Determination of sirolimus in rabbit arteries using liquid chromatography separation and tandem mass spectrometric detection

Abstract

Sirolimus, an effective immunosuppressive agent, is used for drug eluting stents. During stent development, an analytical method for the determination of sirolimus in tissue needs to be established. Normally, tissue samples are homogenized and then analyzed against the calibration standards prepared in a tissue homogenate. This approach provides insufficient control of the homogenization process. In this paper, tissue quality control samples were introduced for the optimization of the homogenization process during method development, but also allowance for the performance evaluation of the entire analytical process. In addition, a new approach using rabbit blood as a homogenization medium was developed to stabilize sirolimus in rabbit tissue homogenates. Calibration standards and quality controls were prepared by spiking different sirolimus working solutions into rabbit blood. Homogenization quality control samples were prepared by injecting other sirolimus working solutions into empty test tubes and pre-cut arteries within pre-defined masses. A high-throughput homogenization procedure was optimized based on the specific chemical properties of sirolimus. The linear dynamic range was between 49.9 pg/mL and 31.9 ng/mL to accommodate the expected artery homogenate concentrations. Additionally, quality controls in rabbit blood were also used in the extraction to support the calibration standards. The accuracy and precision of the quality controls in rabbit blood reflect the extraction performance and the accuracy and precision of the homogenization tissue quality controls reflect the overall performance of the method. The mean bias was between -4.5 and 0.2% for all levels of quality controls in the blood and between 4.8 and 14.9% for all levels of the homogenization tissue quality controls. The CVs of all concentration levels were < or =5.3% for the quality controls in blood and < or =9.2% for the homogenization tissue quality controls. The method was successfully applied to determine the concentration of sirolimus in the rabbit arteries.

Liquid chromatographic method for the determination of sirolimus in blood using electrochemical detection

“…Three techniques of measuring drug concentrations are available: the first technique is high-performance liquid chromatography (HPLC) with mass spectrometry (MS) (Bogusz et al, 2007;Koal et al, 2004;Streit et al, 1996;Taylor and Johnson, 1998;Volosov et al, 2001;Wallemacq et al, 2003;Zhang et al, 2007); the second technique is HPLC with ultraviolet (UV) detection (Campareno et al, 2004;Cattaneo et al, 2002;Di marco et al, 2003;French et al, 2001;Khoschsorur, 2005;Napoli and Kahan, 1994;Svensson et al, 1997); and the third technique is the microparticle enzyme immunoassay (Jones et al, 2000). Although these methods meet many or all of the generally accepted criteria for validated analysis of immunosuppressive drugs, the HPLC/UV assay is cumbersome due to the presence of interfering peaks in the chromatograms, requiring tedious extraction procedures and long running time to resolve the peaks.…”

Development of a validated high‐throughput LC‐ESI‐MS method for determination of sirolimus on dried blood spots

“…At present, two methods of measuring drug concentrations are available: the reference method is high-performance liquid chromatography (HPLC) with ultraviolet (UV) or mass spectrometry (MS) detection and a second technique is an enzyme immunoassay using micro particles coated with anti-sirolimus antibodies (MEIA) (Zhang et al, 2007;Buchberger et al, 2004;Kirchner et al, 1999;Taylor and Johnson, 1998;Morris et al, 2006;Mochizuki et al, 2009;Koal et al, 2004;Christiansa et al, 2000;Cattaneo et al, 2002). The reported chromatographic methods for measuring sirolimus blood levels in patients with organ transplants are time-consuming when large numbers of samples have to be processed.…”