WO2017050096A1 - 一种质谱定量分析的模拟内标方法、装置及应用 - Google Patents

一种质谱定量分析的模拟内标方法、装置及应用 Download PDF

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WO2017050096A1
WO2017050096A1 PCT/CN2016/097261 CN2016097261W WO2017050096A1 WO 2017050096 A1 WO2017050096 A1 WO 2017050096A1 CN 2016097261 W CN2016097261 W CN 2016097261W WO 2017050096 A1 WO2017050096 A1 WO 2017050096A1
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internal standard
sample
mass spectrometry
standard
standard sample
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PCT/CN2016/097261
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English (en)
French (fr)
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朱建雄
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广东联捷生物科技有限公司
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Priority to US15/762,019 priority Critical patent/US10643829B2/en
Priority to EP16847978.0A priority patent/EP3355054A4/en
Priority to JP2018515956A priority patent/JP6678736B2/ja
Priority to CA2999366A priority patent/CA2999366A1/en
Publication of WO2017050096A1 publication Critical patent/WO2017050096A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/0009—Calibration of the apparatus
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
    • 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
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8665—Signal analysis for calibrating the measuring apparatus
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N2030/042—Standards
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8822—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving blood
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/20—Injection using a sampling valve

Definitions

  • the invention relates to the technical field of mass spectrometry, in particular to a method, a device and an application for simulating an internal standard for quantitative analysis of mass spectrometry.
  • the straight line is only the result obtained under ideal conditions.
  • the relationship between the measured signal and the concentration (YC) is only linear in the range where the concentration change is small, and for the larger range, such as the concentration change is more than 10 times.
  • the concentration of the unknown can be calculated by taking the signal obtained by measuring the unknown sample into the obtained curve function, and the concentration of the unknown sample can be directly found on the calibration curve. This is the general process of quantitative analysis.
  • the error introduced by the difference in the sample processing process does not necessarily need to be eliminated by the internal standard method, and the source of error can be discharged by strict operating procedures and automated processing techniques.
  • the error caused by the instability of the instrument is principled. Even if the instrument is made refined, such an error still exists, so the internal standard method is needed to eliminate it.
  • almost all quantitative analyses use the internal standard method.
  • Mass spectrometry is an analytical instrument for the separation and determination of charged particles. At present, mass spectrometry is generally used for quantitative determination of trace chemical components, especially in pharmacokinetics, drug metabolism, toxicology, clinical trials, etc. The use of mass spectrometry in the field is already the best choice. Using mass spectrometry as an analytical means, the substance to be tested must first be ionized into charged particles. The mass spectrometer can distinguish charged particles according to their ratio of mass to charge with a high degree of resolution.
  • the ionization efficiency obtained by the slight difference in structure can be different. It is necessary to find an ionization property and the substance to be tested. Consistent internal standard substances are very difficult.
  • the isotope is to replace some elements in the analyte with heavy isotopes, such as replacing the hydrogen with hydrazine to obtain the molecular weight.
  • Larger but identically structured allogeneic molecules when the analyte and the isotope are simultaneously introduced into the mass spectrometry, the signal of the two can be distinguished by the high resolution function of the mass spectrometer, and the ratio of their signal intensities is The basis of the standardization of the standard.
  • the only way to improve the accuracy of mass spectrometry is to use an isotope as an internal standard, usually called an isotope internal standard.
  • the problems of mass spectrometry quantification are as follows: Although the isotope internal standard can increase the accuracy of mass spectrometry from about 70% to more than 95%, and the accuracy can be achieved by other instruments, but the synthesis cost of the isotope internal standard is very high. In addition to clinical trial analysis in the development of new drugs, most other applications, including preclinical experiments in the pharmaceutical process, are too costly and too long to wait for synthesis, which greatly hampers the wide range of mass spectrometry techniques. Application, while also delaying the biopharmaceutical process and increasing costs. Furthermore, mass spectrometry should be able to play a greater role in clinical testing, environmental analysis, food safety, etc., without the problems of internal standards.
  • a simulated internal standard method for quantitative analysis of mass spectrometry comprising the steps of:
  • the blank substrate of the sample to be tested is configured into one to several concentrations of the internal standard solution, and the concentration range and span of the internal standard series. Consistent with the standard solution;
  • the standard sample and the internal standard sample are successively injected into a chromatography-mass spectrometry system for analysis and determination;
  • the ratio of the peak areas of the measured standard sample to the internal standard sample is plotted against the concentration to obtain a calibration curve that is quantified by a simulated internal standard method
  • the sample to be tested is processed by a sample processing step, injected into a chromatographic-mass spectrometry system for analysis and determination, and then the peak area of the measured signal is compared with a calibration curve to determine the concentration of the sample to be tested.
  • the simulated internal standard method for quantitative analysis of mass spectrometry wherein each time after entering a standard sample, the sample is paused for a while, and after the time required for the standard sample is measured, the internal standard sample is injected, and the internal standard sample is passed through chromatography-mass spectrometry.
  • the column of the combined system, the standard sample and the internal standard sample are separated and analyzed in the same detection cycle.
  • the simulated internal standard method for quantitative analysis of mass spectrometry wherein after each standard sample is entered, the internal standard sample is further input, and the internal standard sample is bypassed by the chromatography-mass spectrometry system.
  • the column is flow-injected directly into the mass spectrometer for measurement.
  • the simulated internal standard method for quantitative analysis of mass spectrometry wherein after each standard sample is taken, after the chromatographic peak of the standard sample is detected, the internal standard sample closest to the standard sample concentration is further input, and The standard sample bypasses the chromatographic-mass spectrometry column and is directly injected into the mass spectrometer for flow measurement.
  • the simulated internal standard method for quantitative analysis of mass spectrometry wherein the injection flow rate is reduced by 20% when the internal standard sample is injected, or the flow rate of the spray gas during injection is increased from 60 liters/min to 70-100 liters/min. .
  • a simulated internal standard device for quantitative analysis of mass spectrometry comprising:
  • An injector connected to the mobile phase delivery device
  • the injector includes a syringe and an injection valve coupled to the syringe for switching between an injection state and a injection state, the syringe being coupled to the injection valve through a sample loop.
  • the simulated internal standard device for quantitative analysis of mass spectrometry wherein the mobile phase switching device is two three-way switching valves connected before and after the column.
  • the simulated internal standard device for quantitative analysis of mass spectrometry wherein the mobile phase switching device is a four-way switching valve connected to the column.
  • the simulated internal standard device for quantitative analysis of mass spectrometry wherein the mobile phase switching device is a six-way switching valve connected to the column.
  • the simulated internal standard device for quantitative analysis of the photograph wherein the flow switching valve connected to the column is a six-way injection valve in the internal standard injector for internal standard sample injection.
  • the simulated internal standard device for quantitative analysis of mass spectrometry further comprising an internal standard sample introduction device connected to the four-way switching valve, wherein the internal standard sample introduction device comprises an internal standard injection connected to the four-way switching valve a second mobile phase transport device coupled to the internal standard injector, and a second mobile phase storage device coupled to the second mobile phase transport device.
  • the present invention uses the standard of the substance to be analyzed itself as an internal standard for mass spectrometry quantitative analysis, and the “internal standard” is not added to the analysis sample, but is separately introduced into the mass spectrometer for measurement, specifically in the sample. Immediately after the appearance of the analyte signal in the mass spectrometer, a standard of a known amount of the analyte is introduced into the mass spectrometer as an internal standard, and the measured signal of the object to be tested and the subsequent internal standard signal are taken.
  • the ratio is quantified as the ordinate value of the calibration curve, and the sensitivity change caused by the change in mass spectrometry ionization is small in a short time interval, so the quantitative method of the present invention is similar to the effect achieved by the usual internal standard method. Basically, the inaccuracy caused by the change in ionization efficiency can be completely eliminated by mass spectrometry.
  • the invention can achieve an accuracy of more than 95% without using an isotope internal standard; the invention broadly extends the linear response range of mass spectrometry quantitative analysis.
  • the method and device of the present invention can accurately determine the concentration of the drug in the patient's blood sample after the patient is administered to determine whether the drug used is effective for a specific patient. The purpose of medication.
  • FIG. 1 is a schematic structural view of Embodiment 1 of the present invention.
  • FIG. 2 is another schematic structural diagram of Embodiment 1 of the present invention.
  • Fig. 3 is a chromatogram of the first embodiment of the present invention.
  • Figure 4 is a standard curve at the beginning of the first embodiment of the present invention.
  • Figure 5 is a standard curve after 2 hours in the first embodiment of the present invention.
  • Figure 6 is a chromatogram of Example 2 of the present invention.
  • Figure 7 is a standard curve at the beginning of Embodiment 2 of the present invention.
  • Figure 8 is a standard curve after 2 hours in the second embodiment of the present invention.
  • Fig. 9 is a view showing the state change of the six-way switching valve in the second embodiment of the present invention.
  • Figure 10 is a diagram showing the state change of the internal standard sample introduction device in the second embodiment of the present invention.
  • Figure 11 is a schematic structural view of a third embodiment of the present invention.
  • Figure 12 is a chromatogram of Example 3 of the present invention.
  • FIG. 13 is a schematic structural diagram of another embodiment of the third embodiment of the present invention.
  • Figure 14 is a standard curve at the beginning of the third embodiment of the present invention.
  • Figure 15 is a standard curve after 7 hours in the third embodiment of the present invention.
  • the present invention provides a method and a device for simulating an internal standard for quantitative analysis of mass spectrometry.
  • an internal standard for quantitative analysis of mass spectrometry In order to make the object, technical solution and effect of the present invention more clear and clear, the following further details the present invention. Detailed description. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
  • the invention provides a simulated internal standard method for quantitative analysis of mass spectrometry, which comprises the steps of:
  • the standard sample and the internal standard sample are successively injected into a chromatography-mass spectrometry system for analysis and determination;
  • the ratio of the peak area of the measured standard sample to the internal standard sample is plotted against the concentration to obtain a calibration curve of the simulated internal standard method
  • the sample to be tested is processed by a sample processing step and injected into a chromatography-mass spectrometry system for measurement, and then the peak area of the measured signal is compared with a calibration curve to determine the concentration of the sample to be tested.
  • each time after entering a standard sample it can be paused for a while (after waiting for the time required to measure the standard sample, such as 30s, to ensure that the standard sample signal does not overlap with the subsequent internal standard sample signal), and then The internal standard sample was injected to pass the internal standard sample through the chromatographic-mass spectrometry column, and the standard sample and the internal standard sample were separated and analyzed in the same detection cycle. It is also possible to enter the internal standard sample after each standard sample is entered, and the internal standard sample is bypassed to the column and flow directly into the mass spectrometer for measurement. Or after each standard sample is entered, the color of the standard sample is detected.
  • the internal standard sample closest to the standard sample concentration is entered, and the internal standard sample is bypassed to the column and flowed directly into the mass spectrometer for measurement.
  • the principle is that the obtained internal standard sample signal is as close as possible to the standard sample signal without overlapping.
  • the injection flow rate is reduced by 20%, or the flow rate of the spray gas at the time of injection is increased from 60 liters/min to 70-100 liters/min (e.g., preferably 80 liters/min).
  • Pre-column injection is more sensitive than post-column injection at the same concentration. The reason is mainly the change of spray ionization efficiency. For this reason, by temporarily improving the spray conditions, the spray efficiency of post-column injection is improved, and the sensitivity is eliminated. The purpose of the difference.
  • the specific method is to reduce the flow rate of the post-column injection, so that the spray is more complete, the flow rate can be reduced by about 20% to eliminate the difference in sensitivity; the second is to increase the flow rate of the spray gas from 60 liters / minute to 70-100 liters / Minutes can reduce the difference to less than 5%.
  • the invention also provides a simulated internal standard device for quantitative analysis of mass spectrometry, comprising:
  • An injector connected to the mobile phase delivery device
  • the injector includes a syringe and an injection valve coupled to the syringe for switching between an injection state and a injection state, the syringe being coupled to the injection valve through a sample loop.
  • the simulated internal standard device for quantitative analysis of mass spectrometry wherein the mobile phase switching device is two A three-way switching valve connected to the front and back of the column.
  • the simulated internal standard device for quantitative analysis of mass spectrometry wherein the mobile phase switching device is a four-way switching valve connected to the column.
  • the simulated internal standard device for quantitative analysis of mass spectrometry wherein the mobile phase switching device is a six-way switching valve connected to the column.
  • the simulated internal standard device for quantitative analysis of the photo wherein the mobile phase switching device is a six-way injection valve for use in an internal standard injector for internal standard sample injection.
  • the simulated internal standard device for quantitative analysis of mass spectrometry further comprises an internal standard sample introduction device connected to the four-way switching valve, wherein the internal standard sample introduction device comprises an internal standard sampler connected to the four-way switching valve, A second mobile phase transport device coupled to the internal standard injector, and a second mobile phase storage device coupled to the second mobile phase transport device.
  • the present invention also provides an application of the analog internal standard device as described above, which is applied to blood sample detection.
  • the hardware used in this embodiment and the usual LC/MS (liquid-mass-mass system) No, but need to add a control software in the system control system to control the injection, so that each time after entering a standard sample (different concentration of standard solution), pause for a while and then inject the internal standard sample, standard sample and internal
  • the standard sample is separated and analyzed in the same test cycle, except that the internal standard sample is introduced into the system after the standard sample lags behind.
  • the lag time of the internal standard sample injection is determined during the development of the analytical method. After the chromatographic separation conditions are optimized, as long as the baseline width of the analyte signal is measured, the lag time is set to be slightly larger than the baseline width. The key is that the signal generated by the post-injected standard does not overlap with the previously injected sample signal.
  • the working process is: 1) The syringe 210 in the injector 200 is first inserted into a sample solution (not shown) driven by a robot (not shown), in the injector 200. A needle pump (not shown) directly connected to the syringe 210 is quickly activated, and the standard sample is quantitatively aspirated through the syringe 210. In this embodiment, 5 ⁇ l (microliter) is taken while the injection valve 230 is switched to the sample loading. status.
  • the robot inserts the sample needle 210 of the sample into the injection port 220 connected to the injection valve 230, and the needle pump immediately pushes the standard sample in the needle through the inlet 220 into the sample ring 240, and the sample ring 240 is looped.
  • the original liquid is pushed into the waste liquid.
  • This process is called “loading” or “loading”; 2) after the sample is loaded, the injection valve is switched to the injection state (as shown by the circular dotted line in Figure 1), and the sample injection ring 240 is loaded. At this time, one end is connected to the high pressure liquid pump 100, and the other end is passed to the column 300.
  • the solvent (mobile phase) in the mobile phase storage device 400 is transported by the high pressure liquid pump 100 through the sample loop 240, and the sample loop is taken.
  • a standard sample of 240 is fed to the column 300.
  • This process is the injection process, the combination process of sample loading and injection is commonly referred to as “injection”; 3) the standard sample is sent to the column 300 through the injection process, and the chemical components in the standard sample are combined with the column.
  • the filling material interaction in 300 generates adsorption, and the mobile phase continuously flushes the column 300 under the push of the high pressure liquid pump 100, so that the components in the adsorbed state are continuously washed out to generate desorption, and each sample component is in the column.
  • the adsorption-desorption is repeated between the stationary phase and the mobile phase, but is always moved by the mobile phase toward the outlet of the column 300. Since the moving speed is related to the distribution coefficient of the substance between the mobile phase and the stationary phase, the standard sample The components in the final phase are separated from the column by the respective distribution coefficients, and are separated from each other; 4) the separated standard sample components are successively ionized into the mass spectrometer ion source, and the generated ions are separated by the mass spectrometer 500 and then One by one measurement.
  • the mass spectrometer 500 is set to monitor only the ions generated by the object to be tested and its internal standard, and other ions are neglected, so that the integration time of the test signal can be increased, thereby improving the test sensitivity and eliminating other substances.
  • the ions interfere with the signal of the object being measured.
  • a sample analysis cycle begins immediately after a sample is injected into the system, including data acquisition.
  • the injector 200 only operates before the start of the analysis cycle, and there are no other tasks besides self-cleaning throughout the cycle.
  • 5 ⁇ l of an internal standard sample of 2.5 ⁇ g/ml is injected, at which time the standard sample analysis cycle is still in progress, and the injection of the internal standard sample is only for the injector 200.
  • the chromatographic data of the sample contains the (standard sample) sample peak and the (internal standard sample) internal standard peak, as shown in Figure 3.
  • the complete separation of the sample peak from the internal standard peak is as close as possible to the basic principle of determining the time interval between the two injections.
  • the specific injection time gap is determined according to this principle in combination with the instrument conditions and signal width.
  • Figures 4 and 5 are images of the first (starting) and tenth (after 2 hours) calibration curves, respectively, using the standard sample.
  • the lower curve (square mark) is the result of plotting the peak area without the internal standard
  • the upper curve (the prism mark) is the standard sample and the internal standard sample after the internal standard is used.
  • the ratio of the peak areas of the two is plotted. It can be seen from the two graphs that since the ionization efficiency changes with time, the sensitivity of the calibration curve without internal standard changes greatly before and after, the coordinate values of the two graphs are the same, and the maximum response value of the calibration curve without the internal standard (peak area) ) From about 1.4 million to less than 1.1 million, the change is 21.4%, and the calibration curve after adding the internal standard has a rate of change of only 4.0%. The effect of the simulated internal standard is evident through the above two calibration curves.
  • the solution used in this example is identical to that of the first embodiment, but the internal standard sample is not directly injected into the mass spectrometer through the column.
  • the calibration curve of mass spectrometry is already curved in the 10-fold concentration range, and the linear range is small.
  • the post-column injection internal standard can adjust the internal standard injection amount according to the signal peak size after the sample signal peak comes out. Nonlinear response.
  • This embodiment uses three internal standard concentrations, as shown in Table 1 below:
  • the instrument used in this embodiment is another set of chromatography-mass spectrometry system.
  • the control software of this embodiment is installed to read the data of the system and control the system according to the read data.
  • a flow injection channel is added to the original liquid-mass-mass system, as shown in Fig. 1, except that two three-way switching valves 310 are added to the system, and the synchronous operation is performed, and the whole system is switched to the upper state.
  • It is a general liquid-mass spectrometer.
  • the instrument When switching to the lower state, the instrument is a flow injection analysis system. When the sample is analyzed, the system is in the upper state.
  • the standard sample is injected by the injector 200 and sent to the column 300 by the high pressure liquid pump 100 for separation.
  • the middle is passed through the additional three-way switching valve 310 in front of the column, and then passes through the column.
  • the three-way switching valve 310 enters the mass spectrometer for measurement.
  • the control software constantly monitors the mass spectrum signal of the analyte (standard sample), and at the same time controls the injector 200 to switch the injection valve 230 to the loading state after the injection is completed, and once the sample peak is completed, the control is performed.
  • the software immediately selects the internal standard sample which is the closest to the sample peak from the three internal standard solutions according to the size of the sample peak, and then directs the injector 200 to load from the selected internal standard, and then the injection valve 230 is loaded. Switching to the injection state, the two three-way switching valves 310 simultaneously switch to the lower state, allowing the internal standard sample to bypass the column 300 and flow directly into the mass spectrometer for measurement.
  • the obtained data is basically the same as that of the first embodiment, as shown in FIG. 6.
  • the two three-way switching valves 310 in FIG. 1 can also be connected by a four-way switching valve 380 as shown in FIG. 2.
  • the four-way switching valve 380 is switched to the chromatogram.
  • the column 300 is short-circuited and the mobile phase does not pass directly through the column 300 into the mass spectrometer, achieving the same effect as described above.
  • Figures 7 and 8 are images of the first (starting) and tenth (after 2 hours) calibration curves, respectively.
  • the internal standard path can also replace the two three-way switching valves 310 by a six-way switching valve, as shown in FIG.
  • the advantage of using a six-way switching valve is that only one valve needs to be controlled to avoid blockages due to hardware failure or software failure. If the two three-way switching valves 310 are not switched synchronously, the mobile phase will be blocked, causing the system to overpressure. The six-way switching valve does not have the need for synchronous switching, and thus does not cause a problem of system overpressure.
  • the A state in Fig. 9 is for sample analysis, in which the sample must be separated by the column 300 and sent to the mass spectrometer for measurement.
  • the six-way switching valve is switched to the B state after the control software detects that the sample peak has come out, and the injector 200 is commanded to enter the appropriate internal standard sample. In the B state, the incoming internal standard sample is directly sent to the mass spectrometer for measurement by the mobile phase without passing through the six-way switching valve.
  • the present invention also designs an internal standard sample introduction device as shown in FIG.
  • This is a flow injection system designed to introduce an internal standard after column 300. The purpose is to use internal standards to eliminate fluctuations in the instrument during sample testing and to correct the nonlinear response of the instrument.
  • the flow injection system is combined with the above system through a four-way switching valve 900, and the working mechanism is: 1) the internal standard sampler 600 (with an internal standard injector) in the device introduces the internal standard to In the pipeline leading to the mass spectrometer; 2) the liquid phase pump (second mobile phase transport device 700) inputs the mobile phase in the mobile phase storage device (ie, the second mobile phase storage device 800) into the internal standard injector 600 Keep the internal standard along the pipeline
  • the mass spectrometer 500 is sent to the mass spectrometer 500; 3) the mass spectrometer 500 measures the internal standard, giving the same signal as the chromatographic peak.
  • the mobile phase In the case of a mass spectrometer connected to a liquid chromatograph, the mobile phase must use a solvent or buffer solution that aids in ionization.
  • the operation of the entire device is fully controlled by the control software.
  • the sample separation analysis is carried out by a common chromatography-mass spectrometry system.
  • the control software continuously monitors the mass spectrometry signal of the analyte (sample). Once the signal of the analyte (sample) is detected, the software is based on The product signal strength selects the internal standard, and then the internal standard sampler 600 is commanded to load the selected internal standard. When the signal peak of the object to be tested is completely present, the software immediately switches the four-way switching valve 900 in the device to FIG.
  • the internal standard injector 600 In the B state, the internal standard injector 600 is instructed to be injected, and the second mobile phase conveying device is activated to send the internal standard to the mass spectrometer 500 for measurement.
  • the entire device is in the A state before the signal of the object to be tested appears, and is always on standby.
  • the four-way switching valve 900 is immediately switched to the A state in FIG.
  • the internal standard injection device does not interfere at all, and it runs completely silently behind the scenes.
  • the biggest advantage of introducing the internal standard after the column is that the injection amount of the internal standard sample can be determined immediately according to the sample signal intensity, so that the signal peak generated by the internal standard sample is close to the signal peak size generated by the standard sample, and the present invention is controlled by such dynamic control. Not only can the problem of inaccurate analysis result due to ionization efficiency change with time in mass spectrometry can be eliminated, but also the linear response range of mass spectrometry can be extended by more than 10 times and even tens of thousands of times.
  • the liquid-mass-mass system is controlled and the data is collected by itself, and the internal standard sample introduction device shown in FIG. 10 is controlled at the same time, so that all the hardware becomes a complete analysis system.
  • Figure 11 is a schematic illustration of the system.
  • the sample analysis process is: 1) the standard sample is introduced into the analysis line by the injector 200; 2) the high pressure liquid pump 100 continuously transports the mobile phase in the mobile phase storage device 400, and the sample is taken into the column 300 for separation, and then sent.
  • the mass spectrometer 500 is measured; 3) the mass spectrometer 500 collects the signal after the system is injected; 4) when the chromatographic peak of the analyte is present, the internal standard injection portion is immediately started, and the four-way switching valve 900 is switched to let the sample
  • the passage leads to the waste liquid, and the internal standard passage is connected to the mass spectrometer 500.
  • the internal standard sample is measured by the internal standard sample introduction device into the mass spectrometer, and an internal standard peak is generated after the peak of the sample appears.
  • the four-way switching valve 900 in the system immediately switches to the internal standard injection position, and the software simultaneously calculates the approximate concentration corresponding to the sample peak.
  • the internal standard injector 600 then injects 5 microliters of the closest internal standard sample. Since the flow injection line is short and the inner diameter of the tube is small, an internal standard peak can be introduced into the mass spectrum in a short time after the peak of the sample appears. The closer the sample peak to the internal standard peak are, the more the internal standard calibration effect is. it is good.
  • the mass spectrometry ionization efficiency is not instantaneous, and the variation range within a time range of about one minute is usually negligible, so the injection time of the internal standard is not necessarily controlled with high precision, and the accuracy is sufficient in the second range.
  • the invention adopts software control to store chromatographic data of multiple sample analyses in the same Keep the timeline continuous in the data file.
  • the test of the sample series was repeated 10 times, with a 15 minute gap added between each pass for a total of nearly 7 hours.
  • the measured chromatogram is shown in Figure 12.
  • the present embodiment exemplifies that the liquid passing through the column has a high column pressure of about 100 MPa (megapascal), the flow velocity in front of the column is reduced by the volume compression of the liquid, and the volume expansion flow rate is increased after the column, but the total flow rate is increased. constant.
  • the line speed at which the mass spectrometer enters is higher than the flow injection. Since the electrospray ionization source is used in this embodiment, the better the ionization efficiency, the higher the ionization efficiency, and the stronger the mass spectrometry signal obtained. The greater the line speed at which the liquid comes out of the spray port, the better the spray effect.
  • the system shown in FIG. 13 is used, and the internal standard sample is directly introduced into the solvent flow after the column 300, and the problem that the above internal standard peak is shorter than the sample peak is successfully solved.
  • the operation process of the system of this embodiment is basically the same as that of the foregoing system, except that the sample valve of the internal standard injector 600 is directly connected to the analysis pipeline, and the internal standard is used at the time of switching the four-way switching valve.
  • the six-way switching valve of the injector 600 is switched to the injection position, and the internal standard sample is driven by the analysis of the mobile phase to obtain the same linear velocity into the mass spectrometer, and the obtained signal intensity is also the same.
  • Figures 14 and 15 are images of the first (starting) and tenth (after 7 hours) calibration curves, respectively.
  • the calibration curve can only use the quadratic fitting curve to represent the obtained data without the internal standard, and the fitting degree is not ideal.
  • the fitting coefficient R 2 has only two 9 after the decimal point, especially Comparing the data collected at the start time with the data collected at 7 hours, the coefficients of the fitting function are far apart, and the shape can clearly see the difference.
  • the internal standard sample signal is almost The fluctuation of the standard sample signal is completely corrected, and the nonlinear problem of the instrument is completely solved, so that the linear response range of the instrument is greatly expanded.
  • the calibration curve after using the internal standard is almost the ideal straight line R value after the decimal point has four 9 and the ideal is 1.
  • the slope of the line changes by less than one thousandth.
  • the slope represents the sensitivity.
  • the change of sensitivity is the key factor that causes the result to be inaccurate.
  • the sensitivity of the analysis changes not only with time but also with concentration.
  • the calibration curve when there is no internal standard in only 10 times the concentration range (as in Examples 1 and 2) is curved. Examples 1 and 2 also show that the sensitivity of the mass spectrum is reduced by 14% in 2 hours, and by 25.4% after 7 hours in Example 3. It can be seen that the change in measurement sensitivity is likely to be caused by a variety of uncertain factors, and the direction of change over time cannot be predicted.
  • the variation factors can be eliminated very effectively, and the standard substance of the object to be tested is directly used as the internal standard, which greatly saves the trouble of finding the internal standard, especially for drug analysis, the invention avoids the use of the isotope.
  • Internal standards can save a lot of money and speed up the process of drug clinical trials.
  • the invention is particularly suitable for the concentration analysis of drugs in blood samples, and is particularly suitable for the establishment of a drug-time curve.
  • a drug-time curve In the development of medication or drug, the patient or the subject takes a blood sample after taking the drug, and the blood sample is measured to obtain the concentration of the drug in each blood sample versus time. Such a spectrum is called a drug-time curve.
  • all sample processing and analysis was done according to the specifications of the blood sample analysis. It is also an established goal of the present invention to open up the application of mass spectrometry in the field of clinical drug detection.

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Abstract

一种质谱定量分析的模拟内标方法、装置及应用，方法包括步骤：取一标准物用待测样品的空白基底配置成不同浓度的标准溶液，再取同样标准物用待测样品的空白基底配置成一至若干个浓浓度的内标溶液；将所述不同浓度的标准溶液和内标溶液经样品处理步骤处理成标准样品和内标样品；将标准样品和内标样品先后注射到色谱-质谱联用系统进行分析测定；将标准样品与内标样品两者的峰面积之比对浓度作图得到模拟内标法定量的校准曲线；将待测样品经样品处理步骤进行处理后注射到色谱-质谱联用系统进行分析测定，然后将测得信号的峰面积与校准曲线比对确定出待测样品的浓度。

Description

一种质谱定量分析的模拟内标方法、装置及应用 技术领域
本发明涉及质谱定量技术领域，尤其涉及一种质谱定量分析的模拟内标方法、装置及应用。
背景技术
定量分析概述：首先用标准物质建立校正曲线，亦即以测定信号(Y)对浓度(C)作图得到一条满足Y＝aC+b函数的直线。但直线只是理想状态下得到的结果，一般情形是，测定信号和浓度(Y-C)之间的关系只有在浓度变化较小的范围内满足线性，对于大一点的范围，如浓度变化在10倍以上，得到的Y-C图形总是弯曲的，可以用Y＝ax2+bx+c这样的曲线函数方程来表达，所以通常把Y-C关系图称之为“校正曲线”或“分析曲线”，而不是“xx直线”。得到校正曲线后，只要把对未知样品测定得到的信号带入得到的曲线函数式就能算出未知物的浓度，也可以直接在校正曲线上根据未知样品的测定值找出它的浓度。这便是定量分析的通用过程。
内标定量方法：在定量分析过程中，如果仪器的灵敏度随着外界条件的变化有所改变，同样浓度的样品在不同时间测试就会产生不同信号强度，计算出的浓度也就不一致，导致误差。配制溶液的过程中由于加入量的不准或样品处理过程中操作上有差异也会造成测定误 差。为了消除这些误差，定量分析时常常在样品和标准中等量加入一个已知浓度的标准物(非待测物)做内标，当仪器响应灵敏度上升或下降时，待测物和内标的信号同时上升或下降，但两者信号的比值却基本保持不变，由此，样品处理过程和仪器相应存在的误差得以消除。因此，内标法定量的校正曲线是信号比值对浓度作图，亦即前面提到的Y应该是：Y＝Ix/Iis，Ix为待测物的信号强度，Iis为内标的信号强度。样品处理过程的差异所引进的误差不一定需要用内标法消除，严格操作程序和采用自动化处理技术都可以排出这个误差来源。然而仪器不稳定造成的误差有的是原理性的，即便把仪器造得再精致，这样的误差还是存在，所以需要内标法加以消除。目前，几乎所有的定量分析都是采用内标法。
质谱定量概述：质谱是一种分离和测定带电粒子的分析仪器，目前，定量测定痕量化学成分时一般要用到质谱，尤其是在药物动力学、药物代谢、毒理毒代、临床试验等领域中采用质谱技术已经是不二选择。用质谱作为分析手段是，待测物质必须首先电离成为带电粒子，质谱能把带电粒子根据它们的质量与电荷的比值以很高的分辨度区分开来，因此，用质谱定量时通常是对单一荷质比的离子进行测定，其它物质由于质量或荷质比不一样而被排除了，对待测信号不产生明显干扰，这是质谱定量的巨大优势。然而，分子电离一般要用强电场或粒子轰击来实现，随着时间的推移电场或轰击粒子子的密度与能量很难保持一成不变，所以，质谱测定灵敏度会随时间推移而发生变化，而且变化幅度可达30％以上，使得质谱测定的准确度只有70％左右。 按理，用前面叙述的内部法应该可以消除这种变化，但是电离效率与物质结构紧密相关，结构上的微细差异得到的电离效率可以是天差地别，要找到一个电离性质与待测物质相一致的内标物质非常困难，目前唯有用待测物的同位素异体才能做内标，同位素异体是把分析物中的某些元素用重的同位素取代，譬如把其中的氢用氘取代，得到分子量大一些但结构一样的异体分子，将待测物与这样的同位素异体同时引入质谱测定时，由于质谱的高分辨功能，可以把二者的信号区分开来，它们的的信号强度之比就是内标法定量的基础。总之，目前提高质谱定量准确度的唯一方法是用同位素异体做内标，通常就叫同位素内标。
目前质谱定量存在的问题如下：同位素内标虽然可以把质谱定量的准确度从70％左右提高到95％以上，和用其它仪器能达到的准确度相当，但是同位素内标的合成成本非常高，目前除了新药开发中的临床试验分析采用这种方法，大部分其它的应用包括制药过程中的临床前实验都因费用太高以及等待合成耗时太长而无法使用，这大大妨碍了质谱技术的广泛应用，同时也延缓生物制药进程并增加费用。再则，如果没有内标难寻的问题，质谱技术在临床检测、环境分析、食品安全等等领域应该能够发挥更大的作用。
因此，现有技术还有待于改进和发展。
发明内容
鉴于上述现有技术的不足，本发明的目的在于提供一种质谱定量 分析的模拟内标方法及装置，旨在解决现有的质谱定量技术存在准确度低、同位素内标成本高的问题。
本发明的技术方案如下：
一种质谱定量分析的模拟内标方法，其中，包括步骤：
取一标准物用待测样品的空白基底配置成不同浓度的标准溶液，再取同样标准物用待测样品的空白基底配置成一至若干个浓度的内标溶液，内标系列的浓度范围和跨度与标准溶液一致；
将所述不同浓度的标准溶液和内标溶液经样品处理步骤处理成标准样品和内标样品；
将标准样品和内标样品先后注射到色谱-质谱联用系统进行分析测定；
将测定到的标准样品与内标样品两者的峰面积之比对浓度作图得到模拟内标法定量的校准曲线；
将待测样品经样品处理步骤进行处理后注射到色谱-质谱联用系统进行分析测定，然后将测得信号的峰面积与校准曲线比对确定出待测样品的浓度。
所述的质谱定量分析的模拟内标方法，其中，每次进完一个标准样品之后停顿片刻，等到测定完标准样品所需的时间之后，再注射内标样品，使内标样品通过色谱-质谱联用系统的色谱柱，标准样品和内标样品在同一个检测周期中完成分离分析。
所述的质谱定量分析的模拟内标方法，其中，每次进完一个标准样品之后，再进内标样品，并使内标样品绕过色谱-质谱联用系统的 色谱柱以流动注射的方式直接进入质谱仪进行测定。
所述的质谱定量分析的模拟内标方法，其中，每次进完一个标准样品之后，在检测到标准样品的色谱峰之后，再进与该标准样品浓度最接近的内标样品，并使内标样品绕过色谱-质谱联用系统的色谱柱以流动注射的方式直接进入质谱仪进行测定。
所述的质谱定量分析的模拟内标方法，其中，在注射内标样品时，使注射流速减低20％，或者提高注射时喷雾气体的流量，从60升/分钟提高到70-100升/分钟。
一种质谱定量分析的模拟内标装置，其中，包括：
色谱流动相及流动相输送装置；
连接于流动相输送装置的进样器；
连接于进样器的色谱柱；
与色谱柱相配合的流动相切换装置；
连接于色谱柱的质谱仪；
控制以上硬件实施模拟内标质谱定量过程的软件系统；
所述进样器包括进样针以及连接于进样针的用于在进样状态和注样状态之间切换的进样阀，所述进样针与进样阀之间通过样品环连接。
所述的质谱定量分析的模拟内标装置，其中流动相切换装置是两个连接在色谱柱前后的三通切换阀。
所述的质谱定量分析的模拟内标装置，其中流动相切换装置是一个连接在色谱柱的四通切换阀。
所述的质谱定量分析的模拟内标装置，其中流动相切换装置是连接在色谱柱的六通切换阀。
所述的照片定量分析的模拟内标装置，其中连接在色谱柱的流动切换阀就是用于内标样品注射的内标进样器中的六通进样阀。
所述的质谱定量分析的模拟内标装置，其中，还包括连接在四通切换阀上的内标进样装置，所述内标进样装置包括一连接于四通切换阀的内标进样器、连接于内标进样器的第二流动相输送装置、连接于第二流动相输送装置的第二流动相存储装置。
一种如上所述的模拟内标装置的应用，其中，将所述模拟内标装置应用于血样检测中。
有益效果：本发明采用待分析物质的标准物本身作为内标进行质谱定量分析，该“内标”不加入到分析样品内，而是与样品分别引入到质谱中进行测定，具体是在样品中的待测物信号在质谱中出现以后立即向质谱仪中引入已知量的待测物的标准物作为内标进行测定，取测得的待测物信号与紧接着的内准信号两者的比值作为校准曲线的纵坐标值进行定量，由于质谱电离情况的变化所引起的灵敏度变化在很短的时间间隔内很小，所以本发明的定量方法与通常的内标法达到的效果也十分相近，基本能完全消除质谱定量由于电离效率变化引起的的不准确问题。本发明可以在不采用同位素内标的情况下，达到95％以上准确度；本发明大范围扩展了质谱定量分析的线性响应范围。另外，本发明的方法和装置可对患者用药后药物在患者血样中的浓度进行准确测定从而确定所用药物对特定患者是否有效，以此达到精准 用药的目的。
附图说明
图1为本发明实施例一的结构示意图。
图2为本发明实施例一的另一结构示意图
图3为本发明实施例一的色谱图。
图4为本发明实施例一起始时的标准曲线。
图5为本发明实施例一2小时后的标准曲线。
图6为本发明实施例二的色谱图。
图7为本发明实施例二起始时的标准曲线。
图8为本发明实施例二2小时后的标准曲线。
图9为本发明实施例二中六通切换阀的状态变化图。
图10为本发明实施例二中内标进样装置的状态变化图。
图11为本发明实施例三的结构示意图。
图12为本发明实施例三的色谱图。
图13为本发明实施例三另一结构示意图。
图14为本发明实施例三起始时的标准曲线。
图15为本发明实施例三7小时后的标准曲线。
具体实施方式
本发明提供一种质谱定量分析的模拟内标方法及装置，为使本发明的目的、技术方案及效果更加清楚、明确，以下对本发明进一步详 细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明所提供的一种质谱定量分析的模拟内标方法，其包括步骤：
取一标准物用待测样品的空白基底配置成不同浓度的标准溶液，再取同样标准物用待测样品的空白基底配置一至若干个固定浓度的内标溶液，内标系列的浓度范围和跨度与标准溶液一致；
将所述不同浓度的标准溶液和内标溶液经样品处理步骤处理成标准样品和内标样品；
将标准样品和内标样品先后注射到色谱-质谱联用系统进行分析测定；
将测定到的标准样品与内标样品两者峰面积之比对浓度作图得到模拟内标法定量的校准曲线；
将待测样品经样品处理步骤进行处理后注射到色谱-质谱联用系统进行测定，然后将测得信号的峰面积与校准曲线比对确定出待测样品的浓度。
在具体实施过程中，可以每次进完一个标准样品之后停顿片刻(等到测定完标准样品所需的时间之后，如30s，以保证标准样品信号与后继的内标样品信号不发生重叠)，再注射内标样品使内标样品通过色谱-质谱联用系统的色谱柱，标准样品和内标样品在同一个检测周期中完成分离分析。也可以每次进完一个标准样品之后，再进内标样品，并使内标样品绕过色谱柱以流动注射的方式直接进入质谱仪进行测定。或者每次进完一个标准样品之后，在检测到标准样品的色 谱峰之后，再进与该标准样品浓度最接近的内标样品，并使内标样品绕过色谱柱以流动注射的方式直接进入质谱仪进行测定。原则是得到的内标样品信号与标准样品信号尽量靠近而又不重叠。
在注射内标样品时，使注射流速减低20％，或者提高注射时喷雾气体的流量，从60升/分钟提高到70-100升/分钟(如优选的80升/分钟)。柱前注射比柱后注射在同样浓度的情况下灵敏度更高，究其原因，主要是喷雾离子化效率的改变，为此通过临时改善喷雾条件，提高柱后注射的喷雾效率，达到了消除灵敏度差异的目的。具体做法一是降低柱后注射的流速，使得喷雾更完全，流速减低20％左右就可以消除掉这个灵敏度的差异；二是提高喷雾气体的流量，从60升/分钟提高到70-100升/分钟可以把差异减少到5％以内。
本发明还提供一种质谱定量分析的模拟内标装置，其包括：
色谱流动相及流动相输送装置；
连接于流动相输送装置的进样器；
连接于进样器的色谱柱；
与色谱柱相配合的流动相切换装置；
连接于色谱柱的质谱仪；
控制以上硬件实施模拟内标质谱定量过程的软件系统；
所述进样器包括进样针以及连接于进样针的用于在进样状态和注样状态之间切换的进样阀，所述进样针与进样阀之间通过样品环连接。
所述的质谱定量分析的模拟内标装置，其中流动相切换装置是两 个连接在色谱柱前后的三通切换阀。
所述的质谱定量分析的模拟内标装置，其中流动相切换装置是一个连接在色谱柱的四通切换阀。
所述的质谱定量分析的模拟内标装置，其中流动相切换装置是连接在色谱柱的六通切换阀。
所述的照片定量分析的模拟内标装置，其中流动相切换装置就是用于内标样品注射的内标进样器中的六通进样阀。
所述的质谱定量分析的模拟内标装置，还包括连接在四通切换阀上的内标进样装置，所述内标进样装置包括一连接于四通切换阀的内标进样器、连接于内标进样器的第二流动相输送装置、连接于第二流动相输送装置的第二流动相存储装置。
本发明还提供一种如上所述的模拟内标装置的应用，将所述模拟内标装置应用于血样检测中。
下面通过具体实施例来对本发明进行详细说明。
实施例一：柱前内标注射
取一标准物以甲醇做溶剂配置7个浓度：0.250、0.500、1.00，2.00，5.00，8.00，10.0μg/ml(微克/毫升)的标准溶液，再取标准物配置一个2.50μg/ml的标准溶液作为内标。标准样品是取50微升上述标准溶液(7个浓度)加入到50微升人血血清并混匀，再加入100微升(冷)甲醇，在震动器上震动5分钟，让血清中的蛋白沉淀，离心分离后取出上层清液注射到色谱-质谱联用系统进行分析测定。本实施例所用到的硬件和通常的LC/MS(液-质联用系统)并 无二至，但需要在系统控制系统中加入一控制软件，用于控制进样，使得每次进完一个标准样品(不同浓度的标准溶液)之后停顿片刻再注射内标样品，标准样品和内标样品在同一个检测周期中完成分离分析，只是内标样品滞后于标准样品被引入到系统。内标样品注入的滞后时间是在分析方法的开发过程中确定的，在色谱分离条件优化好以后，只要测定出待测物信号的基线宽度，滞后时间就设置为比基线宽度稍大一些就可以了，关键是后面注进去的标样所产生的信号不与之前注进去的样品信号产生重叠。
如图1所示，其工作过程是：1)进样器200中的进样针210在机器手(未示出)驱动之下先插到样品溶液(未示出)，进样器200中与进样针210直接相连的针泵(未示出)迅即启动，通过进样针210定量吸取标准样品，本实施例是吸取5μl(微升)，与此同时进样阀230切换到加样状态。接下来机器手将吸好样品的进样针210插入与进样阀230连接的进样口220，针泵迅即把针内的标准样品通过进样口220推送进入样品环240，样品环240环内原有液体则被推进废液。此过程称作“上样”或“加样”；2)完成加样后，进样阀随即切换到注样状态(如图1中圆形虚线所示)，装了样品的进样环240此时一头与高压液相泵100连通、另一头通到色谱柱300，与此同时，流动相存储装置400中的溶剂(流动相)由高压液相泵100输送通过样品环240，把样品环240里的标准样品送入色谱柱300。此过程为注样过程，加样与注样的组合过程通称为“进样”；3)标准样品通过进样过程被送进色谱柱300，标准样品内的各个化学组分会和色谱柱 300内的填充材料相互作用产生吸附，而流动相在高压液相泵100的推动下连续冲洗色谱柱300，使得吸附状态的组分不断被冲脱而产生解吸附，各个样品组分在色谱柱300固定相与流动相之间反复进行吸附-解吸附，但始终被流动相带动往色谱柱300的出口方向移动，由于移动速度与物质在流动相与固定相之间的分布系数相关，标准样品中的组分因各自的分布系数不同最终分先后从色谱柱流出，相互分离开来；4)分离后的标准样品组分先后进入质谱离子源进行离子化，产生的离子被质谱仪500分离然后一一测定。在进行定量分析时，质谱仪500设置在只监测待测物及其内标所产生的离子，其它离子一概忽略，这样既可以增加测试信号的积分时间从而提高测试灵敏度，又可以消除其它物质产生的离子对待测物的信号造成干扰。
通常一个样品注射进系统之后随即开始一个样品分析周期，包括数据采集，进样器200只在分析周期开始之前动作，在整个周期中除了自我清洗没有其他任务。本实施例在每个标准样品注入之后等待大约0.5分钟再注入5μl 2.5μg/ml的内标样品，此时标准样品分析周期仍然处于进行之中，内标样品的注入只是让进样器200再进行一次完整的进样过程，并不引发新的样品分析周期，因此，样品的色谱数据包含了(标准样品)样品峰和(内标样品)内标峰，如图3所示。样品峰与内标峰的完全分离却又尽量靠近是确定两者进样时间间隙的基本原则，具体的进样时间间隙完全根据这一原则结合仪器条件和信号宽度而决定。
在本实施例中为了反映质谱灵敏度随时间变化的情况，对样品的 分析重复了10次，得到10个校准曲线，时间跨度大约两个小时。图4和图5分别是第一个(起始时)和第十个(2小时后)校准曲线的图像，分析物用的是标准样品。
图4和图5中，下方的曲线(正方形标识)是不加内标用峰面积对浓度作图的结果，上方的曲线(棱形标识)是用了内标后用标准样品与内标样品两者峰面积之比作图的结果。由两个图可以看出，由于离子化效率随时间变化，不加内标的校准曲线灵敏度前后变化较大，两个图的坐标值是一样的，没有用内标的校正曲线最大响应值(峰面积)从140万左右降到了110万以下，变化21.4％，加了内标后的校准曲线变化率只有4.0％。模拟内标的作用通过上面两个校准曲线显现无遗。
实施例二：柱后内标注射
本实施例所采用的溶液与实施例一完全一致，但是内标样品不通过色谱柱直接注入质谱仪。质谱定量的校正曲线在10倍浓度范围就已经是弯曲的，线性范围较小，柱后注射内标可以在样品信号峰出来以后，根据信号峰的大小临时调整内标的进样量而校正仪器的非线性响应。本实施例用了三个内标浓度，如下表一所示：
表一
Figure PCTCN2016097261-appb-000001
本实施例所采用用的仪器是另一套色谱-质谱联用系统，经过了改造，安装上本实施例的控制软件，用以即时读取系统的数据并根据读到的数据控制系统中进样器200和外加阀门的动作。硬件方面在原有液-质联用系统中增加了一个流动注射通道，具体如图1所示，只是在系统中增加了两个三通切换阀310，同步动作，切换到上方的状态时整个系统就是一般的液-质联用仪，切换到下方的状态时仪器就是一套流动注射分析系统。样品分析时系统处于上方的状态，标准样品用进样器200注入后被高压液相泵100送进色谱柱300进行分离，中间要通过柱前附加的三通切换阀310，之后再通过柱后的三通切换阀310进入质谱仪得到测定。在此过程中，控制软件一直监视待测物(标准样品)的质谱信号，同时控制进样器200在进样完毕后把进样阀230切换到上样状态等待，一旦样品峰出现完毕，控制软件立即根据样品峰的大小从三个内标溶液中选择能产生色谱峰大小最接近样品峰的内标样品，再指挥进样器200从选择好的内标上样，之后把进样阀230切换到注样状态，两个三通切换阀310同时切换到下方的状态，让内标样品绕过色谱柱300以流动注射的方式直接进入质谱仪进行测定。得到的数据与实施例一基本一致，如图6所示。图1中的两个三通切换阀310也可以由一个四通切换阀380按图2所示连接，在上述步骤中切换两个三通切换阀310的时刻把四通切换阀380切换到色谱柱300被短路，流动相不通过色谱柱300直接进入质谱仪的状态，达到的效果与上述过程完全一样。
本实施例为了反映质谱灵敏度随时间变化的情况，对样品的分析 重复了10次，得到10个校准曲线，时间跨度大约两个小时。图7和图8分别是第一个(起始时)和第十个(2小时后)校准曲线的图像。
内标通路也可以用一个六通切换阀代替上述两个三通切换阀310，接法如图9所示。
用六通切换阀的好处是只需要控制一个阀门，可以避免因硬件故障或软件失灵造成管路堵塞。两个三通切换阀310如果不同步切换，流动相就会被堵住，造成系统超压。用六通切换阀没有同步切换的需要，因而不会造成系统超压的问题。和前面用两个三通切换阀的情形一样，图9中的A状态是用于样品分析，在这个状态下样品必须通过色谱柱300进行分离然后送进质谱仪进行测定。在控制软件监测到样品峰出来后才切换六通切换阀到B状态，同时指挥进样器200进合适的内标样品。在B状态下，进的内标样品不通过六通切换阀直接被流动相送进质谱仪进行测定。
由于仪器市场上有很多品牌的进样器，为了避免软件开发时费很多功夫制作控制各类进样器的驱动程序，本发明还设计了如图10所示的内标进样装置。这是一个流动注射系统，专门用于在色谱柱300之后引入内标，目的是利用内标消除仪器在样品测试过程中的波动和矫正仪器的非线性响应。这个流动注射系统，通过一四通切换阀900与上述系统组合，协同工作，其工作机理是：1)装置中的内标进样器600(内设内标进样器)把内标引进到通往质谱仪的管路中；2)液相泵(第二流动相输送装置700)把流动相存储装置(即第二流动相存储装置800)中的流动相输进内标进样器600把内标沿管路一直 送进质谱仪500；3)质谱仪500测定内标，给出与色谱峰同样的信号。如果是与液相色谱相连接的质谱仪，流动相必须用能帮助电离的溶剂或缓冲溶液。
整个装置的运行由控制软件全部控制。样品分离分析由通常的色谱-质谱联用系统承担，在样品分析过程中，控制软件不断监测分析物(样品)的质谱信号，一旦监测到待测物(样品)的信号出现，软件便根据的产物信号强度选择内标，然后指挥内标进样器600将选定的内标上样，当待测物的信号峰完全出现后，软件立刻把装置中的四通切换阀900切换到图10中的B状态，随即指挥内标进样器600注样，同时启动第二流动相输送装置用第二流动相把内标送进质谱仪500进行测定。在待测物信号出现之前整个装置处于A状态，随时待命。在内标流动注射过程完成以后，四通切换阀900又马上切换到图10中的A状态待命。对于样品分析系统，内标进样装置丝毫不干预，完全是在幕后无声无息地运行。
柱后引入内标的最大好处是可以根据样品信号强度即时确定内标样品的进样量，使得内标样品产生的信号峰与标准样品产生的信号峰大小很接近，通过这样的动态控制，本发明不仅可以消除质谱定量时因为电离效率随时间变化而引起分析结果不准确的问题，同时还能把质谱定量的线性响应范围扩展10倍以上甚至上万倍。
实施例三：动态内标进样
本实施例既控制液-质联用系统并自行采集数据、同时控制上图10所示的内标进样装置，使得所有硬件成为一个完整的分析系统。 图11为系统的示意图。样品分析过程是：1)标准样品用进样器200引入分析管路；2)高压液相泵100连续输送流动相存储装置400中的流动相把样品带进色谱柱300进行分离，进而送进质谱仪500被测定；3)质谱仪500在系统进样后一直采集信号；4)当待测物的色谱峰出现以后内标进样部分立即启动，这时四通切换阀900切换，让样品通路通向废液，而内标通路连接质谱仪500上，内标样品通过内标进样装置进入质谱仪进行测定，在样品峰出现之后产生一个内标峰。
利用这个系统，首先用一个标准物质平行配置两套浓度相差100倍的人血清样品系列：0.0100,0.0500,0.100，1.00，2.00，5.00，10.0，20.0，50.0，100uμg/ml。样品的制备是，吸取50微升样品，加入200微升(冷)甲醇把血清中的蛋白沉淀掉，离心分离后把上层清液移入样品瓶。制备好了的标准样品一个系列放进进样器200，另一个系列放进内标进样器600当作内标样品。标准样品通过上述分析过程每次注射5微升一一进行分析，当样品峰信号出现以后，系统中的四通切换阀900立即切换到内标注射位置，软件同时计算出样品峰对应的大致浓度，内标进样器600随即注入5微升浓度最接近的内标样品。由于流动注射管路短，管路内径又很小，所以样品峰出现以后很短时间里就可以把一个内标峰引入质谱，样品峰和内标峰之间相隔越近，内标的校正效果就越好。但是，质谱电离效率并非瞬息万变，一分钟左右的时间范围之内的变化幅度通常可以忽略不计，所以内标的注入时间没有必要高精度控制，精度在秒范围就足够了。
本发明采用软件控制，让多个样品分析的色谱数据存储在同一个 数据文件中并保持时间轴连续。为了测试出质谱随时间推移灵敏度的变化情况，对样品系列的测试重复了10遍，每遍之间还加了15分钟的间隙，总共历时接近7小时。测得的色谱图如图12所示。
在实施过程中每次内标的选择均没出现错误，亦即，软件每次都准确无误地计算出了用哪个内标样品进行内标注射。然而从得到的结果看，虽然内标样品和标准样品实际上是同样的浓度，用的流速也一样，得到的内标峰却比样品峰低了20％左右，亦即，在质谱测定中，物质通过色谱柱分离比直接注射灵敏度更高。用同样的样品在气-质联用系统上做同样的实验，没有出现同类问题。进一步本实施例证明，通过色谱柱的液体由于柱压很高，100MPa(兆帕)左右，柱前的流速因液体体积压缩而变小，柱后则会产生体积膨胀流速加大，但总流速不变。造成进入质谱仪时的线速度比流动注射要高。因为本实施例用的是电喷雾离子化源，喷雾效果越好离子化效率就越高，得到的质谱信号也就越强。液体从喷雾口出来时的线速度越大，喷雾效果则越好。由此可见，用上述柱后流动注射引进内标的方法不能补偿样品分析过程中因压力变化而产生的信号强度波动。但是，上述系统有一个很大的好处：分析峰后面出现的任何干扰都不会影响到内标峰，因为内标用的是独立的流动注射系统引入质谱仪的。
本实施例采用如图13所示的系统，将内标样品直接引入色谱柱300后的溶剂流中，成功解决了上述内标峰矮于样品峰的问题。本实施例系统的运行过程与前述系统基本一样，只是把内标进样器600的样品阀直接连接到分析管路中，在上述切换四通切换阀的时刻把内标 进样器600的六通切换阀切换到注样位置，让内标样品在分析流动相的推动下得到同样的线速度进入质谱仪，所得到的信号强度也就一样了。
本实施例中对样品的分析重复了10次，得到10个校准曲线，时间跨度大约7小时。图14和图15分别为第一个(起始时)和第十个(7小时后)校准曲线的图像。
从图中可以清楚看到，没有内标时校准曲线只能用二次项拟合曲线去代表所得数据，而且拟合度并不理想，拟合系数R2小数点后面只有两个9，尤其是比较起始时间收集的数据和7小时候收集的数据，拟合函数的系数相差甚远，形状也可以明显看出差异，而使用本实施例的动态内标进样方法后，内标样品信号几乎完全校正了标准样品信号的波动，同时还把仪器的非线性相应问题完全解决，使得仪器的线性响应范围极大地扩展了。用了内标后的校正曲线几乎是理想直线R值小数点后有四个9，理想是1。而且直线的斜率变化不到千分之一，斜率代表灵敏度，灵敏度的变化是造成结果不准确的关键因素，不用内标时，分析灵敏度不仅随时间在变而且随浓度在变。仅仅10倍浓度范围(如实施例一和二)没有内标时的校准曲线就是弯的。实施例一和二还表明2小时内质谱的灵敏度下降了14％，而实施例三7小时后上升了25.4％。由此可见，测定灵敏度的变化很可能是由多种不确定因素造成的，随时间变化的方向无法预测。采用本实施例的内标法能非常有效地消除这些变化因素，而且直接用待测物的标准物质去做内标，大大节省了寻找内标的麻烦，尤其对于药物分析，本发明避免 了使用同位素内标，可以节省大量费用和加快药物临床实验的进程。
本发明特别适合于药物在血样中的浓度分析，尤其适合于药-时曲线的确立。在用药或药物开发中，患者或实验对象用药以后按时间抽取血样，对血样进行测定后得到药物在各个血样中的浓度对时间作图，这样的图谱就叫药-时曲线。在本发明的三个实施例中，所有样品处理与分析都是按血样分析的规范完成的。开辟质谱定量在临床药物检测领域里的应用也是本发明的一个既定目标。
应当理解的是，本发明的应用不限于上述的举例，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进和变换都应属于本发明所附权利要求的保护范围。

Claims (12)

  1. 一种质谱定量分析的模拟内标方法，其特征在于，包括步骤：
    取一标准物用待测样品的空白基底配置成不同浓度的标准溶液，再取同样标准物用待测样品的空白基底配置成一至若干个浓度的内标溶液，内标系列的浓度范围和跨度与标准溶液一致；
    将所述不同浓度的标准溶液和内标溶液经样品处理步骤处理成标准样品和内标样品；
    将标准样品和内标样品注射到色谱-质谱联用系统进行分析测定；
    将测定到的标准样品与内标样品两者的峰面积之比对浓度作图得到模拟内标法定量的校准曲线；
    将待测样品经样品处理步骤进行处理后注射到色谱-质谱联用系统进行分析测定，然后将测得信号的峰面积与校准曲线比对确定出待测样品的浓度。
  2. 根据权利要求1所述的质谱定量分析的模拟内标方法，其特征在于，每次进完一个标准样品之后停顿片刻，等到测定完标准样品所需的时间之后，再注射内标样品使内标样品通过色谱-质谱联用系统的色谱柱，标准样品和内标样品在同一个检测周期中完成分离分析。
  3. 根据权利要求1所述的质谱定量分析的模拟内标方法，其特征在于，每次进完一个标准样品之后，再进内标样品，并使内标样品绕过色谱-质谱联用系统的色谱柱以流动注射的方式直接进入质谱仪进行测定。
  4. 根据权利要求1所述的质谱定量分析的模拟内标方法，其特 征在于，每次进完一个标准样品之后，在检测到标准样品的色谱峰之后，再进与该标准样品浓度最接近的内标样品，并使内标样品绕过色谱-质谱联用系统的色谱柱以流动注射的方式直接进入质谱仪进行测定。
  5. 根据权利要求3所述的质谱定量分析的模拟内标方法，其特征在于，在使用流动注射注入内标样品时，使流动注射流速比样品分析中流动相的流速低20％，或者提高注射时喷雾气体的流量，从60升/分钟提高到70-100升/分钟。
  6. 一种质谱定量分析的模拟内标装置，其特征在于，包括：
    色谱流动相及流动相输送装置；
    连接于流动相输送装置的进样器；
    连接于进样器的色谱柱；
    与色谱柱相配合的流动相切换装置；
    连接于色谱柱的质谱仪；
    控制以上硬件实施模拟内标质谱定量过程的软件系统；
    所述进样器包括进样针以及连接于进样针的用于在进样状态和注样状态之间切换的进样阀，所述进样针与进样阀之间通过样品环连接。
  7. 根据权利要求6所述的质谱定量分析的模拟内标装置，其特征在于，流动相切换装置是两个连接在色谱柱前后的三通切换阀。
  8. 根据权利要求6所述的质谱定量分析的模拟内标装置，其特征在于，流动相切换装置是一个连接在色谱柱的四通切换阀。
  9. 根据权利要求6所述的质谱定量分析的模拟内标装置，其特征在于，流动相切换装置是连接在色谱柱的六通切换阀。
  10. 根据权利要求9所述的质谱定量分析的模拟内标装置，其特征在于，所述六通切换阀是用于内标样品注射的内标进样器中的六通进样阀。
  11. 根据权利要求8所述的质谱定量分析的模拟内标装置，其特征在于，还包括连接在四通切换阀上的内标进样装置，所述内标进样装置包括一连接于四通切换阀的内标进样器、连接于内标进样器的第二流动相输送装置、连接于第二流动相输送装置的第二流动相存储装置。
  12. 一种如权利要求6-11任一项所述的模拟内标装置的应用，其特征在于，将所述模拟内标装置应用于血样检测中。
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