A method for simultaneous detection of the concentration of 6 immunosuppressants in blood

Abstract

This invention relates to a method for simultaneously detecting the concentrations of six immunosuppressants in blood. The method includes: (1) preparing a standard solution and detecting the standard solution using liquid chromatography-mass spectrometry to establish a standard curve; wherein the six immunosuppressants are azathioprine, 6-mercaptopurine, 6-methylmercaptopurine, 6-thioguanine, 6-thioguanine nucleoside, and imidazolidin; (2) mixing the blood sample to be treated, internal standard working solution, and protein precipitant, and diluting the supernatant to obtain the sample to be injected; (3) detecting the sample to be injected using liquid chromatography-mass spectrometry, and determining the content of the six immunosuppressants in the blood sample to be treated based on the detection results and the standard curve. The detection method provided by this invention can determine the content of six immunosuppressants in a single injection, with a short detection time, producing results within 5 minutes, improving detection efficiency while reducing operational difficulty, and is suitable for high-throughput sample detection.

Description

Technical Field

[0001] This invention relates to the field of blood drug detection technology, and in particular to a method for simultaneously detecting the concentrations of six immunosuppressants in blood. Background Technology

[0002] Currently, the levels of immunosuppressants such as azathioprine, 6-mercaptopurine, 6-methylmercaptopurine, 6-thioguanine, 6-thioguanine nucleoside, and imidazoribine in blood can usually be determined using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). However, there is no method for simultaneously detecting these substances. Furthermore, most of the reported methods in the literature have problems such as requiring large sample volumes, adding matrix for standard curve pretreatment, and single-substance detection. These problems increase the difficulty of detection and reduce the detection efficiency, making them unsuitable for high-throughput sample detection. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a method for simultaneously detecting the concentrations of six immunosuppressants in blood.

[0004] In a first aspect, the present invention provides a method for simultaneously detecting the concentrations of six immunosuppressants in blood, the method comprising:

[0005] (1) Prepare at least three different concentrations of standard solutions containing six immunosuppressants and their internal standards, and detect the standard solutions using liquid chromatography-mass spectrometry. Establish standard curves for the six immunosuppressants based on the detection results.

[0006] The six immunosuppressants are azathioprine, 6-mercaptopurine, 6-methylmercaptopurine, 6-thioguanine, 6-thioguanine nucleoside, and imidazolidin.

[0007] (2) Mix the blood sample to be processed, the internal standard working solution and the protein precipitant, and take the supernatant for dilution to obtain the sample to be injected;

[0008] (3) The sample to be injected was detected by liquid chromatography-mass spectrometry, and the content of the six immunosuppressants in the sample was determined according to the detection results and the standard curve.

[0009] The detection method provided by this invention can measure the content of six immunosuppressants in one injection. The detection time is short, with results available within 5 minutes. It improves detection efficiency while reducing operational difficulty and is suitable for high-throughput sample detection.

[0010] As a preferred embodiment of the present invention, in step (2), the protein precipitant is selected from methanol and / or acetonitrile.

[0011] In the sample pretreatment conditions provided by this invention, only a simple protein precipitant is used for treatment, followed by dilution with a diluent, which can reduce matrix effect and solvent effect.

[0012] As a preferred embodiment of the present invention, the volume ratio of the blood sample to be treated to the protein precipitant is 1:(5-10), for example, 1:6, 1:7, 1:8, 1:9, etc.

[0013] As a preferred embodiment of the present invention, the volume ratio of the blood sample to be processed to the internal standard working solution is (5-10):1, for example, 6:1, 7:1, 8:1, 9:1, etc.

[0014] In a preferred embodiment of the present invention, the solvent used for dilution is pure water.

[0015] As a preferred embodiment of the present invention, the volume ratio of the clarified liquid to pure water is 1:(1-5), for example, 1:2, 1:3, 1:4, etc.

[0016] If the dilution factor is too small, the matrix effect and solvent effect cannot be completely avoided; if the dilution is too large, the detection signal of the target analyte will be weak.

[0017] As a preferred embodiment of the present invention, the conditions for liquid chromatography include:

[0018] Mobile phase: Phase A is an aqueous solution containing 0.2-3% formic acid, 1-10 mmol / L ammonium formate or ammonium acetate, and Phase B is methanol or acetonitrile.

[0019] The 0.2-3% formic acid can be 0.5%, 1%, 1.5%, 2%, 2.5%, etc.; the 1-10 mmol / L can be 2 mmol / L, 4 mmol / L, 6 mmol / L, 8 mmol / L, etc.

[0020] Gradient elution:

[0021] 0.00min: Phase A 85-98%, Phase B 15-2%;

[0022] 0.50 min: Phase A 85-98%, Phase B 15-2%;

[0023] 0.60 min: Phase A 50-75%, Phase B 50-25%;

[0024] 1.20 min: Phase A 50-75%, Phase B 50-25%;

[0025] 1.50 min: Phase A 25-45%, Phase B 75-55%;

[0026] 2.00 min: Phase A 25-45%, Phase B 75-55%;

[0027] 2.50 min: Phase A 2-20%, Phase B 98-80%;

[0028] 3.00 min: Phase A 2-20%, Phase B 98-80%;

[0029] 3.10 min: Phase A 85-98%, Phase B 15-2%;

[0030] 5.00 min: Phase A 85-98%, Phase B 15-2%.

[0031] In gradient elution conditions, for 0.00-0.50 min, phase A can be 86%, 88%, 90%, 92%, 95%, 97%, etc., and phase B can be 14%, 12%, 10%, 8%, 5%, 3%, etc.

[0032] For a time interval of 0.60–1.20 min, phase A can be 52%, 55%, 58%, 60%, 62%, 65%, 68%, 70%, 72%, etc., and phase B can be 48%, 45%, 42%, 40%, 38%, 35%, 32%, 30%, 28%, etc.

[0033] For a time of 1.50–2.00 min, phase A can be 28%, 30%, 32%, 35%, 38%, 40%, 42%, etc., and phase B can be 72%, 70%, 68%, 65%, 62%, 60%, 58%, etc.

[0034] For a time of 2.50-3.00 min, phase A can be 5%, 8%, 10%, 12%, 15%, 18%, etc., and phase B can be 95%, 92%, 90%, 88%, 85%, 82%, etc.

[0035] For 3.10-5.00 min, phase A can be 86%, 88%, 90%, 92%, 95%, 97%, etc., and phase B can be 14%, 12%, 10%, 8%, 5%, 3%, etc.

[0036] As a preferred embodiment of the present invention, the column temperature is 35-40℃, such as 36℃, 37℃, 38℃, 39℃, etc.

[0037] As a preferred embodiment of the present invention, the flow rate of the mobile phase is 0.3-0.5 mL / min, for example, 0.35 mL / min, 0.4 mL / min, 0.45 mL / min, etc.

[0038] As a preferred embodiment of the present invention, the conditions for liquid chromatography include: using an Agilent Poroshell 120EC-C18, phenomenex Kinetex, Waters Cortecs T3, or Shimadzu Shim-pack Veiox C18 analytical column.

[0039] As a preferred embodiment of the present invention, the mass spectrometry conditions include: ion source temperature: 450-500℃, such as 460℃, 470℃, 480℃, 490℃, etc.; ion source high voltage: 4500-5000V, such as 4600V, 4700V, 4800V, 4900V, etc.; curtain gas: 12-20L / min, such as 14L / min, 15L / min, 16L / min, 18L / min, etc.; collision gas: 7-10L / min, such as 8L / min, 9L / min, etc.; drying gas 1: 30-80L / min, such as 40L / min, 50L / min, 60L / min, 70L / min, etc.; drying gas 2: 30-80L / min, such as 40L / min, 50L / min, 60L / min, 70L / min.

[0040] In the detection method provided by this invention, imidazoribine is detected in a positive ion mode with a relatively high positive ion signal, which meets the quantitative requirements while reducing the requirements on the instrument. It can be detected simultaneously with five other immunosuppressants, reducing the detection procedure and improving detection efficiency. The detection conditions defined by this invention result in good chromatographic peak shape and chromatographic separation, avoiding ion crosstalk and effectively reducing matrix effects.

[0041] As a preferred embodiment of the present invention, the internal standards of the six immunosuppressants are azathioprine-d3, 6-methylmercaptopurine-d3, and 6-mercaptopurine-13C,15N2.

[0042] The sample pretreatment conditions defined by this invention enable sample detection to be completed using only three internal standards, saving internal standard costs and simplifying pretreatment.

[0043] In this invention, the internal standard corresponding to azathioprine, 6-thioguanine nucleoside and imidazoribine is azathioprine-d3; the internal standard corresponding to 6-methylmercaptopurine and 6-thioguanine is 6-methylmercaptopurine-d3; and the internal standard corresponding to 6-mercaptopurine is 6-mercaptopurine-13C,15N2.

[0044] As a preferred embodiment of the present invention, the method for preparing the standard solution includes: preparing standard working solutions of at least three concentration levels; mixing the standard working solution, internal standard working solution and protein precipitant; taking the supernatant and diluting it to obtain the standard solution.

[0045] In the process of preparing the standard solution, in order to fully simulate the sample pretreatment process, some pure water can be added. The standard working solution + water can be regarded as the plasma to be tested.

[0046] As a preferred embodiment of the present invention, the standard working solutions of each concentration level are obtained by diluting the standard stock solution with a diluent. The standard stock solution is obtained by dissolving the respective standards of the six immunosuppressants in a solvent.

[0047] The internal standard working solution is obtained by diluting the internal standard stock solution with a diluent. The internal standard stock solution is obtained by dissolving the internal standard standards of the six immunosuppressants respectively with a solvent.

[0048] As a preferred embodiment of the present invention, the diluent is an aqueous methanol solution with a concentration of 60-80%, wherein the 60-80% can be 65%, 70%, 75%, 80%, etc.

[0049] The technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art:

[0050] (1) The detection method provided by the present invention can measure the content of 6 immunosuppressants in one injection. The detection time is short, and the results are obtained within 5 minutes. It improves the detection efficiency while reducing the difficulty of operation and is suitable for the detection of high-throughput samples.

[0051] (2) The detection method provided by the present invention can reduce impurity interference, reduce residues and reduce matrix effects, so that the detection of 6 substances can be completed using only 3 isotope internal standards and the standard curves do not require matrix pretreatment.

[0052] (3) The sample pretreatment provided by the present invention requires only 50 μL of serum, which saves costs and simplifies the pretreatment steps. Attached Figure Description

[0053] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0054] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 This is the chromatogram of the standard solution described in Example 3 of the present invention;

[0056] Figure 2 This is the chromatogram of the sample to be injected as described in Embodiment 3 of the present invention;

[0057] Figure 3 The chromatogram of the standard solution described in Example 4 of this invention;

[0058] Figure 4 This is the chromatogram of the standard solution described in Comparative Example 4 of the present invention;

[0059] Figure 5 This is a chromatogram of the standard solution described in Comparative Example 5 of the present invention. Detailed Implementation

[0060] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0061] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0062] Example 1

[0063] This embodiment provides a method for preparing a standard working solution.

[0064] (1) Prepare stock solutions of 6 immunosuppressant standards

[0065] Accurately weigh 5.87 mg of 6-thioguanine (concentration 95.00%) standard into a 10 mL brown glass bottle, add 2 mL of pure water, 900 μL of methanol and 100 μL of 1 mol / L NaOH, dissolve and mix to obtain a mother liquor with a concentration of 1859 μg / mL.

[0066] Accurately weigh 1.974 mg of azathioprine (concentration 99.30%) standard into a 2 mL cryovial, add 1 mL of pure water and 10 μL of 1 mol / L NaOH, dissolve and mix to obtain a mother liquor with a concentration of 1941 μg / mL;

[0067] Accurately weigh 4.038 mg of 6-mercaptopurine (concentration 98.00%) standard into a 2 mL cryovial, add 1.5 mL of pure water and 30 μL of 1 mol / L NaOH, then add 100 μL of DMSO, dissolve and mix to obtain a mother liquor with a concentration of 2428 μg / mL;

[0068] Accurately weigh 1.38 mg of 6-thioguanine nucleoside (concentration 96.00%) into a 2 mL cryovial, add 1 mL of DMSO, dissolve and mix well to obtain a stock solution with a concentration of 1250 μg / mL;

[0069] Accurately weigh 1.382 mg of 6-methylmercaptopurine (concentration 98.00%) standard into a 2 mL cryovial, add 1.5 mL of methanol aqueous solution (methanol:water = 1:1) and 20 μL of 1 mol / L NaOH, dissolve and mix to obtain a mother liquor with a concentration of 1328 μg / mL;

[0070] Accurately weigh 1.202 mg of imidazoribine (concentration 98.00%) standard into a 2 mL cryovial, add 1 mL of methanol and 200 μL of pure water, dissolve and mix to obtain a mother liquor with a concentration of 982 μg / mL.

[0071] (2) Take the stock solutions of the above 6 immunosuppressants to be tested, and prepare standard intermediate solutions using a diluent of methanol:water = 7:3. Mix the standard intermediate solutions and dilute them again using a diluent (methanol:water = 7:3) to obtain standard working solutions with 7 levels of concentration, as shown in Table 1:

[0072] Table 1

[0073]

[0074] Example 2

[0075] This embodiment provides a method for preparing an internal standard working solution.

[0076] (1) Preparation of internal standard stock solution

[0077] 6-Methylmercaptopurine-d3 (concentration 98.00%) standard specification is 2.50 mg. Add 1.98 mL of methanol:water = 1:1 solution and 20 μL of 1 mol / L NaOH, dissolve and mix to obtain a stock solution with a concentration of 1225 μg / mL;

[0078] The standard of azathioprine-d3 (concentration 97.00%) is 1.00 mg. Add 1 mL of methanol:water = 1:1 solution, dissolve and mix to obtain a stock solution with a concentration of 970 μg / mL.

[0079] 1.00 mg of 6-mercaptopurine-13C,15N2 (concentration 96.00%) standard was added to 0.894 mL of methanol:water = 1:1 solution and 10 μL of 1 mol / L NaOH, and the solution was dissolved and mixed to obtain a mother liquor with a concentration of 860 μg / mL.

[0080] (2) Mix the mother liquors and then dilute them with a diluent of methanol:water = 7:3 to obtain the internal standard working solution, as shown in Table 2:

[0081] Table 2

[0082] Internal Standard Internal standard working solution concentration (μg / mL) 6-Methylmercaptopurine-d3 22.5 Azathioprine-D3 3.0 6-Mercaptopurine-13C,15N2 3.0

[0083] Example 3

[0084] This embodiment provides a sample processing method and a detection method.

[0085] (1) Preparation of standard solutions

[0086] a. The internal standard working solution and the standard working solution should be left at room temperature for 30 minutes to equilibrate to room temperature;

[0087] b. Take 8 1.5mL centrifuge tubes and label them L7-L1 and blank respectively. Use a micropipette (range: 0.5-10μL) to add 5μL of internal standard working solution to centrifuge tubes labeled L7-L1. Add 5μL of methanol:water = 7:3 diluent to the blank centrifuge tube.

[0088] c. Using a micropipette (range: 0.5-10μL), pipette 5μL of each standard working solution into the corresponding numbered centrifuge tube. Add 5μL of diluent (methanol:water = 7:3) to the blank centrifuge tube. Accurately pipette 45μL of pure water and 300μL of methanol into each of the above centrifuge tubes and mix at 2000r / min for 1min.

[0089] d. After mixing, take 50 μL of the supernatant into a 1.5 mL plastic centrifuge tube, add 100 μL of pure water, and vortex at 2000 r / min for 1 min.

[0090] e. Take a micropipette (range: 10-100μL), aspirate 100μL of the mixture into a 96-well plate, and wait for injection.

[0091] (2) Sample pretreatment

[0092] a. Sample collection

[0093] Sample type: Various plasma types are acceptable;

[0094] Container and additive types: Various containers are acceptable;

[0095] Collection and processing: Venous blood was collected, centrifuged at 3500 r / min for 10 min, and plasma was promptly separated as the blood sample to be processed;

[0096] Storage method: Keep away from light and refrigerate;

[0097] b. Sample processing

[0098] Transfer 5 μL of internal standard working solution to a 1.5 mL plastic centrifuge tube using a pipette, add 50 μL of the blood sample to be processed and 300 μL of methanol, vortex at 2000 rpm for 5 min, then centrifuge at 14000 rpm for 10 min. Take 50 μL of the supernatant and transfer it to a 1.5 mL plastic centrifuge tube, add 100 μL of pure water and mix, vortex at 2000 rpm for 1 min, and use this as the sample to be injected.

[0099] (3) Detection of standard solutions and test samples

[0100] The standard solutions were detected using high performance liquid chromatography-mass spectrometry, and standard curves for six immunosuppressants were established.

[0101] When establishing a standard curve, the ratio of the peak area of ​​the target analyte to the peak area of ​​its corresponding internal standard is used as Y, and the ratio of the concentration of the target analyte to the concentration of its corresponding internal standard is used as X.

[0102] The test samples were analyzed using high performance liquid chromatography-mass spectrometry, and the concentrations of six immunosuppressants in the test samples were determined using the established standard curve.

[0103] The detection parameters are:

[0104] A. The detection instrument was an AB SCIEX Jasper HPLC MS TRIPLE QUAD 4500MD;

[0105] The chromatographic column used for the chromatographic analysis was a Waters Cortecs T3, T3 3.0×100mm, 2.7μm;

[0106] Mobile phase: Phase A is a 10 mmol / L ammonium formate aqueous solution containing 0.5% formic acid, and Phase B is methanol;

[0107] The analytical column was used with gradient elution at a flow rate of 0.4 mL / min, a column temperature of 40℃, and an injection volume of 1 μL. The analysis time was 5.0 min. The gradient elution conditions are shown in Table 3.

[0108] Table 3

[0109]

[0110] For mass spectrometry conditions, an AB SCIEX 4500MD detector, electrospray ionization (ESI) source, positive ion mode, and multiple reaction monitoring (MRM) were used. Specific parameters are shown in Table 4, and ion pair parameters are shown in Table 5.

[0111] Table 4

[0112]

[0113] Table 5

[0114]

[0115]

[0116] Chromatograms of azathioprine, 6-mercaptopurine, 6-methylmercaptopurine, 6-thioguanine, 6-thioguanine nucleoside, and imidazoribine in the standard solution are shown below. Figure 1 Chromatograms of azathioprine, 6-mercaptopurine, 6-methylmercaptopurine, 6-thioguanine, 6-thioguanine nucleoside, and imidazoribine in plasma samples are shown below. Figure 2 ,exist Figure 1 and Figure 2 In the image, from left to right, are the chromatographic peaks of imidazolidin, 6-thioguanine, 6-mercaptopurine, 6-thioguanine nucleoside, azathiopurine, and 6-methylmercaptopurine.

[0117] As shown in the figure, the sample pretreatment method provided by this invention, combined with liquid chromatography-mass spectrometry detection conditions, can accurately separate and detect the content of six immunosuppressants. The retention times of each immunosuppressant are shown in Table 6 below.

[0118] Table 6

[0119] Immunosuppressants Retention time / min azathioprine 3.05 6-Mercaptopurine 1.98 6-Methylmercaptopurine 3.23 6-Thioguanine 1.76 6-Thioguanine nucleoside 2.31 Imidazolidin 1.27 6-Methylmercaptopurine-d3 3.23 Azathioprine-D3 3.05 6-Mercaptopurine-13C,15N2 1.98

[0120] Example 4

[0121] This embodiment provides a detection method.

[0122] The difference from Example 3 is that, in this example, the chromatographic column used for chromatographic analysis is a Phenomenex Kinetex, and the obtained chromatogram is shown in [reference needed]. Figure 3 .

[0123] Example 5

[0124] This embodiment provides a detection method.

[0125] The difference from Example 3 is that in this example, phase A is a 1 mmol / L ammonium formate aqueous solution containing 3% formic acid, phase B is acetonitrile, the column temperature is 35°C, and the gradient elution conditions are shown in Table 7.

[0126] Table 7

[0127]

[0128] For mass spectrometry conditions, an AB SCIEX 4500MD detector, electrospray ionization (ESI) source, positive ion mode, and multiple reaction monitoring (MRM) were used. Specific parameters are shown in Table 8.

[0129] Table 8

[0130]

[0131]

[0132] Example 6

[0133] This embodiment provides a detection method.

[0134] The difference from Example 3 is that, in this example, the gradient elution conditions are shown in Table 9:

[0135] Table 9

[0136]

[0137] Performance Analysis 1

[0138] The analytical methods provided in Examples 3-6 of this invention were subjected to analyses of linearity, recovery rate, precision, matrix effect, etc., as follows:

[0139] (1) Linear Analysis

[0140] The linearity of the six immunosuppressants obtained in Example 3 was demonstrated using standard curves, and the results are shown in Table 10:

[0141] Table 10

[0142]

[0143] As shown in Table 10, the analytical method provided by this invention has a wide detection range and good linear correlation. The linear range of Examples 4-6 is similar to that of Example 3. The specific detection limit and quantitation limit results are shown in Table 11.

[0144] Table 11

[0145]

[0146] As shown in Tables 10-11, the detection limit and quantitation limit of the detection method provided by the present invention meet the requirements.

[0147] (2) Recovery rate and precision

[0148] Standard working solutions of azathioprine, 6-mercaptopurine, 6-methylmercaptopurine, 6-thioguanine, 6-thioguanine nucleoside, and imidazoribine were prepared into low, medium, and high concentrations for recovery and precision experiments. The methods provided in Examples 3-6 were followed, and five batches were analyzed repeatedly. The recovery rates and precision are shown in Tables 12-13 and 14-15, respectively.

[0149] Table 12: Recovery Rate

[0150]

[0151] Table 13: Recovery Rate

[0152]

[0153] Table 14: Precision

[0154]

[0155]

[0156] Table 15: Precision

[0157]

[0158] As shown in Tables 12-15, the average recovery rate for the three spiking levels (low, medium, and high) was 97.10%-103.25%, and the precision was 0.56%-5.25%. The analytical method provided by this invention meets the requirements for recovery rate and precision, has good reproducibility, high spiking recovery rate, and improves the accuracy of detection results.

[0159] Based on the above verification experiments, the recovery rate, precision, and other technical indicators of this embodiment all meet the requirements, exhibiting good reproducibility and high recovery rate, thus improving the accuracy of the detection results. Adding an internal standard to the plasma sample, precipitating proteins, and then diluting the supernatant before direct injection simplifies the detection process, making it faster and more efficient, and more suitable for high-throughput sample detection.

[0160] (3) Matrix effect

[0161] Different matrix ratios (90% matrix, 80% matrix, 50% matrix, 20% matrix, and 0 matrix) were added to the same high-concentration (concentration ≥ 10 × concentration in the matrix sample) standard solution. Five different matrix ratios were examined for each matrix sample. The study of Example 3 revealed:

[0162] 1. The RSD of the target area / internal standard area for the same matrix ratio in different matrices is ≤10%;

[0163] 2. The bias of the target area / internal standard area ratio for different matrix proportions with the same matrix is ​​less than 10%, which meets the requirements. (X matrix - 0 matrix) / 0 matrix

[0164] Example 7

[0165] This embodiment provides a sample processing method and a detection method.

[0166] The difference from Example 3 is that in step (2)-b of this example, the protein precipitant in the pretreatment of the sample to be tested is acetonitrile, which replaces the protein precipitant used in the standard solution (step (1)-c).

[0167] Examples 8-9

[0168] This embodiment provides a sample processing method and a detection method.

[0169] The difference from Example 3 is that in step (2)-b of this example, the dilution factor of the supernatant and pure water is 1 (Example 8) and 5 (Example 9), respectively, and the dilution factor in the standard solution is changed accordingly.

[0170] Comparative Example 1

[0171] This comparative example provides a sample processing method and a detection method.

[0172] The difference from Example 3 is that in step (2)-b of this comparative example, the protein precipitant in the pretreatment of the sample to be tested is perchloric acid, which replaces the protein precipitant used in the standard solution (step (1)-c).

[0173] Comparative Example 2

[0174] This comparative example provides a sample processing method and a detection method.

[0175] The difference from Example 3 is that in this comparative example, step (2)-b does not include the dilution step, but is as follows: pipette 5 μL of internal standard working solution into a 1.5 mL plastic centrifuge tube, add 50 μL of blood sample to be processed and 300 μL of methanol, vortex at 2000 r / min for 5 min, then centrifuge at 14000 r / min for 10 min, and take the supernatant as the sample to be injected. Correspondingly, the standard solution dilution step is omitted (step (1)-d is omitted).

[0176] Comparative Example 3

[0177] This comparative example provides a sample processing method and a detection method.

[0178] The difference from Example 3 is that in step (2)-b of this comparative example, the dilution factor of the supernatant and pure water is 7 times, and the dilution factor in the standard solution is changed accordingly.

[0179] Performance Analysis 2

[0180] The sample pretreatment methods provided in Examples 3, 7-9, and Comparative Examples 1-3 were subjected to chromatographic analysis using the detection method provided in Example 3. The analytical results are as follows:

[0181] (1) The chromatograms of the standard solutions provided in Examples 7-9 are similar to those provided in Examples 3 and 4. Therefore, it can be seen that the detection conditions provided by the present invention can meet the detection requirements.

[0182] (2) Comparative Example 1 had a low extraction rate for each target analyte, and the subsequent liquid chromatography test signal was low. As can be seen from the comparison between Examples 3 and 7 and Comparative Example 1, the extraction of each substance by acid precipitation of protein was poor and the signal was low, which could not meet the quantitative requirements.

[0183] (3) As can be seen from the comparison between Example 3 and Comparative Example 2, when the pretreatment is performed using the method of Comparative Example 2, the solvent effect of imidazoribine is strong and the peak shape is poor; and the matrix effect of imidazoribine, 6-thioguanine and 6-thioguanine nucleosides is strong, and the accuracy is unqualified.

[0184] (4) As can be seen from the comparison between Example 3 and Comparative Example 3, when the method of Comparative Example 3 is used for pretreatment, the signal of imidazoribine is low and the detection limit is high, which does not meet the quantitative requirements; and the matrix effect of imidazoribine and 6-thioguanine nucleoside is changed, which is inconsistent with the matrix effect of the internal standard, resulting in unqualified accuracy.

[0185] Comparative Example 4

[0186] This comparative example provides a detection method.

[0187] The difference from Example 3 is that, in this comparative example, the gradient elution conditions for chromatographic analysis are shown in Table 16:

[0188] Table 16

[0189]

[0190]

[0191] Comparative Example 5

[0192] This comparative example provides a detection method.

[0193] The difference from Example 3 is that, in this comparative example, the gradient elution conditions for chromatographic analysis are shown in Table 17:

[0194] Table 17

[0195]

[0196] Performance Analysis 3

[0197] The sample pretreatment methods provided in Example 3 and Comparative Examples 4-5 were subjected to chromatographic analysis using the detection method provided in Example 3. The analytical results are as follows:

[0198] (1) The chromatogram of the standard solution described in Comparative Example 4 is shown in [reference needed]. Figure 4 The chromatogram of the standard solution described in Comparative Example 5 is shown in Figure 5. Figure 5 As shown in the figure, the detection methods provided in Comparative Examples 4-5 cannot effectively separate certain substances. In Comparative Example 4, except for imidazoribine, the other five substances clustered together as peaks; in Comparative Example 5, azathioprine and 6-methylmercaptopurine could not be separated, resulting in ion crosstalk (see Figure 5). Figure 5 The accuracy and precision are poor.

[0199] (2) The recovery rate and precision of the detection methods provided in Example 3 and Comparative Examples 4-5 were evaluated, and the results are shown in Tables 18-19:

[0200] Table 18: Recovery Rate

[0201]

[0202] Table 19: Precision

[0203]

[0204] As can be seen from the performance tests of the examples and comparative examples, using the gradient elution conditions provided by the present invention in combination with other conditions can achieve good chromatographic separation and high accuracy and precision.

[0205] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0206] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneously detecting the concentrations of six immunosuppressants in blood, characterized in that, The method includes: (1) Prepare at least three different concentrations of standard solutions containing six immunosuppressants and their internal standards, and detect the standard solutions using liquid chromatography-mass spectrometry. Establish standard curves for the six immunosuppressants based on the detection results. The six immunosuppressants are azathioprine, 6-mercaptopurine, 6-methylmercaptopurine, 6-thioguanine, 6-thioguanine nucleoside, and imidazolidin. (2) Mix the blood sample to be processed, the internal standard working solution and the protein precipitant, and take the supernatant for dilution to obtain the sample to be injected; (3) The sample to be injected was detected by liquid chromatography-mass spectrometry, and the content of the six immunosuppressants in the blood sample to be processed was determined according to the detection results and the standard curve; The conditions for the liquid chromatography include: Mobile phase: Phase A is an aqueous solution containing 0.2-3% formic acid, 1-10 mmol / L ammonium formate or ammonium acetate, and Phase B is methanol or acetonitrile; Gradient elution: 0.00 min: Phase A 85-98%, Phase B 15-2%; 0.50 min: Phase A 85-98%, Phase B 15-2%; 0.60 min: Phase A 50-75%, Phase B 50-25%; 1.20 min: Phase A 50-75%, Phase B 50-25%; 1.50 min: Phase A 25-45%, Phase B 75-55%; 2.00 min: Phase A 25-45%, Phase B 75-55%; 2.50 min: Phase A 2-20%, Phase B 98-80%; 3.00 min: Phase A 2-20%, Phase B 98-80%; 3.10 min: Phase A 85-98%, Phase B 15-2%; 5.00 min: Phase A 85-98%, Phase B 15-2%; The analytical columns used were Agilent Poroshell 120 EC-C18, Waters Cortecs T3, or Shimadzu Shim-pack Veiox C18.

2. The method according to claim 1, characterized in that, In step (2), the protein precipitant is selected from methanol and / or acetonitrile; And / or, the volume ratio of the blood sample to be treated to the protein precipitant is 1:(5-10); And / or, the volume ratio of the blood sample to be processed to the internal standard working solution is (5-10):1; And / or, the solvent used for dilution is pure water.

3. The method according to claim 2, characterized in that, The volume ratio of the clarified liquid to pure water is 1:(1-5).

4. The method according to claim 1 or 2, characterized in that, The column temperature is 35-40℃; And / or, the flow rate of the mobile phase is 0.3-0.5 mL / min.

5. The method according to claim 1 or 2, characterized in that, The conditions for the mass spectrometry include: ion source temperature: 450-500℃; ion source high voltage: 4500-5000V; curtain gas: 12-20 L / min; collision gas: 7-10 L / min; drying gas 1: 30-80 L / min; drying gas 2: 30-80 L / min.

6. The method according to claim 1 or 2, characterized in that, The internal standards for the six immunosuppressants were azathioprine-d3, 6-methylmercaptopurine-d3, and 6-mercaptopurine-13C,15N2, respectively.

7. The method according to claim 1 or 2, characterized in that, The method for preparing the standard solution includes: preparing standard working solutions of at least three concentration levels; mixing the standard working solution, internal standard working solution, and protein precipitant; and diluting the supernatant to obtain the standard solution.

8. The method according to claim 7, characterized in that, The standard working solutions of each concentration level were obtained by diluting the standard stock solution with a diluent. The standard stock solution was obtained by dissolving the respective standards of the six immunosuppressants in a solvent. The internal standard working solution is obtained by diluting the internal standard stock solution with a diluent. The internal standard stock solution is obtained by dissolving the internal standard standards of the six immunosuppressants respectively with a solvent.

9. The method according to claim 8, characterized in that, The diluent is an aqueous methanol solution with a concentration of 60-80%.

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