Chromatography system with detector correction function

Abstract

The present invention provides a chromatographic system with a detector correction function, comprising: an injection flow channel (L1), an analysis flow channel (L2), a waste liquid flow channel (L4), a multi-way valve, and a quantitative loop. The quantitative loop is connected to any two ports of the multi-way valve to form a connection flow channel (L3); the injection flow channel (L1), the analysis flow channel (L2), and the waste liquid flow channel (L4) are respectively connected to any one port of the multi-way valve; and a self-calibration liquid flow channel (L5) is further included. The self-calibration liquid flow channel (L5) includes a self-calibration liquid storage bottle (B) and a self-calibration pump (P1) connected to the self-calibration liquid flow channel, and the self-calibration liquid flow channel is also connected to any one of the remaining ports of the multi-way valve. By introducing a substance identical to the target substance as a self-calibration internal standard, the chromatographic system can well correct the instability of the detection system, and has the advantages of simple operation method, rapid determination, high sensitivity, good accuracy, strong stability, and low cost.

Description

Technical Field

[0001] The present invention relates to chromatographic quantitative analysis, and specifically belongs to the field of chromatographic analysis instruments. Background Art

[0002] Liquid chromatography-tandem mass spectrometry (LC-MS / MS), also known as liquid chromatography-mass spectrometry coupling technology, uses liquid chromatography as the separation system and mass spectrometry as the detection system. After the sample is ionized in the mass spectrometry ion source, the ion fragments are separated by the mass analyzer of the mass spectrometer according to the mass number, and a mass spectrum is obtained through an ion induction device. LC-MS / MS embodies the complementary advantages of chromatography and mass spectrometry, combining the high separation ability of chromatography for complex samples with the high selectivity, high sensitivity of MS and the advantage of being able to provide relative molecular weight, and has been widely used in many fields such as drug analysis, food analysis and environmental analysis.

[0003] LC-MS / MS will encounter problems such as in-source fragmentation of compounds, suppression or enhancement of ionization efficiency, and formation of ion adducts. These problems lead to extremely unstable mass spectrometry measurements. In addition, mass number drift and vacuum degree change will also affect the stability of the mass spectrometry. Common correction methods include conventional internal standard method and stable isotope internal standard method, but the above methods have the following problems:

[0004] 1. It is very difficult to find a compound that is relatively consistent with the target in terms of structural properties and other aspects by the conventional internal standard method.

[0005] 2. For the stable isotope internal standard method, the synthesis process of most isotope internal standards is relatively complex and it is difficult to carry out large-scale industrial synthesis. Therefore, its purchase price is very expensive. Moreover, there are certain differences between the stable isotope and the target in terms of physical and chemical properties. Therefore, when the mass spectrometry state changes greatly, the change in the peak area of the stable isotope is not consistent with the change in the peak area of the target. Therefore, in many cases, the stable isotope is also difficult to play a good correction role.

[0006] Therefore, it has become particularly urgent and important to develop a technical means or method that can correct the instability of the mass spectrometry to the greatest extent. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a chromatographic system with a correction function to correct the instability of the detection system. This chromatographic system introduces the same substance as the target substance as a self-calibration internal standard, and uses the time difference to introduce its own substance into the determination system, thereby correcting the instability of the detection system, greatly reducing the time for finding conventional internal standard substances, improving the R & D speed and efficiency, and also avoiding problems such as the high cost of using stable isotopes as internal standards, and being more accurate and stable. This system is not only applicable to mass spectrometry detectors, but also applicable to the correction of other unstable detectors, such as fluorescence detectors, electrochemical detectors, etc.

[0008] The technical solution of the present invention is as follows:

[0009] A chromatographic system with a detector correction function, comprising:

[0010] An injection flow path for injecting samples;

[0011] An analysis flow path for sample analysis;

[0012] A waste liquid flow path for discharging waste liquid;

[0013] A multi-port valve for connecting and switching each flow path;

[0014] A quantitative loop, which is connected to any two ports of the multi-port valve to form a connection flow path;

[0015] The injection flow path, the analysis flow path, and the waste liquid flow path are respectively connected to any one port of the multi-port valve;

[0016] It further includes a self-calibration liquid flow path, which includes a self-calibration liquid storage bottle and a self-calibration pump connected to the self-calibration liquid flow path. The self-calibration liquid flow path is also connected to any one of the remaining ports of the multi-port valve; the self-calibration pump is a pump with a cavity volume ≤ 200 μL.

[0017] In a preferred embodiment, the multi-port valve is a six-port valve. The injection flow path communicates with the starting port of the six-port valve, the analysis flow path communicates with the first port of the six-port valve, the waste liquid flow path communicates with the third port of the six-port valve, the connection flow path communicates with the second port and the fifth port of the six-port valve, and the self-calibration liquid flow path communicates with the fourth port of the six-port valve.

[0018] In a preferred embodiment, a refrigeration device is provided outside the self-calibration liquid storage bottle, which can cool the self-calibration liquid and make it more stable.

[0019] In a preferred embodiment, the self-calibration liquid flow path is further provided with a filter, which is located on the downstream flow path of the self-calibration pump and can filter impurities in the self-calibration liquid.

[0020] In a preferred embodiment, the self-calibration pump includes an injection pump, a diaphragm pump, a peristaltic pump, a plunger pump, etc., and the cavity volume is preferably ≤ 200 μL.

[0021] In a preferred embodiment, at least one mobile phase storage bottle, a chromatographic pump, and an injector are provided on the injection flow path.

[0022] In a preferred embodiment, an analytical column is provided on the analysis flow path, and a detector is connected to the rear end of the analysis flow path.

[0023] Technical principle: In the chromatographic part of the mass spectrometry system, the principle is that the liquid is used as the mobile phase, and a high-pressure liquid delivery system is adopted. A mobile phase such as a single solvent with different polarities, a mixed solvent with different ratios, or a buffer solution is used to pump a mixture into a chromatographic column filled with a stationary phase. Due to the differences in the properties and structures of the components in the mixture, the forces generated between the components and the stationary phase are of different magnitudes and strengths. As the mobile phase moves, the mixture undergoes repeated distribution equilibria between the two phases, resulting in different retention times of the components by the stationary phase. Thus, the components flow out of the stationary phase in a certain order and enter the detector for detection, thereby realizing the analysis of the sample. After introducing an automated mass spectrometry calibration structure, by controlling the start of the self-calibration pump, the self-calibration liquid is pre-stored in the six-port valve. After injection, after a certain period of time (usually within 1 minute), through the switching of the internal structure of the six-port valve, the self-calibration liquid is added to the system. Finally, the self-calibration liquid and the sample to be measured are separated under the same chromatographic conditions and chromatographic column, and peak out in sequence, thus playing a role in correcting and calibrating the target.

[0024] The self-calibration liquid uses the target substance as the internal standard, the dissolution solution of the self-calibration liquid is the same as the mobile phase output by the chromatographic pump, and the concentration of the self-calibration liquid is within the linear range of the target substance to be measured. Introducing the self-calibration liquid increases the selectivity of the internal standard. Especially for some target substances without isotopes, isomers, and similar structural properties, the self-calibration liquid can well serve as an internal standard to play a corrective role.

[0025] Compared with the prior art, the advantages of the present invention are:

[0026] 1. By introducing a substance identical to the target substance as the self-calibration internal standard, this chromatographic system uses the time difference to introduce its own substance into the measurement system, thereby correcting the instability of the detection system, greatly reducing the time for finding conventional internal standard substances, improving the R & D speed and efficiency, and also avoiding problems such as the high cost of using stable isotopes as internal standards, and being more accurate and stable.

[0027] 2. This automatic calibration system avoids a series of problems such as inconsistent internal standard addition amounts caused by factors such as personnel and measuring instruments in the traditional internal standard addition method, simplifies the process of chromatographic method development, is no longer limited by the types of chromatographic columns and mobile phases, ensures the consistency of internal standard addition, and thus greatly guarantees the corrective effect of the internal standard.

[0028] 3. The chromatographic system of the present invention maximally corrects the detector, improves the stability of mass spectrometry quantitative detection, and enables the mass spectrometry system to be well applied to high-risk and high-difficulty detection projects, such as clinical drug treatment monitoring and other fields. Brief Description of the Drawings

[0029] Figure 1 is a schematic structural diagram of the chromatographic system with a detector correction function of the present invention;

[0030] Figure 2 It is the initial working state diagram of the self-calibration module during the system operation;

[0031] Figure 3 It is the working state diagram of the self-calibration module during the system operation;

[0032] Figure 4 It is the working state diagram of the self-calibration module during the system operation;

[0033] Figure 5 It is the chromatogram of cyclosporine and internal standard. The retention time of cyclosporine is 2.464 min, and the retention times of internal standards are 2.984 (self-calibration internal standard) and 2.919 (cyclosporine A-d4) respectively.

[0034] Figure 6 It is the standard curve diagram of cyclosporine.

[0035] Figure 7 The analysis result diagram of cyclosporine blood drug concentration in blood samples;

[0036] Figure 8 The analysis result diagram of cyclosporine blood drug concentration in blood samples;

[0037] Among them: S1: Mobile phase I, S2: Mobile phase II, S3: Mobile phase III, DGU: Degasser, P1: Self-calibration pump, SIL: Automatic sampler, P2: Chromatography pump, V1: Six-port valve, C: Filter, C2: Chromatographic column, D: Detector, W: Waste liquid bottle, Loop ring: Quantitative loop, A: Refrigeration device, B: Self-calibration liquid, L1: Sampling flow path, L2: Analysis flow path, L3: Connection flow path, L4: Waste liquid flow path, L5: Self-calibration liquid flow path, 0: Starting port, 1: First port, 2; Second port, 3; Third port, 4; Fourth port, 5; Fifth port. Specific implementation mode

[0038] The detailed structure of the present invention will be further described below in conjunction with the attached drawings and specific implementation modes.

[0039] Example 1

[0040] As Figure 1 shown: A chromatographic system with a detector calibration function, including: a sampling flow path L1, an analysis flow path L2, a waste liquid flow path L4, a multi-port valve, and a quantitative loop. The quantitative loop is connected to any two ports of the multi-port valve to form a connection flow path L3; a self-calibration liquid flow path L5.

[0041] The multi-way valve is a six-way valve. The injection flow path L1 is connected to the starting port 0 of the six-way valve. The analysis flow path L2 is connected to the first port 1 of the six-way valve. The waste liquid flow path L4 is connected to the third port 3 of the six-way valve. The connection flow path L3 is connected to the second port 2 and the fifth port 5 of the six-way valve. The self-reference liquid flow path L5 is connected to the fourth port 4 of the six-way valve.

[0042] The self-reference liquid flow path L5 includes a self-reference liquid storage bottle B and a self-reference pump P1 connected to the self-reference liquid flow path. The self-reference pump P1 is a small-volume diaphragm pump with a cavity volume of 25 microliters.

[0043] A refrigeration device A is provided outside the self-reference liquid storage bottle B.

[0044] The self-reference liquid flow path L5 is also provided with a filter C, and the filter C is located on the downstream flow path of the self-reference pump P1.

[0045] Three mobile phase storage bottles, a chromatographic pump P2, and an injector SIL are provided on the injection flow path L1.

[0046] An analytical column C2 is provided on the analysis flow path L2, and a detector D is connected to the rear end of the analysis flow path L2.

[0047] Description of the working process:

[0048] Step 1) As Figure 2 shown: In the initial state, the chromatographic pump P2 is started. The injection flow path LI is connected to the analysis flow path L2 through the six-way valve, and the analysis flow path is connected to the detector D.

[0049] Step 2) As Figure 3 shown; In the working state of the self-reference module, after a certain period of time (generally within 1 minute, for example, 0.5 minutes), the self-reference pump P1 is started. The self-reference liquid flow path L5 is connected to the fourth port 4 of the six-way valve, connected to the connection flow path L3 through the fifth port 5, and then connected to the waste liquid flow path L4 through the second port 2. At this time, the self-reference liquid is stored in the quantitative loop of the connection flow path L3.

[0050] Step 3) As Figure 4 shown; The six-way valve is switched. The starting port 2 is connected to the fifth port 5, and the first port 1 is connected to the second port 2, so that the injection flow path LI is connected to the connection flow path L3 and then to the analysis flow path L2, and the self-reference liquid stored in the quantitative loop is brought into the chromatographic column. The self-reference liquid and the sample to be measured are separated under the same chromatographic conditions and by the same chromatographic column, and peak out in sequence, thereby playing a role in correcting and calibrating the target.

[0051] Example 2, specific application case

[0052] In this experiment, cyclosporine was selected as the test drug. The self-comparison internal standard method, isotope internal standard method and external standard method were used to investigate the linearity, sensitivity, specificity, accuracy, precision and other indicators. The feasibility, reliability and stability of the liquid chromatography / mass spectrometry system with detector calibration function were verified by comparing the results of the three quantitative methods.

[0053] 1. Instrument

[0054] The chromatography system with detector calibration function described in Example 1 was used, and the mass spectrometry detection system adopted was Shimadzu LCMS-8050CL clinical mass spectrometer.

[0055] Chromatographic pump: Shimadzu LC-30AD CL

[0056] Injector: Shimadzu SIL-30AC MP CL

[0057] Controller: Shimadzu CBM-20A CL

[0058] Detector: Shimadzu LCMS-8050CL

[0059] Workstation: Shimadzu Lab-Solution 2. Analytical conditions Chromatographic column: German MN Nucleodur CN-RP column (4.6×100mm, 5μm);

[0060] Mobile phase: water (containing 2mmol / L formic acid): methanol = 35:65 (V / V);

[0061] Flow rate: Chromatographic pump flow rate is 1.0mL·min-1

[0062] Column temperature: 55°C

[0063] Injection volume: 1 μL

[0064] Characteristic ions: 1224.90>1224.75 (quantitative)

[0065] 1224.90>1189.20(qualitative)

[0066] Internal standard: Cyclosporine A-d4 (100 ng / ml); Cyclosporine (self-contained solution 10 ng / ml)

[0067] The system detector calibration function is controlled by the chromatography workstation time program, without human intervention, to achieve highly automated rapid analysis.

[0068] The main component conditions in each process are shown in Table 1.

[0069] Table 1 Time program

[0070]

[0071] 3. Preparation of Solutions

[0072] 3.1 Preparation of Standard Solutions

[0073] Accurately weigh 0.95 mg of cyclosporine reference substance, dissolve it with methanol and dilute to an appropriate volume to obtain a mother liquor of cyclosporine with a concentration of 945.0 μg·mL-1, and store it in a refrigerator at -76 °C. Dilute it to the working solution with the required concentration when in use. Accurately pipette an appropriate amount of the above mother liquor and dilute it with human serum to prepare low-concentration (LQC), medium-concentration (MQC), and high-concentration (HQC) quality control samples with concentrations of 25, 100, and 400 ng·mL-1 respectively.

[0074] 3.2 Preparation of Internal Standard Solutions

[0075] Take a certain amount of the prepared cyclosporine standard solution, use the mobile phase as the matrix, prepare 50 ml of a self-calibration solution with a concentration of 10 ng / ml, and store it in a refrigerator at -24 °C. It can be directly used when needed.

[0076] Accurately weigh 1.0 mg of cyclosporine A-d4 reference substance, dissolve it with methanol and dilute to an appropriate volume to obtain a mother liquor of cyclosporine A-d4 with a concentration of 40.0 μg·mL -1 and store it in a refrigerator at -76 °C. Dilute it to a working solution of 4.0 μg·mL -1 when in use.

[0077] 4. Sample Pretreatment Method

[0078] 4.1 Pretreatment for External Standard Method or Self-Calibration Internal Standard Method

[0079] Accurately pipette 1000 μl of 90% acetonitrile into a 1.5 ml EP tube, then accurately add 400 μl of serum, vortex for 1 min, centrifuge at high speed (14500 r·min-1) for 8 minutes, and take 1000 μl of the supernatant for on-machine testing.

[0080] 4.2 Pretreatment for Isotope Internal Standard Method

[0081] Take 400 μl of serum into a 1.5 ml EP tube, add 10 μl of the cyclosporine A-d4 internal standard working solution with a concentration of 4.0 μg·mL-1, and mix well. Accurately pipette 1000 μl of 90% acetonitrile into the serum, vortex for 1 min, centrifuge at high speed (14500 r·min-1) for 8 minutes, and take 1000 μl of the supernatant for on-machine testing.

[0082] 5. Method Validation

[0083] 5.1 Investigation of Chromatographic Behavior and Specificity

[0084] Under the above chromatographic conditions, the blank blood samples of cyclosporine, the standard solution, the blank blood samples spiked with the standard, and the blood samples of the subjects after taking the drug were measured respectively to investigate the specificity of the method.

[0085] 5.2 Investigation of linear range and limit of quantitation (LOQ)

[0086] Referring to the relevant guidelines of the Clinical and Laboratory Standards Institute (CLSI) of the United States, a series of working solutions of cyclosporine with different concentrations were prepared, processed and measured according to the above method. When quantifying by the external standard method, the peak area of cyclosporine in the spiked serum was used as the ordinate and the concentration as the abscissa to plot the working curve, and the linear regression equation was obtained to investigate the linear range of cyclosporine. When quantifying by the internal standard method, the ratio of the peak area of cyclosporine to the peak area of the internal standard in the spiked serum was used as the ordinate and the concentration as the abscissa to plot the working curve, and the linear regression equation was obtained to investigate the linear range of cyclosporine.

[0087] 5.3 Investigation of within-day precision and between-day precision

[0088] The quality control samples of cyclosporine at high, medium and low concentration levels were processed and measured. Five parallel samples were set for each concentration under each quantification method. The measured results were substituted into the linear regression equation to calculate the concentration, and the calculated concentration was compared with the theoretically added concentration to obtain the recovery rate and coefficient of variation of the method, and to test the accuracy and within-day precision. The above concentration samples were prepared and measured continuously for 3 days to investigate the between-day precision. At the same time, the changes in the peak area of the target substance, the peak area of the internal standard substance, and the ratio of the two peak areas of the quality control samples at the medium concentration level were investigated under the three quantification methods.

[0089] 5.4 Investigation of sample stability

[0090] The quality control samples of cyclosporine at high, medium and low concentration levels were taken and measured after being repeatedly frozen and thawed at -76°C for 0, 1, 2, 3 times to investigate the freeze-thaw stability of the plasma samples; they were measured after being placed at room temperature for 8 h to investigate the storage stability of the plasma samples; the working curve of cyclosporine and the treatment solutions of the quality control samples at three concentrations were placed in the autoinjector at 10°C and analyzed by repeated injection within 12 h to investigate the storage stability of the sample treatment solutions; the quality control samples at three concentrations were stored under frozen conditions at -76°C. Before storage (day 0) and after frozen storage for 30 days, 60 days, and 90 days, the samples were taken out, thawed, processed and analyzed to investigate the frozen storage stability of the plasma samples.

[0091] 6 Results and discussion

[0092] 6.1 Chromatographic behavior and specificity

[0093] The experimental results showed that the plasma endogenous substances and other impurities did not interfere with the separation and determination of the samples. The retention time of cyclosporine was 2.464 min, and the retention times of the internal standards were 2.984 (self-comparative internal standard) and 2.919 (cyclosporine A-d4) respectively. The chromatogram is shown in Figure 5 .

[0094] 6.2 Linear range and limit of quantification

[0095] The prepared series of gradient working solutions of cyclosporine at 9.63, 24.08, 48.16, 96.32, 240.8, 481.6 ng / mL-1 were processed and determined according to the above method respectively. For the external standard method, the concentration of cyclosporine in the spiked serum was taken as the abscissa and the peak area as the ordinate to plot the working curve. For the internal standard method, the ratio of the peak area of cyclosporine to the peak area of the internal standard in the spiked serum was taken as the ordinate and the concentration as the abscissa to plot the working curve. The obtained linear regression equations are as shown in Figure 2 . The results showed that when cyclosporine was in the range of 9.63 - 481.6 ng mL -1 , there was a good linear relationship between the concentration and the peak area (peak area ratio), as shown in Figure 6 .

[0096] 6.3 Accuracy and precision of the method

[0097] The changes in the peak area of the target substance, the peak area of the internal standard substance, and the ratio of their peak areas under the three quantitative methods were investigated for the medium-concentration level quality control samples. The recoveries, within-day precision, and between-day precision of the three concentration level quality control samples under the three quantitative methods were investigated. The results are shown in Table 2-10. The coefficients of variation of the peak area of the target substance, the peak area of the internal standard substance, and the ratio of their peak areas in the test results of the medium-concentration quality control product under the isotope internal standard quantitative method were 13.59%, 8.43%, and 14.90% respectively. The accuracy of the quality control samples at each concentration level was 93.0% - 102.6%, and the within-day and between-day coefficients of variation were both less than 16.9%. The coefficients of variation of the peak area of the target substance, the peak area of the internal standard substance, and the ratio of their peak areas in the test results of the medium-concentration quality control product under the self-comparative internal standard quantitative method were 16%, 17%, and 3% respectively. The accuracy of the quality control samples at each concentration level was 99.2% - 103.7%, and the within-day and between-day coefficients of variation were both less than 3.2%. The coefficient of variation of the peak area of the target substance in the test results of the medium-concentration quality control product under the external standard quantitative method was 25.10%. The accuracy of the quality control samples at each concentration level was 92.6% - 102.1%, and the within-day and between-day coefficients of variation were both less than 34.4%.

[0098] Through the comparison of three quantitative methods, it can be seen that the coefficients of variation of the peak areas of the analyte and the internal standard are relatively large under all three quantitative methods. However, the coefficient of variation of the ratio of the peak area of the analyte to the peak area of the internal standard is the lowest in the self-comparative internal standard quantitative method, with the lowest intra-day and inter-day coefficients of variation. This analytical method has the strongest correction ability and the highest stability, meeting the methodological requirements for bioequivalence studies.

[0099] Table 2 Changes in the peak areas of the analyte, internal standard, and their ratio in the isotope internal standard method

[0100]

[0101] Table 3 Recovery and intra-day precision of the isotope internal standard quantitative method (n = 5)

[0102]

[0103]

[0104] Table 4 Inter-day precision of the isotope internal standard quantitative method (n = 5)

[0105]

[0106] Table 5 Changes in the peak areas of the analyte, internal standard, and their ratio in the self-comparative internal standard method

[0107]

[0108] Table 6 Recovery and intra-day precision of the self-comparative internal standard quantitative method (n = 5)

[0109]

[0110] Table 7 Inter-day precision of the self-comparative internal standard quantitative method (n = 5)

[0111]

[0112]

[0113] Table 8 Changes in the peak area of the analyte in the external standard method

[0114]

[0115] Table 9 Recovery and intra-day precision of the external standard quantitative method (n = 5)

[0116]

[0117] Table 10 Inter-day precision of the external standard quantitative method (n = 5)

[0118]

[0119] 6.4 Sample Stability

[0120] The experimental results show that cyclosporine in plasma does not change significantly during 3 freeze - thaw cycles; the differences between the measured values of the quality control samples after 8 h at room temperature and the control measured values at 0 h are all within ±6%, and the coefficients of variation are all less than 7%, indicating that cyclosporine in plasma is stable within 8 hours at room temperature; the working curve of cyclosporine and the quality control samples in the analysis batches repeatedly analyzed within 12 hours when the processing solution is placed in the auto - sampler (10℃) meet the requirements for accurate quantification, indicating that the sample processing solution is stable within 12 hours when stored in the auto - sampler; the plasma samples of cyclosporine are stable within 90 days under the frozen condition of - 76℃.

[0121] 6.5 Analysis of Actual Samples

[0122] The self - calibration internal standard quantitative method verified by comparison is used to determine the drug concentration of cyclosporine in patients' plasma. The drug concentrations in the plasma of 30 patients collected after using cyclosporine for a certain period of time are measured. The typical chromatograms are as Figure 7 、 8 shown, among which Figure 7 the blood drug concentration of cyclosporine in the blood sample is 87.5 ng·mL - 1, Figure 8 the concentration of cyclosporine in the blood sample is 98.5 ng·mL - 1. The monitoring of the blood drug concentration of cyclosporine can timely guide the clinical implementation of individualized drug administration.

[0123] 7 Conclusion

[0124] In this paper, the feasibility, reliability and stability of the self - calibration internal standard method were verified through three comparative experiments: the external standard method, the isotope internal standard method and the self - calibration internal standard method. A method for the determination of cyclosporine in plasma by a liquid chromatography / mass spectrometry system with detector calibration function was established. This method can complete the detection of cyclosporine in plasma within 3.5 minutes. The (self - calibration) internal standard method was used for quantification. The limit of quantification of the method was 9.63 ng / mL, the linear range was 9.63 - 481.9 ng / mL, and the correlation coefficient was 0.9994. The results of specificity investigation showed that endogenous substances in plasma and other impurities did not cause obvious interference to the analysis, and there was no interference from subsequent chromatographic peaks during consecutive sample injections. The intra - day precision of the method was 1.1 - 2.0%, the inter - day precision was 1.8 - 3.2%, and the accuracy of quality control samples at each concentration level was 99.2% - 103.7%, which could meet the requirements for accurate quantification of drug concentrations in plasma. The results of the stability experiment showed that the samples were stable when placed at room temperature for 8 hours, the concentration of cyclosporine did not change significantly after 3 freeze - thaw cycles, and the cyclosporine did not change significantly when the treated samples were placed in the auto - sampler (at 10 °C) for 12 hours. This method has the characteristics of simple pretreatment, rapid determination, high sensitivity, good accuracy and strong stability, and can meet the requirements of timeliness, accuracy and determination range of clinical reports. It can be used as an effective method for the determination of cyclosporine samples in therapeutic drug monitoring and clinical pharmacy research. The system of the present invention has a powerful drug determination ability, a highly automated technology and a complete reliability technology, making the operation simple and the analysis highly compatible.

[0125] The above is the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and its concept of the present invention, making equivalent substitutions or changes, shall be covered by the protection scope of the claims of the present invention.

Claims

1. A chromatographic system with a detector calibration function, comprising: An injection flow path (L1) for sample injection; An analysis flow path (L2) for sample analysis; A waste liquid flow path (L4) for discharging waste liquid; A multi-way valve for connecting and switching each flow path; A quantitative loop, the quantitative loop is connected to any two ports of the multi-way valve to form a connection flow path (L3); The injection flow path (L1), the analysis flow path (L2), and the waste liquid flow path (L4) are respectively connected to any one port of the multi-way valve; It is characterized in that: it further includes a self-calibration liquid flow path (L5), the self-calibration liquid flow path (L5) includes a self-calibration liquid storage bottle (B) and a self-calibration pump (P1) connected to the self-calibration liquid flow path, and the self-calibration liquid flow path (L5) is also connected to any one of the remaining ports of the multi-way valve; the self-calibration pump (P1) is a pump with a cavity volume ≤ 200 μL; The multi-way valve is a six-way valve, the injection flow path (L1) communicates with the starting port (0) of the six-way valve, the analysis flow path (L2) communicates with the first port (1) of the six-way valve, the waste liquid flow path (L4) communicates with the third port (3) of the six-way valve, the connection flow path (L3) communicates with the second port (2) and the fifth port (5) of the six-way valve, and the self-calibration liquid flow path (L5) communicates with the fourth port (4) of the six-way valve.

2. The chromatographic system with a detector correction function according to claim 1, characterized in that, A refrigeration device (A) is provided outside the self-calibration liquid storage bottle (B).

3. The chromatographic system with a detector correction function according to claim 1, characterized in that, The self-calibration liquid flow path (L5) is further provided with a filter (C), and the filter (C) is located on the downstream flow path of the self-calibration pump (P1).

4. The chromatographic system with a detector correction function according to claim 1, characterized in that, The self-calibration pump (P1) includes an injection pump, a diaphragm pump, a peristaltic pump, and a plunger pump.

5. The chromatographic system with a detector correction function according to claim 1, characterized in that, At least one mobile phase storage bottle, a chromatographic pump (P2), and an injector (SIL) are provided on the injection flow path (L1).

6. The chromatographic system with a detector correction function according to claim 1, characterized in that, An analytical column (C2) is provided on the analysis flow path (L2), and a detector (D) is connected to the rear end of the analysis flow path (L2).

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