DRV concentrations in formulation, cell lysates, mouse plasma, and tissue samples were measured using the established LC-MS/MS method we have previously detailed [28 (link),29 (link)]. Prior to collection for in vitro study, differentiated U1 cells, also known as U1 macrophages, underwent a washing process with PBS and were then harvested using RIPA buffer. Quantification of DRV and the internal standard RTV (at a concentration of 50 ng/mL) was conducted using an AB SCIEX Triple Quad 5500 tandem mass spectrometer, which featured an electron spray ionization source operating in positive mode. The separation of these compounds was achieved through a liquid chromatography system, specifically the LC-20AD XR model from Shimadzu, MD. Data acquisition and analysis were carried out in multiple-reaction monitoring (MRM) mode using MultiQuant® software (Version 3.0.2) by AB Sciex (Foster City, CA, USA). To mitigate matrix effects, calibration curves were constructed using blank control samples, such as plasma or tissue homogenates, tailored to the types of samples being analyzed. These controls showed no significant interference. All samples underwent centrifugation at 10,000 rpm using a Centrifuge SORVALL LEGEND X1R by Thermo Scientific, USA, after which the supernatant was further processed for LC-MS/MS analysis.
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