A detection kit for detecting immunosuppressants in whole blood by high performance liquid chromatography tandem mass spectrometry and its detection method
A high-performance liquid chromatography and immunosuppressive technology, which is applied in the field of biochemical analysis, can solve the problems of easy interference, poor specificity, and large errors in the detection results, so as to improve the detection accuracy and sensitivity, the detection results are accurate and stable, Simple and efficient detection process
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Embodiment 1
[0060] Example 1 Sample preparation, pretreatment and detection
[0061] 1. Sample preparation
[0062] 1. Preparation of standard curve and quality control samples
[0063] The immunosuppressant cyclosporine A, tacrolimus, sirolimus, and everolimus standards were prepared into a mixed solution as a stock solution for standard working solution and quality control working solution, using matrix additives Sterile bovine blood of 1.0 mg / mL vitamin E was used as the matrix to prepare the standard curve and quality control samples.
[0064] The four immunosuppressants have 10 serial concentrations (S1 to S10) in the standard, as shown in Table 1:
[0065] Table 1. 10 series concentrations (S1~S10) of 4 kinds of immunosuppressants in standard products
[0066]
[0067]
[0068] The four immunosuppressants have three series concentrations of low (L), medium (M) and high (H) in the quality control products, as shown in Table 2:
[0069] Table 2. Three series of concentration...
Embodiment 2
[0091] Example 2 The effect of adding antioxidants on the stability of immunosuppressants
[0092] In this example, according to the method provided in Example 1, different types of antioxidants were used to prepare the internal standard solution and system suitability solution of the same concentration, and they were placed at 37°C for 14 days to investigate the effect of different antioxidants on the internal standard solution. Contains Cyclosporine-d4, Tacrolimus-13C-d4, Sirolimus-d3 and Cyclosporine A, Tacrolimus, Sirolimus, Ive Shelf life effects of moss. Antioxidants include 2,6-di-tert-butyl-p-cresol, vitamin E, vitamin C, beta-carotene, sodium metabisulfite. The results are shown in Tables 1 and 2:
[0093] Table 1. Effects of different antioxidants on the stability of cyclosporine A, tacrolimus, sirolimus, and everolimus contained in the system suitability solution for 14 days at 37°C
[0094]
[0095] Table 2. Effects of different antioxidants on the stability ...
Embodiment 3
[0100] The selection of the content of embodiment 3 vitamin E
[0101] In this example, according to the method provided in Example 1, cyclosporine A, tacrolimus, sirolimus, and everolimus standard solution were added to sterile bovine blood, and vitamin E of different concentrations was added respectively, After being placed at 37℃ for 14 days, the effects of different concentrations of vitamin E on the shelf life of cyclosporine A, tacrolimus, sirolimus and everolimus in whole blood were investigated. The results are shown in Table 3.
[0102] Table 3. The influence of vitamin E content on the detection results of samples placed at 37°C for 14 days
[0103]
[0104] As can be seen from Table 3, when the concentration of vitamin E was 0.5 mg / mL, the concentration of everolimus decreased by 22.5% after being placed at 37 °C for 14 days, failing to meet the requirements; when the concentration of vitamin E was 1.0 mg Cyclosporine A, tacrolimus, sirolimus, and everolimus di...
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