Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes.

Luboš Fical,Zuzana Hadysová,Hana Kočová Vlčková,František Švec,Lukáš Červený,Ivan Vokřál,Ivona Lhotská,Lucie Nováková,Maria Khalikova

doi:10.3390/molecules26082123

Abstract

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.

Highlights

RP-ultra-high performance liquid chromatography (UHPLC)-MS/MS has been a method of choice in most previously published methods for the analysis of antiviral drugs [1,9]
Most of the compounds exhibited ionization in both tested modes under RP-UHPLCMS/MS conditions except for abacavir, atazanavir, efavirenz, and tenofovir disoproxil that did not ionize in ESI−
The calibration range in RP-UHPLC-MS/MS was evaluated in the concentration range of 0.1–10,000 ng/mL to cover great differences in method sensitivity for individual analytes (Table 2)

Summary

Introduction

Antiviral drugs are an important class of compounds because many viruses can cause life-threatening diseases, as we are witnessing with the COVID-19 pandemic. Antiviral drugs can act against viruses at different stages of the viral life cycle, including (i) inhibitors of virion fusion or entry, (ii) inhibitors of uncoating, (iii) inhibitors of integrase,. (iv) inhibitors of nucleic acid synthesis, (v) protease inhibitors, and (vi) neuraminidase inhibitors [1]. Most antiviral drugs affect the viral synthesis step, important groups involve the reverse transcriptase inhibitors and the protease inhibitors, acting in the viral replication and maturation step, respectively. A variety of structurally different antiviral drugs has been developed to target these different stages and different types of viruses, including generally four major groups of (i) herpes viruses, (ii) respiratory viruses,

Methods

Results

Conclusion