Simultaneous quantification of nirmatrelvir/ritonavir in human serum by LC-HRMS

In December 2021, the U.S. Food and Drug Administration (FDA) granted emergency authorization for Paxlovid (R) as an antiviral treatment for COVID-19. Paxlovid (R) 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-HRMS method for simultaneous determination of nirmatrelvir/ritonavir in human serum for therapeutic drug monitoring. Internal standard saquinavir was added in 25 mu L 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 mu L, 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(-1), for ritonavir was 15.63-4000 ng mL(-1) (r(2)>0.9900). The accuracy ranged from 87.45 % similar to 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 similar to 112.36 %. The matrix effect of nirmatrelvir was 88.31 similar to 97.73 %, and that of ritonavir was 85.17 similar to 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(-1), 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.