Introduction: Remdesivir (GS-5734) is a nucleoside analog prodrug with antiviral activity against several single-stranded RNA viruses, including the novel severe respiratory distress syndrome virus 2 (SARS-CoV-2). It is currently the only FDA-approved antiviral agent for the treatment of individuals with COVID-19 caused by SARS-CoV-2. However, remdesivir pharmacokinetics/pharmacodynamics (PK/PD) and toxicity data in humans are extremely limited. It is imperative that precise analytical methods for the quantification of remdesivir and its active metabolite, GS-441524, are developed for use in further studies. We report, herein, the first validated anti-viral paper spray-mass spectrometry (PS-MS/MS) assay for the quantification of remdesivir and GS-441524 in human plasma. We seek to highlight the utility of PS-MS/MS technology and automation advancements for its potential future use in clinical research and the clinical laboratory setting.
Methods: Calibration curves for remdesivir and GS-441524 were created utilizing seven plasma-based calibrants of varying concentrations and two isotopic internal standards of set concentrations. Four plasma-based quality controls were prepared in a similar fashion to the calibrants and utilized for validation. No sample preparation was needed. Briefly, plasma samples were spotted on a paper substrate contained within pre-manufactured plastic cassette plates, and the spots were dried for 1 h. The samples were then analyzed directly for 1.2 min utilizing PS-MS/MS. All experiments were performed on a Thermo Scientific Altis triple quadrupole mass spectrometer utilizing automated technology.
Results: The calibration ranges were 20 - 5000 and 100 - 25000 ng/mL for remdesivir and GS-441524, respectively. The calibration curves for the two antiviral agents showed excellent linearity (average R2 = 0.99-1.00). The inter- and intra-day precision (%CV) across validation runs at four QC levels for both analytes was less than 11.2% and accuracy (%bias) was within ± 15%. Plasma calibrant stability was assessed and degradation for the 4 °C and room temperature samples were seen beginning at Day 7. The plasma calibrants were stable at -20 °C. No interference, matrix effects, or carryover was discovered during the validation process.
Conclusions: PS-MS/MS represents a useful methodology for rapidly quantifying remdesivir and GS-441524, which may be useful for clinical PK/PD, therapeutic drug monitoring (TDM), and toxicity assessment, particularly during the current COVID-19 pandemic and future viral outbreaks.
Keywords: ANOVA, A one-way analysis of variance; AUC, area under the curve; Antiviral; CE, collision energy; CES1, carboxylesterase-1; CES2, carboxylesterase-2; CV, coefficient of variation; DMSO, dimethyl sulfoxide; EC50, half maximum effective concentration; ECMO, extracorporal membrane oxygenation; H-ESI, heated electrospray ionization; IRB, institutional review board; IS, internal standard; Inc, Incorporated; LC-MS/MS, liquid chromatography–mass spectrometry; LLC, Limited Liability Company; LLOQ, lower limit of quantitation; LOD, limit of detection; MP, monophosphate; Mass spectrometry; PD, pharmacodynamics; PK, pharmacokinetics; PS-MS/MS, paper spray–mass spectrometry; Paper spray; QC, quality control; QC-LLOQ, quality control-lower limit of quantification; R2, coefficient of determination; RF, radio frequency; Remdesivir; S/B, Signal-to-Blank; SARS-CoV-2; SIL, stable isotopically-labeled; SS, spiking solution; V, volts; kV, kilovolts; m/z, mass-to-charge; mL, milliliter; ng, nanogram; therapeutic drug monitoring, TDM; µL, microliter; µg, microgram.
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