Determination of anti-SARS-CoV-2 virustatic pharmaceuticals in the aquatic environment using high-performance liquid chromatography high-resolution mass spectrometry

The Covid-19 pandemic has affected the global population since 2019. The rapid development and approval of vaccines has brought relief. Yet, effective cures are still being researched. Even if the pandemic situation may end, SARS-CoV-2 will remain and, thus, continued application of the drugs will lead to emissions of the active ingredients into the aquatic environment, as with other anthropogenic micropollutants. However, a general method for trace analysis of antiviral drugs is still missing. To this purpose, favipiravir, remdesivir, its active metabolite GS-441524, molnupiravir and its active metabolite EIDD-1931 were selected as representative analytes. A method was developed based on solid phase extraction and high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight high-resolution mass spectrometry. Optimization comprised the choice of chromatographic columns, elution gradient, mass spectrometry and tandem mass spectrometry parameters. Solid phase extraction proved suitable for increase in limits of detection and quantitation. amelioration of the limits of detection and quantitation. Matrix effects were investigated applying the optimized method to a wastewater sample with added virustatics. All five compounds could be separated with reversed phase chromatography, whereas EIDD-1931 profited from hydrophilic interaction liquid chromatography. The optimized method yielded limits of detection and quantification of 2.1.10-1, 6.9.10(-1) mu g.L-1 for favipiravir, 1.8.10-3, 5.5 .10-3 mu g.L-1 for remdesivir, 1.9.10-3, (-1)7.6.10-3 mu g.L-1 for GS-441524, 2.9.10-3, 8.7.10-3 mu g .L-1 for molnupiravir, and 1.3.10(-1), 3.8.10(-1) mu g. L-1 for EIDD 1931. The method was first applied to compound stability testing at pH 2.8 and 9.7. At pH 2.8, remdesivir, GS-441524 and molnupiravir proved stable, whereas about 14% of EIDD-1931 and favipiravir were degraded. All five antiviral compounds were almost completely decomposed at pH 9.7. The application of the method was further demonstrated for potential transformation product detection on favipiravir ozonation monitoring.

Automated summary

The Covid-19 pandemic has had a global impact since 2019, and although vaccines have been developed and approved, effective cures are still being researched. However, the continued use of drugs will lead to the presence of active ingredients in the aquatic environment, and a general method for trace analysis of antiviral drugs is still missing.