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

Published:7 April 2021 DOI: 10.3390/molecules26082123 PMID: 33917128
Lubo? Fical, Maria Khalikova, Hana Ko?ová Vl?ková, Ivona Lhotská, Zuzana Hadysová, Ivan Vok?ál, Luká? ?erveny, Franti?ek ?vec, Lucie Nováková

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.

Substances (66)

Materials
Procduct Name CAS Molecular Formula Supplier Price
Ritonavir 155213-67-5 C37H48N6O5S2 695 suppliers $5.00-$1624.00
Ritonavir 155213-67-5 C37H48N6O5S2 695 suppliers $5.00-$1624.00
Ritonavir 155213-67-5 C37H48N6O5S2 695 suppliers $5.00-$1624.00
Ritonavir 155213-67-5 C37H48N6O5S2 695 suppliers $5.00-$1624.00
Zidovudine 30516-87-1 C10H13N5O5 596 suppliers $8.00-$2490.00
Zidovudine 30516-87-1 C10H13N5O5 596 suppliers $8.00-$2490.00
Zidovudine 30516-87-1 C10H13N5O5 596 suppliers $8.00-$2490.00
Zidovudine 30516-87-1 C10H13N5O5 596 suppliers $8.00-$2490.00
Lopinavir 192725-17-0 C37H48N4O5 410 suppliers $12.00-$2590.00
Lopinavir 192725-17-0 C37H48N4O5 410 suppliers $12.00-$2590.00

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