Comparison of sample preparation methods, validation of an UPLC–MS/MS procedure for the quantification of cyclosporine A in whole blood sample

Current main methods for therapeutic drug monitoring (TDM) of cyclosporine A (CsA) are immunoassays and liquid chromatography tandem mass spectrometry. The sample pretreatment of these methods is mainly based on extraction of drug which is bound to erythrocytes by divalent heavy metal ions (such as zinc and copper). Although these methods are effective for whole blood drug extraction and measurement, the pollution of heavy metals in sample pretreatment process will have potential negative impact on environment and human health. To overcome the pollution problem, in this study we have developed and validated an UPLC-MS/MS method for CsA determination in whole blood samples using physical pretreatment method. According to the characteristics of erythrocytes, a series of physical pretreatment methods, including sonication, freeze-thaw and osmotic burst, have been developed and evaluated. The results showed that the osmotic burst method was an effective way for drug extraction from erythrocytes. The lower limit of quantitation for CsA was 25 ng/mL, the within-run and between-run coefficient of variations were both less than 11.6 %. The agreement of the UPLC-MS/MS methods using these two sample pretreatment was evaluated by Bland-Altman plot and the two-tailed Student’s T-test. Comparison studies show that the effect of erythrocyte fragmentation by osmotic burst is similar to that of zinc sulfate method. The CsA measurement of 103 whole blood samples obtained by these two UPLC-MS/MS assays were no significant difference. These results demonstrate that the sample pretreatment by osmotic burst method is an eco-friendly and precise method for detecting the whole blood CsA concentration and therapeutic drug monitoring of CsA.