WO2022193697A1 - 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 - Google Patents

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 Download PDF

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WO2022193697A1
WO2022193697A1 PCT/CN2021/130578 CN2021130578W WO2022193697A1 WO 2022193697 A1 WO2022193697 A1 WO 2022193697A1 CN 2021130578 W CN2021130578 W CN 2021130578W WO 2022193697 A1 WO2022193697 A1 WO 2022193697A1
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calibrator
target analyte
sample
natural isotope
calibration curve
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郏征伟
陈铭
谭晓杰
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裕菁科技(上海)有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/64—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using wave or particle radiation to ionise a gas, e.g. in an ionisation chamber
    • 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
    • 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
    • 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

Definitions

  • 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.
  • the main quantitative methods include external standard method, internal standard method and standard addition method. According to different calibration methods, it can be divided into single-point calibration, multi-point calibration and calibration curve method; according to the different solutions used to prepare calibrators, it can be divided into pure solution standards and matrix-matched standards. Among them, the results of matrix-matched internal standard calibration curve method and standard addition method are the most accurate.
  • the standard addition method is mostly used for the confirmation of results due to the difficulty in estimating the amount of the target compound in the sample and the large workload (at least three samples need to be prepared for each sample). Therefore, the matrix-matched internal standard calibration curve method is the most commonly used trace analysis method.
  • the advantage of this method is that the internal standard compound is added during the pretreatment process to parallel the loss of the target compound during the pretreatment process.
  • the internal standard compound is generally selected from the stable isotope-labeled analog of the target compound. Because the two have very similar physical and chemical properties, the internal standard can not only calibrate the loss of target compounds in the pretreatment process, but also calibrate the matrix effect in the process of mass spectrometry; but the main disadvantage of this method is that it is difficult to obtain preparation standards The required blank matrix, especially when the target substance to be analyzed is endogenous, and there are many kinds of sample matrices with great individual differences, it is particularly difficult to obtain the blank matrix.
  • the patent document with application number 201280036810.1 describes a method for quantifying a target analyte in a sample, comprising obtaining a mass spectrometry signal, the mass spectrometry signal including a first calibrator signal, including a second calibrator signal, and possibly containing from A target analyte signal for a single sample comprising a first known amount of the first calibrator, comprising a second known amount of the second calibrator, and potentially comprising the target analyte.
  • the first known quantity and the second known quantity 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 using the first calibrator signal, the second calibrator signal, and the target analyte signal to quantify the target analyte in the single sample.
  • the drawback of this method is that it must contain at least two calibrators, and the first calibrator, the second calibrator and more calibrators are different stable isotope-labeled analogs of the target analyte (analyzed by testosterone).
  • the added calibrators are D2-testosterone, D3-testosterone, D5-testosterone mixed calibrators at different concentrations).
  • each element generally has multiple isotopes
  • the peak intensity of each isotope can be calculated according to the following binomial expansion formula.
  • a and b, c and d are the natural abundances of the M and M+1 isotopes of the first and second elements, respectively (customarily, they can also be called light isotopes and heavy isotopes, for example, taking C as an example, a and b are the natural abundances of 12 C and 13 C, respectively), and n and m are the atomic numbers of the first and second elements, respectively.
  • Most of the current mainstream mass spectrometers are equipped with tool software to calculate the isotopic abundance ratio of organic compounds, as long as the molecular formula of the compound is entered, so the calculation principle of the abundance ratio will not be repeated here.
  • mass spectrometric analysis of stable isotope analogs When mass spectrometric analysis of stable isotope analogs is performed, mass spectral signals for a series of natural isotopes of the stable isotope analogs are obtained simultaneously. However, no one has seen any further utilization of the mass spectrometry signal. There is also no report in the prior art that the mass spectral signals of a series of natural isotopes of stable isotope analogs obtained by mass spectrometry are used in the quantification of target analytes.
  • the purpose of the present invention is to provide a method for quantifying target analytes in a sample by mass spectrometry.
  • the present invention provides a natural isotope calibration curve method for quantifying a target analyte in a sample, comprising the following steps:
  • step C carry out mass spectrometry analysis on the sample to be tested processed in step B to obtain the mass spectrum signal of the calibrator and the natural isotope of the calibrator, and the mass spectrum signal of the target analyte in the sample to be tested;
  • step A there are one or more target analytes, and a known amount of calibrator is added to the sample to be tested for each target analyte.
  • the pretreatment method is selected from solid phase extraction, solid-liquid extraction, liquid-liquid extraction, protein precipitation, direct dilution, solvent extraction, salting out, chemical separation, Any of concentration method, adsorption chromatography, partition chromatography, ion exchange chromatography, and masking method.
  • the natural isotope mass spectrometry signal of the calibrator is one or more.
  • the standard curve drawing method in step D is as follows:
  • the mass-to-charge ratio of the calibrator to the natural isotope of the calibrator is greater than or equal to 1 Dalton mass; when high-resolution mass spectrometry is used, the calibrator The mass-to-charge ratio of the natural isotope of the calibrator is greater than or equal to 0.05 Dalton mass number.
  • the calibrator and target analyte differ from each other in mass spectrometry analysis by at least 2 Dalton masses.
  • the sample to be tested includes any one of biological samples, environmental samples, food samples, synthetic samples, pharmaceutical samples, chemical samples, clinical chemical samples, forensic samples, pharmacological samples, and agricultural samples;
  • the biological sample includes any of plasma, serum, whole blood, urine, tissue, cerebrospinal fluid, sweat, saliva, hair, and skin.
  • the target analyte is an organic molecule comprising at least 3 carbon atoms.
  • the calibrator is a stable isotope-labeled analog of the target analyte, obtained after at least one atom of the target analyte is replaced by its stable isotope
  • stable isotopes include but are not limited to 2 H, 11 Any one or more of B, 13 C, 15 N, 17 O, 18 O, 33 S, 34 S, and 36 S.
  • the ion source used in the mass spectrometry method includes but is not limited to electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), matrix-assisted laser desorption ionization (MALDI), desorption electrospray ionization (DESI), electron impact source (EI), chemical ionization source (CI), mass analyzers include but are not limited to quadrupole mass analyzers, ion trap mass analyzers, sector magnetic mass spectrometers, time-of-flight mass analyzers, Any one of electrostatic field orbitrap mass analyzer and Fourier transform ion cyclotron resonance mass analyzer or a combination of two or more of them; the detection mode of the mass spectrometer is selected from full scan mode, product ion detection mode, mother ion detection mode any one of ion detection mode, multiple reaction detection mode, neutral loss scan, data-dependent scan mode, and data-independent scan mode;
  • ESI electrospray ionization
  • the mass spectrometry analysis also includes the step of separating by chromatography; the chromatography is selected from liquid chromatography, gas chromatography, capillary electrophoresis, affinity chromatography, immunoaffinity chromatography, and supercritical fluid chromatography method, any of the ion mobility methods.
  • step D the step of calibrating the content of the target analyte in the sample to be tested is further included; the target analyte and the stable isotope-labeled calibrator of the same concentration are injected into the mass spectrometer for analysis, and the target analyte and the stable isotope-labeled calibrator are injected into the mass spectrometer for analysis.
  • the response ratio of the calibrator is used as the relative response factor; when the actual sample is analyzed, the content result of the target analyte should be multiplied by the relative response factor as the final result.
  • the present invention has the following beneficial effects:
  • the present invention draws a standard curve using stable isotope-labeled analogs of the target analyte and a series of natural isotopes of the stable isotope-labeled analogs as calibrators for the first time, so as to quantify the 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, which can technically be called a single-sample self-calibration method.
  • results obtained by the method of the present invention are basically consistent with the results obtained by the matrix-matched internal standard calibration curve method, which shows the reliability and accuracy of the method of the present invention; and the method of the present invention does not require any additional blank matrix, which greatly simplifies the detection. procedures, further reducing the cost of testing.
  • the method of the present invention only needs to use one stable isotope-labeled analog for one target analyte, avoids the need to synthesize multiple stable isotope-labeled analogs, and greatly saves the detection cost; and for only one stable isotope-labeled analog
  • the target analyte of the analog can be directly quantified by the method of the present invention, which greatly expands the application range.
  • Figure 1 shows the structures of catecholamine metabolites (metanephrine and norepinephrine) and their stable isotope-labeled analogs (metanephrine-D3 and norepinephrine-D3) in Example 1;
  • Fig. 2 is the mass spectrum of metanephrine detected in Example 1 (from bottom to top, the order is the target compound, the calibrator, the first natural isotope of the calibrator, and the mass spectrum signal of the second natural isotope of the calibrator);
  • Figure 3 is the mass spectrum of norepinephrine detected in Example 1 (from bottom to top, the order is the target compound, the calibrator, the first natural isotope of the calibrator, the mass spectrum signal of the second natural isotope of the calibrator) ;
  • Fig. 4 is the internal standard calibration curve of metanephrine and norepinephrine matrix matching in Example 1;
  • Fig. 5 is the result comparison of two kinds of calibration methods of metanephrine in Example 1;
  • Fig. 6 is the result comparison of two kinds of calibration methods of norepinephrine in embodiment 1;
  • Figure 7 is the structure of 25-hydroxyvitamin D3 and its stable isotope-labeled analog (25-hydroxyvitamin D3-D3) in Example 2;
  • Fig. 8 is the mass spectrogram of detecting 25-hydroxyvitamin D3 in Example 2 (from bottom to top, the order is the target compound, the calibrator, the first natural isotope of the calibrator, the mass spectrum signal of the second natural isotope of the calibrator);
  • Fig. 9 is the calibration curve of 25-hydroxyvitamin D3 matrix matching internal standard in Example 2.
  • Figure 10 is the result of natural isotope calibration curve method and matrix matching internal standard calibration method in Example 2;
  • Figure 11 is the structure of aldosterone and its stable isotope-labeled analog (aldosterone-D8) in Example 3;
  • Figure 12 is a mass spectrogram of detecting aldosterone in Example 3 (from bottom to top, the order is the target compound, the calibrator, the first natural isotope of the calibrator, and the mass spectral signal of the second natural isotope of the calibrator);
  • Figure 13 is an aldosterone matrix-matched internal standard calibration curve in Example 3.
  • Figure 14 is the structure of tacrolimus and its stable isotope-labeled analog (tacrolimus- 13 CD2) in Example 4;
  • Figure 15 is the mass spectrogram of the detection of tacrolimus in Example 4 (from bottom to top, the order is the target compound, the calibrator, the first natural isotope of the calibrator, the mass spectrum signal of the second natural isotope of the calibrator);
  • Figure 16 is the tacrolimus matrix-matched internal standard calibration curve in Example 4.
  • Figure 17 is a schematic diagram of the principle of the natural isotope calibration curve method adopted in the present invention.
  • the following embodiment provides a natural isotope calibration curve method for quantifying target analytes in a sample, comprising the following steps:
  • step C carry out mass spectrometry analysis on the sample to be tested processed in step B to obtain the mass spectrum signal of the calibrator and the natural isotope of the calibrator, and the mass spectrum signal of the target analyte in the sample to be tested;
  • step A there are one or more target analytes, and a known amount of calibrator is added to the sample to be tested for each target analyte.
  • the principle of the natural isotope calibration curve method is shown in Figure 17. According to the content of the calibrator and its molecular formula, the relative abundance and content of its natural isotopes are calculated, and then the content of the calibrator and the content of its natural isotopes are taken as the abscissa, The peak area is the ordinate to draw a standard curve; then the peak area of the target analyte is corresponding to the standard curve, that is, the content value of the target analyte is obtained.
  • Example 1 Content analysis of catecholamine metabolites in human plasma samples
  • This example describes the determination of catecholamine metabolites (metanephrine and norepinephrine) in human plasma samples by adding known amounts of stable isotope-labeled analogs, using the stable isotopes and their known abundances. Natural isotopes were used to construct a calibration curve and quantitatively analyze the content of catecholamine metabolites in samples. The obtained results were also compared with those obtained by the matrix-matched internal standard calibration curve method. This example demonstrates the precise and accurate quantification of catecholamine metabolites in human plasma samples by using stable isotope-labeled calibrators and their natural isotopes of known abundance.
  • Preparation of stable isotope-labeled calibrators use commercially available stable isotope-labeled analogs of the target analytes as calibrators.
  • the target analytes are metanephrine and norepinephrine, respectively
  • the corresponding commercially available stable isotope-labeled analogs are metanephrine-D3 and norepinephrine-D3, respectively, see FIG. 1 .
  • Preparation of a single stock solution of target analyte and calibrator take solid powders of target analyte and stable isotope-labeled calibrator respectively, use methanol as solvent to prepare stock solutions, and the concentration of each stock solution is 1 mg/ mL.
  • metanephrine-D3-F1 is the first natural isotope of metanephrine-D3
  • metanephrine-D3-F2 is the second natural isotope of metanephrine-D3
  • norepinephrine-D3-F1 is norepinephrine-D3-F1.
  • Norepinephrine-D3 is the first natural isotope
  • norepinephrine-D3-F2 is the second natural isotope of norepinephrine-D3.
  • a single stock solution of target analyte is diluted to prepare a mixed target solution of target analytes (metanephrine and norepinephrine), wherein metanephrine and norepinephrine at a concentration of 1 ng/mL, respectively;
  • Table 3 shows the response ratios for the same concentrations of target analytes to stable isotope-labeled calibrators as relative response factors
  • a series of matrices were constructed by adding a series of target analyte solutions of catecholamine metabolites (in this example, metanephrine and norepinephrine calibrators) to blank plasma treated with secondary activated carbon Matched calibrators containing target analytes at concentrations of 1 pg/mL, 2 pg/mL, 10 pg/mL, 20 pg/mL, 100 pg/mL, 200 pg/mL, respectively.
  • catecholamine metabolites in this example, metanephrine and norepinephrine calibrators
  • Plasma samples from 17 normal individuals were used to initially assess the degree of agreement between the results obtained by adding isotopic calibrators and matrix-matched calibrators.
  • the analysis was performed using a Waters ACQUITY UPLC I-Class/Xevo TQ-S triple quadrupole tandem mass spectrometer system, and a Waters ACQUITY UPLC HSS PEP column (1.8 ⁇ m, 2.1 mm ⁇ 100 mm) was used to analyze the samples, with 0.1% formic acid in water and acetonitrile, respectively.
  • Mobile phases A and B were subjected to gradient elution as shown in Table 4. The run time was 4 min, the column temperature was 40 °C, and the injection volume was 10 ⁇ L.
  • the Waters Xevo TQ-S triple quadrupole tandem mass spectrometer system operates in the mode of multiple reaction monitoring. The mass spectrometry conditions are shown in Table 1, and the separation results are shown in Figure 2 and Figure 3.
  • Peak area integration and response calculation were performed using TargetLynx software. By calculating the analyte peak area/internal standard peak area ratio, a six-point external calibration line was generated to calculate the analyte concentration in each plasma sample.
  • the integrated peak area of each channel in Figure 2 was measured by TargetLynx software and imported into Microsoft Excel. Taking the target analyte metanephrine as an example, the concentration of the added calibrator metanephrine-D3 was known to be 0.909ng/mL.
  • the atom substituted by 2 H(D) does not contain other isotopes by default, then calculate the abundance ratio of its main peak, the first and second natural isotope peaks are 100%, 11.44% and 1.21%, respectively, the corresponding main peak, the first and second natural isotope concentrations are 909.0 pg/mL, 104.3 pg/mL and 11.0 pg/mL, respectively.
  • the concentration of the added calibrator norepinephrine-D3 is known to be 0.9625ng/mL, and the molecular formula of the calibrator measured in the mass spectrometer [C 9 H 11 D 3 NO 3 ] + , the atom substituted by 2 H(D) does not contain other isotopes by default, then the abundance ratios of its main peak, the first and second natural isotope peaks are calculated to be 100%, 10.34%, and 1.10%, respectively, then the corresponding The concentrations of the main peak, the first and second natural isotope peaks were 962.5 pg/mL, 99.5 pg/mL and 10.6 pg/mL, respectively.
  • 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 curve (see Figure 4). The results are shown in Tables 7 and 8.
  • this example shows the application principle and implementation process of the natural isotope calibration curve method.
  • the calibrator is the stable isotope analog of the target compound, and the points on the calibration curve are the main peak of the calibrator, the first natural isotope peak of the calibrator, and the second natural isotope peak of the calibrator. They have very similar molecular structures to the target compounds and can be used to correct for loss of recovery during pretreatment, as well as for injection errors and matrix effects during analysis.
  • the only difference is that the ionization efficiency of the calibrator and target analyte is slightly different at the ion source end, so the results obtained 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-matched internal standard calibration curve method, and the former has the convenience of not needing to prepare a blank matrix.
  • Example 1 the preparation of blank matrices is particularly difficult and expensive when the target analyte is ubiquitously present in the sample matrix and at high levels.
  • the content of 25-hydroxyvitamin D3 in normal serum is relatively high, so conventional LC-MS/MS mass spectrometry quantitative methods generally use 1% bovine serum albumin solution instead of blank matrix to prepare a series of calibrators, and then pass the calibrators in the calibrators.
  • the same amount of internal standard was added to the sample to be tested to correct the matrix effect, so as to quantify the 25-hydroxyvitamin D 3 in the sample.
  • This example describes the analysis of 25-hydroxyvitamin D in human serum samples, each of which was spiked with a known amount of a stable isotope-labeled analog, using the stable isotope and its natural isotope of known abundance to A calibration curve was constructed to quantitatively analyze the content of 25-hydroxyvitamin D3 in the samples. The results obtained were also compared with those obtained by the matrix-matched internal standard calibration method. This example demonstrates the precise and accurate quantification of 25-hydroxyvitamin D3 in human serum samples using stable isotope-labeled calibrators and their natural isotopes of known abundance.
  • a commercially available stable isotope-labeled analog of the target analyte is used as the calibrator.
  • the target analyte is 25-hydroxyvitamin D 3
  • the corresponding A commercially available stable isotope labeled analog is 25-hydroxyvitamin D3- D3 , see Figure 7 .
  • Preparation of a single stock solution of target analyte and calibrator take solid powders of target analyte and stable isotope-labeled calibrator respectively, use methanol as solvent to prepare stock solutions, and the concentration of each stock solution is 0.1 mg respectively /mL.
  • a single working solution of the target analyte and calibrator from a single stock solution of the target analyte and its stable isotope-labeled analog, the target analyte (25-hydroxyvitamin D 3 ) and the stable isotope-labeled analog are prepared by dilution A mixed solution of 25-hydroxyvitamin D3-D3, wherein the concentrations of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 - D3 in the mixed solution are both 10 ng/mL.
  • Table 11 shows the response ratios for the same concentrations of target analytes to stable isotope-labeled calibrators as relative response factors
  • bovine serum albumin solution take 1 g of bovine serum albumin into a 100 mL volumetric flask, add phosphate buffer to dissolve, adjust the volume to the mark, and shake well.
  • a series of matrix-matched calibrators were constructed by adding a series of 25-hydroxyvitamin D 3 target analyte solutions to 1% bovine serum albumin solution, which contained target analyte concentrations of 5ng/mL, 20ng/mL, 50ng/mL.
  • TargetLynx software was used to integrate the peak areas, calculate the response, and generate a three-point external calibration line by calculating the analyte peak area/internal standard peak area ratio to calculate the analyte concentration in each serum sample.
  • the corresponding concentrations of the main peak, the first and second natural isotope peaks are 50ng/mL, 14.875ng/mL and 2.34ng/mL, respectively.
  • Peak area integration and response calculation were performed using TargetLynx software.
  • a three-point external calibration curve was generated to calculate the analyte concentration in each serum sample, and the results are shown in Table 14.
  • Example 3 Determination of aldosterone content in human plasma samples by pre-column derivatization
  • This example describes the analysis of aldosterone in human plasma samples.
  • a known amount of a stable isotope-labeled analog is added to each sample, a calibration curve is constructed using this stable isotope and its natural isotope of known abundance, and the quantitative analysis is performed.
  • the content of aldosterone in the sample was compared with those obtained by the conventional matrix calibration curve method.
  • the latter uses 1% bovine serum albumin/phosphate buffer solution to prepare a series of calibrators, and then quantifies the aldosterone content in the samples by adding an internal standard to the calibrators and samples to correct for recovery and matrix effects.
  • Preparation of stable isotope-labeled calibrator use a commercially available stable isotope-labeled analog of the target analyte as the calibrator.
  • the target analyte is aldosterone
  • the labeled analogs include aldosterone-D4, aldosterone-D7 or aldosterone-D8, etc.
  • aldosterone-D8 was selected, see Figure 11 .
  • Preparation of a single stock solution of target analyte and calibrator take solid powders of target analyte and stable isotope-labeled calibrator respectively, use methanol as solvent to prepare stock solutions, and the concentration of each stock solution is 0.1 mg respectively /mL.
  • the single stock solutions of the target analyte and calibrator were diluted to obtain a single solution of 0.1 ⁇ g/mL, which was used to optimize and explore mass spectrometry conditions.
  • the results are shown in Table 15.
  • the aldosterone-D8 derivative-F1 is the first natural isotope of the aldosterone-D8 derivative
  • the aldosterone-D8 derivative-F2 is the second natural isotope of the aldosterone-D8 derivative.
  • a solution of the target analyte is prepared by diluting a single stock solution of the target analyte, wherein the concentration of aldosterone is 1 ng/mL.
  • a working solution of the calibrator was prepared by dilution from a single stock solution of the calibrator with a concentration of 1 ng/mL of aldosterone-D8.
  • Table 16 shows the response ratios for the same concentrations of target analytes to stable isotope-labeled calibrators as relative response factors
  • bovine serum albumin solution take 1 g of bovine serum albumin into a 100 mL volumetric flask, add phosphate buffer to dissolve, adjust the volume to the mark, and shake well.
  • the analysis was performed using a Waters ACQUITY UPLC I-Class/Xevo TQ-S triple quadrupole tandem mass spectrometer system, and a Waters ACQUITY UPLC BEH C8 column (1.8 ⁇ m, 2.1 mm ⁇ 100 mm) was used to analyze the samples, and the samples were analyzed with 0.1% formic acid in water (containing 2 mM) ammonium formate) and methanol were gradient elutions for mobile phases A and B, respectively, as shown in Table 17.
  • the running time was 5.5 min, the column temperature was 40 °C, and the injection volume was 20 ⁇ L.
  • the Waters Xevo TQ-S triple quadrupole tandem mass spectrometer system operates in the mode of multiple reaction monitoring. The mass spectrometry conditions are shown in Table 15, and the separation results are shown in Figure 12.
  • Table 17 UPLC gradient conditions for the analysis of catecholamine metabolites in plasma
  • TargetLynx software was used for area integration and response calculation. By calculating the target analyte peak area/internal standard peak area ratio, a six-point calibration curve was generated to calculate the target analyte concentration in each plasma sample.
  • the integrated peak area of each channel in Figure 12 was measured with TargetLynx software, imported into Microsoft Excel, and the concentration of the added calibrator aldosterone-D8 was known to be 100 pg/mL (converted to the sample volume).
  • the calibrator aldosterone-D8 The molecular formula of the derivative is [C28H27D8O5N3] + , the atom substituted by 2 H does not contain other isotopes by default, then the main peak is calculated.
  • the abundance ratios of the first and second natural isotopes are 100%, 31.88%, 5.94%, respectively,
  • the corresponding main peaks, the first and second natural isotope concentrations were 100 pg/mL, 31.88 pg/mL and 5.94 pg/mL, respectively.
  • Table 18 uses the natural isotope calibration curve method to determine the content of aldosterone in the sample
  • Peak area integration and response calculation were performed using TargetLynx software.
  • 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). The results are shown in Table 19.
  • the matrix matching calibration curve method uses the actual matrix simulated by bovine serum albumin, which still has a certain gap with the actual sample.
  • the target Compounds and calibrators were derivatized.
  • the results listed in the table are also within the acceptable range. Using the natural isotope calibration curve method can obtain results that are basically close to the matrix-matched internal standard calibration curve method. The possible reasons for the slight differences in the results and which method can obtain results closer to the theoretical reality can be further explored in the future.
  • Example 4 Application of high-resolution mass spectrometry to therapeutic drug monitoring of tacrolimus content in whole blood samples
  • This example describes the application of the natural isotope calibration curve method to the 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 at low and high concentrations.
  • a known amount of a stable isotope-labeled analog was added to each sample, and the stable isotope and its natural isotope of known abundance were used to construct a calibration curve to quantify the amount of tacrolimus in the sample.
  • the obtained results were also compared with those obtained by the conventional matrix-matched internal standard calibration curve method.
  • the matrix-matched internal standard calibration curve method can use the same blank matrix as the quality control sample to prepare a series of calibrators, so the results obtained by the matrix-matched calibration curve method should be the same as The theoretical values are basically the same.
  • the method uses high-resolution mass spectrometry as the detector, it can be used to evaluate the applicability of the natural isotope calibration curve method between different mass spectrometry platforms. The final results show that the amount of tacrolimus in the sample can be accurately and accurately quantified using the natural isotope calibration curve method.
  • Preparation of stable isotope-labeled calibrator use a commercially available stable isotope-labeled analog of the target analyte as the calibrator.
  • the target analyte is tacrolimus
  • the corresponding commercially available A stable isotope labeled analog is tacrolimus- 13CD2 , see Figure 14.
  • Preparation of a single stock solution of target analyte and calibrator take solid powders of target analyte and stable isotope-labeled calibrator respectively, use methanol as solvent to prepare stock solutions, and the concentration of each stock solution is 1 mg/ mL.
  • a solution of the target analyte is prepared by diluting a single stock solution of the target analyte, wherein the concentration of tacrolimus is 10 ⁇ g/mL.
  • a working solution of the calibrator was prepared from a single stock solution of the calibrator, diluted at a concentration of 5 ⁇ g/mL of tacrolimus- 13CD2 .
  • the scanning method is a data-independent full scan, and there is no need to optimize the mass spectrometry conditions, and the target and calibrators can be analyzed by UPLC/MS/MS. Relative response factors were calculated by comparing the 6-pin average peak areas from the target analytes to the 6-pin average peak areas from stable isotope-labeled calibrators (Table 21).
  • Table 21 shows the response ratios of stable isotope-labeled calibrators to target analytes at the same concentrations as relative response factors
  • the analysis was performed using a Waters ACQUITY UPLC I-Class/Synapt G2-S Qtof high-resolution mass spectrometer system, and a Waters ACQUITY UPLC BEH C18 column (1.7 ⁇ m, 2.1 mm ⁇ 50 mm) was used to analyze the samples, and the samples were analyzed with 0.1% aqueous formic acid (containing 2 mM ammonium formate). ) and 0.1% methanol formic acid (with 2 mM ammonium formate) for gradient elution for mobile phases A and B, respectively, as shown in Table 22.
  • the running time was 2.5 min, the column temperature was 40 °C, and the injection volume was 5 ⁇ L.
  • the Waters Synapt G2-S Qtof high-resolution mass spectrometer system uses a data-independent full-scan mode for data acquisition, and can extract the response mass spectrum according to the exact molecular weight of the target compound. The results are shown in Figure 15.
  • TargetLynx software was used to integrate peak areas and calculate responses. By calculating the analyte peak area/internal standard peak area ratio, a six-point external calibration line was generated to calculate the analyte concentration in each sample.
  • the integrated peak area of each channel in Figure 15 was measured with TargetLynx software, imported into Microsoft Excel, the concentration of the added calibrator tacrolimus- 13 CD2 was known to be 50ng/mL (converted to the sample volume), and the molecular formula of the calibrator was [ C28H27D8O5N3] + , the atoms substituted by 13 C and 2 H do not contain other isotopes by default, if they are excluded from the abundance calculation, the main peak is calculated.
  • the abundance ratios of the first and second natural isotopes are 100%, 48.51 %, 13.98%, the corresponding main peak, the first and second natural isotope concentrations are 50ng/mL, 24.255ng/mL and 6.99ng/mL, respectively.
  • 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 curve (FIG. 16). The results are shown in Table 24.
  • the comparison results show that the quality control data results of the two methods are basically the same. Since the same blank matrix is used for the quality control sample and the matrix calibration curve in this example, the data of the quality control sample should be relatively accurate. At the same time, the example verifies the feasibility of applying the natural isotope calibration method to a high-resolution mass spectrometry system, using the full-scan mode for quantitative analysis, and directly analyzing the quasi-molecular ion peak of the target compound without additional optimization of mass spectrometry conditions.
  • the natural isotope calibration curve method of the present invention is also applicable to the quantitative analysis of other organic molecules including at least 3 carbon atoms.

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Abstract

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。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，定量待测样品中的目标分析物。具有非常高的可靠性和准确性，且简化了检测程序，拓展了应用范围。

Description

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 技术领域
本发明涉及检测分析技术领域，具体涉及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。
背景技术
以质谱为分析手段的痕量分析方法，主要的定量方法包括有外标法，内标法和标准加入法。根据校准方式的不同，可分为单点校准，多点校准和校准曲线法；根据配制校准品所采用的溶液不同，又分为纯溶液标准品和基质匹配的标准品。这其中以基质匹配内标校准曲线法和标准加入法的结果最为准确。标准加入法由于难以预估样品中目标化合物的量且工作量较大(每个样品，需要制备至少三份样品)，较多用于结果的确认。因而，基质匹配内标校准曲线法是目前最为常用的痕量分析方法。该方法的优点在于前处理的过程中加入了内标化合物用于平行校准前处理过程中目标化合物的损失，如果条件允许的情况下，内标化合物一般选取目标化合物的稳定同位素标记的类似物，由于两者具有极其类似的物理化学性质，该内标不仅可以校准前处理过程中目标化合物的损失，同样可以校准质谱分析过程中的基质效应；但该方法的主要弊端在于较难获取配制标准品所需的空白基质，尤其是当分析的目标物是内源性的，且样品基质的种类繁多，个体差异较大时，空白基质的获取尤为困难。
申请号为201280036810.1的专利文献中记载了一种用于定量样品中目标分析物的方法，包括获得质谱信号，所述质谱信号包括第一校准物信号、包括第二校准物信号，并且可能包含来自单个样品的目标分析物信号，所述单个样品包含第一已知量的第一校准物、包含第二已知量的第二校准物，并且潜在包含目标分析物。所述第一已知量和所述第二已知量不同，并且其中所述第一校准物、所述第二校准物以及所述目标分析物可通过质谱法在所述单个样品中各自区分。所述方法还包括使用所述第一校准物信号、所述第二校准物信号，以及所述目标分析物信号来定量所述单个样品中的所述目标分析物。但该方法的缺陷在于其必须包含至少两个校准 物，且所述第一校准物，第二校准物以及更多的校准物均为目标分析物的不同稳定同位素标记的类似物(以睾酮分析为例，添加的校准物为D2-睾酮，D3-睾酮，D5-睾酮的不同浓度混合校准物)。而在实际应用过程中，稳定同位素标记的类似物获取较为困难，成本较高；且绝大部分目标分析物只有一种稳定同位素标记的类似物可供获取，从而导致大部分目标分析物无法直接采用该专利中的方法进行定量。
在自然界中，所有元素是以同位素的混合物存在着的。有机化合物一般由C、H、O、N、S等元素组成，它们都有同位素，根据自然规律，各元素的同位素都以一定的天然丰度存在，见表1。
表1有机化合物中常见元素的天然同位素丰度表
Figure PCTCN2021130578-appb-000001
考虑到有机化合物分子中，一般每个元素都具有多种同位素，则各同位素的峰强度可按下述二项式的展开式算出。
(a+b) n(c+d) m……
其中a和b、c和d分别为第一、第二种元素的M和M+1同位素的天然丰度(习惯上，也可称为轻质同位素和重质同位素，例如以C为例，a和b分别为 12C和 13C的天然丰度)，n，m分别为第一、第二种元素的原子数目。目前主流的质谱分析仪大多配有工具软件可供计算有机化合物的同位素丰度比，只要输入化合物的分子式即可，故这里就不再赘述其丰度比的计算原理。
对稳定同位素类似物进行质谱分析时，会同时获得稳定同位素类似物的一系列天然同位素的质谱信号。但并未见有任何人对该质谱信号进行进一步的利用。现有技术中也并未有任何报道将质谱分析获得的稳定同位素类似物的一系列天然同位素的质谱信号用于目标分析物的定量中。
发明内容
针对现有技术中的不足，本发明的目的是提供一种通过质谱法定量样品中目标分析物的方法。
本发明的目的是通过以下技术方案实现的：
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，包括以下步骤：
A、在待测样品中添加已知量的校准物，所述校准物为目标分析物的稳定同位素标记的类似物；
B、对待测样品进行前处理；
C、将步骤B处理后的待测样品进行质谱法分析，得到校准物及校准物的天然同位素的质谱信号、待测样品中的目标分析物的质谱信号；
D、根据校准物的分子式计算得到校准物的天然同位素丰度比，从而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已知含量、质谱信号响应的对应关系绘制校准曲线，再根据待测样品中的目标分析物的质谱信号响应，对应到校准曲线中即得到待测样品中的目标分析物含量；
步骤A中，所述目标分析物为一个或多个，每一种目标分析物对应在待测样品中添加一个已知量的校准物。
优选地，步骤B中，所述前处理的方法选自固相萃取法、固液萃取法、液液萃取法、沉淀蛋白法、直接稀释法、溶剂提取法、盐析法、化学分离法、浓缩法、吸附色谱分离、分配色谱分离、离子交换色谱分离、掩蔽法中的任一种。
优选地，步骤C中，所述校准物的天然同位素质谱信号为一个或多个。
优选地，所述校准物的天然同位素质谱信号为多个时，即包括校准物的第一天然同位素、校准物的第二天然同位素，以此类推，步骤D的标准曲线绘制方法如下：
根据校准物的分子式计算得到校准物的天然同位素丰度比，从而得到校准物的第一天然同位素的含量作为第二已知量的校准物、校准物的第二天然同位素的含量作为第三已知量的校准物，以此类推；再根据各校准物的已知含量、质谱信号响应的对应关系绘制校准曲线。
优选地，当采用单位质量分辨率的质谱分析时，所述校准物与校准物的天然同位素的质荷比大于等于1个道尔顿质量数；当采用高分辨质谱分析时，所述校准物与校准物的天然同位素的质荷比大于等于0.05个道尔顿质量数。
优选地，所述校准物与目标分析物在质谱法分析中彼此相差至少2个道尔顿质量数。
优选地，步骤A中，所述待测样品包括生物样品、环境样品、食品样品、合成样品、药物样品、化工样品、临床化学样品、法医样品、药理样品、农业样品中的任一种；所述生物样品包括血浆、血清、全血、尿液、组织、脑脊液、汗液、唾液、毛发、皮肤中的任一种。
优选地，步骤A中，所述目标分析物为包括至少3个碳原子的有机分子。
优选地，步骤A中，所述的校准物为目标分析物的稳定同位素标记类似物，为目标分析物的至少1个原子被其稳定同位素替代后得到，稳定同位素包括但不限于 2H、 11B、 13C、 15N、 17O、 18O、 33S、 34S和 36S中的任一种或多种。
优选地，所述质谱法采用的离子源包括但不限于电喷雾离子源(ESI)、大气压化学电离离子源(APCI)、基质辅助激光解吸电离离子源(MALDI)、解吸电喷雾电离离子源(DESI)、电子轰击源(EI)、化学电离源(CI)，质量分析器包括但不限于四级杆质量分析器、离子阱质量分析器、扇形磁质谱质量分析器、飞行时间质量分析器、静电场轨道阱质量分析器、傅里叶变换离子回旋共振质量分析器中的任一种或其中两种及以上的组合；所述质谱的检测模式选自全扫描模式、子离子检测模式、母离子检测模式、多反应检测模式、中性丢失扫描、数据依赖性扫描模式、非数据依赖性扫描模式中的任一种；
所述质谱法分析之前还包括采用色谱法进行分离的步骤；所述色谱法选自液相色谱法，气相色谱法，毛细管电泳法，亲和色谱法，免疫亲和色谱法，超临界流体色谱法，离子淌度法中的任一种。
优选地，步骤D中，还包括对待测样品中的目标分析物含量进行校正的步骤；将相同浓度的目标分析物与稳定同位素标记的校准物注入质谱仪分析，以目标分析物与稳定同位素标记的校准物的响应比值，作为相对响应因子；实际样品分析时，所得目标分析物的含量结果需乘以相对响应因子来作为最终结果。
与现有技术相比，本发明具有如下的有益效果：
1、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。由于它们在性质上与目标化合物极其类似，又共存于同一样品基质中，可近乎完美地校准回收率和基质效应，在技术上可以称为一种单个样品的自校准方法。
2、本发明方法所得的结果与基质匹配内标校准曲线法获得的结果基本一致，说明 了本发明方法的可靠性和准确性；且本发明方法无需额外制备任何的空白基质，大大简化了检测程序，进一步降低了检测成本。
3、本发明方法针对一种目标分析物只需采用一种稳定同位素标记的类似物，避免了需合成多个稳定同位素标记的类似物，大大节约了检测成本；且对于只有一种稳定同位素标记的类似物的目标分析物可直接采用本发明的方法进行定量，极大的拓展了应用范围。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为实施例1中儿茶酚胺代谢物(变肾上腺素和去甲变肾上腺素)及其稳定同位素标记的类似物(变肾上腺素-D3和去甲变肾上腺素-D3)的结构；
图2为实施例1中检测的变肾上腺素的质谱图(自下而上，依次为目标化合物，校准物，校准物的第一天然同位素，校准物的第二天然同位素的质谱信号)；
图3为实施例1中检测的去甲变肾上腺素的质谱图(自下而上，依次为目标化合物，校准物，校准物的第一天然同位素，校准物的第二天然同位素的质谱信号)；
图4为实施例1中的变肾上腺素和去甲变肾上腺素基质匹配内标校准曲线；
图5为实施例1中变肾上腺素两种校准方法的结果比较；
图6为实施例1中去甲变肾上腺素两种校准方法的结果比较；
图7为实施例2中25-羟基维生素D3及其稳定同位素标记的类似物(25-羟基维生素D3-D3)的结构；
图8为实施例2中检测25-羟基维生素D3的质谱图(自下而上，依次为目标化合物，校准物，校准物的第一天然同位素，校准物的第二天然同位素的质谱信号)；
图9为实施例2中25-羟基维生素D3基质匹配内标校准曲线；
图10为实施例2中天然同位素校准曲线法与基质匹配内标校准法结果；
图11为实施例3中醛固酮及其稳定同位素标记的类似物(醛固酮-D8)的结构；
图12为实施例3中检测醛固酮的质谱图(自下而上，依次为目标化合物，校准物，校准物的第一天然同位素，校准物的第二天然同位素的质谱信号)；
图13为实施例3中醛固酮基质匹配内标校准曲线；
图14为实施例4中他克莫司及其稳定同位素标记的类似物(他克莫司- 13CD2)的结 构；
图15为实施例4中检测他克莫司的质谱图(自下而上，依次为目标化合物，校准物，校准物的第一天然同位素，校准物的第二天然同位素的质谱信号)；
图16为实施例4中他克莫司基质匹配内标校准曲线；
图17为本发明采用的天然同位素校准曲线法的原理示意图。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。
以下实施例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，包括以下步骤：
A、在待测样品中添加已知量的校准物，所述校准物为目标分析物的稳定同位素标记的类似物；
B、对待测样品进行前处理；
C、将步骤B处理后的待测样品进行质谱法分析，得到校准物及校准物的天然同位素的质谱信号、待测样品中的目标分析物的质谱信号；
D、根据校准物的分子式计算得到校准物的天然同位素丰度比，从而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已知含量、质谱信号响应的对应关系绘制校准曲线，再根据待测样品中的目标分析物的质谱信号响应，对应到校准曲线中即得到待测样品中的目标分析物含量；
步骤A中，所述目标分析物为一个或多个，每一种目标分析物对应在待测样品中添加一个已知量的校准物。
所述天然同位素校准曲线法的原理如图17所示，根据校准物的含量及其分子式计算得到其天然同位素的相对丰度和含量，再以校准物含量、其天然同位素的含量为横坐标，峰面积为纵坐标绘制标准曲线；然后将目标分析物的峰面积对应到标准曲线上，即得到目标分析物的含量值。
实施例1人血浆样品中儿茶酚胺代谢物的含量分析
常规的LC-MS/MS质谱定量方法，由于基质效应的存在，需要准备空白基质用于制备一系列基质匹配的校准物，而且该空白基质的成分要尽可能与所分析的各批次样品基质保持一致。当目标分析物普遍存在于基质(例如，内源性激素、维生素、氨基酸等)中时，制备这类空白基质是特别困难的，尤其是当目标分析物在基质中含量较高时，这一目的几乎不可能实现。如本实施例中，正常血浆中儿茶酚胺代谢物的含量较低，但仍需要制备二次活性炭处理的血浆来作为空白血浆基质。本实施例描述了人血浆样品中儿茶酚胺代谢物(变肾上腺素和去甲变肾上腺素)的含量测定，加入已知量的稳定同位素标记的类似物，采用该稳定同位素及其已知丰度的天然同位素来构建校准曲线，定量分析样品中儿茶酚胺代谢物的含量。同时将获得的结果与基质匹配内标校准曲线法所得的结果进行比较。该实施例证明，通过使用稳定同位素标记的校准物及其已知丰度的天然同位素，可精确并准确定量人血浆样品中的儿茶酚胺代谢物。
人血浆样品中儿茶酚胺代谢物(变肾上腺素和去甲变肾上腺素)的含量测定具体步骤如下：
1、稳定同位素标记校准物的制备：使用目标分析物的可商购的稳定同位素标记的类似物作为校准物，本实施例中，目标分析物分别为变肾上腺素和去甲变肾上腺素，与之对应的可商购的稳定同位素标记的类似物分别为变肾上腺素-D3和去甲变肾上腺素-D3，参见图1。
2、目标分析物及校准物的单个储备溶液的制备：分别取目标分析物及稳定同位素标记校准物的固体粉末，以甲醇为溶剂，制成储备溶液，每个储备液的浓度分别为1mg/mL。
3、质谱条件优化：由所述目标分析物及校准物的单个储备溶液，分别稀释得到0.1μg/mL的单个溶液，用于优化摸索质谱条件，结果见表2。其中变肾上腺素-D3-F1为变肾上腺素-D3的第一天然同位素，变肾上腺素-D3-F2为变肾上腺素-D3的第二天然同位素；去甲变肾上腺素-D3-F1为去甲变肾上腺素-D3的第一天然同位素，去甲变肾上腺素-D3-F2为去甲变肾上腺素-D3的第二天然同位素。
表2目标分析物及校准物的质谱条件
目标分析物或校准物 MRM跃迁 锥孔电压(V) 碰撞能量(V)
变肾上腺素 180.1→148.2 30 16
变肾上腺素-D3 183.1→151.2 30 16
变肾上腺素-D3-F1 184.1→152.2 30 16
变肾上腺素-D3-F2 185.1→153.2 30 16
去甲变肾上腺素 166.0→134.0 30 16
去甲变肾上腺素-D3 169.0→137.0 30 16
去甲变肾上腺素-D3-F1 170.0→138.0 30 16
去甲变肾上腺素-D3-F2 171.0→139.0 30 16
4、目标分析物及校准物的单个工作溶液的制备：由目标分析物的单个储备溶液，稀释制备目标分析物(变肾上腺素和去甲变肾上腺素)的混合目标物溶液，其中变肾上腺素和去甲变肾上腺素的浓度分别为1ng/mL；
由稳定同位素标记的类似物(变肾上腺素-D3和去甲变肾上腺素-D3)的单个储备溶液，稀释制备稳定同位素标记的类似物的混合校准物溶液，其中变肾上腺素-D3和去甲变肾上腺素-D3的浓度分别为1ng/mL。
5、计算相对响应因子：使用表1中描述的特定MRM跃迁，通过UPLC/MS/MS分析混合目标物溶液和混合校准物溶液。将来自稳定同位素标记的校准物的6针平均峰面积与目标分析物的6针平均峰面积进行对比，计算相对响应因子(如表3所示，以相同浓度的目标分析物与稳定同位素标记的校准物的响应比值，作为相对响应因子)。
表3显示了相同浓度的目标分析物与稳定同位素标记的校准物的响应比值，作为相对响应因子
Figure PCTCN2021130578-appb-000002
6、二次活性炭处理的空白血浆的制备：取适量的正常人血浆加入一定量的活性炭，振摇，放置过夜，离心取上清液，自上述“加入一定量的活性炭，”起，重复上述操作一次，即得。
7、基质匹配校准物的制备：将一系列儿茶酚胺代谢物的目标分析物溶液(本实施例中指变肾上腺素和去甲变肾上腺素校准品)加入二次活性炭处理的空白血浆来构建一系列基质匹配的校准物，其中所含目标分析物的浓度分别为1pg/mL、2pg/mL、10pg/mL、20pg/mL、100pg/mL、200pg/mL。
5、取来自17份正常人的血浆样品，用于初步评估添加同位素校准物和基质匹配校准物所得结果的一致程度。
5.1采用天然同位素校准曲线的方法进行样品制备：
1)取200μL血浆样品；
2)加入200μL 40mM含校准物的乙酸铵溶液(其中含校准物变肾上腺素-D3和去甲变肾上腺素-D3各自约为1ng/mL)；
3)涡旋混合30S；
4)在10℃下14000转/分钟离心5min；
5)上清液待净化；
6)取WCX固相萃取96孔板，依次用200μL甲醇和200μL水淋洗；
7)取350μL上清液置于固相萃取板上；
8)依次用200μL 20mM乙酸铵溶液和200μL乙腈/异丙醇(50/50)溶液淋洗；
9)用50μL 2％甲酸(85％乙腈)溶液洗脱；
10)氮气吹干洗脱液；
11)用40μL水复溶；
12)将复溶液10μL注入超高效液相色谱-串联质谱仪分析。
使用Waters ACQUITY UPLC I-Class/Xevo TQ-S三重四极杆串联质谱系统进行分析，采用Waters ACQUITY UPLC HSS PEP色谱柱(1.8μm，2.1mm×100mm)分析样品，以0.1％甲酸水溶液和乙腈分别流动相A和B进行梯度洗脱，如表4中所示。运行时间为4min，柱温为40℃，进样体积为10μL。Waters Xevo TQ-S三重四极杆串联质谱系统采用多反应监测的模式操作，质谱条件如表1所示，分离结果显示在图2、图3中。
表4血浆中儿茶酚胺代谢物分析的UPLC梯度条件
时间(min) 流速(mL/min) ％A ％B 曲线
0.0 0.4 98 2 -
1.0 0.4 98 2 6
2.5 0.4 90 10 6
3.0 0.4 10 90 6
3.5 0.4 10 90 6
4.0 0.4 98 2 1
用Waters MassLynx软件采集数据，对于天然同位素校准曲线法，使用TargetLynx软件测定图2中各通道的积分峰面积，导入Microsoft Excel用于构建各血浆样品的单个内部校准曲线(使用线性回归分析)，从而计算各血浆样品中的分析物浓度。
5.2采用基质匹配内标校准的方法进行样品制备：
1)取200μL各浓度的基质匹配校准物(6个)、200μL血浆样品(17个)形成23个待测样品；
2)向各个待测样品中加入200μL 40mM含内标的乙酸铵溶液(其中变肾上腺素-D3和去甲变肾上腺素-D3各自约为1ng/mL，这里仅作为内标使用)；
3)涡旋混合30S；
4)在10℃下14000转/分钟离心5min；
5)上清液待净化；
6)取WCX固相萃取96孔板，依次用200μL甲醇和200μL水淋洗；
7)取350μL上清液置于固相萃取板上；
8)依次用200μL 20mM乙酸铵溶液和200μL乙腈/异丙醇(50/50)溶液淋洗；
9)用50μL 2％甲酸(85％乙腈)溶液洗脱；
10)氮气吹干洗脱液；
11)用40μL水复溶；
12)将23个复溶液以10μL注入超高效液相色谱-串联质谱仪分析。
使用TargetLynx软件进行峰面积积分、计算响应。通过计算分析物峰面积/内标峰面积比，生成六点外部校准线，从而计算各血浆样品中的分析物浓度。
结果：
方法一：天然同位素校准曲线法
用TargetLynx软件测定图2中各通道的积分峰面积，导入Microsoft Excel，以目标分析物变肾上腺素为例，已知加入的校准物变肾上腺素-D3的浓度为0.909ng/mL，质谱中测得校准物的分子式[C 10H 13D 3NO 3] +，默认被 2H(D)取代的原子不含别的同位素，则计算其主峰、第一、第二天然同位素峰的丰度比分别为100％、11.44％、1.21％，则对应的主峰、第一、第二天然同位素的浓度分别为909.0pg/mL、104.3pg/mL和11.0pg/mL。将校准物的第二天然同位素峰定义为ST1，校准物的第一天然同位素峰定义为ST2，校准物主峰定义为ST3，以浓度为X轴，峰面积为Y轴，构建各血浆样品的单个内部校准曲线(使用线性回归分析)，再根据血浆样品中的目标分析物响应在校准曲线上读取相应的浓度。结果见表5和表6。
表5采用天然同位素校准曲线法测定样品中变肾上腺素的含量
Figure PCTCN2021130578-appb-000003
以目标分析物去甲变肾上腺素为例，已知加入的校准物去甲变肾上腺素-D3的浓度为0.9625ng/mL，质谱中测得校准物的分子式[C 9H 11D 3NO 3] +，默认被 2H(D)取代的原子不含别的同位素，则计算其主峰、第一、第二天然同位素峰的丰度比分别为100％、10.34％、1.10％，则对应的主峰、第一、第二天然同位素峰的浓度分别为962.5pg/mL、99.5pg/mL和10.6pg/mL。
表6采用天然同位素校准曲线法测定样品中去甲变肾上腺素的含量
Figure PCTCN2021130578-appb-000004
由于加入的校准物与目标分析物在质谱端的离子化效率略有差异，见表2，上述天然同位素校准曲线法所得的结果需除以相对响应因子后得到上述表5、表6中的最终校准后结果。
方法二：基质匹配内标校准法
用TargetLynx软件进行峰面积积分、计算响应。通过计算分析物峰面积/内标峰面积比，生成六点外部校准曲线(见图4)，从而计算各血浆样品中的分析物浓度，结果见表7和表8。
表7采用基质匹配内标校准曲线法测定样品中变肾上腺素的含量
Figure PCTCN2021130578-appb-000005
表8采用基质匹配内标校准曲线法测定样品中去甲变肾上腺素的含量
Figure PCTCN2021130578-appb-000006
结果的对比：将使用方法一的天然同位素校准曲线法所得结果与使用方法二的基质匹配内标校准曲线法所得结果进行对比(表9，图5和图6)。
表9天然同位素校准曲线法与基质匹配内标校准曲线法的结果比较
Figure PCTCN2021130578-appb-000007
对比结果(图5和图6)显示R2＞0.99，结果非常一致，并且斜率接近于1。表明使用天然同位素校准曲线法可以获得和基质匹配内标校准曲线法基本一致的结果。
由此可见，本实施例显示了天然同位素校准曲线法的应用原理与实施过程。校准物为目标化合物的稳定同位素类似物，校准曲线上的点分别为该校准物的主峰、该校准物 的第一天然同位素峰和该校准物的第二天然同位素峰。它们与目标化合物具有极其类似的分子结构，可以校准前处理过程中的回收率损失，同时也可以校准分析过程中的进样误差和基质效应。唯一的不同在于校准物和目标分析物在离子源端的离子化效率略有差异，故所得结果需要用相对响应因子来校准。结果显示天然同位素校准曲线法测得的结果与传统的基质匹配内标校准曲线法所得结果基本一致，而前者具有不需要制备空白基质的便利性。
实施例2：人血清样品中25-羟基维生素D 3的含量分析
如实施例1所述，当目标分析物普遍存在于样品基质且含量较高时，制备空白基质特别困难且昂贵。25-羟基维生素D 3在正常血清中含量较高，因此常规的LC-MS/MS质谱定量方法普遍采用1％牛血清白蛋白溶液替代空白基质制备一系列浓度的校准品，再通过在校准品及待测样品中加入同样量的内标来校正基质效应，从而定量样品中的25-羟基维生素D 3。本实施例描述了人血清样品中25-羟基维生素D 3的含量分析，其中每个样品中加入已知量的稳定同位素标记的类似物，采用该稳定同位素及其已知丰度的天然同位素来构建校准曲线，定量分析样品中25-羟基维生素D 3的含量。同时将获得的结果与基质匹配内标校准法所得的结果进行比较。该实施例证明，通过使用稳定同位素标记的校准物及其已知丰度的天然同位素，可精确并准确定量人血清样品中的25-羟基维生素D 3。
人血清样品中的25-羟基维生素D 3的含量测定具体步骤如下：
1、稳定同位素标记校准物的准备：使用目标分析物的可商购的稳定同位素标记的类似物作为校准物，本实施例中，目标分析物为25-羟基维生素D 3，与之对应的可商购的稳定同位素标记的类似物为25-羟基维生素D 3-D3，参见图7。
2、目标分析物及校准物的单个储备溶液的制备：分别取目标分析物及稳定同位素标记校准物的固体粉末，以甲醇为溶剂，制成储备溶液，每个储备液的浓度分别为0.1mg/mL。
3、质谱条件的优化：由所述目标分析物及校准物的单个储备溶液，分别稀释得到0.1μg/mL的单个溶液，用于优化摸索质谱条件，结果见表10。其中25-羟基维生素D 3-D3-F1为25-羟基维生素D 3-D3的第一天然同位素，25-羟基维生素D 3-D3-F2为25-羟基维生素D 3-D3的第二天然同位素。
表10目标分析物及校准物的质谱条件
目标分析物或校准物 MRM跃迁 锥孔电压(V) 碰撞能量(V)
25-羟基维生素D 3 383.3→91.2 30 52
25-羟基维生素D 3-D3 386.3→257.3 30 16
25-羟基维生素D 3-D3-F1 387.3→258.3 30 16
25-羟基维生素D 3-D3-F2 388.3→259.3 30 16
4、目标分析物及校准物的单个工作溶液的制备：由目标分析物及其稳定同位素标记的类似物的单个储备溶液，稀释制备目标分析物(25-羟基维生素D 3)和稳定同位素标记类似物(25-羟基维生素D 3-D3)的混合溶液，所述混合溶液中25-羟基维生素D 3和25-羟基维生素D 3-D3的浓度均为10ng/mL。
5、计算相对响应因子，使用表9中描述的特定MRM跃迁，通过UPLC/MS/MS分析混合溶液。将来自目标分析物的6针平均峰面积与稳定同位素标记的类似物的6针平均峰面积进行对比，计算相对响应因子(表11)。
表11显示了相同浓度的目标分析物与稳定同位素标记的校准物的响应比值，作为相对响应因子
Figure PCTCN2021130578-appb-000008
6、1％牛血清白蛋白溶液的制备：取1g牛血清白蛋白至100mL容量瓶中，加入磷酸盐缓冲液溶解，定容至刻度，摇匀，即得。
7、基质匹配校准物的制备：将一系列25-羟基维生素D 3目标分析物溶液加入1％牛血清白蛋白溶液来构建一系列基质匹配的校准物，其中所含目标分析物的浓度分别为5ng/mL,20ng/mL,50ng/mL。
8、取来自20份正常人的血清样品，用于初步评估添加同位素校准物和基质匹配校准物所得结果的一致程度。
8.1采用天然同位素校准曲线法进行样品制备：
1)取200μL基质匹配校准物或血清样品；
2)加入200μL含校准物25-羟基维生素D 3-D3的甲醇+乙腈(1+1)溶液(其中含校准物25-羟基维生素D 3-D3为50ng/mL)；
3)涡旋混合30s；
4)加入1mL正己烷；
5)涡旋混合5min；
6)在10℃下14000转/分钟离心5min；
7)取上清液0.8mL至2mL离心管中，室温氮吹至干；
8)加入100μL85％甲醇水溶液(含0.1％甲酸)复溶；
9)涡旋混合30s；
10)在10℃下14000转/分钟离心3min；
11)吸取上清液至96孔进样板；
12)将上清液20μL注入超高效液相色谱-串联质谱仪分析。
使用Waters ACQUITY UPLC I-Class/Xevo TQ-S三重四极杆串联质谱系统进行分析，采用Waters ACQUITY UPLC HSS T3色谱柱(1.8μm，2.1mm×100mm)分析样品，以0.1％甲酸水溶液和0.1％甲酸甲醇分别为流动相A和B进行梯度洗脱，如表12中所示。运行时间为7.5min，柱温为40℃，进样体积为20μL。Waters Xevo TQ-S三重四极杆串联质谱系统采用多反应监测的模式操作,质谱条件如表10所示，分离结果显示在图8中。
表12血清中25-羟基维生素D 3分析的UPLC梯度条件
时间(min) 流速(mL/min) ％A ％B 曲线
0.0 0.4 30 70 -
1.5 0.4 15 85 6
5.0 0.4 5 95 6
6.0 0.4 5 95 6
7.5 0.4 5 70 1
用Waters MassLynx软件采集数据，对于天然同位素校准曲线法，使用TargetLynx软件测定图8中各通道的积分峰面积，导入Microsoft Excel用于构建各血浆样品的单个内部校准曲线(使用线性回归分析)，从而计算各血浆样品中的分析物浓度。
5.2采用基质匹配内标校准法进行样品制备：
1)取200μL各浓度的基质匹配校准物(3个)、200μL血清样品(20个)形成23个待测样品；
2)向各个待测样品中加入200μL含25-羟基维生素D 3-D3的甲醇+乙腈(1+1)溶液(其中含25-羟基维生素D 3-D3为50ng/mL，这里仅作为内标使用)；
3)涡旋混合30s；
4)加入1mL正己烷；
5)涡旋混合5min；
6)在10℃下14000转/分钟离心5min；
7)取上清液0.8mL至2mL离心管中，室温氮吹至干；
8)加入100μL85％甲醇水溶液(含0.1％甲酸)复溶；
9)涡旋混合30s；
10)在10℃下14000转/分钟离心3min；
11)吸取上清液至96孔进样板；
12)将各个复溶液20μL注入超高效液相色谱-串联质谱仪分析。
使用TargetLynx软件进行峰面积积分、计算响应，通过计算分析物峰面积/内标峰面积比，生成三点外部校准线，从而计算各血清样品中的分析物浓度。
结果：
方法一：天然同位素校准曲线法
用TargetLynx软件测定图7中各通道的积分峰面积，导入Microsoft Excel，已知加入的校准物25-羟基维生素D 3-D3的浓度为50ng/mL，质谱中测得校准物的分子式[C 27H 42D 3O 2]+，默认被2H(D)取代的原子不含别的同位素，则计算其主峰、第一、第二天然同位素峰的丰度比分别为100％，29.75％、4.68％，则对应的主峰、第一、第二天然同位素峰的浓度分别为50ng/mL、14.875ng/mL和2.34ng/mL。将校准物的第二天然同位素峰定义为ST1，校准物的第一天然同位素峰定义为ST2，校准物主峰定义为ST3，以浓度为X轴，峰面积为Y轴，构建各血浆样品的单个内部校准曲线(使用线性回归分析)，再根据血清样品中的目标分析物响应在校准曲线上读取相应的浓度。结果见表13。
表13采用天然同位素校准曲线法测定样品中25-羟基维生素D 3的含量
Figure PCTCN2021130578-appb-000009
由于加入的校准物与目标分析物在质谱端的离子化效率略有差异，见表11，上述天然同位素校准曲线法所得的结果需除以相对响应因子后得到最终的结果(即表13中校正后的结果)。
方法二：基质匹配内标校准法
用TargetLynx软件进行峰面积积分、计算响应。通过计算分析物峰面积/内标峰面积比，生成三点外部校准曲线(见图9)，从而计算各血清样品中的分析物浓度，结果见表14。
表14采用基质匹配内标校准曲线法测定样品中25-羟基维生素D 3的含量
Figure PCTCN2021130578-appb-000010
将采用方法一的天然同位素校准曲线法所得结果与方法二的基质匹配内标校准法所得结果进行对比，见图10。两种方法所得结果接近，回归分析r 2>0.99并且斜率接近于1。
实施例3：柱前衍生化法测定人血浆样品中醛固酮的含量分析
该实例描述了人血浆样品中醛固酮的含量分析，在每个样品中加入已知量的稳定同位素标记的类似物，采用该稳定同位素及其已知丰度的天然同位素来构建校准曲线，定量分析样品中醛固酮的含量。同时将获得的结果与常规的基质校准曲线法所得的结果进行比较。后者采用1％牛血清白蛋白/磷酸缓冲溶液来制备一系列浓度的校准品，再通过在校准品及样品中加入内标来校正回收率和基质效应，从而定量样品中的醛固酮的含量。由于该实施例的前处理涉及衍生化的操作，进一步验证了稳定同位素和目标分析物之间物理化学性质的一致性，结果显示使用天然同位素校准曲线法，即可精确并准确定量样本中的醛固酮的含量。
人血浆样品中的醛固酮的含量测定具体步骤如下：
1、稳定同位素标记校准物的准备：使用目标分析物的可商购的稳定同位素标记的类似物作为校准物，该实施例中，目标分析物为醛固酮，与之对应的可商购的稳定同位素标记的类似物有醛固酮-D4，醛固酮-D7或醛固酮-D8等，本次实验选择的是醛固酮-D8，参见图11。
2、目标分析物及校准物的单个储备溶液的制备：分别取目标分析物及稳定同位素标记校准物的固体粉末，以甲醇为溶剂，制成储备溶液，每个储备液的浓度分别为0.1mg/mL。
3、质谱条件优化：由所述目标分析物及校准物的单个储备溶液，分别稀释得到0.1μg/mL的单个溶液，用于优化摸索质谱条件，结果见表15。其中醛固酮-D8衍生物-F1为醛固酮-D8衍生物的第一天然同位素，醛固酮-D8衍生物-F2为醛固酮-D8衍生物的第二天然同位素。
表15目标分析物及校准物的质谱条件
目标分析物或校准物 MRM跃迁 锥孔电压(V) 碰撞能量(V)
醛固酮衍生物 494.3→415.2 20 35
醛固酮-D8衍生物 502.3→423.2 20 35
醛固酮-D8衍生物-F1 503.3→424.2 20 35
醛固酮-D8衍生物-F2 504.3→425.2 20 35
4、目标分析物及校准物的单个工作溶液的制备：由目标分析物的单个储备溶液，稀释制备目标分析物的溶液，其中醛固酮的浓度为1ng/mL。由校准物的单个储备溶液，稀释制备校准物的工作溶液，其中醛固酮-D8的浓度为1ng/mL。
5、计算相对响应因子：分别取1ng/mL的醛固酮和醛固酮-D8的工作溶液10uL，按下述步骤8.1的前处理方案，自“9)依次加入乙酸…”起，同法操作，得到目标分析物和校准物的衍生产物。使用表14中描述的特定MRM跃迁，通过UPLC/MS/MS分析目标物和校准物的衍生产物。将来自目标分析物的6针平均峰面积与校准物的6针平均峰面积进行对比，计算相对响应因子(表16)。
表16显示了相同浓度的目标分析物与稳定同位素标记的校准物的响应比值，作为相对响应因子
Figure PCTCN2021130578-appb-000011
6、1％牛血清白蛋白溶液的制备：取1g牛血清白蛋白至100mL容量瓶中，加入磷酸盐缓冲液溶解，定容至刻度，摇匀，即得。
7、基质匹配校准物的制备：以1％牛血清白蛋白溶液为稀释剂，将1ng/mL醛固酮目标分析物溶液稀释成一系列模拟基质匹配的校准物，其中所含目标分析物的浓度分别为2、5、10、20、50、100pg/mL。
8、取来自8份正常人的血浆，用于评估天然同位素校准曲线法和基质匹配校准法所得结果的一致程度。
8.1采用天然同位素校准曲线法进行样品制备：
1)取200μL血浆样品置2mL离心管中；
2)加入20μL含校准物醛固酮-D8的溶液(其中醛固酮-D8的浓度为1ng/mL)；
3)涡旋混合30s；
4)加入1mL甲基叔丁基醚；
5)上下剧烈震荡3min；
6)在10℃下14000转/分钟离心5min；
7)尽量吸取上清液，转移至另一1.5mL离心管中
8)氮气吹干
9)依次加入乙酸，衍生化试剂
10)室温反应10min
11)氮气吹干，流动相初始溶液50uL复溶；
12)将各个复溶液20μL注入超高效液相色谱-串联质谱仪分析。
使用Waters ACQUITY UPLC I-Class/Xevo TQ-S三重四极杆串联质谱系统进行分析，采用Waters ACQUITY UPLC BEH C8色谱柱(1.8μm，2.1mm×100mm)分析样品，以0.1％甲酸水溶液(含2mM甲酸铵)和甲醇分别为流动相A和B进行梯度洗脱，如表17中所示。运行时间为5.5min，柱温为40℃，进样体积为20μL。Waters Xevo TQ-S三重四极杆串联质谱系统采用多反应监测的模式操作,质谱条件如表15所示，分离结果显示在图12中。
表17血浆中儿茶酚胺代谢物分析的UPLC梯度条件
时间(min) 流速(mL/min) ％A ％B 曲线
0.0 0.4 85 15 -
0.2 0.4 85 15 6
2.5 0.4 60 40 6
3.5 0.4 10 90 6
4.5 0.4 10 90 6
4.6 0.4 85 15 6
5.5 0.4 85 15 1
用Waters MassLynx软件采集数据，对于天然同位素校准曲线法，使用TargetLynx软件测定图12中各通道的积分峰面积，导入Microsoft Excel用于构建各血浆样品的单个内部校准曲线(使用线性回归分析)，从而计算各血浆样品中的目标分析物浓度。
8.2对于基质匹配内标校准法，采用基质匹配内标校准的方法进行样品制备：
1)取200μL各浓度的基质匹配校准物(6个)、200μL血浆样品(8个)形成14个待测样品；
2)向各个待测样品中加入20μL含校准物醛固酮-D8的溶液(其中醛固酮-D8的浓度为1ng/mL，这里仅作为内标使用)；
3)涡旋混合30s；
4)加入1mL甲基叔丁基醚；
5)上下剧烈震荡3min；
6)在10℃下14000转/分钟离心5min；
7)尽量吸取上清液，转移至另一1.5mL离心管中
8)氮气吹干
9)依次加入乙酸，衍生化试剂
10)室温反应10min
11)氮气吹干，流动相初始溶液50uL复溶；
12)将各个复溶液20μL注入超高效液相色谱-串联质谱仪分析。
用Waters MassLynx软件采集数据，对于基质匹配内标校准法，使用TargetLynx软件进行峰面积积分、计算响应。通过计算目标分析物峰面积/内标峰面积比，生成六点校准曲线，从而计算各血浆样品中的目标分析物浓度。
结果：
方法一：天然同位素校准曲线法
用TargetLynx软件测定图12中各通道的积分峰面积，导入Microsoft Excel，已知加入的校准物醛固酮-D8的浓度为100pg/mL(折合入样品体积)，经前处理后，校准物醛固酮-D8的衍生物分子式为[C28H27D8O5N3] +，默认被 2H取代的原子不含别的同位素，则计算其主峰，第一，第二天然同位素的丰度比分别为100％，31.88％，5.94％，则对应的主峰，第一，第二天然同位素的浓度分别为100pg/mL，31.88pg/mL和5.94pg/mL。将校准物的第二天然同位素定义为ST1，校准物的第一天然同位素定义为ST2，校准物主峰定义为ST3，以浓度为X轴，峰面积为Y轴，构建各血浆样品的单个内部校准曲线(使用线性回归分析)，再根据血浆样品中的目标分析物响应在校准曲线上读取相应的浓度。结果见表18。
表18采用天然同位素校准曲线法测定样品中醛固酮的含量
Figure PCTCN2021130578-appb-000012
如实施例1和2所述，由于加入的校准物与目标分析物在质谱端的离子化效率略有差异，见表16，上述天然同位素校准曲线法所得的结果需除以相对响应因子后得到最终校准后的结果。
方法二：基质匹配内标校准法
用TargetLynx软件进行峰面积积分、计算响应。通过计算目标分析物峰面积/内标峰面积比，生成六点外部校准曲线(图13)，从而计算各血浆样品中的分析物浓度，结果见表19。
表19采用基质匹配内标校准曲线法测定样品中醛固酮的含量
Figure PCTCN2021130578-appb-000013
结果的对比：将使用天然同位素校准曲线法所得结果与使用基质匹配内标校准曲线法所得结果进行对比(表20)。
表20天然同位素校准曲线法与基质匹配内标校准曲线法的结果比较
Figure PCTCN2021130578-appb-000014
对比结果显示，两组数据较为接近。由于该组数据本身是在超痕量分析的范围，基质匹配校准曲线法采用的是牛血清白蛋白模拟的实际基质，跟实际的样品还是存有一定的差距，另外样品前处理过程中，目标化合物和校准品进行了衍生化反应。表格所列结果亦在可接受的范围内。使用天然同位素校准曲线法可以获得和基质匹配内标校准曲线法基本接近的结果，后续可进一步探讨结果产生微小差异的可能原因以及何种方法获得的结果更接近理论实际。
实施例4：高分辨质谱应用于治疗药物监测全血样品中他克莫司的含量
该实例描述了将天然同位素校准曲线法应用于人全血样品中他克莫司的治疗药物监测，以低浓度和高浓度两组质控样品来评估方法的准确性和精密度。在每个样品中加入已知量的稳定同位素标记的类似物，采用该稳定同位素及其已知丰度的天然同位素来构建校准曲线，定量分析样品中他克莫司的含量。同时将获得的结果与常规的基质匹配内标校准曲线法所得的结果进行比较。由于他克莫司为外源性的药物，基质匹配内标校准曲线法可采用与制备质控样品相同的空白基质来制备一系列浓度的校准物，故基质匹配校准曲线法所得的结果应与理论值基本一致。可供进一步评估天然同位素校准曲线法的准确性。另外由于该方法采用高分辨质谱作为检测器，可用于评估天然同位素校准曲线法在不同质谱平台之间的适用性。最终结果显示使用天然同位素校准曲线法，即可精确并准确定量样本中他克莫司的含量。
人全血样品中的他克莫司的含量测定具体步骤如下：
1、稳定同位素标记校准物的准备：使用目标分析物的可商购的稳定同位素标记的类似物作为校准物，该实施例中，目标分析物为他克莫司，与之对应的可商购的稳定同位素标记的类似物为他克莫司- 13CD2，参见图14。
2、目标分析物及校准物的单个储备溶液的制备：分别取目标分析物及稳定同位素标记校准物的固体粉末，以甲醇为溶剂，制成储备溶液，每个储备液的浓度分别为1mg/mL。
3、目标分析物及校准物的单个工作溶液的制备：由目标分析物的单个储备溶液，稀释制备目标分析物的溶液，其中他克莫司的浓度为10μg/mL。由校准物的单个储备溶液，稀释制备校准物的工作溶液，其中他克莫司- 13CD2的浓度为5μg/mL。
4、计算相对响应因子：分别取10μg/mL的他克莫司和他克莫司- 13CD2溶液，用乙腈稀释得到各含他克莫司和他克莫司- 13CD2为10ng/mL溶液，即得。用以考察两者之间的相对响应因子。
由于采用高分辨质谱作为检测器，扫描方式为数据非依赖的全扫描，无需额外优化质谱条件，通过UPLC/MS/MS分析目标物和校准物。将来自目标分析物的6针平均峰面积与稳定同位素标记的校准物的6针平均峰面积进行对比，计算相对响应因子(表21)。
表21显示了相同浓度的稳定同位素标记的校准物与目标分析物的响应比值，作为相对响应因子
Figure PCTCN2021130578-appb-000015
5、基质匹配校准物的制备：以空白全血样品为稀释剂，将10μg/mL他克莫司目标分析物溶液稀释成一系列基质匹配的校准物，其中所含目标分析物的浓度分别为10、20、50、100、200、500ng/mL。经样品前处理后，基质溶液稀释约10倍，折合后的目标分析物的浓度分别为1、2、5、10、20、50ng/mL。
6、高低浓度质控样品的制备：以同样的空白全血样品为稀释剂，将10μg/mL他克莫司目标分析物溶液稀释成50和200ng/mL的低浓度和高浓度质控样品。经样品前处理后，折合后的高低浓度质控样品中目标分析物的浓度分别为5和20ng/mL。
7、取高低浓度的质控样品，各一式三份，共6份样品，用于评估天然同位素校准曲线法和基质匹配内标校准法所得结果的一致程度。
7.1采用天然同位素校准曲线法进行样品制备：
1)取5μL5μg/mL他克莫司- 13CD2校准品溶液置2mL离心管中，氮气吹干液体；
2)加入50μL全血质控样品
3)加入100μL0.1M硫酸锌溶液；
4)加入400μL乙腈；
5)上下剧烈震荡3min；
6)在10℃下，14000转/分钟离心5min；
7)吸取上清液100μL转移至全回收进样小瓶；
8)将上清液5μL注入超高效液相色谱-串联质谱仪分析。
使用Waters ACQUITY UPLC I-Class/Synapt G2-S Qtof高分辨质谱系统进行分析，采用Waters ACQUITY UPLC BEH C18色谱柱(1.7μm，2.1mm×50mm)分析样品，以0.1％甲酸水溶液(含2mM甲酸铵)和0.1％甲酸甲醇(含2mM甲酸铵)分别为流动相A和B进行梯度洗脱，如表22中所示。运行时间为2.5min，柱温为40℃，进样体积为5μL。Waters Synapt G2-S Qtof高分辨质谱系统采用数据非依赖的全扫描模式进行数据采集,可根据目标化合物的精确分子量提取响应的质谱图，结果显示在图15中。
表22全血样品中他克莫司分析的UPLC梯度条件
时间(min) 流速(mL/min) ％A ％B 曲线
0.0 0.5 50 50 -
0.1 0.5 50 50 6
1.0 0.5 0 100 6
1.5 0.5 0 100 6
2.5 0.5 50 50 1
用Waters MassLynx软件采集数据，对于天然同位素校准曲线法，使用TargetLynx软件测定图14中各通道的积分峰面积，导入Microsoft Excel用于构建各血浆样品的单个内部校准曲线(使用线性回归分析)，从而计算各血浆样品中的分析物浓度。
7.2对于基质匹配内标校准法，采用基质匹配内标校准的方法进行样品制备：
1)取5μL5μg/mL他克莫司- 13CD2校准品溶液置2mL离心管中，氮气吹干液体；
2)加入50μL各浓度的基质匹配校准物(6个)、高低浓度质控样品(各一式三份，6个)形成12个待测样品；
3)加入100μL0.1M硫酸锌溶液；
4)加入400μL乙腈；
5)上下剧烈震荡3min；
6)在10℃下，14000转/分钟离心5min；
7)吸取上清液100μL转移至全回收进样小瓶；
8)将上清液5μL注入超高效液相色谱-串联质谱仪分析。
对于基质匹配内标校准法，使用TargetLynx软件进行峰面积积分、计算响应。通过计算分析物峰面积/内标峰面积比，生成六点外部校准线，从而计算各样品中的分析物浓度。
结果：
方法一：天然同位素校准曲线法
用TargetLynx软件测定图15中各通道的积分峰面积，导入Microsoft Excel，已知加入的校准物他克莫司- 13CD2的浓度为50ng/mL(折合入样品体积)，校准物的分子式为[C28H27D8O5N3] +，默认被 13C和 2H取代的原子不含别的同位素，排除在丰度计算之外，则计算其主峰，第一，第二天然同位素的丰度比分别为100％，48.51％，13.98％，则对应的 主峰，第一，第二天然同位素的浓度分别为50ng/mL，24.255ng/mL和6.99ng/mL。将校准物的第二天然同位素定义为ST1，校准物的第一天然同位素定义为ST2，校准物主峰定义为ST3，以浓度为X轴，峰面积为Y轴，构建各质控样品的单个内部校准曲线(使用线性回归分析)，再根据质控样品中的目标分析物响应在校准曲线上读取相应的浓度。结果见表23。
表23采用天然同位素校准曲线法测定样品中他克莫司的含量
Figure PCTCN2021130578-appb-000016
如实施例1和2所述，由于加入的校准物与目标分析物在质谱端的离子化效率略有差异，见表21，上述天然同位素校准曲线法所得的结果需除以相对响应因子后得到最终校准后的结果。
方法二：基质匹配内标校准法
用TargetLynx软件进行峰面积积分、计算响应。通过计算分析物峰面积/内标峰面积比，生成六点外部校准曲线(图16)，从而计算各血浆样品中的分析物浓度，结果见表24。
表24采用基质匹配内标校准曲线法测定样品中他克莫司的含量
Figure PCTCN2021130578-appb-000017
结果的对比：将使用天然同位素校准曲线法所得结果与使用基质匹配内标校准曲线法所得的质控样的结果进行对比(表25)。
表25天然同位素校准曲线法与基质匹配内标校准曲线法的结果比较
Figure PCTCN2021130578-appb-000018
对比结果显示，两个方法的质控数据结果基本一致。由于该实施例中质控样品和基质校准曲线采用的是相同的空白基质，所以质控样品的数据应较为准确。同时实施例验 证了将天然同位素标曲法应用于高分辨质谱系统的可行性，采用全扫描的模式进行定量分析，直接分析目标化合物的准分子离子峰，而无需额外的优化质谱条件。
基于与上述实施例相同的原理，本发明的天然同位素校准曲线法同样适用于其他包括至少3个碳原子的有机分子的定量分析。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。

Claims (11)

  1. 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，其特征在于，包括以下步骤：
    A、在待测样品中添加已知量的校准物，所述校准物为目标分析物的稳定同位素标记的类似物；
    B、对待测样品进行前处理；
    C、将步骤B处理后的待测样品进行质谱法分析，得到校准物及校准物的天然同位素的质谱信号、待测样品中的目标分析物的质谱信号；
    D、根据校准物的分子式计算得到校准物的天然同位素丰度比，从而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已知含量、质谱信号响应的对应关系绘制校准曲线，再根据待测样品中的目标分析物的质谱信号响应，对应到校准曲线中即得到待测样品中的目标分析物含量；
    步骤A中，所述目标分析物为一个或多个，每一种目标分析物对应在待测样品中添加一个已知量的校准物。
  2. 根据权利要求1所述的用于定量样品中目标分析物的天然同位素校准曲线法，其特征在于，步骤B中，所述前处理的方法选自固相萃取法、固液萃取法、液液萃取法、沉淀蛋白法、直接稀释法、溶剂提取法、盐析法、化学分离法、浓缩法、吸附色谱分离、分配色谱分离、离子交换色谱分离、掩蔽法中的任一种。
  3. 根据权利要求1所述的用于定量样品中目标分析物的天然同位素校准曲线法，其特征在于，步骤C中，所述校准物的天然同位素质谱信号为一个或多个。
  4. 根据权利要求3所述的用于定量样品中目标分析物的天然同位素校准曲线法，其特征在于，所述校准物的天然同位素质谱信号为多个时，即包括校准物的第一天然同位素、校准物的第二天然同位素，以此类推，步骤D的标准曲线绘制方法如下：
    根据校准物的分子式计算得到校准物的天然同位素丰度比，从而得到校准物的第一天然同位素的含量作为第二已知量的校准物、校准物的第二天然同位素的含量作为第三已知量的校准物，以此类推；再根据各校准物的已知含量、质谱信号响应的对应关系绘制校准曲线。
  5. 根据权利要求1或4所述的用于定量样品中目标分析物的天然同位素校准曲线法，其特征在于，当采用单位质量分辨率的质谱分析时，所述校准物与校准物的天然同 位素的质荷比大于等于1个道尔顿质量数；当采用高分辨质谱分析时，所述校准物与校准物的天然同位素的质荷比大于等于0.05个道尔顿质量数。
  6. 根据权利要求1或4所述的用于定量样品中目标分析物的天然同位素校准曲线法，其特征在于，所述校准物与目标分析物在质谱法分析中彼此相差至少2个道尔顿质量数。
  7. 根据权利要求1所述的用于定量样品中目标分析物的天然同位素校准曲线法，其特征在于，步骤A中，所述待测样品包括生物样品、环境样品、食品样品、合成样品、药物样品、化工样品、临床化学样品、法医样品、药理样品、农业样品中的任一种；所述生物样品包括血浆、血清、全血、尿液、组织、脑脊液、汗液、唾液、毛发、皮肤中的任一种。
  8. 根据权利要求1所述的用于定量样品中目标分析物的天然同位素校准曲线法，其特征在于，步骤A中，所述目标分析物为包括至少3个碳原子的有机分子。
  9. 根据权利要求1所述的用于定量样品中目标分析物的天然同位素校准曲线法，其特征在于，步骤A中，所述的校准物为目标分析物的稳定同位素标记类似物，为目标分析物的至少1个原子被其稳定同位素替代后得到；所述稳定同位素包括 2H、 11B、 13C、 15N、 17O、 18O、 33S、 34S和 36S中的任一种或多种。
  10. 根据权利要求1所述的用于定量样品中目标分析物的天然同位素校准曲线法，其特征在于，所述质谱法采用的离子源包括电喷雾离子源、大气压化学电离离子源、基质辅助激光解吸电离离子源、解吸电喷雾电离离子源、电子轰击源、化学电离源，质量分析器包括四级杆质量分析器、离子阱质量分析器、扇形磁质谱质量分析器、飞行时间质量分析器、静电场轨道阱质量分析器、傅里叶变换离子回旋共振质量分析器中的任一种或其中两种及以上的组合；所述质谱的检测模式选自全扫描模式、子离子检测模式、母离子检测模式、多反应检测模式、中性丢失扫描、数据依赖性扫描模式、非数据依赖性扫描模式中的任一种；
    所述质谱法分析之前还包括采用色谱法进行分离的步骤；所述色谱法选自液相色谱法，气相色谱法，毛细管电泳法，亲和色谱法，免疫亲和色谱法，超临界流体色谱法，离子淌度法中的任一种。
  11. 根据权利要求1所述的用于定量样品中目标分析物的天然同位素校准曲线法，其特征在于，步骤D中，还包括对待测样品中的目标分析物含量进行校正的步骤；具体包括：将相同浓度的目标分析物与稳定同位素标记的校准物注入质谱仪分析，以目标分 析物与稳定同位素标记的校准物的响应比值，作为相对响应因子；实际样品分析时，所得目标分析物的含量结果需乘以相对响应因子来作为最终结果。
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