阅读说明：本技术 通过引入系列不同同位素标签用于快速实时定量样品中目标分析物的检测方法 (Detection method for rapid real-time quantification of target analytes in a sample by introducing a series of different isotopic labels ) 是由 谭晓杰 郏征伟 陈铭 廖承慧 徐永威 余琴 于 2021-04-01 设计创作，主要内容包括：本发明提供了一种通过引入系列不同同位素标签用于快速实时定量样品中目标分析物的检测方法,以已知系列浓度的目标分析物分别连接不同的同位素衍生试剂a作为内标物,待测样品中的待测目标分析物连接同位素衍生试剂b,将所述内标物加入待测样品后进行质谱法分析,得到内标物的信号和待测目标分析物的信号,然后通过内标物的信号来计算得到待测目标分析物的浓度。本发明无需配制外标回归标准曲线,待测样品实时随行2个及以上内标参照或者内标标准标准曲线,实现了对基质的实时校正；通过采用系列不同同位素衍生试剂,在衍生试剂上加不同同位素来将不同同位素引入目标分析物中,解决了多个目标分析物测定时各目标分析物均需重标同位素内标的问题。(The invention provides a detection method for quickly quantifying a target analyte in a sample in real time by introducing a series of different isotope labels, which is characterized in that the target analyte with known series of concentrations is respectively connected with different isotope derivative reagents a to serve as internal standard substances, the target analyte to be detected in the sample to be detected is connected with an isotope derivative reagent b, the internal standard substances are added into the sample to be detected, mass spectrometry analysis is carried out to obtain signals of the internal standard substances and signals of the target analyte to be detected, and then the concentration of the target analyte to be detected is calculated through the signals of the internal standard substances. According to the method, an external standard regression standard curve is not required to be prepared, the sample to be detected follows 2 or more internal standard reference or internal standard curves in real time, and the real-time correction of the matrix is realized; different isotopes are added to the derivatization reagent to introduce different isotopes into target analytes by adopting a series of different isotope derivatization reagents, so that the problem that each target analyte needs heavy standard isotope internal standards when a plurality of target analytes are measured is solved.)

1. A detection method for rapid real-time quantification of a target analyte in a sample by introducing a series of different isotopic labels, comprising the steps of:

connecting target analytes with known series of concentrations with different isotope derivative reagents a respectively to serve as internal standard substances, connecting target analytes to be detected in a sample to be detected with isotope derivative reagents b, adding the internal standard substances into the sample to be detected, performing mass spectrometry analysis to obtain signals of the internal standard substances and signals of the target analytes to be detected, and calculating the concentration of the target analytes to be detected through the signals of the internal standard substances;

the known series of concentrations of target analyte comprises more than 2 different concentrations of target analyte, each concentration of target analyte being linked to an isotopically-derivatized reagent a.

2. The detection method according to claim 1, wherein the target analytes of the known series of concentrations are pure target analyte solutions of the known series of concentrations or mixed sample solutions containing the target analytes of the known series of concentrations.

3. The detection method according to claim 1, wherein the different isotope derivative reagents a and b are each selected from one of a light isotope derivative reagent, a heavy isotope derivative reagent, a different fluorescent labeling reagent, a different luminescent labeling reagent, and a different chemical group reaction reagent, and specific compounds used in the isotope derivative reagents a and b are different from each other.

4. The detection method according to claim 1, wherein the target analytes include any one or more types of steroid hormones, fatty acids, organic acids, amino acids and amino acid metabolites, phosphatidylethanolamine compounds, carbohydrates, thiols, oxidized thiols, peptides and proteins, vitamins, fats, cholesterol and derivatives thereof, steroids and derivatives thereof;

when the target analytes are of various types, different isotope derivative reagents a and b are correspondingly connected to each type of target analytes.

5. The detection method according to claim 1, wherein the mass spectrometry is performed by mass spectrometry selected from the group consisting of quadrupole mass spectrometry, high resolution mass spectrometry; the detection mode of the mass spectrum is selected from any one of a full scan mode, an ion detection mode, a parent ion detection mode, a multi-reaction detection mode, a neutral loss scan, a data-dependent scan mode and a data-independent scan mode.

6. The detection method according to claim 1 or 5, wherein the mass spectrometry analysis further comprises a step of separation by chromatography; the chromatography is selected from any one of liquid chromatography, gas chromatography, capillary electrophoresis, affinity chromatography, supercritical fluid chromatography, and ion mobility.

7. The detection method according to claim 1 or 5, characterized by further comprising a step of pretreating the sample to be detected before the mass spectrometry analysis; the pretreatment method is any one selected from solid phase extraction, solid-liquid extraction, liquid-liquid extraction, protein precipitation, direct dilution, solvent extraction, immunoaffinity enrichment, salting out, concentration and masking.

8. The detection method according to claim 1, wherein the sample to be detected comprises any one of a biological sample, an environmental sample, a food sample, a synthetic sample, a pharmaceutical sample, and a chemical sample; the biological sample comprises any one of plasma, serum, whole blood, urine, tissue, cerebrospinal fluid, sweat, saliva, hair, and skin.

9. A product for use in the detection method of claim 1 for rapid real-time quantification of a target analyte in a sample by introducing a series of different isotopic labels.

10. An apparatus for the detection method for rapid real-time quantification of a target analyte in a sample by introducing a series of different isotope labels according to claim 1, comprising an apparatus for introducing a series of different isotope labels, a pretreatment apparatus for a sample, a sample storage apparatus and a transport apparatus, wherein the apparatus is on-line or off-line with respect to the sample.

11. A detection kit for rapid real-time quantification of a target analyte in a sample based on the detection method of claim 1, wherein the kit comprises different isotope derivative reagents a, b, a blank matrix, a series of pure target analytes with known concentrations or a series of mixed samples containing target analytes with known concentrations, and a quality control product.

Technical Field

The invention relates to the technical field of detection and analysis, in particular to a detection method for quickly quantifying a target analyte in a sample in real time, and particularly relates to a detection method for quickly quantifying the target analyte in the sample in real time by introducing a series of different isotope labels.

Background

Chromatography-mass spectrometry is increasingly used in biological samples, environmental samples, food samples, synthetic samples, pharmaceutical samples, chemical samples, clinical chemical samples, forensic samples, pharmacological samples, agricultural samples, and the like. In the current analysis process, an external standard curve method is generally used, and an internal standard one-point method is also used. The main disadvantage of the internal standard one-point method is that only one-point method can be used for correction, and two-point or multi-point or internal standard curve correction cannot be used.

Although the existing analysis method and operation flow can also be used for relatively accurately quantifying a sample to be tested, the following problems always exist, and the defects greatly limit the rapid analysis, accurate quantification and wider application of the chromatography-mass spectrometry technology.

The following lists the current analytical procedures and methods, and the deficiencies and limitations that exist.

1. The extraction efficiency, extraction error and sampling error of the sample in the pretreatment process need to be corrected, the purpose can be realized by a following internal standard correction compound, but the internal standard correction compound and a compound with the same property as the object to be detected are needed, but the interference to the object to be detected in the determination process can not be caused. The method for correcting the compound by the following internal standard is used for correcting the extraction efficiency, the extraction error and the sampling error, is commonly used in the measurement of biological samples, and because the matrix in the biological samples is more complex and the content of the compound to be measured is very low, the error brought in the sample treatment process or the error brought by different matrixes can have very obvious influence on the accurate measurement of the compound to be measured; the selection of isotopes of the compounds to be tested as the accompanying internal standard calibrants is the most common and industry recommended method in the determination of biological samples. The method of the internal standard correction substance improves the control on the detection accuracy, reduces the difference and the instability of the result caused by the operation, but needs to find a proper internal standard substance, generally selects an analog of a compound to be detected, but in practice, finds that even the analog has different properties from the compound to be detected, is most suitable for the internal standard isotope of the compound to be detected at present, basically defaults that the isotope of the compound to be detected is basically identical with the property of the compound to be detected, but the synthesis of the internal standard isotope is difficult, the purchase price is very high because the synthesis cost is high, when a plurality of compounds are simultaneously detected, the corresponding internal standard isotope of each compound is needed, the cost is higher, and even if a large number of internal standard isotopes are purchased, the problems of the sample extraction efficiency, the extraction error and the sampling error correction can only be solved, the problem that the measurement calibration object is required to be calibrated by external calibration cannot be solved.

2. The chromatographic-mass spectrometry technology is determined by the detection principle, although the specificity and the sensitivity are high, the response of a mass spectrometer to a compound to be detected is unstable and fluctuates up and down to a certain extent, and the content determination of a sample to be detected needs to be corrected and calculated by taking the response of the compound to be detected with known concentration as a reference. Using a sample with known concentration as a reference, and calculating the concentration of the compound to be detected in the sample to be detected by adopting an external standard one-point method, an external standard two-point method, an external standard curve and the like; it is necessary to determine the response of the test compound in one or more samples of known concentration to form a regression curve of the reference or concentration response, which is the most common method of plotting, and then to calculate the concentration of the test sample. External standard curve method: before testing samples to be tested, a series of samples with known concentration are prepared in advance to draw a standard curve, 1) the process is very time-consuming, the samples are processed and prepared, 2) and the most troublesome is that even if only one sample to be tested needs to be tested, the standard curve needs to be prepared and tested, which is not economical, 3) if the samples received each day are different and the compounds to be tested are different, each standard curve needs to be prepared, all the time is wasted on preparing the standard curve, the testing speed is very slow, so that currently, in the industry, only one item is tested on one instrument in one day, but the actual market demands are different, which causes that the items with small number of samples are not developed, or the samples are collected, and the samples with small number are collected together to be tested, thus the period of detection becomes long; 4) even if only one item is detected every day, because the external standard method, equipment and environment are changed, the standard curve prepared in the morning is difficult to accurately determine the sample to be detected as the reference for content determination in the afternoon and evening.

3. In the above, the chromatography-mass spectrometry technology is determined by the detection principle, and although the specificity and sensitivity are high, the response of the mass spectrometer to the compound to be detected is unstable and fluctuates up and down to a certain extent, and the content determination of the sample to be detected must be corrected and calculated by taking the response of the compound to be detected with known concentration as a reference. The reference is usually made by the one-point method using a heavy standard isotope of known concentration as an internal standard. Internal standard one-point method: the internal standard one-point method can usually use a re-standard isotope with a known concentration as a reference, but the concentration difference of the compounds to be detected in the sample to be detected is large, and it is difficult to reference and correct the compounds to be detected with different concentrations in the sample to be detected by using only one internal standard with one concentration.

4. The matrix effect of different samples to be detected is different, and the matrix effect can influence the ionization efficiency of a mass spectrum, generate ion inhibition or ion enhancement and further influence the detection result. In order to solve the problem, a sample with known concentration is used as a reference substance, and a one-point method, a two-point method, a standard curve and other methods are adopted to calculate the concentration of the compound to be detected in the sample to be detected; it is necessary to determine the response of the test compound in one or more samples of known concentration to form a regression curve of the reference or concentration response, which is the most common method of plotting, and then to calculate the concentration of the test sample. However, the external standard or external standard regression standard curve configured by this method can only be used with a specific substrate, and for example, in biological sample analysis, plasma or blank biological sample of a certain person or a certain animal, or mixed plasma of 6-10 persons or animals, or simulated plasma is usually used, but neither method can completely represent unknown amounts of samples to be tested, because the substrate in the plasma of each person is very different, and the external standard method cannot solve the problem.

The current industrial method has the limitation that the real-time and faster and accurate measurement of a sample to be measured is limited, a brand new method is necessary to solve the problem, but the analysis cost cannot be increased, and the strong performability needs to be ensured.

A composition, method and kit for quantifying a target analyte in a sample is described in the patent application No. 201280036810.1, which uses a method of purchasing or synthesizing a series of calibrators to a test compound, including different analogs, derivatives, metabolites or isotopes of the test compound, to quantify the test compound by adding these calibrators to the sample. This patent, although published for many years, has not been able to be performed, with the major drawback that each test compound requires the purchase or synthesis of a series of calibrators, which is a significant challenge: 1) the patent proposes using different analogues of the test compound as calibrators, but all these analogues are difficult and costly to purchase and synthesize, and it is not enough to synthesize only one internal standard in the usual application scenario, and 3 or more analogues need to be synthesized, which makes the synthesis more difficult, makes it very difficult to find suitable analogues, or makes it difficult to find an analogue, and may be very different from the properties of the test compound; 2) usually, the stable heavy-mark isotope is the first choice for the internal standard of different sample determination, because the isotope heavy-mark isotope and the compound to be detected have almost the same structure, and even if other similar compounds are synthesized by spending much time and expense, even if only one chemical group is changed, the property of the compound to be detected still has larger difference with the compound to be detected, and the difference makes the calibration object difficult to perform accurate calibration on the compound to be detected. Therefore, throughout the industry, whether biological, environmental, food, synthetic or other analysis, or the like, all require the use of stable heavy-gauge isotopes as calibrators; 3) the synthesis of stable heavy-mark isotopes needs to replace C12 or H on the original structure with C13 or D, but because the structures of compounds to be tested are different and the synthesis routes are different, the structures are usually complex, and a stable heavy-mark isotope calibrator corresponding to the compound needs to be synthesized for each specific compound, for different compounds to be tested, not only is the acquisition of heavy-mark isotope raw materials difficult (no commercialized raw materials are likely to be available), the whole synthesis route needs to redesign and determine the time and the position for introducing the heavy-mark isotopes, but also the synthesis route is long, so that the synthesis cost is higher (the synthesis cannot be completed within 2-3 steps generally), the synthesis route is complex, and the difficulties of introducing light-mark isotope impurities in the synthesis process (otherwise, the separation is difficult later, and the most direct interference is brought to the content measurement) are very high, therefore, it has been a great challenge to synthesize only one isotope internal standard, but according to this patent, at least 3 different stable heavy isotope calibrators are synthesized, which is very challenging to synthesize because 3 different isotope calibrators must be replaced by heavy calibrators at different positions of the compound to be tested; 4) The method according to this patent requires the corresponding synthesis of steroid hormones or other analogues of at least 60 heavy isotopes, the synthesis of such large heavy isotope steroid hormones and analogues is very costly and expensive, requiring only one project of at least several million investments, which is uneconomical and irrational for the analytical work.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a detection method for quickly quantifying a target analyte in a sample in real time by introducing a series of different isotope labels, which can realize internal standard 2-point correction, internal standard multi-point correction or an internal standard curve.

The purpose of the invention is realized by the following technical scheme:

the invention provides a detection method for rapid real-time quantification of a target analyte in a sample by introducing a series of different isotope labels, which comprises the following steps:

connecting different isotope derivative reagents a with target analytes with known series of concentrations to serve as internal standard substances, connecting the target analytes to be detected in the samples to be detected with isotope derivative reagents b, adding the internal standard substances into the samples to be detected, performing mass spectrometry analysis to obtain signals of the internal standard substances and signals of the target analytes to be detected, and then calculating the concentration of the target analytes to be detected through the signals of the internal standard substances;

the known series of concentrations of target analyte comprises more than 2 different concentrations of target analyte, each concentration of target analyte being linked to an isotopically-derivatized reagent a.

According to the invention, different isotope derivative reagents a are connected to target analytes with different concentrations to serve as internal standard substances, for example, when two target analytes with known concentrations are adopted, the target analyte with a first concentration is connected with one isotope derivative reagent a1, the target analyte with a second concentration is connected with another isotope derivative reagent a2 (if the target analytes with known concentrations are more than 2, the process is repeated), the two internal standard substances formed in the process are used as follower internal standard substances and are mixed with a sample to be detected (wherein the target analyte to be detected is connected with another isotope derivative reagent b), mass spectrometry is carried out together, a standard curve (the concentration is an abscissa and the peak area is an ordinate) is drawn according to signals of the target analytes with the first concentration and the second concentration, and then the concentration values of the target analytes are obtained through calculation according to the signals of the target analytes to be detected.

The detection method further comprises the step of correcting by adopting a conventional correction method.

Preferably, the target analytes of the known series of concentrations are pure target analyte solutions of the known series of concentrations or mixed sample solutions containing target analytes of the known series of concentrations.

Preferably, the concentration range of the target analyte of the known series of concentrations should cover the concentration of the target analyte to be measured.

Preferably, the different isotope derivative reagents a and b are each selected from one of a light isotope derivative reagent, a heavy isotope derivative reagent, a different fluorescence labeling reagent, a different luminescence labeling reagent, and a different chemical group reaction reagent, and the specific compounds used in the isotope derivative reagents a and b are different from each other. The purpose of using different isotope derivative reagents in the invention is to distinguish target analytes with different concentrations from target analytes to be detected, and therefore, in principle, the isotope derivative reagents are different from each other and do not need to be particularly limited.

Preferably, the target analyte includes any one or more types of compounds having a carbonyl group, compounds having a carboxyl group, compounds having an amine group, compounds having a hydroxyl group or an alcohol group or phenols, peptides and proteins, vitamins, lipids such as fats, cholesterol and derivatives thereof, steroids and derivatives, and industrial polymers.

Preferably, the target analytes include any one or more types of steroid hormones, fatty acids, organic acids, amino acids and amino acid metabolites, phosphatidylethanolamine, carbohydrates, thiols, oxidized thiols, peptides and proteins, vitamins, fats, cholesterol and derivatives thereof, steroids and derivatives thereof;

when the target analytes are of various types, different isotope derivative reagents a and b are correspondingly connected to each type of target analytes.

For example, when the target analytes are a plurality of compounds of the same type, the isotope derivative reagents a and b can react with a series of target analytes, so that when a plurality of target analytes of the same type are simultaneously measured, only one set of isotope derivative reagents a and b are needed to be respectively introduced into different target analytes, and then the simultaneous detection of the plurality of target analytes of the same type can be realized at one time.

When the target analytes are a plurality of different types of compounds, each type of compound adopts a set of isotope derivatization reagents a and b, and is respectively introduced into the different types of target analytes, so that the simultaneous detection of the plurality of different types of target analytes at one time can be realized.

Preferably, the mass spectrometry employs a mass spectrum selected from the group consisting of quadrupole mass spectrometers, high resolution mass spectrometers; the detection mode of the mass spectrum is selected from any one of a full scan mode, an ion detection mode, a parent ion detection mode, a multi-reaction detection mode, a neutral loss scan, a data-dependent scan mode and a data-independent scan mode.

Preferably, the mass spectrometry analysis further comprises a step of separation by chromatography; the chromatography is selected from any one of liquid chromatography, gas chromatography, capillary electrophoresis, affinity chromatography, supercritical fluid chromatography, and ion mobility.

Preferably, the mass spectrometry analysis further comprises a step of pretreating the sample to be detected; the pretreatment method is any one selected from solid phase extraction, solid-liquid extraction, liquid-liquid extraction, protein precipitation, direct dilution, solvent extraction, immunoaffinity enrichment, salting out, concentration and masking.

Preferably, when the method further comprises a step of separation by chromatography, the pretreatment step precedes the step of separation by chromatography.

Preferably, the sample to be tested comprises any one of a biological sample, an environmental sample, a food sample, a synthetic sample, a drug sample and a chemical sample; the biological sample comprises any one of plasma, serum, whole blood, urine, tissue, cerebrospinal fluid, sweat, saliva, hair, and skin.

The invention also provides a product for the method for detecting the target analyte in the sample in a rapid and real-time manner.

The invention also provides a device for the detection method for rapidly quantifying the target analyte in the sample in real time by introducing the series of different isotope labels, which comprises a device for introducing the series of different heavy-label isotopes, a sample pretreatment device, a sample storage device and a conveying device, wherein the sample is online or offline.

The invention also provides a detection kit for rapidly quantifying the target analyte in the sample in real time based on the detection method, wherein the kit comprises different isotope derivative reagents a, isotope derivative reagents b, a blank matrix, a series of pure target analytes with known concentrations or a series of mixed samples containing the target analytes with known concentrations and a quality control product.

The invention realizes that specific different isotope labels are introduced on target analytes with different known concentrations by designing a series of (usually 2-3, but not limited to) isotope derivative reagents, designing a series of known concentrations of target analytes (substrates), and connecting different isotope derivative reagents through the target analytes with known concentrations to form a series of internal standards marked by different isotopes; in the experimental process, a series of isotope derivative reagents with known properties and structures which are extremely similar but have different structures are connected with target analytes with different known concentrations to form an internal standard as a reference substance or form a standard curve, and the target analytes in unknown samples to be detected are quantified, so that the purpose of following the internal standard reference substance or the internal standard curve with known concentrations in the same sample is realized, an external standard curve is not needed, the analysis speed is greatly improved, and the purpose of real-time detection is realized; and an internal standard reference substance or an internal standard curve with known concentration is followed in the same sample, and system errors such as instrument response fluctuation, matrix effects of different samples, sample injection errors and the like are corrected to the maximum extent, so that the accuracy of determination is greatly improved.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention realizes that the sample to be detected follows 2 and more than 2 internal standard reference or internal standard curves in real time, 1) an external standard regression standard curve is not required to be prepared before the sample to be detected is detected every time, so that a large amount of time is saved, 2) the problem that the external standard curve of the self needs to be prepared every day when various different items are crossed is solved, so that the real-time detection is realized, and 3) the problem that the external standard curve cannot accurately correct the sample to be detected with longer time interval because the internal standard follows the standard curve in the sample to be detected is solved; 4) because 2 and more than 2 internal standard reference or standard curves follow in the sample to be detected, the real-time correction of the matrix is realized, and the influence on the determination result due to the matrix difference among different samples is avoided;

2. the invention introduces different isotopes into target analytes by adopting a series of different isotope derivative reagents and adding a series of different isotopes on the derivative reagents, has simple and convenient operation, solves the problem that each target analyte needs to purchase or synthesize heavy-label isotope internal standards when a plurality of target analytes are measured, greatly reduces the analysis cost, can react with a series of target analytes, can ensure that a plurality of compounds are quickly connected with the isotopes at one time, has high efficiency and low cost, and solves the problems of difficulty and high cost of obtaining the isotope internal standards by a plurality of compounds.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a graph of three heavy-gauge isotope derivatization reagents used in example 1 of the present invention; wherein: c13 or N15; d is H2;

FIG. 2 is the structural formula of aldosterone in example 1 of the present invention;

FIG. 3 is the structural formula of aldosterone itself heavy isotope aldosterone D-8 in example 1 of the present invention;

FIG. 4 is a specific synthetic route for three heavy-label isotope derivatization reagents used in example 1 of the present invention; wherein: c13 or N15; d is H2;

FIG. 5 is a reaction scheme showing the derivatization of aldosterone with three heavy-label isotope derivatization reagents according to the invention in example 1;

FIG. 6 is a reaction scheme of derivatization of aldosterone's own heavy-label isotope with three heavy-label isotope derivatization reagents in accordance with example 1 of the present invention;

FIG. 7 is a reaction scheme of derivatization of aldosterone with a light standard derivatizing agent in example 1 of the present invention;

FIG. 8 is a reaction scheme of derivatization of a heavy isotope compound of aldosterone itself with a light label derivatizing agent in accordance with example 1 of the present invention;

FIG. 9-1 is a schematic illustration of the operation of step one therein;

FIG. 9-2 is a schematic diagram of the operation and extraction correction principle of step two;

FIG. 10-1 is a schematic diagram comparing an internal standard curve method and a conventional external standard curve method according to an embodiment of the present invention;

FIG. 10-2 is a graph showing correlation analysis results of comparison between the measurement results of the same sample by the internal standard curve method according to the embodiment of the present invention and the measurement results by the conventional external standard curve method;

FIG. 11 shows the structural formulas and mass-to-charge ratios of parent ions and daughter ions detected by mass spectrometry, taking aldosterone light standard product A + P as an example, in example 1 of the present invention;

fig. 12 is a schematic diagram of an internal standard of a series of heavy-mark isotopes (the heavy-mark isotopes of the compound to be detected are only used for correcting extraction efficiency, extraction errors, etc., and are not labeled in the schematic diagram);

FIG. 13 is a schematic view of a process for testing a sample;

fig. 14 is a schematic diagram of calculating the concentration of a compound to be detected in a sample to be detected by using an internal standard curve, which can be an internal standard 2 point or an internal standard multiple point or an internal standard curve, by using a series of different isotope labels introduced;

FIG. 15 is a schematic representation of the reaction scheme for introducing different isotopic labels into the carbonyl-containing compound of example 2;

FIG. 16 illustrates the general structural formulae of steroid and glucocorticoid (for example only and not for limitation, the steroid and glucocorticoid in this figure);

FIG. 17 shows the reaction sequence for the introduction of testosterone into different isotopic labels.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

The present invention will be further illustrated by the following specific example 1 of measuring aldosterone, which is one of carbonyl group-containing compounds, wherein the present invention is not limited to this example, the heavy-label isotopic derivatization reagents mentioned in the examples are suitable for carbonyl group-containing compounds, and the present invention is not limited to this derivatization reagent, which is suitable for carbonyl group-containing compounds.

The invention focuses on explaining that a series of different heavy standard isotope labels are rapidly and conveniently introduced, and a 2-point or multi-point or standard curve which can be used for internal standard calibration is prepared according to different concentrations of a substrate (aldosterone in the embodiment 1) to realize real-time rapid internal standard calibration of a sample to be detected.

The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.

Example 1

The invention focuses on explaining that a series of different heavy standard isotope labels are rapidly and conveniently introduced, and a 2-point or multi-point or standard curve which can be used for internal standard calibration is prepared according to different concentrations of a substrate (in example 1, aldosterone is not added), so that the real-time rapid internal standard calibration of a sample to be treated is realized.

In this embodiment, 3 different heavy-label isotope derivative tags are introduced as an example, but not limited to 3, which may be 1,2,3,4,5,6,7,8,9,10, etc. In this embodiment, 3 different heavy-label isotope derivative reagents are added for example, but not limited to, only 3 different heavy-label isotope derivative reagents are added, and 1,2,3,4,5,6,7,8,9,10, etc. or more heavy-label isotope derivative reagents may be added to generate more series of heavy-label isotope derivative products, such as 1,2,3,4,5,6,7,8,9,10, etc.

Step one, introducing heavy-mark isotopes by using a series of different heavy-mark isotope derivatization reagents to prepare a heavy-mark isotope internal standard solution:

1) in 3 aldosterone standard solutions (aldosterone is named as a1, a2, A3, and the concentration is 5, 20, 100pg/ml in the following figures and calculation formulas) with known concentration (3 known concentrations are to cover the concentration of aldosterone in the sample to be tested, the concentration is respectively 5, 20, 100 pg/ml), a heavy-label isotope solution of aldosterone with fixed concentration (the heavy-label isotope compound of aldosterone is subjected to heavy-label substitution on the structure of aldosterone compound, so as to correct extraction efficiency, extraction error and sampling error of aldosterone in the sample pretreatment process, different from the heavy-label isotope internal standard in the present invention used for accurate quantification of internal standard of aldosterone, the heavy-label isotope compound of aldosterone is named as a', the heavy-label isotope compound of aldosterone compound of the present embodiment is adopted as aldosterone-D8, the structural formula is shown in figure 3).

2) Adding 10 microliter of 3 different heavy isotope derivative reagents (the structures of the heavy isotope derivative reagent 1, the heavy isotope derivative reagent 2, the heavy isotope derivative reagent 3 and the 3 heavy isotope derivative reagents are shown in figure 1 and are respectively named as P ', P ' ', P ' ' ') into the solution A1, A2 and A3 treated in the step 1), standing for 10 minutes to complete the derivatization reaction, introducing different heavy isotope group labels through the series of heavy isotope derivative reagents to generate 3 different aldosterone heavy isotope labeling products, namely A1+ P ', A2+ P ' ', A3+ P ' ' ', and 3 aldosterone self heavy isotope labeling products, namely A ' + P ', A ' + P ' ', A ' + P ' ' '. The 3 solutions after reaction are mixed by vortex to prepare a series of mixtures of heavy-mark isotope compounds (hereinafter referred to as heavy-mark isotope internal standard solutions) for later use. FIG. 9-1 shows a schematic diagram of the operation step one.

The synthesis system of the invention has the advantages of easy realization of the heavy-standard isotope derivative reagent, simple raw materials required for synthesis, easy purchase, simple synthesis method and short route, adopts a simple method (only 2 steps of reaction), and can realize the rapid preparation and synthesis of the heavy-standard isotope derivative reagent by adopting commercial raw materials. The starting materials, synthetic pathways and products of the synthesis of the 1-3 heavy-label isotopically-derived reagents are shown in FIG. 4, with the products being designated P ', P ", P' '', respectively.

Fig. 5 shows a reaction formula of generating 3 different heavy-label isotope products by introducing 3 different heavy-label isotope derivatization reagents, wherein the reaction formula is a reaction formula, and the reaction product has a structural formula, and the reaction products are respectively named as a1+ P ', a2+ P ", and A3+ P' '' (1, 2,3 indicate different concentrations, a1, a2, A3, and 3 known concentrations are to cover the concentration of aldosterone in a sample to be tested).

Fig. 6 shows the reaction formula of the reaction generated by introducing 3 different heavy isotope derivatization reagents to generate a series of heavy isotope products of 3 different aldosterone self heavy isotopes, the reaction products have structural formulas, and the reaction products are respectively named as a ' + P ', a ' + P ", a ' + P ' ″ (the aldosterone self heavy isotope solution has a fixed concentration, which in this example is 1 ng/ml).

Note that: the heavy-mark isotope internal standard solution prepared in the step does not need to be prepared in each detection, can be frozen or stored at ultralow temperature after being subpackaged, can be used in each determination, and greatly reduces the workload.

And step two, measuring true samples to be measured, such as biological samples, environmental samples, food samples, synthetic samples, medicine samples, chemical samples, clinical chemical samples, forensic samples, pharmacological samples, agricultural samples and the like, wherein the biological samples include but are not limited to plasma, serum, whole blood, urine, tissues, cerebrospinal fluid, sweat, saliva, hair and skin samples. In the following, plasma is taken as an example, but the present invention is not limited to plasma.

1) Extraction and purification of aldosterone from plasma: extracting aldosterone by conventional solid phase extraction method. 200. mu.l of plasma was taken and placed in a2 ml Eppendorf tube, 20. mu.l of the heavy-isotope compound A' of the aldosterone compound itself, 200. mu.l of methanol, 0.2M zinc sulfate solution, 450. mu.l of 0.05% phosphoric acid solution were added, mixed, vortexed for 2 minutes, and centrifuged (centrifugal force 1000g, 5 minutes, 4 ℃). Collecting supernatant to a solid phase extraction column, washing off the matrix with 20% methanol solution containing 5% formic acid, discarding, eluting with 50 μ l methanol, and collecting the eluate.

2) Light-label derivatization of aldosterone in plasma: adding 5 microliters of acetic acid and 10 microliters of derivatization reagent (common light standard derivatization reagent, named as P, with the concentration of 1 mg/ml, and the reaction formula shown in figure 7) into the eluate collected in the step 1), standing for 10 minutes to complete the derivatization reaction, and naming the product as A + P. The heavy isotope compound A 'of the aldosterone compound itself is also subjected to derivatization simultaneously, the reaction formula is shown in figure 8, and the product is named A' + P.

3) Adding the series of heavy-mark isotope internal standard solutions (mixed solution of A1+ P ', A2+ P' ', A3+ P' '', A '+ P', A '+ P' ', A' + P '' ') prepared in the step one into the products A + P and A' + P obtained in the step 2), and uniformly mixing by vortex.

And thirdly, blowing the mixture by using nitrogen, redissolving the mixture by using 50 microliter of 50% methanol, and carrying out liquid chromatography tandem mass spectrometry on 20 microliter of sample.

Step four, calculating the content of aldosterone in the sample to be detected:

taking the known concentrations of a1+ P ', a2+ P ", A3+ P'" as abscissa x, the peak area ratio of a1+ P '/a' + P ', a2+ P'/a '+ P ", A3+ P'/a '+ P'" as ordinate y, the standard curve is usually a linear standard curve (in particular cases also a non-linear standard curve), and the regression equation is y = ax + b.

Sample to be tested aldosterone concentration = ((peak area of A + P/peak area of A' + P in sample to be tested) -b)/a

FIG. 9-2 shows the operation steps of step two and the principle of extraction correction.

When the sample obtained after the pretreatment method is subjected to the liquid chromatography tandem mass spectrometry (i.e., the third step in the operation method), the liquid chromatography parameters and the analysis conditions are as follows:

mobile phase A of water

Mobile phase B methanol

Chromatographic column BEH C8, 100mm x 2.1mm, 1.7 μm

Column oven temperature 35 ℃

Sample injection volume of 20 microliter

The flow rate of the mobile phase is 0.40mL/min

The mobile phase gradients are shown in table 1.

TABLE 1

Taking the structure of parent ion and daughter ion of A + P as an example, the structure and mass-to-charge ratio of parent ion and daughter ion in mass spectrum of A + P, A '+ P, A + P', A + P '', A + P '' ', A' + P ', A' + P '', A '+ P' '' will be described, as shown in FIG. 11. The information of A + P, A '+ P, A1+ P', A2+ P '', A3+ P '' ', A' + P ', A' + P '', A '+ P' '' on the mass spectrometric detection is tabulated in Table 2.

The mass spectrum detection parameters and analysis conditions are as follows:

ionization mode ES +

Capillary Voltage (kV) 2.40

The cone voltage (V) is given in Table 2 below

Ion source temperature (C) 150

Desolventizing temperature (C) 600

Taper hole air flow rate (L/Hr) 200

Desolventizing gas flow rate (L/Hr) 1000

MSMS Collision Voltage see Table 2 below

TABLE 2 aldosterone liquid chromatography mass spectrometry Mass Spectrometry parameters and analysis conditions

Interscan Scan Delay (secs):0.02

Interscan Channel Delay (secs):0.01

Other response factors that need to be used:

1) the formula for calculating the response factor of the mass spectrum to a + P, a ' + P, a1+ P ', a2+ P ' ', A3+ P ' ' ', a ' + P ' ' ' (the same concentration of compound, without extraction, was measured using a standard solution, calculated using peak area):

mass response factor = mass response (usually peak area) of compound 1 at equal concentration/mass response of compound 2 at equal concentration

Mass Spectrometry Response abbreviated MRf (MS Response factor), Peak Area (Area)

Namely:

MRf (A+P')=Area(A+P') /Area(A+P)

MRf (A+P'')=Area(A+P'') /Area(A+P)

MRf (A+P''')=Area(A+P''') /Area(A+P)

MRf (A'+P)=Area(A'+P) /Area(A+P)

MRf (A'+P')=Area(A'+P') /Area(A+P)

MRf (A'+P'')=Area(A'+P'') /Area(A+P)

MRf (A'+P''')=Area(A'+P''') /Area(A+P)

calculating relative mass spectrum response factors of A '+ P, A1+ P', A2+ P '', A3+ P '' ', A' + P ', A' + P '', A '+ P' '' by dividing the peak areas of A '+ P, A3+ P' '', A '+ P', A '+ P' ', A' 1+ P ', A2+ P' ', A3+ P' '', A '+ P' '', respectively, with the mass spectrum response of A + P as 100%; the mass spectrum response factors of the above compounds in this example are all 1.

2) And (3) investigating derivatization efficiency: this example 1 gave 100% derivatization efficiency, and other examples, if derivatization efficiency needs to be calculated, can detect the concentration of unreacted compound after the derivatization reaction, as follows:

a. derivatization efficiency = 100% - (concentration of unreacted compound after derivatization/total concentration of compound before derivatization)

b. Or the derivatization efficiency was investigated using standard addition methods.

3) Correction Factor for Extraction Efficiency Correction Factor (EECF), Peak Area (Area)

Calculation of each correction factor (peak area measurement after same concentration same extraction method):

EECF(A+P')=Area(A+P') /Area(A+P)

EECF(A+P'')=Area(A+P'') /Area(A+P)

EECF (A+P''')=Area(A+P''') /Area(A+P)

EECF(A'+P)=Area(A'+P) /Area(A+P)

EECF（A'+P')=Area(A'+P') /Area(A+P)

EECF(A'+P'')=Area(A'+P'') /Area(A+P)

the correction factors for the extraction efficiency of the above compounds in this example were all 1.

In this example, the content of aldosterone in 3 plasma samples was determined, and the content of the same sample was calculated simultaneously by using the conventional external standard curve method, and the determination results are shown in table 3. FIG. 10-1 shows a schematic comparison of an internal standard curve method and a conventional external standard curve method according to an embodiment of the present invention. For the same sample, the correlation analysis results obtained by comparing the measurement results of the internal standard curve method and the traditional external standard curve method in the embodiment of the invention are shown in fig. 10-2, and the results show that the results of the two methods are consistent and the correlation coefficient is 0.998.

TABLE 3 measurement results of examples and conventional external standard method

The above examples illustrate standard curves using aldosterone standards, and a series of samples of known aldosterone concentrations can also be used to prepare mixed internal standard solutions. A substrate (e.g., a biological sample substrate, plasma, serum, tissue, etc., a food sample substrate, pork, fruit, vegetable, etc., and other substrates) corresponding to the sample to be tested is taken and pre-treated corresponding to the sample to be tested, as in example 1, to prepare a heavy-label isotope-mixed internal standard solution.

To summarize: the embodiment provides a detection method for rapidly quantifying a target analyte in a sample in real time by introducing a series of different heavy-label isotope labels, wherein the series of heavy-label isotope labels are used for distinguishing from each other, different heavy-label isotope internal labels with a series of concentrations are prepared based on the series of concentrations of a compound (substrate) to be detected, and the sample to be detected is added as the internal label, so that 2-point correction, multi-point correction or an internal label standard curve of the internal label can be realized;

by the method, 2 and more than 2 heavy-mark isotope internal standard calibration substances are contained in a sample to be detected, the sample to be detected is analyzed by adopting a chromatography-mass spectrometry method, signals of light-mark isotopes of a compound to be detected in the sample to be detected and signals of different heavy-mark isotopes with 2 and more than 2 known concentrations can be simultaneously obtained, and the concentration of the compound to be detected is internally calculated by using a series of heavy-mark isotope signals with known concentrations;

for clarity of presentation, a brief schematic of the key steps is given.

Fig. 12 is a schematic diagram of an internal standard of a series of heavy-mark isotopes (the heavy-mark isotopes of the compound to be detected are only used for correcting extraction efficiency, extraction errors, etc., and are not labeled in the schematic diagram);

FIG. 13 is a schematic view of a process for testing a sample;

fig. 14 is a schematic diagram of calculating the concentration of a compound to be detected in a sample to be detected by using an internal standard curve, which can be an internal standard 2-point or internal standard multi-point or internal standard curve, by using a series of heavy standard isotopes introduced.

It can be seen that, although example 1 only measures the aldosterone content, those skilled in the art can deduce that the method is also applicable to other target analytes to be measured according to the measurement method and principle of the present invention.

Example 2

The same method as in example 1 can be used for simultaneous detection of multiple compounds because the derivatization reaction occurs at specific chemical groups, as long as the compounds with the specific chemical groups react. The invention is further illustrated below in connection with a series of steroid hormone and glucocorticoid compounds.

Many of the steroid hormone and glucocorticoid compounds contain carbonyl groups, which can be reacted as in example 1, and both steroid hormones and glucocorticoids containing carbonyl groups can be rapidly linked with a series of isotopic labels, which can be analyzed by the above methods. The general reaction formula of the compound containing carbonyl is shown in figure 15, and the compound containing carbonyl and heavy-mark isotope derivatization reagents P ', P' '', P respectively carry out derivatization reaction to generate corresponding derivatization products. Steroid hormones and glucocorticoids are very common compounds and usually need to be measured simultaneously, typically 5 to hundreds of steroid hormones and glucocorticoids need to be measured simultaneously.

The general structural formulae of steroid hormones and glucocorticoids are illustrated in fig. 16 (for example only and not limited to steroid hormones and glucocorticoids in this figure). For example, in this embodiment, testosterone, 11-deoxycorticosterone, 11-deoxycorticosterol, corticosterone, dihydrotestosterone, progesterone, cortisone, and cortisol in the sample to be tested are simultaneously quantified in a rapid and real-time manner. Taking testosterone as an example, as shown in fig. 17, testosterone is subjected to derivatization reaction with different re-labeling derivatization reagents P ', P ", P'", P by the method of example 1 to generate corresponding derivatization products, so as to form B + P, B1+ P ', B2+ P ", B3+ P'" (where B is testosterone in a sample to be tested, and B1, B2, and B3 are testosterone with different known concentrations). And by analogy, derivative products are formed, wherein the derivative products are obtained by performing derivatization reaction on the testosterone, the 11-deoxycorticosterone, the 11-deoxycorticosterol, the corticosterone, the dihydrotestosterone, the progesterone, the cortisone and the cortisol respectively with different heavy standard derivatization reagents P ', P' '', and P.

The concentrations of testosterone, 11-deoxycorticosterone, 11-deoxycorticosterol, corticosterone, dihydrotestosterone, progesterone, cortisone, and cortisol can be obtained simultaneously in the same manner as in example 1. Simultaneous quantitative determination of multiple target analytes is achieved.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.