Development and Validation of A Semi-Automated Assay for the Highly Sensitive Quantification of Biolimus A9 in Human Whole Blood Using High Performance Liquid Chromatography-Tandem Mass Spectrometry

摘要

Drug-eluting stents are sustained-release intra-coronary devices that are usually coated with a few hundred micrograms of drug. Measuring the drugs that are released over weeks in order to assess human pharmacokinetics is a challenge that requires assays with high sensitivity.We developed and validated a semi-automated LC-MS/MS assay for the quantification of Biolimus A9, a proliferation signal inhibitor that was specifically developed for coating on drug-eluting stents in human EDTA blood. The only manual step was the addition of a zinc sulfate/ methanol protein precipitation solution which included the internal standard. Samples were injected into the HPLC and extracted online. The assay had the following performance characteristics: range of reliable response 0.01 – 100 ng/mL (r²>0.99), inter-day accuracy (0.033 ng/mL): 111.7%, and inter-day precision: 8.6%. There was no ion suppression, matrix interferences or carry-over. Extracted samples were stable in the autosampler at +4°C for at least 24 hours and could undergo three freeze-thaw cycles. The assay, with a lower limit of detection of 333 fg/mL and a lower limit of quantitation of 10 pg/mL, was sufficiently sensitive and robust for quantifying Biolimus A9 in clinical trials after IV injection and after stent implantation.