Objective: To establish the national standard materials of cyclosporine A, tacrolimus, sirolimus, and everolimus in frozen human whole blood. Methods: Anticoagulated whole blood samples were collected without obvious hemolysis, jaundice, or lipemia. The whole blood samples that had tested negative for four infectious diseases were pooled. They were frozen at - 20 ℃ for more than 48 h. After thawing, the samples were centrifuged at 4 000×g for 10 min, the precipitate was discarded, and pure raw materials of cyclosporine A, tacrolimus, sirolimus, and everolimus were added to prepare the target concentrations. The mixture was mixed thoroughly and then aliquoted into ampoules at two concentration levels. The homogeneity and stability of the standard materials were evaluated using one-way analysis of variance and linear regression methods. The assigned values were determined by reference methods, and uncertainty was calculated. Finally, the commutability of the standards was evaluated. Results: The F values of the homogeneity for cyclosporine A, tacrolimus, sirolimus, and everolimus at level I were 1.492 1,1.395 6, 1.432 8, and 1.060 2, respectively, and at level Ⅱ were 1.441 2, 1.476 1, 1.458 0, and 1.454 6, respectively. All of them were less than F0.05. Level Ⅰ standard materials were stable for 10 d at 20-25 ℃ and 2-8 ℃, and for 30 d at - 20 ℃; Level Ⅱstandard materials were stable for 10 d at 20-25 ℃ and for 30 d at 2-8 ℃ and - 20 ℃. The assigned values of standard (k=2) were as follows: cyclosporine A level Ⅰwas (105.6±8.9) ng · mL-1, level Ⅱ was (419.8±32.8) ng · mL-1; tacrolimus level Ⅰwas (5.4±0.4) ng · mL-1, level Ⅱ was (17.0±1.8) ng · mL-1; sirolimus level Ⅰ was (5.0±0.3) ng · mL-1, level Ⅱ was (15.7±1.1) ng · mL-1; everolimus level Ⅰ was (4.8±0.2) ng · mL-1, level Ⅱ was (10.2±0.6) ng · mL-1. The two levels of standard concentration are within the 95% confidence interval of the regression line for fresh whole blood samples, indicating good commutability. Conclusion: The batch of the national standard material of cyclosporine A, tacrolimus, sirolimus, and everolimus in frozen human whole blood is homogeneous, stable, and commutable. The assigned values are accurate and reliable, and can be used as national standard for calibration of these four immunosuppressive agents, thereby promoting the standardization and consistency of test results.
YU Ting
,
SHEN min
,
HUANG Jie
,
ZHANG Tian-jiao
. Development of the national standard materials of cyclosporine A, tacrolimus, sirolimus, and everolimus in frozen human whole blood*[J]. Chinese Journal of Pharmaceutical Analysis, 2025
, 45(5)
: 843
-850
.
DOI: 10.16155/j.0254-1793.2024-0108
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