Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome using combined sorbent composed of C18 and ion exchange moiety, which finally allowed to cover the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity of two orders of magnitude for many compounds. Method validation results indicated that HILIC mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes leading to signal enhancement in RP and signal suppression in HILIC.

• Keywords
• UHPLC-MS/MS, antiviral drug, hydrophilic interaction chromatography, microextraction, reversed phase, solid phase extraction,
• MeSH
• Antiviral Agents chemistry   pharmacokinetics   MeSH
• Chromatography, Reverse-Phase * MeSH
• Humans MeSH
• Solid Phase Microextraction * MeSH
• Drug Monitoring MeSH
• Reproducibility of Results MeSH
• Tandem Mass Spectrometry * MeSH
• Chromatography, High Pressure Liquid * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antiviral Agents MeSH

P-glycoprotein (ABCB1), an ATP-binding-cassette efflux transporter, limits intestinal absorption of its substrates and is a common site of drug-drug interactions (DDIs). ABCB1 has been suggested to interact with many antivirals used to treat HIV and/or chronic hepatitis C virus (HCV) infections. Using bidirectional transport experiments in Caco-2 cells and a recently established ex vivo model of accumulation in precision-cut intestinal slices (PCIS) prepared from rat ileum or human jejunum, we evaluated the potential of anti-HIV and anti-HCV antivirals to inhibit intestinal ABCB1. Lopinavir, ritonavir, saquinavir, atazanavir, maraviroc, ledipasvir, and daclatasvir inhibited the efflux of a model ABCB1 substrate, rhodamine 123 (RHD123), in Caco-2 cells and rat-derived PCIS. Lopinavir, ritonavir, saquinavir, and atazanavir also significantly inhibited RHD123 efflux in human-derived PCIS, while possible interindividual variability was observed in the inhibition of intestinal ABCB1 by maraviroc, ledipasvir, and daclatasvir. Abacavir, zidovudine, tenofovir disoproxil fumarate, etravirine, and rilpivirine did not inhibit intestinal ABCB1. In conclusion, using recently established ex vivo methods for measuring drug accumulation in rat- and human-derived PCIS, we have demonstrated that some antivirals have a high potential for DDIs on intestinal ABCB1. Our data help clarify the molecular mechanisms responsible for reported increases in the bioavailability of ABCB1 substrates, including antivirals and drugs prescribed to treat comorbidity. These results could help guide the selection of combination pharmacotherapies and/or suitable dosing schemes for patients infected with HIV and/or HCV.

• Keywords
• Caco-2 cells, P-glycoprotein, antiviral drugs, drug-drug interactions, intestinal absorption, precision-cut intestinal slices, rhodamine 123,
• MeSH
• Antiviral Agents pharmacology   MeSH
• Atazanavir Sulfate pharmacology   MeSH
• Benzimidazoles pharmacology   MeSH
• Caco-2 Cells drug effects   metabolism   MeSH
• Fluorenes pharmacology   MeSH
• Hepatitis C complications   drug therapy   virology   MeSH
• HIV Infections complications   drug therapy   virology   MeSH
• Imidazoles pharmacology   MeSH
• Carbamates MeSH
• Rats MeSH
• Anti-HIV Agents pharmacology   MeSH
• Drug Interactions MeSH
• Middle Aged MeSH
• Humans MeSH
• Lopinavir pharmacology   MeSH
• Maraviroc pharmacology   MeSH
• ATP Binding Cassette Transporter, Subfamily B, Member 1 antagonists & inhibitors   metabolism   MeSH
• Rats, Wistar MeSH
• Pyrrolidines MeSH
• Ritonavir pharmacology   MeSH
• Saquinavir pharmacology   MeSH
• Aged MeSH
• Intestines drug effects   MeSH
• Valine analogs & derivatives   MeSH
• Zidovudine pharmacology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antiviral Agents MeSH
• Atazanavir Sulfate MeSH
• Benzimidazoles MeSH
• daclatasvir MeSH Browser
• Fluorenes MeSH
• Imidazoles MeSH
• Carbamates MeSH
• Anti-HIV Agents MeSH
• ledipasvir MeSH Browser
• Lopinavir MeSH
• Maraviroc MeSH
• ATP Binding Cassette Transporter, Subfamily B, Member 1 MeSH
• Pyrrolidines MeSH
• Ritonavir MeSH
• Saquinavir MeSH
• Valine MeSH
• Zidovudine MeSH