Simultaneous determination of six immunosuppressants by liquid chromatography-mass spectrometry

Abstract

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

Description

Technical Field

[0001] This disclosure relates to the field of blood drug detection technology, and in particular to a liquid chromatography-mass spectrometry method for simultaneously detecting the concentrations of six immunosuppressants. 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 issues, this disclosure provides a liquid chromatography-mass spectrometry method for simultaneously detecting the concentrations of six immunosuppressants.

[0004] In a first aspect, this disclosure provides a liquid chromatography-mass spectrometry (LC-MS) method for simultaneously detecting the concentrations of six immunosuppressants, the LC-MS method comprising:

[0005] (1) Prepare at least three different concentrations of standard solutions containing six immunosuppressants, 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) The sample to be tested was detected by liquid chromatography-mass spectrometry, and the content of the six immunosuppressants in the sample to be tested was determined based on the detection results and the standard curve.

[0008] The detection method disclosed herein can measure the content of six immunosuppressants in a single injection. The detection time is short, with results available within 5 minutes. This improves detection efficiency while reducing operational difficulty, making it suitable for high-throughput sample detection.

[0009] As a preferred embodiment of this disclosure, the conditions for liquid chromatography include:

[0010] 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;

[0011] 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.

[0012] Gradient elution:

[0013] 0.00min: Phase A 85-98%, Phase B 15-2%;

[0014] 0.50 min: Phase A 85-98%, Phase B 15-2%;

[0015] 0.60 min: Phase A 50-75%, Phase B 50-25%;

[0016] 1.20 min: Phase A 50-75%, Phase B 50-25%;

[0017] 1.50 min: Phase A 25-45%, Phase B 75-55%;

[0018] 2.00 min: Phase A 25-45%, Phase B 75-55%;

[0019] 2.50 min: Phase A 2-20%, Phase B 98-80%;

[0020] 3.00 min: Phase A 2-20%, Phase B 98-80%;

[0021] 3.10 min: Phase A 85-98%, Phase B 15-2%;

[0022] 5.00 min: Phase A 85-98%, Phase B 15-2%.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] As a preferred technical solution of this disclosure, the conditions for liquid chromatography include: column temperature of 35-40℃, such as 36℃, 37℃, 38℃, 39℃, etc.

[0029] As a preferred embodiment of this disclosure, the flow rate of the mobile phase is 0.3-0.5 mL / min, such as 0.35 mL / min, 0.4 mL / min, 0.45 mL / min, etc.

[0030] As a preferred technical solution of this disclosure, the analytical chromatographic column used in the liquid chromatography is Agilent Poroshell 120EC-C18, phenomenex Kinetex, Waters Cortecs T3, or Shimadzu Shim-pack Veiox C18.

[0031] As a preferred embodiment of this disclosure, 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.

[0032] The detection method provided in this disclosure uses imidazoribine in positive ion mode, and the positive ion signal is relatively good and high, which can meet 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 specified in this disclosure can obtain good chromatographic peak shape and chromatographic separation, avoid ion crosstalk, and effectively reduce matrix effects.

[0033] As a preferred embodiment of this disclosure, the standard solution further contains internal standards of six immunosuppressants.

[0034] As a preferred technical solution of this disclosure, the sample to be tested contains internal standards of six immunosuppressants.

[0035] As a preferred embodiment of this disclosure, the concentrations of the internal standards of the six immunosuppressants in the test sample are the same as those of the internal standards of the six immunosuppressants in the standard solution.

[0036] As a preferred embodiment of this disclosure, the internal standards of the six immunosuppressants are azathioprine-d3, 6-methylmercaptopurine-d3, and 6-mercaptopurine-13C,15N2.

[0037] As a preferred embodiment of this disclosure, 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.

[0038] The sample pretreatment conditions specified in this disclosure enable sample testing to be completed using only three internal standards, saving internal standard costs and simplifying pretreatment.

[0039] As a preferred embodiment of this disclosure, 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.

[0040] 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.

[0041] As a preferred technical solution of this disclosure, 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.

[0042] 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.

[0043] As a preferred embodiment of this disclosure, the diluent is an aqueous methanol solution with a concentration of 60-80%, wherein the 60-80% can be 65%, 70%, 75%, 80%, etc.

[0044] The technical solution provided in this disclosure has the following advantages compared with the prior art:

[0045] (1) The detection method provided in this disclosure can determine 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.

[0046] (2) The detection method provided in this disclosure 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 without adding matrix pretreatment to the standard curve. Attached Figure Description

[0047] The accompanying drawings, which are incorporated in and form a part of this specification, illustrate embodiments consistent with this disclosure and, together with the description, serve to explain the principles of this disclosure.

[0048] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the accompanying 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.

[0049] Figure 1 This is a chromatogram of the standard solution described in Example 3 of this disclosure;

[0050] Figure 2 This is the chromatogram of the sample to be injected as described in Embodiment 3 of this disclosure;

[0051] Figure 3 This is a chromatogram of the standard solution described in Example 4 of this disclosure;

[0052] Figure 4 The chromatogram of the standard solution described in Comparative Example 1 of this disclosure;

[0053] Figure 5 This is a chromatogram of the standard solution described in Comparative Example 2 of this disclosure. Detailed Implementation

[0054] To better understand the above-mentioned objectives, features, and advantages of this disclosure, the solutions disclosed herein will be further described below. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other.

[0055] Numerous specific details are set forth in the following description in order to provide a full understanding of this disclosure, but this disclosure may also be implemented in other ways different from those described herein; obviously, the embodiments in the specification are only some, and not all, of the embodiments of this disclosure.

[0056] Example 1

[0057] This embodiment provides a method for preparing a standard working solution.

[0058] (1) Prepare stock solutions of 6 immunosuppressant standards

[0059] 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.

[0060] 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;

[0061] 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;

[0062] 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;

[0063] 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;

[0064] 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.

[0065] (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:

[0066] Table 1

[0067]

[0068] Example 2

[0069] This embodiment provides a method for preparing an internal standard working solution.

[0070] (1) Preparation of internal standard stock solution

[0071] 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;

[0072] 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.

[0073] 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.

[0074] (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:

[0075] Table 2

[0076] Internal Standard Internal standard working solution concentration (μg / mL) 6-Methylmercaptopurine-d3 22.5 Azathioprine-D3 3.0 6-Mercaptopurine-13C,15N2 3.0

[0077] Example 3

[0078] This embodiment provides a sample processing method and a detection method.

[0079] (1) Preparation of standard solutions

[0080] 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;

[0081] 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.

[0082] 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.

[0083] d. After mixing, take 50 μL of the supernatant into a 1.5 mL plastic centrifuge tube, add 150 μL of pure water, and vortex at 2000 r / min for 1 min.

[0084] e. Take a micropipette (range: 10-100μL), aspirate 100μL of the mixture into a 96-well plate, and wait for injection.

[0085] (2) Sample pretreatment

[0086] a. Sample collection

[0087] Sample type: Various plasma types are acceptable;

[0088] Container and additive types: Various containers are acceptable;

[0089] 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;

[0090] Storage method: Keep away from light and refrigerate;

[0091] b. Sample processing

[0092] 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 150 μL of pure water and mix, vortex at 2000 rpm for 1 min, and use this as the sample to be injected.

[0093] (3) Detection of standard solutions and test samples

[0094] The standard solutions were detected using high performance liquid chromatography-mass spectrometry, and standard curves for six immunosuppressants were established.

[0095] 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.

[0096] 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.

[0097] The detection parameters are:

[0098] A. The detection instrument is an AB SCIEX Jasper HPLC MS TRIPLE QUAD 4500MD.

[0099] The chromatographic column used for the chromatographic analysis was a Waters Cortecs T3, T3 3.0×100mm, 2.7μm;

[0100] Mobile phase: Phase A is a 10 mmol / L ammonium formate aqueous solution containing 0.5% formic acid, and Phase B is methanol;

[0101] 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.

[0102] Table 3

[0103]

[0104]

[0105] 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.

[0106] Table 4

[0107]

[0108] Table 5

[0109]

[0110] 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.

[0111] As shown in the figure, the sample pretreatment method provided in this disclosure, 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.

[0112] Table 6

[0113] 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

[0114] Example 4

[0115] This embodiment provides a detection method.

[0116] 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 .

[0117] Example 5

[0118] This embodiment provides a detection method.

[0119] 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.

[0120] Table 7

[0121]

[0122] 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.

[0123] Table 8

[0124]

[0125] Example 6

[0126] This embodiment provides a detection method.

[0127] The difference from Example 3 is that, in this example, the gradient elution conditions are shown in Table 9:

[0128] Table 9

[0129]

[0130] Performance Analysis 1

[0131] The analytical methods provided in Examples 3-6 of this disclosure are analyzed for linearity, recovery and precision, matrix effects, etc., as follows:

[0132] (1) Linear Analysis

[0133] The linearity of the six immunosuppressants obtained in Example 3 was demonstrated using standard curves, and the results are shown in Table 10:

[0134] Table 10

[0135]

[0136]

[0137] As shown in Table 10, the analytical method provided in this disclosure 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 limits of detection and limits of quantitation results are shown in Table 11.

[0138] Table 11

[0139]

[0140] As shown in Table 10-11, the detection limit and quantitation limit of the detection method provided in this disclosure meet the requirements.

[0141] (2) Recovery rate and precision

[0142] 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.

[0143] Table 12: Recovery Rate

[0144]

[0145]

[0146] Table 13: Recovery Rate

[0147]

[0148] Table 14: Precision

[0149]

[0150] Table 15: Precision

[0151]

[0152]

[0153] 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 in this disclosure meets the requirements for recovery rate and precision, has good reproducibility, and high recovery rate, thus improving the accuracy of the detection results.

[0154] 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.

[0155] (3) Matrix effect

[0156] 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:

[0157] 1. The RSD of the target area / internal standard area for the same matrix ratio in different matrices is ≤10%;

[0158] 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

[0159] Comparative Example 1

[0160] This comparative example provides a detection method.

[0161] The difference from Example 3 is that, in this comparative example, the gradient elution conditions for chromatographic analysis are shown in Table 16:

[0162] Table 16

[0163]

[0164]

[0165] Comparative Example 2

[0166] This comparative example provides a detection method.

[0167] The difference from Example 3 is that, in this comparative example, the gradient elution conditions for chromatographic analysis are shown in Table 17:

[0168] Table 17

[0169]

[0170] Performance Analysis 2

[0171] The sample pretreatment methods provided in Example 3 and Comparative Examples 1-2 were subjected to chromatographic analysis using the detection method provided in Example 3. The analytical results are as follows:

[0172] (1) The chromatogram of the standard solution described in Comparative Example 1 is shown below. Figure 4 The chromatogram of the standard solution described in Comparative Example 2 is shown below. Figure 5 As shown in the figure, the detection methods provided in Comparative Examples 1-2 cannot effectively separate certain substances. In Comparative Example 1, except for imidazoribine, the other five substances clustered together as peaks; in Comparative Example 2, azathioprine and 6-methylmercaptopurine could not be separated, resulting in ion crosstalk (see Figure 1). Figure 5 The accuracy and precision are poor.

[0173] (2) The recovery rate and precision of the detection methods provided in Example 3 and Comparative Examples 1-2 were evaluated, and the results are shown in Tables 18-19:

[0174] Table 18: Recovery Rate

[0175]

[0176] Table 19: Precision

[0177]

[0178] As can be seen from the performance tests of the examples and comparative examples, using the gradient elution conditions provided in this disclosure in combination with other conditions can achieve good chromatographic separation and high accuracy and precision.

[0179] 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.

[0180] The above description is merely a specific embodiment of this disclosure, enabling those skilled in the art to understand or implement it. 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 this disclosure. Therefore, this disclosure 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 liquid chromatography-mass spectrometry method for simultaneously detecting the concentrations of six immunosuppressants, characterized in that, The liquid chromatography-mass spectrometry analysis method includes: (1) Prepare at least three different concentrations of standard solutions containing six immunosuppressants, and detect the standard solutions using liquid chromatography-mass spectrometry. Based on the detection results, establish standard curves for the six immunosuppressants respectively. The six immunosuppressants are azathioprine, 6-mercaptopurine, 6-methylmercaptopurine, 6-thioguanine, 6-thioguanine nucleoside, and imidazolidin. (2) The sample to be tested was detected by liquid chromatography-mass spectrometry, and the content of the six immunosuppressants in the sample to be tested was determined according to the detection results and the standard curve. The sample to be tested was plasma; 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 in the liquid chromatography were Agilent Poroshell 120 EC-C18, Waters Cortecs T3, or Shimadzu Shim-pack Veiox C18.

2. The liquid chromatography-mass spectrometry analysis method according to claim 1, characterized in that, The conditions for the liquid chromatography include: column temperature of 35-40℃; And / or, the flow rate of the mobile phase is 0.3-0.5 mL / min.

3. The liquid chromatography-mass spectrometry analysis 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.

4. The liquid chromatography-mass spectrometry analysis method according to claim 1 or 2, characterized in that, The standard solution also contains internal standards for six immunosuppressants.

5. The liquid chromatography-mass spectrometry analysis method according to claim 1 or 2, characterized in that, The sample to be tested contained internal standards for six immunosuppressants.

6. The liquid chromatography-mass spectrometry analysis method according to claim 5, characterized in that, The concentrations of the internal standards for the six immunosuppressants in the test sample were the same as those in the standard solution.

7. The liquid chromatography-mass spectrometry analysis method according to claim 4, characterized in that, The internal standards for the six immunosuppressants were azathioprine-d3, 6-methylmercaptopurine-d3, and 6-mercaptopurine-13C,15N2, respectively.

8. The liquid chromatography-mass spectrometry analysis method according to claim 7, characterized in that, 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; the internal standard corresponding to 6-mercaptopurine is 6-mercaptopurine-13C,15N2.

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