Detection Kit for Detecting Immunosuppressors in Whole Blood by High Performance Liquid Chromatography-Tandem Mass Spectrometry and Detection Method Thereof

Abstract

A detection kit for detecting an immunosuppressor in whole blood by high performance liquid chromatography-tandem mass spectrometry and a detection method thereof is provided. An internal standard solution is added with an antioxidant, vitamin E, and mixed with an internal standard diluent containing zinc sulfate heptahydrate, purified water and methanol for sample pretreatment, which not only exerts the function of the internal standard, but also synchronously achieves erythrocyte treatment, protein precipitation and target substance extraction. Various embodiments enable the immunosuppressor to be more stable in a solution matrix, thus promoting the detection accuracy and sensitivity. Various embodiments adopt isotopically-labeled sirolimus as an internal standard of everolimus to substitute isotopically-labeled everolimus, thus overcoming the interference of everolimus on isotopically-labeled everolimus and satisfying the detection requirements. Various embodiments detect four immunosuppressors simultaneously to reduce the cost of the internal standard, and has a lower detection cost, more accurate and stable detection results.

Claims

1. A detection kit for detecting an immunosuppressor in whole blood by high performance liquid chromatography-tandem mass spectrometry, comprising an internal standard solution, wherein the internal standard solution comprises an additive, and the additive is one or more of 2,6-di-tert-butyl-p-cresol, vitamin E, vitamin C, β-carotene, and sodium metabisulfite.

2. The detection kit of claim 1, wherein the additive is vitamin E.

3. The detection kit of claim 2, wherein the vitamin E has a concentration of 0.5-1.5 mg/m L.

4. The detection kit of claim 3, wherein the vitamin E has a concentration of 1.0 mg/m L.

5. The detection kit of claim 4, wherein the immunosuppressor is one or more of cyclosporine A, tacrolimus, sirolimus and everolimus.

6. The detection kit of claim 5, wherein the internal standard solution further comprises an immunosuppressor internal standard and acetonitrile, and the acetonitrile is a solvent of the internal standard solution.

7. The detection kit of claim 6, wherein the immunosuppressor is an isotopically-labeled immunosuppressor, wherein an internal standard of cyclosporine A is isotopically-labeled cyclosporine A, an internal standard of tacrolimus is isotopically-labeled tacrolimus, and internal standards of sirolimus and everolimus are isotopically-labeled sirolimus.

8. The detection kit of claim 1, wherein the detection kit further comprises an internal standard diluent, and the internal standard diluent comprises zinc sulfate heptahydrate, purified water and methanol.

9. The detection kit of claim 8, wherein in the internal standard diluent, a volume ratio of purified water to methanol is 3:7, and zinc sulfate heptahydrate has a content of 60 mM.

10. The detection kit of claim 9, wherein a volume ratio of the internal standard solution and the internal standard diluent is 1:24.

11. The detection kit of claim 10, characterized by further comprising a system suitability solution, wherein the system suitability solution comprises one or more of cyclosporine A, tacrolimus, sirolimus, and everolimus, as well as one or more of isotopically-labeled cyclosporine A, tacrolimus and sirolimus, and further comprises a solvent and an additive; the solvent is additive-containing acetonitrile; and the additive is any one or more of 2,6-di-tert-butyl-p-cresol, vitamin E, vitamin C, β-carotene, and sodium metabisulfite.

12. The detection kit of claim 11, wherein the additive of the system suitability solution is vitamin E having a concentration of 0.5-1.5 mg/mL.

13. The detection kit of claim 12, wherein the detection kit further comprises a standard sample and a quality control sample; the standard sample and the quality control sample are prepared by using a sterile bovine blood containing a matrix additive as a matrix; the matrix additive comprises vitamin E; the standard sample is a sample comprising any one of more of cyclosporine A, tacrolimus, sirolimus and everolimus at a standard concentration; and the quality control sample is a sample comprising three different levels (low, medium and high) of concentrations.

14. The detection kit of claim 13, wherein the vitamin E in the matrix addictive has a concentration of 0.5-1.5 mg/mL.

15. The detection kit of claim 14, wherein the detection kit further comprises a mobile phase of liquid chromatography (LC); the mobile phase of LC comprises a mobile phase A and a mobile phase B; the mobile phase A is a 2 mM aqueous solution of ammonium acetate-0.1% formic acid; and the mobile phase B is 2 mM methanol solution of ammonium acetate-0.1% formic acid.

16. A method for detecting an immunosuppressor in whole blood, comprising the steps: providing a detection kit of claim 1, performing a system suitability test, preparing a sample, pre-treating the sample and detecting the sample; wherein the sample pretreatment comprises the following steps: taking the internal standard solution and internal standard diluent from the detection kit for mixing according to a ratio of 1:24 to obtain an internal standard working solution; taking a sample and the internal standard working solution for mixing evenly according to a ratio of 1:3-1:7, and performing centrifugation, then taking supernatant for detection by high performance liquid chromatography-tandem mass spectrometry.

17. The method of claim 16, wherein during the sample pretreatment, a volume ratio of the sample to the internal standard solution is 1:5.

18. The method of claim 17, wherein during the sample detection, isotopically-labeled sirolimus serves as an internal standard of everolimus, thus detecting a content of everolimus.

19. The method of claim 18, wherein gradient elution is used in the detection by high performance liquid chromatography-tandem mass spectrometry, and the gradient elution time is 2 min, and the gradient elution procedure is as follows: TABLE-US-00015 Mobile Mobile Flowrate Time (min) phase A % phase B % (ml/min) 0.00 80 20 0.5 0.2 80 20 0.5 1 2 98 0.5 1.6 2 98 0.5 2.0 80 20 0.5

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIG. 1A-FIG. 1D are a chromatogram of a standard curve S1 in Example 1.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0061] The present invention will be further described in detail with reference to the drawings and examples. It should be indicated that the examples below aim at facilitating the understanding of the present invention, but not limiting the present invention.

Example 1 Sample Preparation, Pretreatment and Detection

[0062] I. Sample Preparation

[0063] 1. Preparation of a Standard Curve and a Quality Control Sample

[0064] Standard substances of immunosuppressors cyclosporine A, tacrolimus, sirolimus and everolimus were prepared into a mixed solution as a stock solution of a standard working solution and a quality control working solution; sterile bovine blood containing a matrix additive of 1.0 mg/mL vitamin E was used as a matrix for preparation, thus preparing a standard curve and a quality control sample.

[0065] 4 immunosuppressors had 10 series concentrations (S1-S10) in the standard substance, as shown in Table 1:

TABLE-US-00004 TABLE 1 10 series concentrations (S1-S10) of the 4 immunosuppressors in the standard substance ng/ml Cyclosporine A Tacrolimus Sirolimus Everolimus S1 10 1 1 1 S2 15 1.5 1.5 1.5 S3 25 2.5 2.5 2.5 S4 50 5 5 5 S5 75 7.5 7.5 7.5 S6 100 10 10 10 S7 250 25 25 25 S8 500 50 50 50 S9 750 75 75 75 S10 1000 100 100 100

[0066] 4 immunosuppressors had 3 series concentrations at low (L), medium (M) and high (H) levels in the quality control substance, as shown in Table 2:

TABLE-US-00005 TABLE 2 Three series concentrations of the 4 immunosuppressors in the quality control substance ng/ml Cyclosporine A Tacrolimu Sirolimus Everolimus L 30 3 3 3 M 300 30 30 30 H 800 80 80 80

[0067] 2. Preparation of an Internal Standard Solution and an Internal Standard Diluent

[0068] (1) Preparation of an Internal Standard Solution

[0069] A mixed internal standard working solution was prepared, and concentrations of cyclosporine A-d4, tacrolimus-13C-d4, and sirolimus-d3 were respectively 1 μg/mL, 50 ng/mL and 50 ng/mL; and an additive 1.0 mg/mL vitamin E was added, acetonitrile served as a solvent, and mixed into an internal standard solution.

[0070] (2) Preparation of an Internal Standard Diluent

[0071] 17.8 g zinc sulfate heptahydrate, 300 ml purified water and 700 ml methanol were respectively weighed and prepared into an internal standard diluent.

[0072] 3. Preparation of a System Suitability Solution

[0073] 20 μL of the mixed internal standard solution containing 100 μg/mL cyclosporine A-d4, 5 μg/mL tacrolimus-13C-d4 and 5 μg/mL sirolimus-d3, 100 μL of the mixed standard solution containing 8 μg/mL cyclosporine A, 0.8 μg/mL tacrolimus, 0.8 μg/mL sirolimus and 0.8 μg/mL everolimus, and 59.88 mL of acetonitrile solution containing 1.0 mg/mL VE were respectively taken and mixed into a system suitability solution.

[0074] II. Sample Pretreatment

[0075] (1) The internal standard solution and internal standard diluent were taken and mixed evenly according to a ratio of 1:24 to obtain an internal standard working solution;

[0076] (2) 50 μL sample was taken and added to a 96-well plate or a centrifugal tube;

[0077] (3) 250 μL internal standard working solution was added for vortex mixing for 5 min, subjected to standing for 5 min at room temperature, then vortex mixed for 5 min, and centrifuged for 10 min at a revolving speed of 4000 rpm, then supernatant was taken for further detection.

[0078] III. Sample Detection

[0079] Supernatant was taken and detected by high performance liquid chromatography-tandem mass spectrometry; and a spectrogram of the standard curve at the lowest point was shown in FIG. 1. Specific analysis conditions were as follows: gradient elution was used to an instrumental method; and the tandem mass spectrum: electrospray ionization (ESI) ion source and positive ion Multiple Reaction Monitoring (MRM) Mode were taken. The chromatographic column was a C18 chromatographic column, the mobile phase had a flow rate of 0.5 mL/min and column temperature was 55° C., and the gradient elution procedure was as follows:

TABLE-US-00006 Mobile Mobile Time phase phase Flow rate (min) A % B % (ml/min) 0.00 80 20 0.5 0.2 80 20 0.5 1 2 98 0.5 1.6 2 98 0.5 2.0 80 20 0.5

[0080] The mass spectrometry conditions were as follows:

TABLE-US-00007 Multiple Electrospray Reaction ionization Monitoring ion source (MRM) Ionization mode (ESI+) Scan Mode Mode Curtain gas 20 L/min Temperature 400° C. (TEM) Collision gas  6 L/min

[0081] Four immunosuppressors for detection and a mass-to-charge ratio (m/z) of parent ion/daughter ion pair of the internal standards were shown in the table below:

TABLE-US-00008 Analyte/internal standard Q1 Q3 Tacrolimus 821.0 768.5 821.0 786.4 Cyclosporine A 602.1 100.2 602.1 156.2 Everolimus 975.3 908.5 975.3 926.5 Sirolimus 931.3 864.5 931.3 882.6 Tacrolimus-13C-d4 826.3 773.5 826.3 791.5 Cyclosporine-d4 604.1 100.3 604.1 156.1 Sirolimus-d3 934.5 864.6 934.5 882.5

[0082] The detection of the 4 immunosuppressors could be determined through the ion pair detected by selective reaction monitoring and the corresponding dwell time; and quantification could be performed through the internal standard of each immunosuppressor.

[0083] After the sample was separated by liquid chromatography, different immunosuppressor appeared peaks at different elution time, and were detected by mass spectrometry MRM, thus detecting the content. According to the series concentrations of the standard sample S1, sterile bovine blood containing an additive 1.0 mg/mL vitamin E served as a matrix to prepare a sample to be detected for detection, and the detection spectrogram was shown in FIG. 1. As shown in FIG. 1, the 4 immunosuppressors could be detected simultaneously and accurately according to the method provided by the example.

Example 2 Influence of Adding an Antioxidant on the Stability of the Immunosuppressors

[0084] In this example, according to the method provided in Example 1, different kinds of antioxidants were respectively used and prepared into an internal standard solution and a system suitability solution having the same concentration; then the solution was put for 14 d at 37° C. to survey the influences of different antioxidants on the service life of cyclosporine-d4, tacrolimus-13C-d4, sirolimus-d3 contained in the internal standard solution and the cyclosporine A, tacrolimus, sirolimus, and everolimus contained in the system suitability solution. Antioxidants included 2,6-di-tert-butyl-p-cresol, vitamin E, vitamin C, β-carotene, and sodium metabisulfite. The results were shown in Tables 1 and 2:

TABLE-US-00009 TABLE 1 Influences of the different antioxidants on the 14 d stability of cyclosporine A, tacrolimus, sirolimus, and everolimus contained in the system suitability solution at 37° C.: Peak area of system suitability solution Antioxidant Cyclosporine A Tacrolimus Sirolimus Everolimus Initial values 209326 74430 115492 111146 Free of adding 216288 7408 4216 2401 antioxidant 2,6-di-tert- 214654 27771 5499 3533 butyl-p-cresol Vitamin E 221593 69879 98087 98290 Vitamin C 210444 30470 5167 11782 β-carotene 198460 8659 8219 3599 Sodium 222696 8044 7075 6256 metabisulfite

TABLE-US-00010 TABLE 2 Influences of the different antioxidants on the 14 d stability of cyclosporine-d4, tacrolimus-13C-d4, sirolimus-d3, and everolimus-d4 contained in the internal standard solution at 37° C.: Peak area of internal standard solution Antioxidant CyclosporineA-d4 Tacrolimus-13C-d4 Sirolimus-d3 Initial values 140935 60178 71424 Free of adding 129898 6069 3768 antioxidant 2,6-di-tert- 136211 28579 7033 butyl-p-cresol Vitamin E 143337 53397 62889 Vitamin C 147298 25618 6664 P-carotene 147555 5494 3308 Sodium 139515 5764 5301 metabisulfite

[0085] It can be seen both in Tables 1 and 2 that in case of not adding an antioxidant, and after the solutions were put for 14 d at 37° C., the content of the immunosuppressors in the system suitability solution and isotopically-labeled immunosuppressors in the internal standard solution significantly decreased, especially, the content of tacrolimus, everolimus and sirolimus almost dropped dramatically, indicating that the 4 immunosuppressors or isotope internal standards thereof in the system suitability solution and the internal standard solution were rather unstable.

[0086] The addition of different antioxidants had different influences on the stability of the 4 immunosuppressors or isotope internal standards thereof in the system suitability solution and the internal standard solution. Experimental results showed that the addition of vitamin E can obviously improve the stability of the four immunosuppressors, after the solution was put for 14 d at 37° C., the 4 immunosuppressors or isotope internal standards thereof in the system suitability solution and the internal standard solution had a content change within 15%, and had better stability.

[0087] Through further experiments, the example further verified that after being put for 2 years at 2-8° C., the 4 immunosuppressors or isotope internal standards thereof in the system suitability solution and the internal standard solution added with vitamin E still kept a stable content.

Example 3 Selection for the Content of Vitamin E

[0088] In this example, according to the method provided in Example 1, standard solutions of cyclosporine A, tacrolimus, sirolimus, and everolimus were added to sterile bovine blood, and vitamin E at different concentrations was added, then the remaining solution was put for 14 d at 37° C. to survey the influences of the vitamin E at different concentrations on the service life of cyclosporine A, tacrolimus, sirolimus, and everolimus in whole blood. The results were shown in Table 3:

TABLE-US-00011 TABLE 3 Influences of the vitamin E content on the detection results of the sample after being put for 14 d at 37° C.: Concentration of Sample detection concentration (ng/mL) vitamin E Cyclosporine A Tacrolimus Sirolimus Everolimus Initial values 786.5 79.7 76.3 80.1 0.5 mg/mL 801.2 73.3 72.2 62.1 1.0 mg/mL 758.5 80.1 75.7 75.3 1.5 mg/mL 775.2 79.0 75.4 75.7

[0089] It can be seen from Table 3 that when the concentration of vitamin E was 0.5 mg/mL, after the solution was put for 14 d at 37° C., the concentration of everolimus decreased by 22.5%, not being up to the requirement; and when the concentration of vitamin E was 1.0 mg/mL and 1.5 mg/mL, cyclosporine A, tacrolimus, sirolimus, and everolimus had no obvious decline; therefore, vitamin E had a preferred concentration of 1.0 mg/mL.

Example 4 Interference of Everolimus on Everolimus Isotopes

[0090] 1. Detection Results of Everolimus Standards

[0091] In the example, according to the method provided by Example 1, a standard solution of everolimus was taken for detection by high performance liquid chromatography-tandem mass spectrometry; and the detection results were shown in Table 4.

Example 4 Interference of Everolimus on an Internal Standard of Everolimus-d4

[0092]

TABLE-US-00012 Test results of Peak area of Peak area of everolimus everolimus everolimus-d4 channel standards 1373689 15205

[0093] It can be seen from Table 4 that no everolimus-d4 was added to the everolimus standard solution, but a signal was found in the everolimus-d4 channel in the detection result, indicating that everolimus would disturb everolimus isotopes. Therefore, if everolimus isotopes were used as internal standards for detection, the detection result would be influenced due to the existing interference, such that the accuracy of everolimus would be seriously affected.

[0094] 2. Detection Results of Everolimus Standard Solutions at Different Concentrations

[0095] Clinical samples were taken and respectively added with everolimus standard solutions at different concentrations; and everolimus-d4 served as an internal standard to detect the recovery rate of the sample after adding the internal standard; 6 samples were set in parallel, and the results were shown in Table 5.

TABLE-US-00013 5. Detection results of everolimus standard solutions at different concentrations Adding amount of everolimus (ng/mL) Sample 4 16 50 1 3.95 14.23 39.72 2 3.48 13.37 39.84 3 3.82 13.90 40.45 4 3.87 14.04 38.60 5 3.98 13.61 37.68 6 3.97 13.59 38.69 Mean value 3.85 13.79 39.17 CV 4.9% 2.32% 2.59% Recovery rate 96.1% 86.18% 78.34%

[0096] It can be seen from Table 5 that in everolimus standards at different concentrations, when everolimus-d4 served as an internal standard for detection, with the increase of the everolimus concentration, the detected recovery rate of everolimus became lower and lower; and when the adding amount was up to 50 ng/mL, the recovery rate was lower than 85%, which completely could not satisfy the accuracy requirements. Therefore, the addition of everolimus-d4 as an internal standard would seriously influence the detection accuracy of the everolimus content.

[0097] Sirolimus and everolimus have a very similar structure. A large number of experiments (data omitted) proved that an isotope internal standard of sirolimus served as an internal standard of everolimus, which completely could satisfy the detection requirements of everolimus, and could reduce the cost of the internal standard, and further promote the accuracy and sensitivity of the detection result.

Example 5 Clinical Verification Experiment

[0098] Clinical samples were taken and respectively added with standard solutions of cyclosporine A, tacrolimus, sirolimus, and everolimus to prepare into recovery rate samples at high, medium, and low levels of concentrations for detection according to the method provided in Example 1; 6 samples were taken in parallel for pretreatment, then the recovery rate was calculated. Recovery rate results were shown in Table 6:

Table 6 Results of Clinical Verification Experiment

[0099]

TABLE-US-00014 Recovery rate sample Tacrolimus Cyclosporine Everolimus Sirolimus LQC Adding 4 40 4 4 amount (ng/mL) Sample 1 3.88 39.08 3.95 3.37 Sample 2 3.44 39.31 3.48 3.92 Sample 3 3.77 41.04 3.82 3.6 Sample 4 4.03 38.09 3.87 3.87 Sample 5 3.59 38.36 3.98 4.05 Sample 6 3.8 39.75 3.97 3.76 Mean 3.75 39.27 3.85 3.76 value (ng/mL) CV 5.6% 2.7% 4.9% 6.5% Recovery 93.8% 98.2% 96.1% 94.0% rate MQC Adding 16 160 16 16 amount (ng/mL) Sample 1 15.87 167.63 15.65 14.01 Sample 2 16.19 163.29 14.71 13.92 Sample 3 13.75 168.83 15.29 14.81 Sample 4 15.52 163.54 15.44 13.63 Sample 5 15.67 164.38 14.97 14.41 Sample 6 13.93 165.45 14.95 14.7 Mean 15.16 165.52 15.17 14.25 value (ng/mL) CV 6.9% 1.4% 2.3% 3.3% Recovery 94.7% 103.5% 94.8% 89.0% rate HQC Adding 50 500 50 50 amount (ng/mL) Sample 1 47.02 495.22 44.88 50.27 Sample 2 49.23 489.83 45.02 47.67 Sample 3 51.89 494.37 45.71 46.66 Sample 4 46.92 503.76 43.62 46.2 Sample 5 47.42 495.44 42.58 50.65 Sample 6 49.22 501.16 43.72 45.36 Mean 48.62 496.63 44.26 47.80 value (ng/mL) CV 3.9% 1.0% 2.6% 4.6% Recovery 97.2% 99.3% 88.5% 95.6% rate

[0100] To summarize the above data, we have surveyed the accuracy and recovery rate of the 4 immunosuppressors at three (high, medium, and low) quality control levels; and the results indicate that the accuracy of the method is less than 7%, and the recovery rate is basically 90% above, indicating that the method accords with the clinical detection requirements.

[0101] Even though the present invention is disclosed above, but it is not limited thereto. A person skilled in the art can make various alterations and modifications within the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be subjected to the scope defined by the claims.