Determination of nelfinavir free drug concentrations in plasma by equilibrium dialysis and liquid chromatography/tandem mass spectrometry: important factors for method optimization

• Published in: European journal of pharmaceutical sciences 2002-03, Vol.15 (2), p.185-195
• Main Authors: Herforth, Claudia, Stone, Judith A, Jayewardene, Anura L, Blaschke, Terrence F, Fang, Fang, Motoya, Toshiro, Aweeka, Francesca T
• Format: Article
• Language: English
• Subjects: Adsorption; Analysis; Antibiotics. Antiinfectious agents. Antiparasitic agents; Antiviral agents; Biological and medical sciences; Calibration; Chemical Phenomena; Chemistry, Physical; Chromatography, Liquid - methods; Dialysis - methods; Equilibrium dialysis; Free fraction; General pharmacology; HIV; HIV Protease Inhibitors - blood; Humans; Hydrogen-Ion Concentration; Mass Spectrometry - methods; Medical sciences; Molecular Structure; Nelfinavir; Nelfinavir - blood; Pharmacology. Drug treatments; Plasma protein binding; Protease inhibitor; Protein Binding; Reproducibility of Results; Sensitivity and Specificity
• ISSN: 0928-0987; 1879-0720
• DOI: 10.1016/S0928-0987(01)00220-2

A method was developed and validated for measuring the free fraction of nelfinavir in plasma employing equilibrium dialysis for the separation of free (unbound) drug and liquid chromatography/tandem mass spectrometry for quantitation. Nelfinavir, widely used to treat HIV infection, is a highly bound HIV protease inhibitor with the fraction bound in plasma being greater than 98%. Thus variations in the free fraction may be clinically important when interpreting total drug concentrations. Optimization of the method was carried out considering the influence of sample matrix and physicochemical and absorptive properties of nelfinavir. Nelfinavir free fraction averaged 0.41±0.094, 0.43±0.087 and 0.41±0.063% at nelfinavir plasma concentrations of 1000, 2000 and 3000 ng/ml, respectively. Free nelfinavir concentrations were underestimated with this assay by ∼25% because of unavoidable losses to adsorption. However, the adsorptive loss was reproducible and consistent across the concentration range of the assay. Within-day and between-day precisions ranged from 6.0 to 9.4% and 15.2 to 27.3%, respectively. The lower limit of quantitation of the unbound concentration of nelfinavir was 1.0 ng/ml, permitting analysis of samples with total concentrations of nelfinavir in plasma that are ≥400 ng/ml. This developed method proves reproducible and sensitive and its application to patient plasma samples is also reported.