Liquid chromatography–mass spectrometry methods for the intracellular determination of drugs and their metabolites: a focus on antiviral drugs

Abstract

Understanding the efficacy and/or toxicity of most drugs requires effective intracellular measurements of the drug and its metabolites. Nevertheless, the most common plasma marker of the biological effect of the drug is the area under the curve. Compared with drug determination in whole blood or urine, various difficulties occur in the development of analytical methods for intracellular measurements. We propose step-by-step guidelines to develop an analytical method exploring intracellular concentrations of antivirals and/or their metabolites. These guidelines are illustrated with the most sensitive liquid chromatography-mass spectrometry methods developed for human in vivo and in vitro studies. We summarize 18 studies that provided methods to explore intracellular concentrations of antivirals since 2002. To explore intracellular metabolites, two different approaches can be envisaged. The direct approach, most frequently using ion-pairing agents, is fast and requires only a small sample but is expensive. The indirect approach is the more widely used approach, but is cumbersome and time-consuming. In both cases, liquid chromatography-mass spectrometry has become the method of choice to determine intracellular drug concentrations with high sensitivity. These methods may increase our understanding of drug behavior in organisms. This is true for preclinical studies where the mechanism of action, the metabolism, and the toxicity of drugs are explored. It is also true for clinical applications when dose adjustment is needed and cannot rely on blood concentrations. Graphical Abstract Direct and indirect approaches to measure intracellular concentrations.

A PBPK model to evaluate zebrafish eleutheroembryos’ actual exposure: bisphenol A and analogs’ (AF, F, and S) case studies

“…At the regulatory level, specific OECD test guidelines (TG) based on zfe assays have been recently adopted either to determine acute toxicity (fish embryotoxicity assay, TG N°236) (OCDE 2013) or endocrine activity of xenobiotics acting as an agonist of estrogen receptors (EASZY assay, TG 250) (Brion et al 2019;Pinto et al 2019). Although these assays provide relevant information on the hazard of xenobiotics, a better characterization of their toxicity or endocrine effects requires accounting for the toxicokinetic processes of the active test substance in the whole organism but also in target tissues (Péry et al 2013) and even in cells (Billat and Saint-Marcoux 2017). In this regard, it is important to consider the spatiotemporal estimation of internal concentrations in a biological model that is undergoing rapid morphological and physiological changes during the first 120 h post-fertilization (hpf).…”

A naturally occurring G11S mutation in the 3C‐like protease from the SARS‐CoV‐2 virus dramatically weakens the dimer interface

“…Achieving the same effect on the WT protein with a PPI inhibitor would require [I]/ K i of 25, that is, a concentration of drug 25‐fold higher than the dissociation constant. Since antiviral drugs can reach intracellular concentrations of tens of micromolars (Billat & Saint‐Marcoux, 2017 ), the dissociation constant, K i , of the PPI inhibitor should be less than 400 nM to destabilize the WT dimer to the same level as G11S. Meanwhile, several PPI inhibitors that entered clinical trials have reported IC 50 values of around 1 nM or less (Cossar et al, 2020 ).…”