Fast and simultaneous analysis of two immunosuppressants in whole blood using high-performance liquid chromatography

<!-- [if !mso]--> <!--[endif]--> ARTICLE INFO Received: 04 May 2024 | Accepted: 25 June 2024 | Available online: 15 July 2024 CITATION Huang Y, Tang H, Meng X, et al. Fast and simultaneous analysis of two immunosuppressants in whole blood using high-performance liquid chromatography. Advances in Analytic Science 2024; 5(1): 2120. doi: 10.54517/aas.v5i1.2120 COPYRIGHT Copyright © 2024 by author(s). Advances in Analytic Science is published by Asia Pacific Academy of Science Pte. Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), permitting distribution and reproduction in any medium, provided the original work is cited. <!-- [if !mso]--> <!--[endif]--> Cyclosporine A and sirolimus are commonly used immunosuppressants in organ transplantation, known for their complementary and synergistic mechanisms of action. However, due to their narrow therapeutic index and critical dosing requirements, maintaining their concentrations within a specific blood range is essential for optimal efficacy and safety. This study systematically compared the chromatographic behaviors of cyclosporine A and sirolimus using a biological liquid chromatography (BioLC) column and traditional liquid chromatography (TraLC) columns under identical conditions. The results indicated that the BioLC column, specifically ZORBAX 300SB C8 (250 mm × 4.6 mm, 5.0 μm), provided the highest peak heights and the narrowest peak widths for both drugs. The number of theoretical plates for both drugs significantly increased on this column when acetonitrile in the mobile phase exceeded 70%. Retention times on both column types were minimally affected by formic acid and trifluoroacetic acid in the mobile phases. Additionally, the ZORBAX 300SB C8 column exhibited a higher number of theoretical plates and a distinct relationship between retention factor and column temperature compared to the TraLC columns. For analysis, a 50 μL whole blood sample was prepared through protein precipitation with 1 mol/L sodium hydroxide, extracted into 500 μL ether-methanol (95:5, v/v), and then centrifuged. The organic layer was evaporated under nitrogen at 50 ℃, and the residue was reconstituted in 200 μL methanol. Cyclosporine A and sirolimus were then separated via isocratic elution on the ZORBAX 300SB C8 column.