CN113252818B - Method for quantifying and evaluating compounds of same series by adopting reference sample - Google Patents
Method for quantifying and evaluating compounds of same series by adopting reference sample Download PDFInfo
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- G01N30/02—Column chromatography
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
Abstract
The invention provides a method for quantifying and evaluating compounds of the same series by adopting a reference sample; preparing a reference sample, wherein the matrix of the reference sample is the same as that of the sample to be detected, and all target analytes in each sample to be detected are covered; respectively adding isotope labels b into samples to be detected, and adding isotope labels a into a reference sample; carrying out mass spectrometry after mixing to obtain a series of internal standard substances which are in one-to-one correspondence with the target analytes and peak area signals of the target analytes; meanwhile, all series of target analytes and corresponding internal standard substance signals are extracted through specific mass spectrum signals generated by deriving reagent labels, a series of compounds in a complex sample form one-to-one corresponding standard data sets, and a sample to be detected is evaluated through an algorithm, mathematical transformation or a model. The method solves the problems that the determination process of the complex sample series compound reference interval is complex, and series such as standard products, internal standards, matrix interference and the like are needed, and has the advantages of high sensitivity, high reliability and the like.
Description
Technical Field
The invention belongs to the technical field of sample detection, relates to a method for quantifying and evaluating compounds of the same series by using a reference sample, in particular to a method for quantifying and evaluating all compounds of the same series in a complex sample by using a reference sample, which is used for realizing real-time rapid quantification and evaluation of known and unknown compounds of the same series in the sample.
Background
In samples, especially biological samples, there are usually a large number of compounds with similar characteristics, such as organic acids, amino acids, fatty acids, lipids, etc., which are self-organized and numerous, and although the molecular formulas and structural formulas of some compounds are known according to earlier scientific researches, more unknown compounds are discovered with the progress of science, and these compounds also have biological significance and play an important role for life bodies.
For sample analysis, it is becoming more and more challenging to characterize a sample feature and property with only a limited number of compounds, and as the understanding of life becomes more and more, to characterize a sample comprehensively by analytical means, the determination of all related compounds (both known and unknown) in a sample becomes a necessary trend and a requirement of the industry for analysis.
However, the following challenges and problems exist for analyzing all related compounds: 1) Typically, a series of compounds is present in a sample in large numbers, each of which is at least several tens, and often hundreds of compounds. To determine the content of these compounds, corresponding standards of all these compounds are generally required. For known compounds, purchasing all standards is itself a challenge, not all compounds are necessarily available as commercial standards, and usually only common compounds are available for commercial companies to develop standards for sale, and further, standards are expensive, purchasing all standards usually requires a large amount of money, and standards usually have a shelf life, usually within 2 years; 2) in order to accurately quantify, except for the standard product of the compound, it is very important to need an isotope internal standard substance of each compound, because the structure of each target compound is different, the corresponding isotope internal standard substance is also different, the synthesis cost of the isotope internal standard substance is far higher than that of the common compound standard product, therefore, the price is higher than that of the compound, the synthesis is difficult, and the purchase of the isotope internal standard substance corresponding to each compound is very difficult and generally impossible to realize; 3) In order to eliminate the influence of the matrix effect, the content of the sample to be detected is calculated by adopting the standard substance of all compounds with the same matrix, which is accurate and reliable, and the content of the sample is calculated by adopting the standard substance without the matrix or the standard substance with different matrixes, which can cause larger deviation on the result; 4) in addition to the problems with known compounds above, it is more difficult to quantify compounds that do not have standards or are not known under consideration in this family of compounds. Since in this case firstly the unknown compound in the sample needs to be identified and secondly this portion of the compound has no standard for comparison and assay, not to mention nor any isotopic internal standard. In this case, not only cannot these compounds be measured, but also significant amounts of these compounds can be easily overlooked, resulting in incomplete sample information and a leaf-shadow. 5) In addition, it is important to determine the Reference interval for each compound in Clinical biological sample analysis, and the industry generally complies with the CLSI C28-A or EP28-A3C De fi ning, observing, and Verifying references in the Clinical Laboratory, applied guided guiding-Third Edition to establish the Reference interval for each marker, but for a series of compounds in a biological sample, such as hundreds of compounds, the work load is very enormous, and in the case of simultaneous quantification of such many markers, it is more valuable to change the compounds rather than to absolute content of the compounds, and therefore, the inventors believe that it is actually not necessary to establish the Reference interval for each compound, and unnecessarily increase the work load.
Taking the compound containing a carboxyl functional group as an example, in a living body, the compound containing a carboxyl functional group is various, and includes organic acids (such as methylmalonic acid, quinolinic acid, homovanillic acid, urovanillylmandelic acid, and the like), amino acids, and fatty acids (such as linoleic acid, arachidonic acid, and the like). Organic acids, amino acids, fatty acids, and the like are important metabolic components in a living body, and metabolic disorders of the components are often accompanied by pathological changes of the living body. Therefore, the compounds containing carboxyl functional groups are biomarkers of a plurality of diseases, and comprehensive analysis of the components is very helpful for improving understanding of the occurrence and development mechanism of the diseases, so that the compounds can be developed into a new means for preventing and treating the diseases. The detection of these components is therefore of crucial importance for the assessment of health and disease status. However, because of the above-mentioned problems, all of them exist, so that it has not been possible to evaluate the contents of the series of organic acids on a large scale in clinical practice and to guide clinical diagnosis and intervention measures to be carried out as soon as possible.
Disclosure of Invention
The invention aims to provide a method for quantifying and evaluating compounds of the same series by adopting a reference sample, which comprises the steps of preparing the reference sample, adding an isotope label b into a sample to be tested, and adding an isotope label a into the reference sample; carrying out mass spectrometry after mixing to obtain a series of internal standard substance signals corresponding to the target analytes one by one and a series of peak area signals of the target analytes; all series of target analytes and corresponding series of internal standard substance peak area signals are extracted through specific mass spectrum signals generated by isotope derivative reagent labels, series of compounds in a complex sample form one-to-one corresponding standard data sets, and a sample to be tested can be evaluated through an algorithm, mathematical transformation or a model so as to realize quick identification, extraction, real-time quantification and evaluation of series of known and unknown target analytes in the sample. Wherein the peak area is the area under the curve of the mass spectral signal of the compound relative to a baseline. As shown in fig. 7, each compound in fig. 7 shows a signal curve with a gaussian distribution-like distribution that rises first and then falls on the mass spectrum, and the area under the curve formed by the signal curve to the base line at the bottom is the peak area of each compound. The area under the curve can be read by the data processing software of the mass spectrometer and given a specific value, i.e. the peak area of the compound. A particular mass spectral signal refers to a neutral loss acquisition mode or a daughter ion acquisition mode. The standard data set refers to a data set in which the peak areas of a series of target compounds in a series of samples to be measured are respectively calculated for the peak areas of a series of target compounds (internal standard substances) in a reference sample, and each target compound is listed by taking the number of the samples to be measured as a row, and because the data set is corrected by the internal standard of each compound (internal standard) in the reference sample, the errors of the system and the influence of the matrix are removed, so that the data set is called as a standard data set.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the present invention relates to a method for quantifying and evaluating a series of compounds in a test sample using a reference sample, said method comprising the steps of:
s1, preparing a standard sample which is the same as the matrix of the sample to be detected and covers all target analytes in each sample to be detected;
s2, adding an isotope label b into the sample to be detected, and adding an isotope label a into the reference sample; performing mass spectrometry after mixing; and identifying and extracting all series of target analytes and series of internal standard substances which are in one-to-one correspondence with the target analytes by adopting a neutral loss acquisition mode or a sub-ion acquisition mode to obtain peak area signals of the series of target analytes and the corresponding series of internal standard substances, and calculating to obtain the relative content of the series of target analytes of the sample to be detected relative to the corresponding internal standard substances in the reference sample.
In one embodiment of the present invention, when the method is used for simultaneously detecting a plurality of samples to be detected, in step S2, the reference samples are respectively added to each sample to be detected and mixed, and then mass spectrometry is performed. For example, for a second large class of target compounds, a label a 'and a label b' are respectively added to the same reference sample and the same sample to be detected, and the label a 'and the label b' are characterized in that the main structures are the same, and only the heavy and light isotope states of individual elements are different. Similarly, for the third major class of target compounds, a label a ″ and a label b ″ are added to the same reference sample and the same sample to be tested, respectively, and the label a ″ and the label b ″ are characterized by the same main structure and different states of heavy and light isotopes of only individual elements. Pairwise pairs of tags a and b (first set), a 'and b' (second set), a '' and b '' (third set), each set of tags being structurally different from each other, corresponding different tags being generated for different types of compounds, and so on for further types of compounds.
As an embodiment of the present invention, step S2 further includes a step of calculating the relative content or relative content ratio of the corresponding series of target analytes through the peak area signals of the series of internal standard substances.
The invention is directed to all series of compounds in the sample to be tested, each compound being given a corresponding internal standard compound one by one, whether a compound is known or not.
According to the invention, the used specific isotope label a or isotope label b is utilized, so that the one-to-one corresponding series of compounds in the sample to be detected and the reference sample are automatically extracted.
According to the invention, the isotope label a or the isotope label b is utilized to extract a series of compounds corresponding to each other in the sample to be detected and the reference sample, so that a data matrix or a data set can be conveniently formed for subsequent content calculation, data transformation, model construction, judgment and the like.
In the invention, the same series of target analytes in a sample to be detected are respectively named as x '1, x '2 and … … x ' n; the corresponding series of internal standards in the reference sample were designated x1, x2, … … xm, respectively; m > n, and x1 is the same compound as x '1, x2 is the same compound as x '2, … … xn is the same compound as x ' n; after introducing the isotope label b into the same series of target analytes in the sample to be detected, marking as x '1+ b, x'2+ b, x '3+ b, … … and x' n + b; the corresponding series of internal standards in the reference sample are labeled x1+ a, x2+ a, x3+ a … … xm + a, m > n after introduction of the isotopic label a.
As an embodiment of the present invention, a ratio of peak areas of each target analyte in a sample to be measured to each corresponding internal standard substance in a reference sample is calculated in a one-to-one correspondence manner, and the calculation formula is as follows:
Areax'1+b/Areax1+a
Areax'2+b/Areax2+a
Areax'3+b/Areax3+a
……
Areax'n+b/Areaxn+a
(compounds not contained in the sample to be tested, i.e., m-n compounds in a ratio of 0/xm + a =0 (labeled as no compounds contained in the sample to be tested); known compounds and unknown compounds are contained in x1 and x2 … … xm.)
The content of each target analyte in the sample to be detected relative to the corresponding internal standard substance in the reference sample is calculated, and the reference value is 1.0.
As an embodiment of the invention, the ratio of the relative content of each compound and other compounds in the sample to be detected and the series of target analytes is respectively calculated in a one-to-one correspondence manner; the relative content is the content of each compound of the sample to be detected relative to the corresponding internal standard substance in the reference sample; the calculation formula comprises:
calculating the ratio of the relative contents of the target analytes x '1, x '2 and … … x ' n in the sample to be detected:
（Areax'1+b/Areax1+a）/( Areax'2+b/Areax2+a)
（Areax'1+b/Areax1+a）/(Areax'3+b/Areax3+a)
（Areax'1+b/Areax1+a）/(Areax'4+b/Areax4+a)
……
（Areax'1+b/Areax1+a）/(Areax'n+b/Areaxn+a)
calculating the ratio of the relative contents of the target analytes x '2 and x '3 and … … x ' n in the sample to be detected:
（ Areax'2+b/Areax2+a）/( Areax'3+b/Areax3+a)
（ Areax'2+b/Areax2+a）/(Areax'4+b/Areax4+a)
……
（ Areax'2+b/Areax2+a）/(Areax'n+b/Areaxn+a)
calculating the ratio of the relative contents of the target analytes x '3 and x '4 and … … x ' n in the sample to be detected:
（Areax'3+b/Areax3+a）/(Areax'4+b/Areax4+a)
……
（Areax'3+b/Areax3+a）/(Areax'n+b/Areaxn+a)；
the target analytes x '4, x'5, x '6 … … x' (n-1) in the sample to be detected are analogized in turn;
(m-n compounds not contained in the sample to be tested in a ratio of 0 (labeled as the sample to be tested does not contain the compound), x1, x2, and … … xm containing known compounds and unknown compounds;)
As an embodiment of the present invention, the sample to be tested is evaluated based on the content of each target analyte in the sample to be tested relative to the corresponding internal standard substance in the reference sample, and/or the ratio of the relative content of each compound and other compounds in the target analytes in the same series of the sample to be tested. The method for evaluating the sample to be tested comprises the steps of neural network, logistic regression, model training, pattern recognition, machine learning and deep learning.
As an embodiment of the invention, the series of compounds includes a known compound and an unknown compound of the same series.
As an embodiment of the present invention, the preparing includes mixing all samples to be tested, or mixing typical conventional samples, in step S1. The typical conventional sample may be an ex vivo sample of a population that meets the requirements of the system.
As one embodiment of the present invention, the target analytes include any one or more types of steroid hormone compounds, fatty acid compounds, organic acid compounds, amino acids and amino acid metabolites, phosphatidylethanolamine compounds, saccharide compounds, thiols, oxidative thiols, peptide and protein compounds, vitamin compounds, fatty compounds, cholesterol and derivatives thereof, steroids and derivatives thereof; when the target analytes are of various types, each type of target analyte is correspondingly connected with a group of isotope labels a and b; different sets of isotopic labels a and b correspond to different types of target analytes.
Preferably, for the same type of target analyte, the selected compounds of the isotope label a and the isotope label b have the same main structure, and only the heavy and light isotope states of the individual elements are different.
Further, the different sets of isotope labels a and b are respectively selected from light-label isotope derivative reagents and heavy-label isotope derivative reagents; or respectively selected from one of different fluorescence labeling reagents, one of different luminescence labeling reagents, or one of different chemical group reaction reagents.
As an embodiment of the present invention, the sample to be tested includes 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.
As an embodiment of the present invention, 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. Wherein the pattern of the neutral loss scan is better at rapidly and specifically extracting the signals of a known and unknown series of target compounds.
As an embodiment of the invention, the mass spectrometry further comprises a separation step by chromatography; the chromatography is selected from any one of liquid chromatography, gas chromatography, capillary electrophoresis, affinity chromatography, supercritical fluid chromatography, and ion mobility.
As an embodiment of the invention, the mass spectrometry further comprises the step of pretreating the sample to be tested; 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.
In one embodiment of the present invention, when the target analyte is a compound containing a carboxyl functional group, the isotopic labels a and b are encoded and constructed based on oxalyl chloride, dimethylaminoethanol and light or heavy isotopic methyl iodide derivatives, respectively.
As an embodiment of the present invention, the compound having a carboxyl functional group is a compound having a carboxyl functional group in a living sample; including organic acids, amino acids, fatty acids. Respectively adopting light isotope (CH) for a sample to be analyzed and a reference sample3I) And heavy isotope (CD)3I) Derivatization; the derivatization reaction comprises acyl chloride activated carboxyl, N-dimethylAminoethanol esterification, and methyl iodide methylation amino reaction marked by light or heavy isotopes. When combined, provide an isotopic internal standard for real-time analysis of all carboxyl-functional compounds in complex biological samples.
The above derivatization reaction is divided into two cases: 1. all samples were individually acyl chloride activated, N-dimethylaminoethanol esterified, light isotope labeled methyl iodide (CH)3I) Performing methylation amido reaction, wherein the samples are to-be-analyzed samples to be detected; 2. mixing all samples, and subjecting the mixed sample to acyl chloride activation, N-dimethylaminoethanol esterification, heavy isotope labeled methyl iodide (CD)3I) The methylated amine groups were reacted, and this sample was the reference sample. As before, the number of carboxyl-functional compounds in the reference sample covers all of the carboxyl-functional compounds in the samples to be tested. Before the derivatization reaction, the method also comprises the step of extracting the compound containing carboxyl functional groups in the biological sample; the extraction method comprises liquid-liquid extraction and solid-phase extraction. The present invention relates to the simultaneous derivatization of all carboxyl functional group-containing compounds extracted from a sample.
The structural formula of the derivative product of the carboxyl functional group-containing compound is as follows:
light isotope derivative product:
，
heavy isotope derivative product:
。
based on the isotope labels a and b constructed by the codes, accurate relative quantitative analysis of all compounds containing carboxyl functional groups in a biological sample can be realized by combining a liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method. Such as the detection mode of neutral loss 59 (NC3H9) using tandem mass spectrometry, to identify the structure of a compound containing a carboxyl functional group. The relative quantification of specific carboxyl functional group containing compounds was performed using tandem mass spectrometry.
In a second aspect, the invention provides a method for constructing an isotope internal standard of a compound containing a carboxyl functional group, which is constructed based on oxalyl chloride, dimethylaminoethanol and light or heavy isotope methyl iodide derivation.
As an embodiment of the present invention, light isotope (CH) is used for a sample to be analyzed and a reference sample, respectively3I) And heavy isotope (CD)3I) And (4) derivation.
As an embodiment of the invention, the derivatization reaction comprises acyl chloride activated carboxyl, N-dimethylaminoethanol esterification and methyl iodide methylated amine reaction marked by light or heavy isotopes. When combined, provide an isotopic internal standard for real-time analysis of all carboxyl-functional compounds in complex biological samples.
In a third aspect, the invention further relates to an application of the construction method of the isotope internal standard of the compound containing the carboxyl functional group in mass spectrometry detection and analysis of the compound containing the carboxyl functional group in a biological sample.
As an embodiment of the invention, the derivative products of the carboxyl-functional compounds are analyzed using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS).
In a fourth aspect, the invention relates to a product for quantifying a series of compounds in a sample to be tested by using the method.
In a fifth aspect, the present invention relates to a device for quantifying a series of compounds in a sample to be tested by using the above method, which comprises a device for introducing a series of different isotope labels to the sample online or offline, a sample pretreatment device, a sample storage device and a conveying device.
In a sixth aspect, the invention relates to a detection kit for quantifying a series of compounds in a sample to be detected by using the method, wherein the kit comprises a plurality of groups of isotope labels a, isotope labels b, a blank matrix, a series of pure target analytes or a series of mixed samples containing the target analytes, a reference sample and a quality control product.
Each set of isotope labels a and b corresponds to one type of target analyte; the different sets of isotopic labels a and b correspond to different specific compounds. For the same type of target analyte, the compounds of the connected isotope label a and the isotope label b have the same main structure, and only the heavy and light isotope states of individual elements are different. And in the mixed sample of the sample to be detected and the reference sample, only the same series of compounds with the same chemical characteristics can be coded with the corresponding group of isotope labels.
The method can be used for quickly identifying, extracting, quantifying in real time and evaluating the known and unknown target analytes of the same series in the sample by preparing the reference sample and connecting the isotope codes on all the target compounds of the same series in the reference sample, and is suitable for detecting a plurality of compounds of the same series in a complex sample. The advantages are that:
1) extracting all series of target analytes and corresponding series of internal standard substance peak area signals (including known and unknown compounds of the same series) through specific mass spectrum signals generated by isotope labels, forming one-to-one corresponding standard data sets for the series of compounds in the complex sample, and evaluating the sample to be detected through an algorithm, mathematical transformation or a model;
2) by connecting isotope codes, the problem that the same series of compounds need to synthesize corresponding different isotope internal standards aiming at different compounds in the determination is solved, the cost is greatly reduced, and the efficiency is improved;
3) the problem that only a small part of compounds with commercial standard products can be measured in the same series of compounds in a complex sample is solved, and the problem of measuring compounds without standard products, even unknown compounds, is solved;
4) the reference sample covers all compounds of the same series in the sample to be detected, and can provide reference control for all compounds of the same series in the sample to be detected;
5) all the compounds of the same series for reference are from a reference sample instead of a standard solution, so that the problem that the content of the compounds of the series to be detected is influenced under different matrixes and systems, so that the detection is inaccurate is solved;
6) by utilizing an isotope encoding mode, the detection sensitivity of all compounds in the same series is improved, and the problems that a plurality of compounds in the same series in a complex sample are low in content and difficult to accurately detect are solved;
7) the isotope coding mode brings the same structural unit for the same series of compounds, and by utilizing the specific structural characteristic, the mass spectrum neutral loss scanning or the daughter ion scanning mode is adopted, so that all the same series of compounds can be quickly searched and extracted, whether the compounds are known or not is solved, the condition that the unknown compounds are ignored in the previous determination and the sample is analyzed for a single-leaf fault is solved, and the complete information of the same series of compounds is obtained;
8) all compounds of the same series in the reference sample and the compounds of the same series in the sample to be detected are respectively coded by isotopes, and the coded isotopes have completely same structures except the heavy and light isotope states of individual elements, so that the retention time on a chromatogram is almost completely the same, and the accurate comparison of the contents of all compounds of the same series in the sample to be detected and the reference sample can be realized by simultaneous detection. Referring to figure 7 in the examples, the classical map of the same series of compound fatty acids in the sample, and the solid line is the series of fatty acids in the sample to be tested; the dotted line is the series of fatty acids in the reference sample, and the retention time of the series of fatty acid chromatograms in the sample to be detected and the reference sample is almost completely consistent and can be accurately compared;
9) the invention also provides a method for analyzing the compound containing the carboxyl functional group in the living body, which is a method for trimethyl aminoethyl ester derivatization and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS); taking 16:0 and 18:0 fatty acids as examples, the detection Limit (LOD) of the derivatives is about 0.6 pg, and very high sensitivity is achieved; after derivatization, N (CH) may be used3)3(59 Da) identifying the compound derivative containing the carboxyl functional group in the real sample in a Neutral Loss Scanning (NLS) mode, and quantitatively analyzing the compound derivative containing the carboxyl functional group in a Multiple Reaction Monitoring (MRM) mode;
10) the invention prepares a reference sample for a compound containing a carboxyl functional group and stabilizes heavy isotope (CD)3I) Code, andwith the sample to be tested being stabilized by a light isotope (CH)3I) Encoding, then mixing with a reference sample and detecting simultaneously; the method provides an isotope internal standard for real-time analysis of all compounds containing carboxyl functional groups in a biological sample, realizes accurate comparison of the concentrations of all compounds containing carboxyl functional groups among very complex samples, and has the advantages of high sensitivity, high reliability, simple operation, commercialized reagents and low price;
11) it should be noted that, the invention adopts the reference sample to carry out one-to-one corresponding quantitative analysis on the same series of compounds in the sample to be detected, the reference sample is usually the sample to be detected or the mixed sample of the isolated sample of the healthy person, if the isolated sample of the healthy person is adopted for preparation, the isolated sample can be prepared in advance for standby, and the preparation is not needed each time;
12) the invention solves the problem that the reference interval for simultaneously measuring a large number of compounds is difficult to determine by adopting the reference sample. Standard specifications such as CLSI C28-A or EP28-A3C De fi ning, updating, and Verifying Reference Intervals in the Clinical Laboratory, and applied guided diagnosis-Third Edition are established by using a Reference interval to prepare a Reference sample, biological samples of the population are collected according to the requirements of the specification system, and the Reference sample is prepared by mixing according to different requirements. Then extracting all series of target analytes and corresponding series of internal standard substance peak area signals (including all known and unknown compounds of the same series), forming one-to-one corresponding standard data sets for the series of compounds in the complex sample, and evaluating the sample to be detected through an algorithm, mathematical transformation or a model. Thus, the comparison (algorithm, mathematical transformation or model) between the sample to be tested and the reference sample can be used to determine the health or pathological state, so that the disease and health state can be judged, and the problem of the reference interval for simultaneously measuring a large number of compounds can be solved.
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 (a) is a schematic diagram of the introduction of stable isotope label a into the homologous compound in the reference sample (containing m homologous compounds); FIG. 1 (b) is a schematic diagram of stable isotope label b introduced into the compounds of the same series in a sample to be tested (containing n compounds of the same series, m > n);
FIG. 2 is a schematic diagram of a reference sample and a sample to be tested mixed to form a one-to-one corresponding data set after stable isotope labels are introduced into compounds of the same series;
FIG. 3 is a schematic diagram of the derivatization reaction of a compound containing a carboxyl functional group;
FIG. 4 shows 20. mu.l of 1. mu.M fatty acid standard (16: 0 FA, 17:0 FA, 18:0 FA) in a test tube, dried and derivatized; dissolving the final product in 1 ml of methanol, and performing direct sample injection analysis on fatty acid and fatty acid derivatives by using a Sciex QTrap 4500 mass spectrometer negative ion scanning detection mode (a) and a Sciex positive ion scanning detection mode (b) respectively;
FIG. 5 Sciex QTrap 4500 Mass Spectroscopy of daughter ions detection (a) with neutral loss 59 detection of fatty acid derivatives; a set of signals indicating a loss of neutrality of 59 upon detection of the fatty acid derivative daughter ion (e.g., about 59 difference between the 16:0 FA derivative product parent ion 342.3 and the fragment ion 283.3); (b) by using the detection mode of neutral loss 59, compounds containing carboxyl functional groups in the biological sample can be rapidly identified (such as 16:0 FA, 17:0 FA, and 18:0 FA-derived parent ions 342.5, 356.4 and 370.2 in the sample);
FIG. 6 is a graph showing the rapid identification of compounds containing carboxyl functional groups in plasma samples using the mode of detection of neutral loss 59 (e.g., 14 fatty acid-derived parent ions such as 12:0 FA, 16:2 FA, 18:3 FA, etc. are identified in the samples);
FIG. 7 liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) analysis of a mixture of specific plasma samples (light isotope-derived products) and reference sample (heavy isotope-derived products) derivatives; solid line: a specific sample total ion flow graph; dotted line: a reference sample total ion flow graph. Wherein the target analyte and the series of internal standard substance signals correspond one to one, and the series of compounds in the complex sample form a one-to-one corresponding standard data set. Of these, 17:0 FA is a fatty acid not contained in the human body and is used here as a calculation of a correction factor.
Detailed Description
The invention provides a method for quantifying compounds of the same series by adopting a reference sample, which comprises the following steps:
a. the same series of compounds refers to a series of compounds having the same chemical group;
b. the compounds of the same series in the sample to be detected are respectively named as x '1, x '2 and … … x ' n;
c. the compounds of the same series in the reference sample are respectively named as x1, x2 and … … xm;
ensuring that the number of the compounds of the same series in the reference sample covers the compounds of the same series in all samples to be detected, namely m is larger than n, and x1 and x '1, x2 and x '2, and … … xn and x ' n are the same compounds;
d. after all compounds of the same series in the reference sample have been introduced with the stable isotope label a, they are recorded as: x1+ a, x2+ a, x3+ a … … xm + a (m > n)
e. After all compounds of the same series in the sample to be tested are introduced with the stable isotope label b, the stable isotope label b is recorded as: x '1+ b, x'2+ b, x '3+ b, … …, x' n + b;
step one, preparing a reference sample, wherein the reference sample can be prepared by adopting the following method: mixing typical conventional samples, such as biological samples of healthy persons, the emphasis of this method is to cover all compounds that are likely to be present in the sample with a sufficient amount of a typical sample; the actual real sample is used for preparing a reference sample, and the key point is that all the same series compounds (including known same series compounds, same series compounds with standard substances, unknown same series compounds and same series compounds without standard substances) in the sample to be tested can be covered to the maximum extent.
The standard sample is prepared by adopting the sample of the healthy population which is established according to the reference interval (meeting the requirements of the CLSI CA28-A and EP 28-A3C), and the indexes of the healthy population can be directly compared to establish a model for evaluation and quantification.
Introducing a stable isotope label a into the reference sample, and introducing a corresponding stable isotope label b into the sample to be detected, wherein the isotope labels a and b usually only have difference of corresponding heavy and light element isotopes, so that the retention time of each compound in the reference sample and the sample to be detected is basically consistent after the stable isotope is introduced;
step three, uniformly mixing the reference sample with each sample to be detected respectively, and jointly entering a mass spectrometer for analysis;
the operation diagrams of the first step, the third step are shown in fig. 1 and fig. 2, wherein the symbols a and b in fig. 1 and fig. 2 are respectively different isotope codes of a reference sample and a sample to be detected, only compounds of the same series with the same chemical characteristics can be coded with the isotopes, and compounds capable of being coded with the isotopes are compounds of the same series, no matter whether the compounds are known or not.
Step four, calculating the relative content of each compound in the sample to be detected and the proportional relation of the relative content based on each compound in the reference sample;
taking triple quadrupole mass spectrometry as an example, triple quadrupole mass spectrometers can extract and determine peak areas of compounds through a variety of data acquisition modes. Because the reference sample and the sample to be detected are respectively coded by isotopes, the coded isotopes are respectively knocked out by collision energy in a mass spectrum, so that the same neutral loss mass or the same daughter ions can be respectively generated, an unknown compound is extracted in a neutral loss acquisition mode or a daughter ion scanning mode by utilizing the characteristic, the peak area of the unknown compound is recorded, and the proportional relation between the relative content and the relative content is further calculated; the characteristic neutral loss signals of the reference sample and the sample to be tested correspond to a and b in fig. 1 and 2, respectively.
a) Calculating the peak Area ratio of each compound in the sample to be detected to each compound in the reference sample, Areax'1+b/Areax1+a, Areax'2+b/Areax2+a, Areax'3+b/Areax3+a, ……, Areax'n+b/Areaxn+aObtaining the content of the sample to be detected relative to the reference sample; the content of the individual compounds gives the state of the sample, and the application scenarios are exemplified, for example, a long-term elevated plasma fatty acid level may lead to muscle insulin resistance, fatThe cells are insensitive to the adipogenic action of insulin, diabetes and hepatic steatosis, and plasma fatty acids are also associated with cachexia caused by cancer, asthma, cystic fibrosis, sudden cardiac death, and the like. In particular examples, lung cancer is the most lethal cancer, and the incidence and mortality of Chinese lung cancer are 53.57/10 ten thousand and 45.57/10 ten thousand, respectively, early diagnosis allows patients a better chance of long-term survival. The relationship between the fatty acids C16:1, C18:1, C18:3, C18:2, C20:4 and C22:6 and non-small cell lung Cancer was reported by Zhili Li (Journal of Cancer, 2014, 5(8): 706-714) and the like, and the relative contents of C16:1, C18:3, C18:2, C18:1, C20:4 and C22:6 and the ratio of the relative contents of C18:2/C18:1 of patients with non-small cell lung Cancer were significantly reduced compared with healthy people. Therefore, the relative contents of C16:1, C18:3, C18:2, C18:1, C20:4 and C22:6 and the ratio of the relative contents of C18:2/C18:1 are very likely to serve as important marker compounds for early detection and diagnosis of non-small cell lung cancer. After further study and validation, the corresponding reference range is established, and the clinical application is possible.
Further, for example, in 2020, Perminder Sachdev (aging Research Reviews 60, 2020, 101043) et al report the use of fatty acids for the assessment of mild cognitive impairment and alzheimer's disease, Perminder Sachdev reported that in the assessment of mild cognitive impairment and alzheimer's disease a significant decrease in the relative levels of most plasma/serum fatty acids was observed, and that in alzheimer's disease the number of fatty acids was observed in the significantly altered relative levels was the greatest; in general, mild cognitive impairment and alzheimer's disease are deficient in fatty acids in healthy people, and after further clinical research and validation, the relative amounts of fatty acids can be used for clinical early diagnosis of mild cognitive impairment and alzheimer's disease. b) Calculating the ratio of the relative content between each two compounds in the sample to be detected, wherein the calculation formula is as follows:
calculating the ratio of the relative contents of the target analytes x '1 and x '2 and … … x ' n in the sample to be detected:
（Areax'1+b/Areax1+a）/( Areax'2+b/Areax2+a)
（Areax'1+b/Areax1+a）/(Areax'3+b/Areax3+a)
（Areax'1+b/Areax1+a）/(Areax'4+b/Areax4+a)
……
（Areax'1+b/Areax1+a）/(Areax'n+b/Areaxn+a)
calculating the ratio of the relative contents of the target analytes x '2 and x '3 and … … x ' n in the sample to be detected:
（ Areax'2+b/Areax2+a）/( Areax'3+b/Areax3+a)
（ Areax'2+b/Areax2+a）/(Areax'4+b/Areax4+a)
……
（ Areax'2+b/Areax2+a）/(Areax'n+b/Areaxn+a)
calculating the ratio of the relative contents of the target analytes x '3 and x '4 and … … x ' n in the sample to be detected:
（Areax'3+b/Areax3+a）/(Areax'4+b/Areax4+a)
……
（Areax'3+b/Areax3+a）/(Areax'n+b/Areaxn+a)；
and analogizing the relative content ratios of the target analytes x '4, x'5, x '6 … … x' (n-1) in the sample to be tested. The state of the sample is characterized by the ratio of the contents between two. For example, long chain fatty acids have been considered as specific biomarkers for peroxisome disorders, such as X-linked adrenoleukodystrophy (X-ALD), adrenomyeloneuropathy, and Zellweger syndrome (brain-liver-kidney syndrome). Taking X-ALD as an example, the prior study of X-ALD is much, patients are asymptomatic at birth, the narrow therapeutic window is often overlooked, and the optimal time for treatment is missed, and the determination of fatty acid is one of the key to solve the problem in the future. According to previous studies on X-ALD, X-linked adrenoleukodystrophy (X-ALD) is characterized by elevated plasma levels of straight-saturated ultra-long chain fatty acids, which can be measured by 26:0 FA/22: 0 FA and 24:0 FA/22: a ratio of 0 FA in combination with clinical indications to identify disease states. In 2019, reference ranges of ratios of Chinese fatty acids C24:0/C22:0 and C26:0/C22:0 are established for the first time by Ling Qiu (Clinica Chimica Acta 495, 2019, 185-190) and the like, serum of 187 healthy people is collected, and the reference ranges of ratios of C24:0/C22:0 and C26:0/C22:0 are determined to be 0.75-1.28 and 0.005-0.0139 respectively according to the specification of CLSI C28-A and the upper and lower limits respectively adopt 2.5% and 97.5% confidence intervals. Since X-ALD was studied for many years, its biomarkers (C24: 0/C22:0 and C26:0/C22: 0) and evaluation methods (C24: 0/C22:0 and C26:0/C22:0 ratios in the range of 0.75-1.28 and 0.005-0.0139 can be considered healthy, above which X-ALD is suspected) are relatively specific, even though for many years X-ALD was studied, we have just established a reference range from 187 samples in 2019. Besides C24:0, C22:0 and C26:0, the medicine also contains various fatty acids in vivo, and the proportional relation of the relative contents of the fatty acids can bring a great deal of valuable information for people and play a role in more disease diagnosis. Based on the invention, more and more relative contents of fatty acid and the rule of the proportional relation of the relative contents can be found, calculated and verified, and more disease detection and treatment methods can be found.
Further, for example, Marina Poll n et al (Nutrients, 2020, 12(10): 3132) reported that fatty acids assessed the risk of breast cancer in 2020 investigating 1017 breast cancer and healthy women (CI below indicates the reference range), stearic Acid (95% confidence interval, CI = 0.30-0.66), linoleic Acid (95% confidence interval, CI = 0.49-0.90), arachidonic Acid (95% confidence interval, CI = 0.48-0.84) calculated ratios to Dihomo- γ -linolenic Acid (20:3, n-6), respectively, higher ratios indicating lower breast cancer risk. Palmitoleic acid (95% confidence interval, CI = 1.20-2.26), antipalmitoleic acid (95% confidence interval, CI = 1.12-2.02), trans industrial palmitic acid (95% confidence interval, CI = 1.14-2.03), oleic acid (95% confidence interval, CI = 1.45-2.87) were each compared to stearic acid, and a high ratio indicated a high risk of breast cancer. But this reference range requires further verification.
By adopting the method, the relative content of each target compound relative to a reference sample and the proportional relation of the relative content of each compound are respectively calculated, and then the sample to be tested is evaluated by adopting the following method:
1) for a few cases, such as for example cases with a reference range like X-linked adrenoleukodystrophy (X-ALD) (Ling Qiu, clinical Chimica Acta 495, 2019, 185-190), a diagnosis can be made with reference to the existing reference range.
2) For most cases, there may be no reference range for a while, the method provided by the present invention ensures that all target compounds (including known and unknown compounds) can be monitored and observed, that the relationship between these compounds and the disease state is observed, that effective compound markers for the disease are determined, and that a large number of verifications and verifications of the effectiveness of the compound markers are performed. In the process, the invention can completely achieve the aim and solve the problems of no standard substance, no internal standard, no reference range, low detection sensitivity, large matrix influence, large system error and the like. After all the compounds are discovered, verified and proved and the relation and the rule with the disease are completely determined, a reference range can be correspondingly established according to the method reported by Ling Qiu, 2019 and used for diagnosis.
3) Further, in the near future, it is likely that it will be found in the later stage of the verification that the reference range of a single index is not necessarily suitable for evaluation, and there may be a case where a plurality of indexes are higher or lower than the reference range but are still healthy, or because the reference range is too large due to large individual differences among people, the too large reference range is no longer suitable for evaluation of health. Therefore, it is likely that a novel algorithm will be involved instead of the conventional evaluation method in which the measurement result is compared only with the reference range. The novel algorithm can also be used in conjunction with reference ranges to assess health and disease status. The above studies, based on the method of the present invention, can be conveniently carried out in the later stage.
The number of fatty acids is very large, far from the limited fatty acids reported so far, and the relative content and proportion relationship thereof are crucial for the diagnosis of diseases. With the development of technology, more disease-related markers are certainly available step by step, and specific reference ranges and evaluation methods are established. The invention, as a technical innovation of the detection method, can provide a powerful technical method for the analysis of the samples, so that the analysis is simpler and more effective, the sensitivity is higher, a series of target compounds (no known compounds or unknown compounds) are covered as much as possible, the system errors and the influence of a matrix on the detection accuracy are removed, and the target compounds in the samples to be detected are quickly and accurately evaluated.
According to the invention, all compounds of all samples to be detected are aligned to a reference sample, the content is calculated, all compounds of the same series in the reference sample are not assigned with a standard product, the content of all compounds in the same series in the samples to be detected is calculated relative to the content of all compounds in the reference sample, if the reference sample is a mixed sample of healthy people, the content of all compounds in the samples to be detected relative to all compounds in the reference sample is the value of the samples to be detected relative to the healthy people, effective analysis results can be obtained, and the states of the samples to be detected, such as the health state, the disease state and the change of the disease course, can be judged according to the analysis results and clinical indications.
The method is simple, sensitive and reliable in operation, is beneficial to implementation, can be used for analyzing the compounds of the same series in a complex sample, can accurately represent the sample, particularly a biological sample by comprehensively analyzing all the compounds of the same series, and can be particularly used for comprehensively and deeply knowing the state, the course and the pathogenesis of certain diseases of human beings. Meanwhile, in the embodiment of the invention, the isotope internal standard which can be used for real-time analysis of all compounds containing carboxyl functional groups in a biological sample is provided by performing light and heavy stable isotope-coded trimethyl aminoethyl derivatization on the compounds containing carboxyl functional groups. The strategy is combined with a liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method to realize accurate relative quantitative analysis of all compounds containing carboxyl functional groups in a biological sample. The method has the characteristics of high sensitivity, high reliability, simple operation, commercialized reagents, low price and the like, and is recommended to be used for analysis of all compounds containing carboxyl functional groups in biological samples and future clinical analysis.
The invention will be further illustrated with reference to the following specific examples of fatty acids (representative of compounds containing carboxyl functional groups) in plasma, but the invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.
Extraction of fatty acids in plasma: the fatty acid is extracted from blood plasma by methanol precipitation method. Briefly, 10 microliters of plasma, 80 microliters of PBS (1X), 100 microliters of methanol, 2 microliters (1N) of hydrochloric acid, and 200 microliters of isooctane were mixed, vortexed for 1 minute, centrifuged (centrifugal force 3000g, 10 min, 4 degrees celsius), the supernatant was collected, and extracted once more with 200 microliters of isooctane. The supernatants were combined and dried at room temperature using a vacuum centrifugal drier.
Fatty acid derivatization: adding 50 microliters of the extract of fatty acid in dried plasma into a centrifuge tube, incubating in a water bath at 50 ℃ for 5 min, and carrying out vacuum centrifugal drying; adding dimethylamino ethanol (60 microliters) into a centrifugal tube, standing for 5 minutes at room temperature, and carrying out vacuum centrifugal drying; then adding [3H ] into a specific sample centrifuge tube to be analyzed respectively]Methyl iodide (CH)3I, 100 μ l), adding [3D ] to the reference sample]Methyl iodide (CD)3I, 100 microliters), and standing for 10 min at room temperature to obtain light and heavy isotope labeled Trimethylaminoethyl (TMAE) ester derivative (FA-TMAE), and performing vacuum centrifugal drying. FIG. 3 is a schematic diagram of the derivatization reaction of a compound containing a carboxyl functional group. The structural formula of the derivative product is as follows:
light isotope derivative product:
，
heavy isotope derivative product:
。
liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) analysis: the above-mentioned dried components were dissolved in 100. mu.l of methanol. After 10. mu.l of each of the specific sample and the reference sample were mixed, the mixture was further diluted 50 times with methanol, and 2. mu.l of the sample was subjected to liquid chromatography electrospray ionization tandem mass spectrometry.
The instruments used in the following examples were Waters I-class UPLC and Sciex QTrap 4500 MS.
The detection parameters of the liquid chromatography electrospray ionization tandem mass spectrometer of the common fatty acid derivatives are shown in the table 1:
TABLE 1 detection parameters of liquid chromatography electrospray ionization tandem mass spectrometer for common fatty acid derivatives
When the liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is used for analysis, the mass spectrometry detection mode comprises a neutral loss detection mode, an ion detection mode, a parent ion detection mode, a multi-reaction detection mode and a full-scan detection mode; the specific neutral loss information of the compound can be rapidly identified by utilizing a neutral loss detection mode, the parent ion information of the carboxyl functional group-containing compound extracted from a biological sample can be rapidly identified by utilizing a parent ion detection mode, and the structures of the carboxyl functional group-containing compounds can be effectively identified by utilizing an daughter ion detection mode; in the multi-reaction detection mode, the parent ions and the specific fragment ions thereof are utilized to effectively realize the quantitative analysis of the specific carboxyl functional group-containing compound.
Example 1
Derivatization efficiency studies using fatty acid standards
20 μ M of 1 μ M fatty acid standards (16: 0 FA, 17:0 FA, 18:0 FA) were taken in a test tube, dried and derivatized. The final product (light isotope derivative product) was dissolved in 1 ml of methanol, and direct sample analysis of fatty acids and fatty acid derivatives was performed using a negative ion scanning detection mode and a positive ion scanning detection mode of a Sciex QTrap 4500 mass spectrometer, respectively.
Figure 4 shows that the negative ion scan detection mode does not detect a signal of no derivatized fatty acid, whereas the positive ion scan detection mode detects a signal of a very strong fatty acid-derivatized product. Indicating that the derivatization efficiency is close to 100%.
Example 2
Qualitative analysis of fatty acid derivatives was performed using a Sciex QTrap 4500 mass spectrometer:
20 μ M of 1 μ M fatty acid standards (16: 0 FA, 17:0 FA, 18:0 FA) were taken in a test tube, dried and derivatized. The final product (light isotope derivative product) was dissolved in 1 ml of methanol and analyzed by direct injection of fatty acid derivatives using a Sciex QTrap 4500 mass spectrometer for daughter ion and neutral loss detection mode.
FIG. 5 shows that a set of signals for neutral loss 59 occurs when the fatty acid derivative is subjected to daughter ion detection (e.g., the difference between the 16:0 FA derivative product parent ion 342.3 and the fragment ion 283.3 is about 59); by using the neutral loss 59 detection mode, compounds containing carboxyl functional groups in the biological sample can be rapidly identified (such as 16:0 FA, 17:0 FA, and 18:0 FA-derived parent ions 342.5, 356.4 and 370.2 in the sample).
Qualitative analysis of fatty acid derivatives in plasma was performed using a Sciex QTrap 4500 mass spectrometer:
after extraction and derivatization of 10 μ l of plasma, the final product (light isotope derivative) was dissolved in 1 ml of methanol and analyzed by direct injection of fatty acid derivatives using the Sciex QTrap 4500 mass spectrometer neutral loss detection mode.
FIG. 6 shows that compounds containing carboxyl functionality in biological samples can be rapidly identified using the mode of detection of neutral loss 59 (e.g., 14 fatty acid-derived parent ions such as 12:0 FA, 16:2 FA, 18:3 FA, etc. are identified in the sample).
Example 3
Analysis of mixtures of specific plasma samples and reference samples using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)
After 10 microliters of a specific plasma sample is extracted and derivatized, the final product (light isotope derivative product) is dissolved in 100 microliters of methanol; after extraction and derivatization of a 10. mu.l reference plasma sample, the final product (heavy isotope derivative) was dissolved in 100. mu.l methanol; after 10. mu.l of each of the specific sample and the reference sample were mixed, the mixture was further diluted 50 times with methanol, and 2. mu.l of the sample was subjected to liquid chromatography electrospray ionization tandem mass spectrometry.
The solid line in FIG. 7 is the series of fatty acids in the sample to be tested; the dotted line is the series of fatty acids in the reference sample, and the retention time of the series of fatty acid chromatograms in the sample to be detected and the reference sample is almost completely consistent and can be accurately compared. It is shown that: the peak-off time of the heavy isotope derived product is consistent with that of the light isotope derived product, and the reference sample provides a real-time isotope internal standard for a specific sample to be analyzed. Wherein the target analyte and the series of internal standard substance signals correspond one to one, and the series of compounds in the complex sample form a one-to-one corresponding standard data set.
In conclusion, the invention provides a method for quantifying and evaluating all compounds of the same series in a complex sample by adopting a reference sample; mixing samples of people meeting the system requirements to prepare a reference sample, wherein the reference sample and a sample matrix to be detected are the same and cover all target analytes in each sample to be detected; respectively adding an isotope derivative reagent b into each sample to be detected, and adding an isotope derivative reagent a into a reference sample; respectively adding the reference sample into each sample to be detected, and then carrying out mass spectrometry to obtain signals of a series of internal standard substances and signals of a series of target analytes which are in one-to-one correspondence with the target analytes; meanwhile, extracting all series of target analytes and corresponding series of internal standard substance signals (including known and unknown compounds of the same series) by deriving specific mass spectrum signals generated by reagent labels, forming one-to-one corresponding standard data sets for the series of compounds in the complex sample, and evaluating the sample to be detected through an algorithm, mathematical transformation or a model; and/or calculating the relative content or the proportional relation of the relative content of the corresponding series of target analytes through the signals of the series of internal standard substances. The invention introduces stable isotope coding labels into the reference samples, realizes the accurate comparison of all compounds of the same series among very complex samples, establishes a data set of all compounds of the same series and one corresponding internal standard of the compounds of the same series, can be used for various data models and algorithms, solves the problems that the determination process of the reference interval of the compounds of the complex samples is complex, the compounds of the series in the complex samples need a standard product, the internal standard (high price and difficult obtaining), matrix interference and other series, and has the advantages of high sensitivity, high reliability, simple operation and the like, wherein all the used reagents are commercialized and have low price.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (9)
1. A method for quantifying and evaluating a series of compounds in a sample to be tested by using a reference sample, which is characterized by comprising the following steps:
s1, preparing a standard sample which is the same as the matrix of the sample to be detected and covers all target analytes in each sample to be detected; the preparation is carried out by mixing typical conventional samples, wherein the typical conventional samples are in vitro samples of people meeting the requirements of a system;
s2, adding an isotope label b into the sample to be detected, and adding an isotope label a into the reference sample; performing mass spectrometry after mixing; and identifying and extracting all series of target analytes and series of internal standard substances which are in one-to-one correspondence with the target analytes by adopting a neutral loss acquisition mode or a sub-ion acquisition mode to obtain peak area signals of the series of target analytes and the corresponding series of internal standard substances, and calculating to obtain the relative content of the series of target analytes of the sample to be detected relative to the corresponding internal standard substances in the reference sample.
2. The method of claim 1, wherein the same series of target analytes in the sample to be tested are designated x '1, x '2, … … x ' n, respectively; the corresponding series of internal standards in the reference sample were designated x1, x2, … … xm, respectively; m is>n, and x1 and x '1, x2 and x '2, … … xn and x ' n are the same compound; after introducing the isotope label b into the same series of target analytes in the sample to be detected, marking as x '1+ b, x'2+ b, x '3+ b, … … and x' n + b; after the corresponding series of internal standards in the reference sample is introduced with isotope label a, the labels are x1+ a, x2+ a, x3+ a … … xm + a, m>n; calculating the ratio of the peak area of each target analyte in the sample to be detected to the peak area of each corresponding internal standard substance in the reference sample in a one-to-one correspondence manner: areax'1+b/Areax1+a,Areax'2+b/Areax2+a,Areax'3+b/Areax3+a,……,Areax'n+b/Areaxn+aAnd obtaining the content of each target analyte in the sample to be detected relative to the corresponding internal standard substance in the reference sample.
3. The method according to claim 2, wherein the ratio of the relative content of each compound to the other compounds in the sample to be tested and the series of target analytes is calculated in a one-to-one correspondence manner; the relative content is the content of each target analyte in the sample to be detected relative to the corresponding internal standard substance in the reference sample; the calculation formula is as follows:
calculating the ratio of the relative contents of the target analytes x '1, x '2 and … … x ' n in the sample to be detected:
(Areax'1+b/Areax1+a)/(Areax'2+b/Areax2+a)
(Areax'1+b/Areax1+a)/(Areax'3+b/Areax3+a)
(Areax'1+b/Areax1+a)/(Areax'4+b/Areax4+a)
……
(Areax'1+b/Areax1+a)/(Areax'n+b/Areaxn+a)
calculating the ratio of the relative contents of the target analytes x '2 and x '3 and … … x ' n in the sample to be detected:
(Areax'2+b/Areax2+a)/(Areax'3+b/Areax3+a)
(Areax'2+b/Areax2+a)/(Areax'4+b/Areax4+a)
……
(Areax'2+b/Areax2+a)/(Areax'n+b/Areaxn+a)
calculating the ratio of the relative contents of the target analytes x '3 and x '4 and … … x ' n in the sample to be detected:
(Areax'3+b/Areax3+a)/(Areax'4+b/Areax4+a)
……
(Areax'3+b/Areax3+a)/(Areax'n+b/Areaxn+a)；
and analogizing the relative content ratios of the target analytes x '4, x'5, x '6 … … x' (n-1) in the sample to be tested.
4. The method of claim 1, wherein the target analytes comprise any one or more 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, each type of target analyte is correspondingly connected with a group of isotope labels a and b; different sets of isotopic labels a and b correspond to different types of target analytes.
5. The method of claim 4, wherein each set of isotope labels a, b is selected from light isotope derivative reagents, heavy isotope derivative reagents; or each group of isotope labels a and b is respectively selected from one of different fluorescence labeling reagents, one of different luminescence labeling reagents or one of different chemical group reaction reagents.
6. The method as claimed in any one of claims 1 to 5, wherein when the target analyte is a compound containing a carboxyl functional group, the sample to be measured and the reference sample each use a light isotope CH3I. Heavy isotope CD3I labeling a target compound; the labeling process comprises acyl chloride activated carboxyl, N-dimethyl aminoethanol esterification and methyl iodide methylated amino reaction marked by light or heavy isotopes.
7. A product for quantifying and evaluating a series of compounds in a sample to be tested using the method of claim 1.
8. An apparatus for quantifying and evaluating a series of compounds in a sample to be tested using the method of claim 1, comprising means for introducing a series of different isotope labels into the sample either on-line or off-line, means for pre-treating the sample, means for storing the sample and means for transporting the sample.
9. A test kit for quantifying and evaluating a series of compounds in a sample to be tested by the method according to claim 1, wherein the test kit comprises a plurality of sets of isotope labels a, isotope labels b, a blank matrix, a series of pure target analytes or a series of mixed target analyte-containing samples, a reference sample and a quality control product.
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