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. 2021 Mar 20:196:113935.
doi: 10.1016/j.jpba.2021.113935. Epub 2021 Jan 28.

Simultaneous quantification of seven repurposed COVID-19 drugs remdesivir (plus metabolite GS-441524), chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin by a two-dimensional isotope dilution LC-MS/MS method in human serum

Katharina Habler  1 , Mathias Brügel  1 , Daniel Teupser  1 , Uwe Liebchen  2 , Christina Scharf  2 , Ulf Schönermarck  3 , Michael Vogeser  1 , Michael Paal  4
Affiliations

Simultaneous quantification of seven repurposed COVID-19 drugs remdesivir (plus metabolite GS-441524), chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin by a two-dimensional isotope dilution LC-MS/MS method in human serum

Katharina Habler et al. J Pharm Biomed Anal. .

Abstract

Background: The present COVID-19 pandemic has prompted worldwide repurposing of drugs. The aim of the present work was to develop and validate a two-dimensional isotope-dilution liquid chromatrography tandem mass spectrometry (ID-LC-MS/MS) method for accurate quantification of remdesivir and its active metabolite GS-441524, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in serum; drugs that have gained attention for repurposing in the treatment of COVID-19.

Methods: Following protein precipitation, samples were separated with a two-dimensional ultra-high performance liquid chromatography (2D-UHPLC) setup, consisting of an online solid phase extraction (SPE) coupled to an analytical column. For quantification, stable isotope-labelled analogues were used as internal standards for all analytes. The method was validated on the basis of the European Medicines Agency bioanalytical method validation protocol.

Results: Detuning of lopinavir and ritonavir allowed simultaneous quantification of all analytes with different concentration ranges and sensitivity with a uniform injection volume of 5 μL. The method provided robust validation results with inaccuracy and imprecision values of ≤ 9.59 % and ≤ 11.1 % for all quality controls.

Conclusion: The presented method is suitable for accurate and simultaneous quantification of remdesivir, its metabolite GS-441525, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in human serum. The quantitative assay may be an efficient tool for the therapeutic drug monitoring of these potential drug candidates in COVID-19 patients in order to increase treatment efficacy and safety.

Keywords: Antiviral therapy; Isotope dilution liquid chromatography tandem mass spectrometry (ID-LC–MS/MS); Therapeutic drug monitoring.

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Conflict of interest statement

Declaration of Competing Interest The authors have no conflict of interest to declare.

Figures

Fig. 1
Fig. 1
Two-dimensional chromatography configuration. A) Online clean up: The sample protein precipitation extract is pumped to the online SPE column with mobile phase A1 for further extraction. B) Analytic separation: The sample is transferred onto the analytical column by backflush with mobile phase A2 and separated with gradient elution using mobile phase A2 and B2. Meanwhile, the online SPE column is washed and reconditioned with mobile phase A1 and B1.
Fig. 2
Fig. 2
Representative ID-LC–MS/MS chromatogram of the analytes in this study with MRM acquisition using the lowest calibrator.
Fig. 3
Fig. 3
Post-column infusion experiment for the analytes in this study: Green line, methanol-water (1:1; v/v), black line, processed blank sample.
See this image and copyright information in PMC

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