Determination of Sirolimus in Blood by High-Performance Liquid Chromatography with Ultraviolet Detection –Experience and Review
FE-LIN LIN WU1,2, RUSSEL RHEI-LONG CHEN3, SHOEI-SHENG LEE1 AND SHAO-WEN SUN1*
1. School of Pharmacy, College of Medicine, National Taiwan University, 1 Jen-Ai Rd., Sec. 1, Taipei City 100, Taiwan, R.O.C.
2. Department of Pharmacy, National Taiwan University Hospital, Taiwan, R.O.C.
3. School of Pharmacy, Tajen Institute of Technology, Taiwan, R.O.C.
(Received: June 28, 2004; Accepted: November 22, 2004)
ABSTRACT
Sirolimus (SRL) is a potent immunosuppressant. Therapeutic drug monitoring (TDM) of SRL is required to optimize the therapy. Immunoassay, high-performance liquid chromatography with ultraviolet detection (HPLC-UV), and HPLC with mass-spectrometric detection (HPLC-MS or HPLC/MS/MS) have been used in the analysis of SRL. The purpose of this study was to share our experience in validating a HPLC-UV method for analyzing SRL and to have an overview of HPLC-UV methods used in SRL assay. A validated HPLC/UV method developed by Wyeth-Ayerst Research with minor modification was use to determine SRL concentration in human whole blood. An Alltima C18 column (5 μm, 150 × 2.1 mm) was used as the stationary phase. The mobile phase was 60%acetonitrile in water, and the flow rate was 0.5 mL/min. Samples were prepared by spiking human whole blood (0.5 mL) with the internal standard (IS) and designated amount of SRL, except blank. Zinc sulfate (50 g/L, 1 mL) and acetone (1 mL) were used for hemolysis and deproteinization. After alkalizing supernatant with 100 mM NaOH (0.2 or 0.3 mL), 1-chlorobutane (2 mL) was used for extraction. The 1-chlorobutane layer was dried, reconstituted with mobile phase and back extracted with 0.5 mL of n-hexane. The limitation of quantification was 2.5 ng/mL and the standard curve was linear at the concentration range of 2.5-75 ng/mL. The intraday and interday coefficients of variation were 2.1-5.2% and 2.8-5.7%, respectively. The intraday and interday relative errors were -0.03-5.3% and 0.7-3.3%, respectively. The recoveries for SRL and the IS were 78.5-92.8% and 76.9 ± 3.9 %, respectively. The samples were proven to be stable after 3 freeze/thaw cycles, and the extract was stable over 24 hr at an autosampler set to 4°C. In addition to an overview of the chemical properties of SRL, different HPLC-UV methods for the quantification of SRL were provided to identify analytic parameters that are critical for the establishment of HPLC-UV assay for SRL.
Key words: immunosuppressive, high-performance liquid chromatography, sirolimus, therapeutic drug monitoring, transplantation