ABSTRACT
In December 2021, the U.S. Food and Drug Administration (FDA) granted emergency authorization for Paxlovid® as an antiviral treatment for COVID-19. Paxlovid® is composed of two tablets, nirmatrelvir and ritonavir. Dose adjustment is necessary in cases of renal insufficiency. The aim of present study is to establish a LC-MS/MS method for simultaneous determination of nirmatrelvir/ritonavir in human serum for therapeutic drug monitoring. Internal standard saquinavir was added in 25 μl human serum samples, and then the samples were precipitated with methanol. The analytes were separated by gradient elution on a C18 column, using a mobile phase of 0.1% formic acid-water and methanol, at a flow rate of 0.4 ml/min. The injection volume was 2 μl, and the analysis time was 5 min. The determination of the analytes was performed by electrospray ionization in positive mode by full mass monitoring. The detected ions of nirmatrelvir, ritonavir and saquinavir were m/z 500.24792, 721.32004 and 671.39155, respectively. The linear concentration range for nirmatrelvir was 78.13~20000 ng/ml, for ritonavir was 15.63~4000 ng/ml (r2>0.9900). The accuracy ranged from 87.45%~104.63%, and the intra-day and inter-day precision RSD was < 15%. The recovery of Nirmatrelvir ranged from 98.72%~109.83%, and that of ritonavir was 95.41%~112.36%. The matrix effect of Nirmatrelvir was 88.31%~97.73%, and that of ritonavir was 85.17%~103.05%. This method was used to measure the trough concentrations of nirmatrelvir/ritonavir in 17 patients. The trough concentration of nirmatrelvir was 1331.7~8352.5 ng/ml, and that of ritonavir was 53.4~1325.5 ng/ml, with large individual differences. The method is simple, sensitive, specific, and reproducible, and can be used for monitoring the blood concentration and pharmacokinetic study of nirmatrelvir/ritonavir in COVID-19 patients.