Adding 30 mu L of water into each group, and incubating for 10min at 25 ℃; after each group was accumulated with 140. Mu.L of water to 500. Mu.L, 200. Mu.L of each was subjected to liquid chromatography-tandem mass spectrometry analysis, and the chromatogram and the mass spectrum response were recorded.
Example 6:
1mg of the weighed juglone was dissolved with 1mL of DMSO solution and diluted to 1. Mu.g/mL with acetonitrile-water (v/v=1/1) solution; mercaptoethanol was formulated with acetonitrile-water (v/v=1/1) solution to 1mg/mL; 1mg of internal standard menaquinone was weighed out, dissolved with acetonitrile-water (v/v=1/1) solution and diluted to 1 μg/mL;
taking 300 mu L of an object solution to be detected, 300 mu L of an internal standard solution and 600 mu L of a mercaptoethanol solution, and carrying out derivatization reaction at 50 ℃ for 1h respectively; two groups of 300. Mu.L of each were added with 30. Mu.L of 3%H 2 O 2 Solution and 30. Mu.L of 30% H 2 O 2 Adding 30 mu L of water into each group, and incubating for 10min at 25 ℃; after each group was accumulated with 140. Mu.L of water to 500. Mu.L, 200. Mu.L of each was subjected to liquid chromatography-tandem mass spectrometry analysis, and the chromatogram and the mass spectrum response were recorded.
Example 7:
300. Mu.L of juglone at a concentration of 1. Mu.g/mL was mixed with 300. Mu.L of menaquinone solution (internal standard) at a concentration of 1. Mu.g/mL and 600. Mu.L of mercaptoethanol solution at a concentration of 1mg/mL;
adding 1 mu L formic acid solution to make the system acidic, adjusting pH to 4, and carrying out derivatization reaction for 1h at 50 ℃; 200 mu L of the sample was subjected to liquid chromatography-tandem mass spectrometry, and a chromatogram and a mass spectrum response value were recorded.
Example 8:
300. Mu.L of juglone at a concentration of 1. Mu.g/mL was mixed with 300. Mu.L of menaquinone solution (internal standard) at a concentration of 1. Mu.g/mL and 600. Mu.L of mercaptoethanol solution at a concentration of 1mg/mL;
the pH value of the system is regulated to 10 by 1 mu L of ammonia water, and derivatization is carried out for 1h at 50 ℃; 200 mu L of the sample was subjected to liquid chromatography-tandem mass spectrometry, and a chromatogram and a mass spectrum response value were recorded.
Example 9:
300. Mu.L of juglone at a concentration of 1. Mu.g/mL was mixed with 300. Mu.L of menaquinone solution (internal standard) at a concentration of 1. Mu.g/mL and 600. Mu.L of mercaptoethanol solution at a concentration of 1mg/mL;
adding 1 mu L of water to make the solution system neutral, and carrying out derivatization reaction for 1h at 50 ℃; 200 mu L of the sample was subjected to liquid chromatography-tandem mass spectrometry, and a chromatogram and a mass spectrum response value were recorded.
Example 10:
1mg of juglone is weighed and dissolved by 1mLDMSO, and the stock solution of juglone is diluted to 100ng/mL, 300ng/mL, 1 mug/mL and 3 mug/mL respectively by acetonitrile-water (v/v=1/1) solution; an internal standard solution (menaquinone, 1 μg/mL) and a mercaptoethanol solution (1 mg/mL) were prepared separately using acetonitrile-water (v/v=1/1) solutions;
taking 300 mu L of a solution of an object to be detected, 300 mu L of an internal standard solution and 600 mu L of mercaptoethanol, and carrying out derivatization reaction for 1h at the temperature of 25 ℃; 200 mu L of the sample is taken to enter LC-MS/MS for analysis, and a chromatogram is recorded; and (3) taking the concentration of the juglone solution as an abscissa, taking the ratio of the peak area of the object to be detected to the peak area of the internal standard solution as an ordinate, and carrying out regression operation by using a weighted W=1/x 2 least square method to obtain a linear regression equation, namely a standard curve.
Comparative example 1:
1mg of juglone was weighed and dissolved with 1mL of ldmso, and the juglone stock solution was diluted to 10 μg/mL with acetonitrile-water (v/v=1/1) solution; preparing an internal standard menaquinone solution with the concentration of 1 mu g/mL by using acetonitrile-water (v/v=1/1); the juglone and menadione were mixed in 200. Mu.L each, 400. Mu.L acetonitrile-water (v/v=1/1) was added and reacted at 50℃for 1 hour, and 200. Mu.L each of the solutions was subjected to liquid chromatography-tandem mass spectrometry analysis, and the chromatogram and mass spectrometry response were recorded.
Comparative example 2:
1mg of juglone was weighed and dissolved with 1mL of ldmso, and the juglone stock solution was diluted to 10 μg/mL with acetonitrile-water (v/v=1/1) solution; an internal standard solution (menaquinone, 1 μg/mL) and a mercaptoethanol solution (1 mg/mL) were prepared separately using acetonitrile-water (v/v=1/1) solutions; respectively taking 200 mu L of a substance to be detected, 200 mu L of menadione and 400 mu L of mercaptoethanol solution, and carrying out derivatization reaction for 1h at 50 ℃; 200 mu L of each group of solutions is taken for liquid chromatography-tandem mass spectrometry analysis, and a chromatogram and a mass spectrum response value are recorded.
FIG. 1 is a secondary mass spectrum and a chromatogram of a juglone compound obtained in example 1 and example 10 after the derivatization with mercaptoethanol.
FIG. 2 is a scanning mass spectrum of juglone compound obtained in example 1 after derivatization with mercaptoethanol, wherein the specific parent ion of the derivatization product is m/z251.0 and the fragment ion is m/z190.9, 121.0.
FIG. 3 is a graph of the results of optimized derivatization reaction time conditions of juglone compounds based on mercaptoethanol derivatization strategy obtained in examples 1-3, showing that the optimal reaction time for derivatization of juglone with mercaptoethanol is 1 hour by experiment and data processing; adding an oxidant H 2 O 2 The mass spectral response can be improved.
FIG. 4 is a graph showing the results of the optimized derivatization reaction temperature conditions of juglone compounds based on the mercaptoethanol derivatization strategy obtained in examples 4-6, and the optimal temperature for the derivatization reaction of juglone and mercaptoethanol was found to be 50℃by experiment and data processing.
FIG. 5 is a graph showing the results of the pH conditions of the optimized derivatization reaction of juglone compounds based on the mercaptoethanol derivatization strategy obtained in examples 7-9, wherein the optimal reaction system conditions of juglone and mercaptoethanol are neutral through experiments and data processing.
FIG. 6 is a typical chromatogram of the juglone derived from example 10.
Fig. 7 is a standard curve of the water sample obtained in example 10 after the derivatization of juglone and mercaptoethanol, and in view of the good linear relationship between the typical standard curves of the derivatization of juglone and mercaptoethanol, it can be known that the absolute quantitative method of mass spectrum of the juglone compound based on the mercaptoethanol derivatization strategy of the invention has good linear relationship, high accuracy, good reproducibility, sensitivity and reliability, and can be used for absolute quantitative analysis of the naphthoquinone compound.
FIG. 8 is a graph comparing the mass spectral response of the underivatized and derivatized juglone compounds obtained in comparative examples 1-2, comparing the mass spectral response values of mercaptoethanol derivatized juglone with those of underivatized juglone, it is clear that the absolute quantification method of mass spectrum of a mercaptoethanol derivatization strategy-based juglone compound of the present invention has a higher sensitivity of mass spectral detection, resulting in a 750-fold improvement in mass spectral response value. The method can be used for absolute quantitative analysis of the naphthoquinone compounds.
The examples described above represent only embodiments of the invention and are not to be understood as limiting the scope of the patent of the invention, it being pointed out that several variants and modifications may be made by those skilled in the art without departing from the concept of the invention, which fall within the scope of protection of the invention.
Claims (10)
1. The analysis and detection method of naphthoquinone compounds based on mercaptoethanol derivatization strategy is characterized by mainly comprising the following steps:
(1) Carrying out derivatization reaction on mercaptoethanol and naphthoquinone compounds;
(2) Separating by liquid chromatography to obtain derivatized naphthoquinone compounds;
(3) And converting the derivatized naphthoquinone compounds into charged gas-phase ions in an ion source, and analyzing and detecting by triple quadrupole-tandem mass spectrometry.
2. The analytical test method of claim 1 wherein the naphthoquinone compound comprises juglone.
3. The analytical test method according to claim 1, wherein the derivatization reaction of step (1) is specifically: dissolving naphthoquinone compounds, internal standard and mercaptoethanol in the reaction solution, adjusting the pH of the reaction solution to 4-10, and reacting for 0.5-8 hours at 25-80 ℃.
4. According to claimThe analytical test method according to claim 1, wherein the conditions for liquid chromatography in step (2) are as follows: chromatographic column: waters Acquity UPLC BEH C 18 Column, 2.1mm x 50mm i.d.,1.7 μm particle size; mobile phase: phase A: water containing 0.1% formic acid by volume, phase B: acetonitrile; gradient elution; column temperature: 40 ℃.
5. The analytical test method of claim 1 wherein the specific process of step (3) is: converting the derivatized naphthoquinone compound into charged gas-phase ions in an ion source, entering a mass spectrum first heavy mass analyzer Q1, screening by an electric field, enabling ions with specific mass-to-charge ratio to enter a second heavy mass analyzer Q2, performing collision induction cracking to obtain a series of fragment ions, enabling the fragment ions to enter a third heavy mass analyzer rod Q3 or an ion trap, selecting the ions with stable high signal response again under the action of the specific electric field as sub-ions to enter a detector for detection, and performing quantitative analysis detection on an object to be detected based on an analysis method for establishing multiple reaction monitoring by using ion pairs formed by parent ions and the sub-ions.
6. The analytical test method according to claim 5, wherein the collision energy is 5 to 15eV.
7. The analytical test method according to claim 5, wherein the parent ion of the naphthoquinone compound is m/z174.15; the secondary fragment ions are m/z 88.9, 116.8, 117.4 and 144.8; specific parent ions of naphthoquinone compounds after derivatization with mercaptoethanol are m/z251.0, and fragment ions are m/z190.9 and 121.0.
8. The analytical test method according to claim 5, wherein the specific ion pair of the thiol-ethanol-derivatized naphthoquinone compound is m/z251.0/190.9; the ion pair of the naphthoquinone compound is m/z 174.15/144.8.
9. The analytical test method of claim 5 wherein the mass spectrometry conditions are: a Q-trap 6500 tandem mass spectrometer equipped with an ESI ionization source and analysis data processing software; detecting juglone in a negative ion mode; detecting the naphthoquinone compound after the derivatization of mercaptoethanol in a positive ion mode; ion ejection voltage 5000V; the temperature is 50 ℃; source internal gas 1: nitrogen pressure 40psi; gas 2: nitrogen pressure 40psi; air curtain gas: nitrogen pressure 35psi; MRM scan mode, declustering voltage: 60V.
10. The analytical test method of claim 1 wherein the steps of sample pretreatment and standard curve preparation are included before determining the juglone in the biological sample and after derivatization with mercaptoethanol.
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