Determination of total and unbound concentrations of lopinavir in plasma using liquid chromatography-tandem mass spectrometry and ultrafiltration methods

Lopinavir is an HIV protease inhibitor with high protein binding (98-99%) in human plasma. This study was designed to develop an ultrafiltration method to measure the unbound concentrations of lopinavir overcoming the non-specific binding issue. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of total concentrations of lopinavir in plasma was developed and validated, and an adaptation was also optimized and validated for the determination of unbound concentrations. The chromatographic separation was performed with a C₁₈ column (100mm×2.1mm i.d., 5μm particle size) using a mobile phase containing deionized water with formic acid, and acetonitrile, with gradient elution at a flow-rate of 350μLmin^-1. Identification of the compounds was performed by multiple reaction monitoring, using electrospray ionization in positive ion mode. The method was validated over a clinical range of 0.01-1μg/mL for human plasma ultrafiltrate and 0.1-15μg/mL in human plasma. The inter and intra-assay accuracies and precisions were between 0.23% and 11.37% for total lopinavir concentrations, and between 3.50% and 13.30% for plasma ultrafiltrate (unbound concentration). The ultrafiltration method described allows an accurate separation of the unbound fraction of lopinavir, circumscribing the loss of drug by nonspecific binding (NSB), and the validated LC-MS/MS methodology proposed is suitable for the determination of total and unbound concentrations of lopinavir in clinical practice.