Parallel ultra high pressure liquid chromatography-mass spectrometry for the quantification of HIV protease inhibitors using dried spot sample collection format.

An assay was developed and validated for the quantification of eight protease inhibitors (indinavir (IDV), ritonavir (RTV), lopinavir (LPV), saquinavir (SQV), amprenavir (APV), nelfinavir (NFV), atazanavir (AZV) and darunavir (DRV)) in dried plasma spots using parallel ultra-high performance liquid chromatography and mass spectrometry detection in the multiple reaction monitoring mode. For each analyte an isotopically labeled internal standard was used and the assay based on liquid-solid extraction the area response ratio (analyte/IS) was found to be linear; from 0.025 μg/ml to 20 μg/ml for IDV, SQV, DRV, AZV, LPV, from 0.025 μg/ml to 10 μg/ml for NFV, APV and from 0.025 μg/ml to 5 μg/ml for RTV using 15 μl of plasma spotted on filter paper placed in a sample tube. The total analysis time was of 4 min and inter-assay accuracies and precisions were in the range of 87.7-109% and 2.5-11.8%, respectively. On dried plasma spots all analytes were found to be stable for at least 7 days. Practicability of the assay to blood was also demonstrated. The sample drying process could be reduced to 5 min using a commercial microwave system without any analyte degradation. Together with quantification, confirmatory analysis was performed on representative clinical samples.