Simultaneous determination of nirmatrelvir, ritonavir, and beta-D-N4-hydroxycytidine in human plasma and epithelial lining fluid using LC-MS/MS and its clinical application to compare rates of achieving effective concentrations

Currently, the trials found that the clinical efficacy of molnupiravir is lower than ritonavir-boosted nirmatrelvir. An explanation for these different efficacies in clinical treatments is still limited. The analysis method was developed and validated to simultaneously quantify nirmatrelvir, ritonavir, and beta-D-N4-hydroxycytidine (NHC) in human plasma and bronchoalveolar lavage fluid (BALF) by electrospray ionization mass spectrometry. Our method was validated over a linear range of 30-10000 ng/mL for both matrices, with precision and accuracy within 15 % across four concentrations. Recovery rates for both analytes from plasma and BALF were between 90.7-102.2 % and 90.5-107.7 %, respectively. The analytical method was then applied to monitor therapeutic drug concentrations in 59 plasma samples from 23 patients receiving ritonavir-boosted nirmatrelvir or molnupiravir. By setting target plasma concentrations of 292 ng/mL for nirmatrelvir and 1205 ng/mL for NHC, based on in vitro antiviral 90 % virus inhibitory concentrations (EC₉₀), the drug's molecular weight and its binding to human plasma proteins, we observed that ritonavir-boosted nirmatrelvir had substantially greater rates of achieving target plasma concentrations. Additionally, we monitored epithelial lining fluid in 4 BALF samples from 4 patients and observed that NHC exhibited higher permeability in lung tissue (approximately 20 % higher than nirmatrelvir). However, subtherapeutic antiviral concentrations of NHC were also present in epithelial lining fluid. These findings highlight the importance of considering these factors in determining the efficacy of these drugs in treating coronavirus disease 2019 (COVID-19).