阅读说明：本技术 一种用于定量样品中目标分析物的天然同位素校准曲线法 (Natural isotope calibration curve method for quantifying target analyte in sample ) 是由 郏征伟 陈铭 谭晓杰 于 2021-03-18 设计创作，主要内容包括：本发明提供了一种用于定量样品中目标分析物的天然同位素校准曲线法,步骤为：在待测样品中添加已知量的校准物；对待测样品进行前处理后进行质谱法分析,得到校准物及校准物的天然同位素和目标分析物的质谱信号；通过计算得到校准物的天然同位素的含量,以校准物和校准物的天然同位素的含量、质谱信号响应的对应关系绘制校准曲线,目标分析物的质谱信号响应对应到校准曲线中即得到目标分析物含量。本发明首次以目标分析物的稳定同位素标记的类似物和稳定同位素标记的类似物的一系列天然同位素来作为校准物绘制标准曲线,从而定量待测样品中的目标分析物。该方法具有非常高的可靠性和准确性,且简化了检测程序,极大拓展了应用范围。(The invention provides a natural isotope calibration curve method for quantifying a target analyte in a sample, which comprises the following steps: adding a known amount of calibrator to the sample to be tested; performing mass spectrometry analysis after pretreating a sample to be detected to obtain a calibrator, a natural isotope of the calibrator and a mass spectrum signal of a target analyte; and calculating to obtain the content of the natural isotope of the calibrator, drawing a calibration curve according to the corresponding relation between the content of the natural isotope of the calibrator and the mass spectrum signal response of the calibrator, and corresponding the mass spectrum signal response of the target analyte to the calibration curve to obtain the content of the target analyte. The invention uses stable isotope labeled analogue of target analyte and a series of natural isotopes of stable isotope labeled analogue as calibrators to draw standard curve for the first time, thereby quantifying target analyte in the sample to be tested. The method has very high reliability and accuracy, simplifies the detection procedure and greatly expands the application range.)

1. A natural isotope calibration curve method for quantifying a target analyte in a sample, comprising the steps of:

A. adding a known amount of a calibrator, which is a stable isotope label of a target analyte, to a sample to be tested;

B. pretreating a sample to be detected;

C. b, performing mass spectrometry analysis on the sample to be detected processed in the step B to obtain mass spectrometry signals of the calibrator and the natural isotope of the calibrator and the target analyte in the sample to be detected;

D. calculating to obtain the natural isotope abundance ratio of the calibrator according to the molecular formula of the calibrator so as to obtain the content of the natural isotope of the calibrator, drawing a calibration curve according to the corresponding relation between the known content of the natural isotopes of the calibrator and the mass spectrum signal response, and corresponding to the calibration curve according to the mass spectrum signal response of the target analyte in the sample to be detected so as to obtain the content of the target analyte in the sample to be detected;

in the step A, one or more target analytes are adopted, and a known amount of calibrator is added into a sample to be tested corresponding to each target analyte;

in step C, the natural isotope mass spectrum signals of the calibrator are one or more;

when the mass spectrum signals of the natural isotopes of the calibrant are multiple, namely the mass spectrum signals comprise a first natural isotope of the calibrant, a second natural isotope of the calibrant, and so on, the standard curve drawing method of the step D is as follows:

calculating the natural isotope abundance ratio of the calibrator according to the molecular formula of the calibrator, thereby obtaining the content of the first natural isotope of the calibrator as a second known amount of calibrator, the content of the second natural isotope of the calibrator as a third known amount of calibrator, and so on; and then drawing a calibration curve according to the corresponding relation between the known content of each calibrator and the mass spectrum signal response.

2. The method according to claim 1, wherein the pre-treatment in step B is selected from any one of solid phase extraction, solid-liquid extraction, liquid-liquid extraction, protein precipitation, direct dilution, solvent extraction, salting-out, concentration, and masking.

3. The method of claim 1, wherein the mass-to-charge ratio of the natural isotope of the calibrator to the natural isotope of the calibrator is 1 or more daltons mass number when mass spectrometry with unit mass resolution is employed; when high resolution mass spectrometry is used, the mass to charge ratio of the calibrant to the natural isotope of the calibrant is greater than or equal to 0.05 daltons mass number.

4. The method according to claim 1, wherein the calibrator and the target analyte differ from each other by at least 2 mass daltons in the mass spectrometry analysis.

5. The method according to claim 1, wherein in step a, the sample to be tested comprises any one of a biological sample, an environmental sample, a food sample, a pharmaceutical sample, and a chemical sample; the biological sample comprises any one of plasma, serum, whole blood, urine, tissue, cerebrospinal fluid, saliva and hair.

6. The method according to claim 1, wherein in step a, the target analyte is an organic molecule comprising at least 3 carbon atoms.

7. The method according to claim 1, wherein in step a, the calibrator is a stable isotope label of the target analyte, and is obtained by replacing at least 1 atom of the target analyte with a stable isotope thereof; the stable isotope comprises²H、¹¹B、¹³C、¹⁵N、¹⁷O、¹⁸O、³³S、³⁴S and³⁶any one or more of S.

8. The method according to claim 1, wherein the mass spectrometry employs an ion source comprising any one of or a combination of two or more of an electrospray ion source, an atmospheric pressure chemical ionization ion source, a matrix assisted laser desorption ionization ion source, a desorption electrospray ionization ion source, an electron bombardment source, and a chemical ionization source, and the mass analyzer comprises a quadrupole mass analyzer, an ion trap mass analyzer, a magnetic sector mass spectrometer, a time-of-flight mass analyzer, an electrostatic field orbital trap mass analyzer, and a fourier transform ion cyclotron resonance mass analyzer; 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;

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, immunoaffinity chromatography, supercritical fluid chromatography, and ion mobility method.

9. The method according to claim 1, wherein the step D further comprises the step of correcting the content of the target analyte in the sample to be tested; the method specifically comprises the following steps: injecting the target analyte and the stable isotope labeled calibrator with the same concentration into a mass spectrometer for analysis, and taking the response ratio of the target analyte and the stable isotope labeled calibrator as a relative response factor; when the actual sample is analyzed, the content result of the target analyte is multiplied by the relative response factor to be used as the final result.

Technical Field

The invention relates to the technical field of detection and analysis, in particular to a natural isotope calibration curve method for quantifying a target analyte in a sample.

Background

The trace analysis method using mass spectrum as analysis means includes external standard method, internal standard method and standard addition method. According to different calibration modes, the method can be divided into a single-point calibration method, a multi-point calibration method and a calibration curve method; according to different solutions adopted for preparing the calibration product, the calibration product is divided into a pure solution standard product and a matrix-matched standard product. The most accurate results are obtained by a matrix matching internal standard calibration curve method and a standard addition method. The standard addition method is more useful for confirmation of results due to the difficulty in estimating the amount of the target compound in the sample and the large amount of work (at least three samples need to be prepared per sample). Thus, the matrix matching internal standard calibration curve method is the most commonly used trace analysis method at present. The method has the advantages that the internal standard compound is added in the pretreatment process for parallel calibration of the loss of the target compound in the pretreatment process, if the conditions allow, the internal standard compound generally selects the stable isotope labeled analogue of the target compound, and because the internal standard compound and the analogue have extremely similar physicochemical properties, the internal standard not only can calibrate the loss of the target compound in the pretreatment process, but also can calibrate the matrix effect in the mass spectrometry process; however, the main disadvantage of this method is that it is difficult to obtain blank matrix required for preparing standard, especially when the target substance to be analyzed is endogenous, and the variety of sample matrix is wide, and the individual difference is large.

In patent document No. 201280036810.1, a method for quantifying a target analyte in a sample is described, comprising obtaining a mass spectra signal comprising a first calibrator signal, comprising a second calibrator signal, and possibly comprising a target analyte signal from a single sample comprising a first known amount of a first calibrator, comprising a second known amount of a second calibrator, and potentially comprising a target analyte. The first known amount and the second known amount are different, and wherein the first calibrator, the second calibrator, and the target analyte are each distinguishable in the single sample by mass spectrometry. The method also includes quantifying the target analyte in the single sample using the first calibrator signal, the second calibrator signal, and the target analyte signal. However, this method has the disadvantage that it must comprise at least two calibrators, and that the first, second and further calibrators are each different stable isotope-labelled analogues of the target analyte (in the case of testosterone analysis, the calibrators added are mixed calibrators of different concentrations of D2-testosterone, D3-testosterone, D5-testosterone). In the practical application process, the stable isotope labeled analogue is difficult to obtain and has higher cost; and most of the target analytes are only available as a stable isotope labeled analogue, so that most of the target analytes cannot be directly quantified by the method in the patent.

In nature, all elements are present as mixtures of isotopes. The organic compound generally consists of C, H, O, N, S and other elements, which all have isotopes, and the isotopes of each element exist in certain natural abundance according to the natural law, as shown in table 1.

TABLE 1 Natural isotopic abundance of common elements in organic compounds

Considering that each element generally has a plurality of isotopes in an organic compound molecule, the peak intensity of each isotope can be calculated by the following expansion of binomial equation.

(a+b)ⁿ(c+d)^m……

Wherein a and b, c and d are eachThe natural abundance of the M and M +1 isotopes of the first and second elements (which may also be conventionally referred to as light and heavy isotopes, e.g. C, a and b being respectively¹²C and¹³natural abundance of C), n, m are the number of atoms of the first and second elements, respectively. Most of the current mainstream mass spectrometers are equipped with tool software for calculating the isotopic abundance ratio of the organic compound, and the calculation principle of the isotopic abundance ratio is not repeated here as long as the molecular formula of the compound is input.

When mass spectrometry is performed on the stable isotope analogs, a series of mass spectrum signals of the natural isotopes of the stable isotope analogs are obtained simultaneously. No one has ever seen further use of the mass spectral signal. There is no report in the prior art of using mass spectra signals of a series of natural isotopes of stable isotope analogues obtained by mass spectrometry for the quantification of target analytes.

Disclosure of Invention

In view of the deficiencies of the prior art, it is an object of the present invention to provide a method for quantifying a target analyte in a sample by mass spectrometry.

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

the invention provides a natural isotope calibration curve method for quantifying a target analyte in a sample, which comprises the following steps:

A. adding a known amount of a calibrator, which is a stable isotope labeled analog of a target analyte, to a sample to be tested;

B. pretreating a sample to be detected;

C. b, performing mass spectrometry analysis on the sample to be detected processed in the step B to obtain mass spectrometry signals of the calibrator and the natural isotope of the calibrator and the target analyte in the sample to be detected;

D. calculating to obtain the natural isotope abundance ratio of the calibrator according to the molecular formula of the calibrator so as to obtain the content of the natural isotope of the calibrator, drawing a calibration curve according to the corresponding relation between the known content of the natural isotopes of the calibrator and the mass spectrum signal response, and corresponding to the calibration curve according to the mass spectrum signal response of the target analyte in the sample to be detected so as to obtain the content of the target analyte in the sample to be detected;

in step a, one or more target analytes are selected, and each target analyte corresponds to a known amount of calibrator added to the sample to be tested.

Preferably, in step B, the pretreatment method is any one selected from the group consisting of solid-phase extraction, solid-liquid extraction, liquid-liquid extraction, protein precipitation, direct dilution, solvent extraction, salting-out, chemical separation, concentration, adsorption chromatography, partition chromatography, ion exchange chromatography, and masking.

Preferably, in step C, the natural isotope mass spectrometry signal of the calibrator is one or more.

Preferably, when the mass spectrum signal of the natural isotope of the calibrator is multiple, that is, the mass spectrum signal includes a first natural isotope of the calibrator, a second natural isotope of the calibrator, and so on, the standard curve plotting method of step D is as follows:

calculating the natural isotope abundance ratio of the calibrator according to the molecular formula of the calibrator, thereby obtaining the content of the first natural isotope of the calibrator as a second known amount of calibrator, the content of the second natural isotope of the calibrator as a third known amount of calibrator, and so on; and then drawing a calibration curve according to the corresponding relation between the known content of each calibrator and the mass spectrum signal response.

Preferably, the mass to charge ratio of the calibrant to the natural isotope of the calibrant is greater than or equal to 1 daltons mass number when mass spectrometry with unit mass resolution is employed; when high resolution mass spectrometry is used, the mass to charge ratio of the calibrant to the natural isotope of the calibrant is greater than or equal to 0.05 daltons mass number.

Preferably, the calibrator and target analytes differ from each other by a mass number of at least 2 daltons in mass spectrometry analysis.

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

Preferably, in step a, the target analyte is an organic molecule comprising at least 3 carbon atoms.

Preferably, in step A, the calibrator is a stable isotope labeled analog of the target analyte obtained by replacing at least 1 atom of the target analyte with a stable isotope thereof, including but not limited to²H、¹¹B、¹³C、¹⁵N、¹⁷O、¹⁸O、³³S、³⁴S and³⁶any one or more of S.

Preferably, the mass spectrometry employs an ion source including, but not limited to, an electrospray ion source (ESI), an atmospheric pressure chemical ionization ion source (APCI), a matrix assisted laser desorption ionization ion source (MALDI), a desorption electrospray ionization ion source (DESI), an electron impact source (EI), a chemical ionization source (CI), a mass analyzer including, but not limited to, a quadrupole mass analyzer, an ion trap mass analyzer, a sector magnetic mass spectrometer, a time-of-flight mass analyzer, an electrostatic field orbitrap mass analyzer, a fourier transform ion cyclotron resonance mass analyzer, or a combination of two or more thereof; 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;

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, immunoaffinity chromatography, supercritical fluid chromatography, and ion mobility method.

Preferably, step D further comprises the step of correcting the content of the target analyte in the sample to be tested; injecting the target analyte and the stable isotope labeled calibrator with the same concentration into a mass spectrometer for analysis, and taking the response ratio of the target analyte and the stable isotope labeled calibrator as a relative response factor; when the actual sample is analyzed, the content result of the target analyte is multiplied by the relative response factor to be used as the final result.

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

1. the invention uses stable isotope labeled analogue of target analyte and a series of natural isotopes of stable isotope labeled analogue as calibrators to draw standard curve for the first time, thereby quantifying target analyte in the sample to be tested. Since they are very similar in nature to the target compounds and coexist in the same sample matrix, the recovery and matrix effects can be calibrated almost perfectly, technically termed a single sample self-calibration method.

2. The result obtained by the method is basically consistent with the result obtained by the matrix matching internal standard calibration curve method, thereby illustrating the reliability and accuracy of the method; in addition, the method does not need to prepare any blank matrix additionally, greatly simplifies the detection procedure and further reduces the detection cost.

3. The method only needs to adopt one stable isotope labeled analogue for one target analyte, avoids synthesizing a plurality of stable isotope labeled analogues, and greatly saves the detection cost; and the target analyte of only one stable isotope labeled analogue can be directly quantified by the method, so that the application range is greatly expanded.

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 the structure of catecholamine metabolites (metaadrenaline and noradrenaline) and stable isotope-labeled analogs thereof (metaadrenaline-D3 and noradrenaline-D3) of example 1;

FIG. 2 is a mass spectrum of the epinephrine detected in example 1 (from bottom to top, mass spectra signals of the target compound, the calibrator, the first natural isotope of the calibrator, and the second natural isotope of the calibrator);

FIG. 3 is a mass spectrum of noradrenaline detected in example 1 (from bottom to top, mass spectra signals of the target compound, the calibrator, the first natural isotope of the calibrator, and the second natural isotope of the calibrator in that order);

FIG. 4 is a calibration curve for the adrenaline and noradrenaline matrix matching internal standard in example 1;

FIG. 5 is a comparison of the results of the two calibration methods for epinephrine in example 1;

FIG. 6 is a comparison of the results of the two calibration methods for norepinephrine in example 1;

FIG. 7 is the structure of 25-hydroxyvitamin D3 and its stable isotope labeled analog (25-hydroxyvitamin D3-D3) in example 2;

FIG. 8 is a mass spectrum of 25-hydroxyvitamin D3 detected in example 2 (from bottom to top, mass spectra signals of target compound, calibrant, first natural isotope of calibrant, second natural isotope of calibrant);

FIG. 9 is a matrix matching internal standard calibration curve for 25-hydroxyvitamin D3 in example 2;

FIG. 10 shows the results of the calibration curve method for natural isotopes and the calibration method for matrix-matched internal standards in example 2;

FIG. 11 is the structure of aldosterone and its stable isotope labeled analog (aldosterone-D8) in example 3;

FIG. 12 is a mass spectrum of aldosterone detected in example 3 (from bottom to top, mass spectra signals of a target compound, a calibrator, a first natural isotope of the calibrator, and a second natural isotope of the calibrator);

FIG. 13 is a calibration curve for aldosterone matrix matching internal standard in example 3;

FIG. 14 is the Tacrolimus and stable isotope labeled analog thereof in example 4 (Tacrolimus-¹³CD 2);

FIG. 15 is a mass spectrum of tacrolimus detected in example 4 (from bottom to top, mass spectrum signals of target compound, calibrant, first natural isotope of calibrant, second natural isotope of calibrant);

FIG. 16 is a calibration curve for matrix matching of tacrolimus in example 4 with an internal standard;

FIG. 17 is a schematic diagram of the natural isotope calibration curve method employed in the present invention.

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 following example provides a natural isotope calibration curve method for quantifying a target analyte in a sample, comprising the steps of:

A. adding a known amount of a calibrator, which is a stable isotope labeled analog of a target analyte, to a sample to be tested;

B. pretreating a sample to be detected;

C. b, performing mass spectrometry analysis on the sample to be detected processed in the step B to obtain mass spectrometry signals of the calibrator and the natural isotope of the calibrator and the target analyte in the sample to be detected;

D. calculating to obtain the natural isotope abundance ratio of the calibrator according to the molecular formula of the calibrator so as to obtain the content of the natural isotope of the calibrator, drawing a calibration curve according to the corresponding relation between the known content of the natural isotopes of the calibrator and the mass spectrum signal response, and corresponding to the calibration curve according to the mass spectrum signal response of the target analyte in the sample to be detected so as to obtain the content of the target analyte in the sample to be detected;

in step a, one or more target analytes are selected, and each target analyte corresponds to a known amount of calibrator added to the sample to be tested.

The principle of the natural isotope calibration curve method is shown in fig. 17, the relative abundance and the content of the natural isotope thereof are calculated according to the content of the calibrator and the molecular formula thereof, and then a standard curve is drawn by taking the content of the calibrator and the content of the natural isotope thereof as horizontal coordinates and taking the peak area as vertical coordinates; and then, corresponding the peak area of the target analyte to a standard curve to obtain the content value of the target analyte.

EXAMPLE 1 analysis of the content of catecholamine metabolites in human plasma samples

Conventional LC-MS/MS mass spectrometry quantitation methods require the preparation of a blank matrix for the preparation of a series of matrix-matched calibrators due to the presence of matrix effects, and the composition of the blank matrix should be as consistent as possible with the batch of sample matrices being analyzed. The preparation of such blank matrices is particularly difficult when the target analyte is ubiquitous in the matrix (e.g., endogenous hormones, vitamins, amino acids, etc.), and this goal is almost impossible to achieve, especially when the target analyte is present in high amounts in the matrix. As in this example, the normal plasma is low in catecholamine metabolites, but it is still necessary to prepare a secondary activated carbon-treated plasma as a blank plasma matrix. This example describes the determination of the content of catecholamine metabolites (metaadrenaline and noradrenaline) in human plasma samples, adding known amounts of stable isotope-labeled analogues, constructing a calibration curve using the stable isotope and its natural isotope of known abundance, and quantitatively analyzing the content of the catecholamine metabolites in the samples. And simultaneously comparing the obtained result with the result obtained by a matrix matching internal standard calibration curve method. This example demonstrates that catecholamine metabolites in human plasma samples can be accurately and accurately quantified by using stable isotope-labeled calibrators and their natural isotopes of known abundance.

The content determination method of catecholamine metabolites (adrenaline and noradrenaline) in a human plasma sample comprises the following specific steps:

1. preparation of stable isotope labeled calibrators: commercially available stable isotope labeled analogs of the target analyte, which in this example were metaepinephrine and noradrenaline, respectively, and the corresponding commercially available stable isotope labeled analogs were metaepinephrine-D3 and noradrenaline-D3, respectively, were used as calibrators, see fig. 1.

2. Preparation of individual stock solutions of target analyte and calibrator: respectively taking solid powder of the target analyte and the stable isotope labeling calibrator, and preparing stock solutions by using methanol as a solvent, wherein the concentration of each stock solution is 1 mg/mL.

3. Optimizing mass spectrum conditions: from the individual stock solutions of the target analyte and calibrator, individual solutions of 0.1. mu.g/mL were diluted separately and used to optimize the fumbling mass spectrometry conditions, the results are shown in Table 2. Wherein norepinephrine-D3-F1 is a first natural isotope of norepinephrine-D3, and norepinephrine-D3-F2 is a second natural isotope of norepinephrine-D3; noradrenaline-D3-F1 is the first natural isotope of noradrenaline-D3, and noradrenaline-D3-F2 is the second natural isotope of noradrenaline-D3.

TABLE 2 Mass Spectrometry conditions for target analytes and calibrators

4. Preparation of single working solutions for target analyte and calibrator: preparing a mixed target solution of target analytes (metaadrenaline and normetaadrenaline) by dilution from a single stock solution of the target analytes, wherein the concentration of the metaadrenaline and the normetaadrenaline is 1ng/mL respectively;

a mixed calibrant solution of stable isotope labeled analogs (metaepinephrine-D3 and noradrenaline-D3) was prepared from a single stock solution of stable isotope labeled analogs at a concentration of 1ng/mL for metaepinephrine-D3 and noradrenaline-D3, respectively.

5. Calculating a relative response factor: the mixed target solution and mixed calibrant solution were analyzed by UPLC/MS using the specific MRM transitions described in table 1. The 6 pin average peak area from the stable isotope labeled calibrator was compared with the 6 pin average peak area of the target analyte to calculate a relative response factor (as shown in table 3, the ratio of the responses of the target analyte and the stable isotope labeled calibrator at the same concentration was used as the relative response factor).

Table 3 shows the ratio of the responses of the same concentration of target analyte to stable isotope labeled calibrator as relative response factors

6. Preparation of secondary activated carbon treated blank plasma: adding a certain amount of active carbon into a proper amount of normal human blood plasma, shaking, standing overnight, centrifuging to obtain a supernatant, and repeating the above steps once from the step of adding a certain amount of active carbon.

7. Preparation of matrix matching calibrators: a series of matrix-matched calibrators were constructed by adding a series of catecholamine metabolite target analyte solutions (referred to as metaadrenaline and noradrenaline calibrators in this example) to double activated carbon-treated blank plasma, containing target analyte concentrations of 1 pg/mL, 2 pg/mL, 10 pg/mL, 20 pg/mL, 100pg/mL, 200pg/mL, respectively.

5. Plasma samples from 17 normal persons were taken for preliminary assessment of the degree of agreement of the results obtained with the addition of the isotopic calibrators and the matrix-matched calibrators.

5.1 sample preparation by means of a natural isotope calibration curve:

1) taking 200 mu L of plasma sample;

2) add 200. mu.L of 40mM calibrant-containing ammonium acetate solution (wherein calibrant-containing epinephrine-D3 and norepinephrine-D3 are each about 1 ng/mL);

3) vortex mixing for 30S;

4) centrifuging at 14000 rpm at 10 ℃ for 5 min;

5) the supernatant is to be purified;

6) taking a WCX solid phase extraction 96-well plate, and leaching with 200 mu L methanol and 200 mu L water in sequence;

7) putting 350 mu L of supernatant on a solid phase extraction plate;

8) sequentially leaching with 200 μ L of 20mM ammonium acetate solution and 200 μ L of acetonitrile/isopropanol (50/50) solution;

9) eluted with 50 μ L of 2% formic acid (85% acetonitrile);

10) blowing the eluent by nitrogen;

11) redissolving with 40 μ L water;

12) and injecting 10 mu L of the double solution into an ultra performance liquid chromatography-tandem mass spectrometer for analysis.

Analysis was performed using a Waters ACQUITY UPLC I-Class/Xevo TQ-S triple quadrupole tandem mass spectrometry system, samples were analyzed using a Waters ACQUITY UPLC HSS PEP chromatography column (1.8 μm, 2.1 mm. times.100 mm) with a gradient elution with 0.1% aqueous formic acid and acetonitrile in mobile phases A and B, respectively, as shown in Table 4. The running time was 4min, the column temperature was 40 ℃ and the injection volume was 10. mu.L. The Waters Xevo TQ-S triple quadrupole tandem mass spectrometry system was operated in the multiple reaction monitoring mode with mass spectrometry conditions as shown in table 1 and the separation results are shown in fig. 2, 3.

TABLE 4 UPLC gradient conditions for catecholamine metabolite analysis in plasma

Data were collected using Waters MassLynx software, integrated peak areas for each channel in figure 2 were determined using TargetLynx software for the native isotope calibration curve method, Microsoft Excel was introduced for constructing a single internal calibration curve (using linear regression analysis) for each plasma sample, and the analyte concentration in each plasma sample was calculated.

5.2 sample preparation by matrix matching internal standard calibration method:

1) taking 200 mu L of matrix matching calibrators (6) with each concentration and 200 mu L of plasma samples (17) to form 23 samples to be detected;

2) to each test sample was added 200 μ L of 40mM ammonium acetate solution containing an internal standard (where norepinephrine-D3 and norepinephrine-D3 were each about 1ng/mL, and used herein as an internal standard only);

3) vortex mixing for 30S;

4) centrifuging at 14000 rpm at 10 ℃ for 5 min;

5) the supernatant is to be purified;

6) taking a WCX solid phase extraction 96-well plate, and leaching with 200 mu L methanol and 200 mu L water in sequence;

7) putting 350 mu L of supernatant on a solid phase extraction plate;

8) sequentially leaching with 200 μ L of 20mM ammonium acetate solution and 200 μ L of acetonitrile/isopropanol (50/50) solution;

9) eluted with 50 μ L of 2% formic acid (85% acetonitrile);

10) blowing the eluent by nitrogen;

11) redissolving with 40 μ L water;

12) the 23 complex solutions were injected into an ultra performance liquid chromatography-tandem mass spectrometer at 10 μ L for analysis.

Peak area integration and response calculation were performed using TargetLynx software. The analyte concentration in each plasma sample was calculated by calculating the analyte peak area/internal standard peak area ratio to generate a six-point external calibration line.

As a result:

the method comprises the following steps: calibration curve method for natural isotope

The integrated peak area of each channel in FIG. 2 was determined using TargetLynx software, Microsoft Excel was introduced, and the concentration of the added calibrator epinephrine-D3 was known to be 0.909ng/mL, as exemplified by the target analyte epinephrine, and the molecular formula of the calibrator [ C ] was determined in mass spectrometry₁₀H₁₃D₃NO₃]⁺Default quilt²If the substituted atom of H (D) does not contain other isotopes, the abundance ratios of the main peak, the first natural isotope peak and the second natural isotope peak are calculated to be 100%, 11.44% and 1.21%, respectively, and the concentrations of the corresponding main peak, the first natural isotope peak and the second natural isotope are calculated to be 909.0pg/mL, 104.3pg/mL and 11.0pg/mL, respectively. Defining the second natural isotope peak of the calibrator as ST1, the first natural isotope peak of the calibrator as ST2, the main calibrator peak as ST3, and the concentration as X-axis and peak area as Y-axis, a single internal calibration curve (using linear regression analysis) was constructed for each plasma sample, which was then analyzed from the plasma samplesThe target analyte response in (a) is read on the calibration curve for the corresponding concentration. The results are shown in tables 5 and 6.

TABLE 5 determination of epinephrine content in samples using natural isotope calibration curve method

Taking the target analyte noradrenaline as an example, the calibrator was known to be added at a concentration of 0.9625ng/mL noradrenaline-D3 and the molecular formula of the calibrator [ C ] was determined in mass spectrometry₉H₁₁D₃NO₃]⁺Default quilt²If the substituted atom of H (D) does not contain other isotope, the abundance ratio of the main peak, the first natural isotope peak and the second natural isotope peak is calculated to be 100%, 10.34% and 1.10%, respectively, and the concentrations of the corresponding main peak, the first natural isotope peak and the second natural isotope peak are calculated to be 962.5pg/mL, 99.5pg/mL and 10.6pg/mL, respectively.

TABLE 6 determination of norepinephrine content in samples using natural isotope calibration curve method

Since the ionization efficiency of the added calibrant and the target analyte at the mass spectrum end is slightly different, see table 2, the results obtained by the above-mentioned natural isotope calibration curve method need to be divided by the relative response factor to obtain the final calibrated results in tables 5 and 6.

The second method comprises the following steps: matrix matching internal standard calibration method

The peak area was integrated by TargetLynx software, and the response was calculated. The analyte concentration in each plasma sample was calculated by calculating the analyte peak area/internal standard peak area ratio to generate a six point external calibration curve (see fig. 4) and the results are shown in tables 7 and 8.

TABLE 7 determination of epinephrine content in samples using matrix matching internal standard calibration curve method

TABLE 8 determination of norepinephrine content in samples using matrix matching internal standard calibration curve method

Comparison of results: the results obtained using the native isotope calibration curve method of method one were compared to the results obtained using the matrix-matched internal standard calibration curve method of method two (table 9, fig. 5 and 6).

TABLE 9 comparison of results of the calibration curve method for natural isotopes with that for the matrix-matched internal standard

The comparison results (fig. 5 and 6) show that R2 > 0.99, the results are very consistent, and the slope is close to 1. It is shown that the results substantially consistent with the matrix matching internal standard calibration curve method can be obtained using the native isotope calibration curve method.

Therefore, the present embodiment shows the application principle and implementation process of the natural isotope calibration curve method. The calibrant is a stable isotope analog of the target compound, and the points on the calibration curve are the major peak of the calibrant, the first natural isotope peak of the calibrant, and the second natural isotope peak of the calibrant, respectively. The compounds have extremely similar molecular structures with target compounds, and can be used for calibrating the recovery rate loss in the pretreatment process and simultaneously calibrating the sampling error and the matrix effect in the analysis process. The only difference is that the ionization efficiencies of the calibrant and target analyte at the ion source end are slightly different, so the results need to be calibrated with relative response factors. The results show that the results obtained by the natural isotope calibration curve method are basically consistent with the results obtained by the traditional matrix matching internal standard calibration curve method, and the former method has the convenience of not preparing blank matrix.

Example 2: 25-hydroxyvitamin D in human serum samples₃Content analysis of (2)

As described in example 1, preparation of a blank matrix is particularly difficult and expensive when the target analyte is present in the sample matrix in a relatively high amount. 25-hydroxy vitamin D₃The content of the standard substance in normal serum is high, so that the conventional LC-MS/MS mass spectrum quantitative method generally adopts 1% bovine serum albumin solution to replace blank matrix to prepare a series of calibrators with concentration, and then internal standards with the same amount are added into the calibrators and samples to be detected to correct matrix effect, so that 25-hydroxyvitamin D in the samples is quantified₃. This example describes 25-hydroxyvitamin D in human serum samples₃Wherein a known amount of a stable isotope-labeled analog is added to each sample, a calibration curve is constructed using the stable isotope and a natural isotope thereof having a known abundance, and 25-hydroxyvitamin D in the sample is quantitatively analyzed₃The content of (a). And simultaneously comparing the obtained result with the result obtained by a matrix matching internal standard calibration method. This example demonstrates that by using a stable isotope labeled calibrator and its natural isotope of known abundance, 25-hydroxyvitamin D in human serum samples can be accurately and accurately quantified₃。

25-hydroxyvitamin D in human serum samples₃The content determination method comprises the following specific steps:

1. preparation of stable isotope labeled calibrators: commercially available stable isotope-labeled analogs of the target analyte, in this example 25-hydroxyvitamin D, were used as calibrators₃The corresponding commercially available stable isotope labeled analogue is 25-hydroxyvitamin D₃D3, see fig. 7.

2. Preparation of individual stock solutions of target analyte and calibrator: preparing the solid powder of the target analyte and the stable isotope labeling calibrator respectively, and preparing stock solutions by using methanol as a solvent, wherein the concentration of each stock solution is 0.1mg/mL respectively.

3. Optimizing mass spectrum conditions: from the single stock solutions of the target analyte and calibrator, dilutions were made separately to give 0.1. mu.g/mL of the singleThe solutions were used to optimize the mass spectrometry conditions and the results are shown in Table 10. Wherein 25-hydroxy vitamin D₃-D3-F1 is 25-hydroxyvitamin D₃The first natural isotope of D3, 25-hydroxyvitamin D₃-D3-F2 is 25-hydroxyvitamin D₃-a second natural isotope of D3.

TABLE 10 Mass Spectrometry conditions for target analytes and calibrators

4. Preparation of single working solutions for target analyte and calibrator: preparation of target analyte (25-hydroxyvitamin D) from a single stock solution of target analyte and its stable isotope labeled analog by dilution₃) And stable isotope labeled analogue (25-hydroxy vitamin D)₃-D3), in which solution 25-hydroxyvitamin D is present₃And 25-hydroxyvitamin D₃The concentrations of-D3 were all 10 ng/mL.

5. The relative response factors were calculated and the mixed solutions were analyzed by UPLC/MS using the specific MRM transitions described in table 9. The 6 pin average peak area from the target analyte was compared to the 6 pin average peak area of the stable isotope labeled analogue and the relative response factor was calculated (table 11).

Table 11 shows the ratio of the responses of the same concentration of target analyte to stable isotope labeled calibrator as relative response factors

6. Preparation of 1% bovine serum albumin solution: and (3) taking 1g of bovine serum albumin to a 100mL volumetric flask, adding phosphate buffer solution to dissolve, fixing the volume to a scale, and shaking up to obtain the bovine serum albumin.

7. Preparation of matrix matching calibrators: mixing a series of 25-hydroxy vitamin D₃Target analyte solutions were added to a 1% bovine serum albumin solution to construct a series of matrix-matched calibrators containingThe target analyte concentrations were 5ng/mL,20ng/mL, and 50ng/mL, respectively.

8. Serum samples from 20 normal persons were taken for preliminary assessment of the degree of agreement of the results obtained with the addition of the isotopic calibrators and the matrix-matched calibrators.

8.1 sample preparation is carried out by adopting a natural isotope calibration curve method:

1) taking 200 μ L of matrix matching calibrator or serum sample;

2) adding 200 μ L of 25-hydroxy vitamin D containing calibrator₃-a methanol + acetonitrile (1 + 1) solution of D3 (containing calibrant 25-hydroxyvitamin D)₃-D3 is 50 ng/mL);

3) vortex mixing for 30 s;

4) adding 1mL of n-hexane;

5) vortex mixing for 5 min;

6) centrifuging at 14000 rpm at 10 ℃ for 5 min;

7) taking 0.8mL to 2mL of supernatant, and blowing nitrogen at room temperature until the supernatant is dry;

8) adding 100 μ L of 85% methanol water solution (containing 0.1% formic acid) for redissolution;

9) vortex mixing for 30 s;

10) centrifuging at 14000 rpm at 10 ℃ for 3 min;

11) sucking supernatant to 96-hole sample feeding plate;

12) and injecting 20 mu L of the supernatant into an ultra performance liquid chromatography-tandem mass spectrometer for analysis.

The analysis was performed using a Waters ACQUITY UPLC I-Class/Xevo TQ-S triple quadrupole tandem mass spectrometry system, and samples were analyzed using a Waters ACQUITY UPLC HST 3 column (1.8 μm, 2.1 mm. times.100 mm) with a gradient elution with 0.1% aqueous formic acid and 0.1% methanol formic acid as mobile phases A and B, respectively, as shown in Table 12. The running time was 7.5min, the column temperature was 40 ℃ and the injection volume was 20. mu.L. The Waters Xevo TQ-S triple quadrupole tandem mass spectrometry system was operated in the multiple reaction monitoring mode with mass spectrometry conditions as shown in table 10 and the separation results are shown in figure 8.

TABLE 12 serum 25-hydroxyvitamin D₃UPLC gradient conditions of analysis

Data were collected using Waters MassLynx software, integrated peak areas for each channel in figure 8 were determined using TargetLynx software for the native isotope calibration curve method, Microsoft Excel was introduced for constructing a single internal calibration curve (using linear regression analysis) for each plasma sample, and the analyte concentration in each plasma sample was calculated.

5.2 sample preparation by adopting a matrix matching internal standard calibration method:

1) taking 200 mu L of matrix matching calibrators (3) with each concentration and 200 mu L of serum samples (20) to form 23 samples to be detected;

2) adding 200 μ L of 25-hydroxy vitamin D into each sample₃-a methanol + acetonitrile (1 + 1) solution of D3 (containing 25-hydroxyvitamin D)₃-D3 at 50ng/mL, used here only as an internal standard);

3) vortex mixing for 30 s;

4) adding 1mL of n-hexane;

5) vortex mixing for 5 min;

6) centrifuging at 14000 rpm at 10 ℃ for 5 min;

7) taking 0.8mL to 2mL of supernatant, and blowing nitrogen at room temperature until the supernatant is dry;

8) adding 100 μ L of 85% methanol water solution (containing 0.1% formic acid) for redissolution;

9) vortex mixing for 30 s;

10) centrifuging at 14000 rpm at 10 ℃ for 3 min;

11) sucking supernatant to 96-hole sample feeding plate;

12) 20 mu L of each complex solution is injected into an ultra high performance liquid chromatography-tandem mass spectrometer for analysis.

Peak area integration and response calculation were performed using TargetLynx software, and a three-point external calibration line was generated by calculating the analyte peak area/internal standard peak area ratio, thereby calculating the analyte concentration in each serum sample.

As a result:

the method comprises the following steps: calibration curve method for natural isotope

Mapping with TargetLynx software7, the integrated peak area of each channel, introduced into Microsoft Excel, known as the added calibrator, 25-hydroxyvitamin D₃Concentration of-D3 was 50ng/mL, molecular formula [ C ] of calibrant measured in mass spectrometry₂₇H₄₂D₃O₂]And the default is that the atom substituted by 2H (D) contains no other isotope, the abundance ratio of the main peak, the first and second natural isotope peaks is calculated to be 100%, 29.75% and 4.68%, respectively, and the concentrations of the corresponding main peak, the first and second natural isotope peaks are calculated to be 50ng/mL, 14.875ng/mL and 2.34 ng/mL, respectively. The second natural isotope peak of the calibrator was defined as ST1, the first natural isotope peak of the calibrator was defined as ST2, the main calibrator peak was defined as ST3, the concentration was taken as the X-axis and the peak area was taken as the Y-axis, a single internal calibration curve (using linear regression analysis) was constructed for each plasma sample, and the corresponding concentrations were read on the calibration curve according to the target analyte response in the serum sample. The results are shown in Table 13.

TABLE 13 determination of 25-hydroxyvitamin D in samples using natural isotope calibration Curve method₃In an amount of

Since the ionization efficiency of the added calibrant and the target analyte at the mass spectrum end are slightly different, see table 11, the result obtained by the above-mentioned natural isotope calibration curve method needs to be divided by the relative response factor to obtain the final result (i.e. the corrected result in table 13).

The second method comprises the following steps: matrix matching internal standard calibration method

The peak area was integrated by TargetLynx software, and the response was calculated. The analyte concentration in each serum sample was calculated by calculating the analyte peak area/internal standard peak area ratio to generate a three-point external calibration curve (see fig. 9), the results of which are shown in table 14.

TABLE 14 determination of 25-hydroxyvitamin D in samples using matrix matching internal standard calibration curve method₃In an amount of

The results obtained using the natural isotope calibration curve method of method one were compared with the results obtained using the matrix matching internal standard calibration method of method two, see fig. 10. The results obtained by the two methods are approximate, and the regression analysis r²>0.99 and the slope is close to 1.

Example 3: analysis for determining content of aldosterone in human plasma sample by pre-column derivatization method

This example describes the analysis of the aldosterone content in human plasma samples, adding to each sample a known amount of an analogue labelled with a stable isotope, using this stable isotope and its natural isotope of known abundance to construct a calibration curve, and quantifying the aldosterone content in the sample. And comparing the obtained result with the result obtained by the conventional matrix calibration curve method. The latter uses 1% bovine serum albumin/phosphoric acid buffer solution to prepare a series of calibrators with concentration, and then corrects recovery and matrix effect by adding internal standard in the calibrators and samples, thereby quantifying the content of aldosterone in the samples. Since the pretreatment of this example involves a derivatization operation, the consistency of physicochemical properties between the stable isotope and the target analyte is further verified, and the results show that the aldosterone content in the sample can be accurately and precisely quantified using a natural isotope calibration curve method.

The method for measuring the content of aldosterone in the human plasma sample comprises the following specific steps:

1. preparation of stable isotope labeled calibrators: commercially available stable isotope labeled analogs of the target analyte, in this example aldosterone, corresponding to commercially available stable isotope labeled analogs such as aldosterone-D4, aldosterone-D7 or aldosterone-D8, were used as calibrators, and aldosterone-D8 was selected for this experiment, see fig. 11.

2. Preparation of individual stock solutions of target analyte and calibrator: preparing the solid powder of the target analyte and the stable isotope labeling calibrator respectively, and preparing stock solutions by using methanol as a solvent, wherein the concentration of each stock solution is 0.1mg/mL respectively.

3. Optimizing mass spectrum conditions: from the individual stock solutions of the target analyte and calibrator, dilutions were made separately to give 0.1. mu.g/mL of individual solutions for optimization of the fumbling mass spectrometry conditions, the results are given in Table 15. Wherein aldosterone-D8 derivative-F1 is a first natural isotope of an aldosterone-D8 derivative and aldosterone-D8 derivative-F2 is a second natural isotope of an aldosterone-D8 derivative.

TABLE 15 Mass Spectrometry conditions for target analytes and calibrators

4. Preparation of single working solutions for target analyte and calibrator: a solution of the target analyte was prepared by dilution from a single stock solution of the target analyte, with an aldosterone concentration of 1 ng/mL. A working solution of calibrator was prepared by dilution from a single stock solution of calibrator with aldosterone-D8 concentration of 1 ng/mL.

5. Calculating a relative response factor: taking 10uL of 1ng/mL of working solution of aldosterone and aldosterone-D8 respectively, and carrying out the same operation from the step 9) of sequentially adding acetic acid … according to the pretreatment scheme of the following step 8.1 to obtain the derivative products of the target analyte and the calibrator. The target and calibrator derivatization products were analyzed by UPLC/MS using the specific MRM transitions described in table 14. The 6 pin average peak area from the target analyte was compared to the 6 pin average peak area of the calibrator and the relative response factor was calculated (table 16).

Table 16 shows the ratio of the responses of the same concentration of target analyte to stable isotope labeled calibrator as relative response factors

6. Preparation of 1% bovine serum albumin solution: and (3) taking 1g of bovine serum albumin to a 100mL volumetric flask, adding phosphate buffer solution to dissolve, fixing the volume to a scale, and shaking up to obtain the bovine serum albumin.

7. Preparation of matrix matching calibrators: 1ng/mL of aldosterone target analyte solution is diluted into a series of simulated matrix-matched calibrators by taking 1% bovine serum albumin solution as a diluent, wherein the target analyte concentrations are 2, 5, 10, 20, 50 and 100pg/mL respectively.

8. Plasma from 8 normal persons was taken to evaluate the degree of agreement between the results obtained by the native isotope calibration curve method and the matrix matching calibration method.

8.1 sample preparation is carried out by adopting a natural isotope calibration curve method:

1) putting 200 mu L of plasma sample into a 2mL centrifuge tube;

2) add 20. mu.L of a solution containing calibrator aldosterone-D8 (with aldosterone-D8 concentration of 1 ng/mL);

3) vortex mixing for 30 s;

4) 1mL of methyl tert-butyl ether was added;

5) shaking for 3 min;

6) centrifuging at 14000 rpm at 10 ℃ for 5 min;

7) the supernatant was aspirated as much as possible and transferred to another 1.5mL centrifuge tube

8) Blow-drying with nitrogen

9) Sequentially adding acetic acid and a derivatization reagent

10) Reacting at room temperature for 10min

11) Blowing by using nitrogen, and redissolving the initial mobile phase solution by 50 uL;

12) 20 mu L of each complex solution is injected into an ultra high performance liquid chromatography-tandem mass spectrometer for analysis.

The analysis was carried out using a Waters ACQUITY UPLC I-Class/Xevo TQ-S triple quadrupole tandem mass spectrometry system, and samples were analyzed using a Waters ACQUITY UPLC BEH C8 column (1.8 μm, 2.1 mM. times.100 mM) with a gradient elution with 0.1% aqueous formic acid (containing 2mM ammonium formate) and methanol as mobile phases A and B, respectively, as shown in Table 17. The running time was 5.5min, the column temperature was 40 ℃ and the injection volume was 20. mu.L. The Waters Xevo TQ-S triple quadrupole tandem mass spectrometry system was operated in the multiple reaction monitoring mode with mass spectrometry conditions as shown in table 15 and the separation results are shown in figure 12.

TABLE 17 UPLC gradient conditions for catecholamine metabolite analysis in plasma

Data were collected using Waters MassLynx software, integrated peak areas for each channel in figure 12 were determined using TargetLynx software for the native isotope calibration curve method, Microsoft Excel was introduced for constructing a single internal calibration curve (using linear regression analysis) for each plasma sample, and the target analyte concentration in each plasma sample was calculated.

8.2 for the matrix matching internal standard calibration method, adopting the matrix matching internal standard calibration method to prepare samples:

1) taking 200 mu L of matrix matching calibrators (6) with each concentration and 200 mu L of plasma samples (8) to form 14 samples to be detected;

2) to each test sample was added 20. mu.L of a solution containing the calibrator aldosterone-D8 (where the concentration of aldosterone-D8 is 1ng/mL and is used herein as an internal standard only);

3) vortex mixing for 30 s;

4) 1mL of methyl tert-butyl ether was added;

5) shaking for 3 min;

6) centrifuging at 14000 rpm at 10 ℃ for 5 min;

7) the supernatant was aspirated as much as possible and transferred to another 1.5mL centrifuge tube

8) Blow-drying with nitrogen

9) Sequentially adding acetic acid and a derivatization reagent

10) Reacting at room temperature for 10min

11) Blowing by using nitrogen, and redissolving the initial mobile phase solution by 50 uL;

12) 20 mu L of each complex solution is injected into an ultra high performance liquid chromatography-tandem mass spectrometer for analysis.

Data were collected using Waters MassLynx software, and peak area integration and response calculation were performed using TargetLynx software for the matrix matching internal standard calibration method. A six-point calibration curve was generated by calculating the target analyte peak area/internal standard peak area ratio, thereby calculating the target analyte concentration in each plasma sample.

As a result:

the method comprises the following steps: calibration curve method for natural isotope

The integrated peak area of each channel in FIG. 12 was determined using TargetLynx software, Microsoft Excel was introduced, and the concentration of the added calibrant aldosterone-D8 was known to be 100pg/mL (reduced to sample volume), and after pretreatment, the derivative of the calibrant aldosterone-D8 had the formula [ C28H27D8O5N3 ]]⁺Default quilt²The H substituted atom contains no other isotope and its main peak is calculated, the abundance ratio of the first and second natural isotopes is 100%, 31.88%, 5.94%, respectively, and the concentrations of the first and second natural isotopes are 100pg/mL, 31.88pg/mL and 5.94pg/mL, respectively. Defining the second natural isotope of the calibrator as ST1, the first natural isotope of the calibrator as ST2, the major peak of the calibrator as ST3, and the concentration as X-axis and peak area as Y-axis, a single internal calibration curve (using linear regression analysis) was constructed for each plasma sample, and the corresponding concentrations were read on the calibration curve according to the target analyte response in the plasma sample. The results are shown in Table 18.

TABLE 18 determination of aldosterone content in samples using natural isotope calibration Curve method

As described in examples 1 and 2, since the ionization efficiency of the added calibrant and the target analyte at the mass spectrum end is slightly different, the result obtained by the above-mentioned natural isotope calibration curve method is divided by the relative response factor to obtain the final calibrated result, as shown in table 16.

The second method comprises the following steps: matrix matching internal standard calibration method

The peak area was integrated by TargetLynx software, and the response was calculated. The analyte concentration in each plasma sample was calculated by calculating the target analyte peak area/internal standard peak area ratio to generate a six point external calibration curve (fig. 13) and the results are shown in table 19.

TABLE 19 determination of aldosterone content in samples using matrix matching internal standard calibration curve method

Comparison of results: the results obtained using the native isotope calibration curve method were compared to those obtained using the matrix-matched internal standard calibration curve method (table 20).

TABLE 20 comparison of results of the calibration curve method for natural isotopes with that for the matrix-matched internal standard

The comparison result shows that the two groups of data are relatively close. Because the group of data is in the ultra trace analysis range, the matrix matching calibration curve method adopts the practical matrix simulated by the bovine serum albumin, and a certain difference still exists between the practical matrix and the practical sample, and in addition, the target compound and the calibrator are subjected to derivatization reaction in the sample pretreatment process. The results listed in the table are also within acceptable ranges. The result which is basically similar to that of the matrix matching internal standard calibration curve method can be obtained by using the natural isotope calibration curve method, and the possible reason of slight difference of the result and the result obtained by which method can be further discussed later are closer to theoretical reality.

Example 4: application of high-resolution mass spectrometry in monitoring content of tacrolimus in whole blood sample of therapeutic drug

This example describes the application of the natural isotope calibration curve method to therapeutic drug monitoring of tacrolimus in human whole blood samples to evaluate the accuracy and precision of the method with two sets of quality control samples of low and high concentration. Adding a known amount of stable isotope labeled analogue into each sample, constructing a calibration curve by using the stable isotope and natural isotope with known abundance thereof, and quantitatively analyzing the content of tacrolimus in the sample. And simultaneously comparing the obtained result with the result obtained by the conventional matrix matching internal standard calibration curve method. Because tacrolimus is an exogenous drug, the matrix matching internal standard calibration curve method can adopt the blank matrix which is the same as that used for preparing the quality control sample to prepare a series of calibrators with concentration, so the result obtained by the matrix matching calibration curve method is basically consistent with the theoretical value. The accuracy of the natural isotope calibration curve method can be further evaluated. In addition, the method adopts high-resolution mass spectrum as a detector, and can be used for evaluating the applicability of a natural isotope calibration curve method between different mass spectrum platforms. The final result shows that the content of the tacrolimus in the sample can be accurately and accurately quantified by using a natural isotope calibration curve method.

The content determination method of tacrolimus in the human whole blood sample comprises the following specific steps:

1. preparation of stable isotope labeled calibrators: as calibrator, a commercially available stable isotope-labeled analogue of the target analyte, in this example tacrolimus, was used, and the corresponding commercially available stable isotope-labeled analogue was tacrolimus-¹³CD2, see fig. 14.

2. Preparation of individual stock solutions of target analyte and calibrator: respectively taking solid powder of the target analyte and the stable isotope labeling calibrator, and preparing stock solutions by using methanol as a solvent, wherein the concentration of each stock solution is 1 mg/mL.

3. Preparation of single working solutions for target analyte and calibrator: a solution of the prepared target analyte was diluted from a single stock solution of the target analyte, wherein the concentration of tacrolimus was 10 μ g/mL. Dilution of the working solutions for the preparation of calibrators from a single stock solution of calibrators, in which tacrolimus-¹³The concentration of CD2 was 5. mu.g/mL.

4. Calculating a relative response factor: taking 10 mu g/mL of tacrolimus and tacrolimus-¹³CD2 solution, diluted with acetonitrile to obtain tacrolimus and tacrolimus-¹³And the CD2 is a 10ng/mL solution, thus obtaining the CD-modified starch. To examine the relative response factor between the two.

Because the high-resolution mass spectrum is used as a detector, the scanning mode is data-independent full scanning, the mass spectrum condition is not required to be optimized additionally, and the target object and the calibrator are analyzed through the UPLC/MS/MS. The 6 pin average peak area from the target analyte was compared to the 6 pin average peak area of the stable isotope labeled calibrator and the relative response factor was calculated (table 21).

Table 21 shows the ratio of the responses of stable isotope labeled calibrator and target analytes at the same concentrations as the relative response factors

5. Preparation of matrix matching calibrators: a blank whole blood sample was used as a diluent to dilute a 10. mu.g/mL solution of tacrolimus target analyte into a series of matrix-matched calibrators containing target analyte concentrations of 10, 20, 50, 100, 200, 500ng/mL, respectively. After sample pretreatment, the matrix solution is diluted by about 10 times, and the concentrations of the converted target analytes are 1, 2, 5, 10, 20 and 50ng/mL respectively.

6. Preparing a high-low concentration quality control sample: the same blank whole blood sample was used as the diluent to dilute 10. mu.g/mL tacrolimus target analyte solutions to low and high concentration quality control samples of 50 and 200 ng/mL. After sample pretreatment, the concentration of the target analyte in the converted high-concentration and low-concentration quality control sample is 5ng/mL and 20ng/mL respectively.

7. And taking quality control samples with high and low concentrations in triplicate, wherein 6 samples are used for evaluating the consistency degree of results obtained by a natural isotope calibration curve method and a matrix matching internal standard calibration method.

7.1 sample preparation is carried out by adopting a natural isotope calibration curve method:

1) taking 5 μ L of 5 μ g/mL tacrolimus-¹³Placing the CD2 calibrator solution in a 2mL centrifuge tube, and blowing the liquid by nitrogen;

2) adding 50 mu L of whole blood quality control sample

3) Adding 100 mu L0.1M zinc sulfate solution;

4) adding 400 mu L of acetonitrile;

5) shaking for 3 min;

6) centrifuging at 14000 rpm for 5min at 10 ℃;

7) sucking 100 mu L of supernatant liquid and transferring the supernatant liquid to a full recovery sample injection vial;

8) and 5 mu L of the supernatant is injected into an ultra performance liquid chromatography-tandem mass spectrometer for analysis.

Analysis was performed using a Waters ACQUITY UPLC I-Class/Synapt G2-S Qtof high resolution Mass Spectrometry system, analyzing samples using a Waters ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 mM. times.50 mM) with a gradient elution with 0.1% aqueous formic acid (containing 2mM ammonium formate) and 0.1% methanol formic acid (containing 2mM ammonium formate) as mobile phases A and B, respectively, as shown in Table 22. The running time was 2.5min, the column temperature was 40 ℃ and the injection volume was 5. mu.L. The Waters synapse G2-S Qtof high resolution mass spectrometry system employs a data-independent full scan mode for data acquisition, and can extract the mass spectra of the response according to the precise molecular weight of the target compound, with the results shown in fig. 15.

TABLE 22 UPLC gradient conditions for tacrolimus analysis in whole blood samples

Data were collected using Waters MassLynx software, integrated peak areas for each channel in figure 14 were determined using TargetLynx software for the native isotope calibration curve method, Microsoft Excel was introduced for constructing a single internal calibration curve (using linear regression analysis) for each plasma sample, and the analyte concentration in each plasma sample was calculated.

7.2 for the matrix matching internal standard calibration method, adopting the matrix matching internal standard calibration method to prepare samples:

1) taking 5 μ L of 5 μ g/mL tacrolimus-¹³Placing the CD2 calibrator solution in a 2mL centrifuge tube, and blowing the liquid by nitrogen;

2) adding 50 mu L of matrix matching calibrators (6) with each concentration and high-low concentration quality control samples (each in triplicate, 6) to form 12 samples to be tested;

3) adding 100 mu L0.1M zinc sulfate solution;

4) adding 400 mu L of acetonitrile;

5) shaking for 3 min;

6) centrifuging at 14000 rpm for 5min at 10 ℃;

7) sucking 100 mu L of supernatant liquid and transferring the supernatant liquid to a full recovery sample injection vial;

8) and 5 mu L of the supernatant is injected into an ultra performance liquid chromatography-tandem mass spectrometer for analysis.

For the matrix matching internal standard calibration method, peak area integration and response calculation were performed using TargetLynx software. The analyte concentration in each sample was calculated by calculating the analyte peak area/internal standard peak area ratio to generate a six point external calibration line.

As a result:

the method comprises the following steps: calibration curve method for natural isotope

The integrated peak area of each channel in FIG. 15 was determined using the TargetLynx software and introduced into Microsoft Excel, knowing that the added calibrator, tacrolimus-¹³CD2 at a concentration of 50ng/mL (reduced to sample volume) and a calibrator of the formula [ C28H27D8O5N3]⁺Default quilt¹³C and²the H-substituted atom contains no other isotope, and excluding the abundance calculation, the main peaks are calculated, the abundance ratios of the first and second natural isotopes are 100%, 48.51%, and 13.98%, respectively, and the corresponding main peaks, the concentrations of the first and second natural isotopes are 50ng/mL, 24.255ng/mL, and 6.99ng/mL, respectively. Defining the second natural isotope of the calibrator as ST1, the first natural isotope of the calibrator as ST2, the main peak of the calibrator as ST3, constructing a single internal calibration curve (using linear regression analysis) for each quality control sample with the concentration as X-axis and the peak area as Y-axis, and reading the corresponding concentration on the calibration curve according to the target analyte response in the quality control sample. The results are shown in Table 23.

TABLE 23 determination of Tacrolimus content in samples by Natural isotope calibration Curve method

As described in examples 1 and 2, since the ionization efficiency of the added calibrant and the target analyte at the mass spectrum end is slightly different, the result obtained by the above-mentioned natural isotope calibration curve method is divided by the relative response factor to obtain the final calibrated result, as shown in table 21.

The second method comprises the following steps: matrix matching internal standard calibration method

The peak area was integrated by TargetLynx software, and the response was calculated. The analyte concentration in each plasma sample was calculated by calculating the analyte peak area/internal standard peak area ratio to generate a six point external calibration curve (fig. 16) and the results are shown in table 24.

TABLE 24 determination of Tacrolimus content in samples by matrix matching internal standard calibration curve method

Comparison of results: the results obtained using the native isotope calibration curve method were compared to the results of quality control samples obtained using the matrix-matched internal standard calibration curve method (table 25).

TABLE 25 comparison of results of the calibration curve method for natural isotopes with that for the matrix-matched internal standard

The comparison result shows that the quality control data results of the two methods are basically consistent. In this embodiment, the same blank matrix is used for the calibration curve of the quality control sample and the matrix, so the data of the quality control sample should be accurate. Meanwhile, the embodiment verifies the feasibility of applying the natural isotope labeling method to a high-resolution mass spectrum system, and the quantitative analysis is carried out by adopting a full-scanning mode, so that the excimer ion peak of the target compound is directly analyzed without additionally optimizing mass spectrum conditions.

The method of the present invention is also applicable to the quantitative analysis of other organic molecules including at least 3 carbon atoms based on the same principle as the above-described examples.

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.