Development and Validation of a Method of Liquid Chromatography Coupled with Tandem Mass Spectrometry for Quantification of ST-246 (Tecovirimat) in Human Plasma

Abstract

The aim of this work was to develop and validate a sensitive and robust method of liquid chromatography coupled with tandem mass spectrometry to quantitate ST-246 (tecovirimat) in plasma using an internal standard (2-hydroxy-N-{3,5-dioxo-4-azatetracyclo [5.3.2.02.6.08.10]dodec-11-en-4-yl}-5-methylbenzamide). The method was validated in negative multiple reaction monitoring mode following recommendations of the European Medicines Agency for the validation of bioanalytical methods. The calibration curve for the analyte was linear in the 10−2500 ng/mL range with determination coefficient R2 > 0.99. Intra- and inter-day accuracy and precision for three concentrations of quality control were <15%. Testing of long-term stability of ST-246 (tecovirimat) in plasma showed no degradation at −20 °C for at least 3 months. The method was applied to a clinical assay of a new antipoxvirus compound, NIOCH-14. Thus, the proposed method is suitable for therapeutic drug monitoring of ST-246 (tecovirimat) itself and of NIOCH-14 as its metabolic precursor.

Keywords: LC-MS/MS; MRM; NIOCH-14; ST-246; plasma; tecovirimat.

Figure 1

Chemical structure of ST-246 (tecovirimat) (a) and IS (b).

Figure 2

Representative chromatograms of (a) transitions m/z 375.1 → 283.1, 375.1 → 188.2, and 375.1 → 96.1 for the LOQ at 10 ng/mL ST-246 (tecovirimat) and (b) transitions m/z 337.2 → 245.2, 337.2 → 188.1, and 337.2 → 111.0 of 30 ng/mL IS with the corresponding retention time (RT) and signal-to-noise (S/N) ratio.

Figure 3

Metabolic conversion of NIOCH-14 into ST-246 (tecovirimat).

Figure 4

The mean plasma concentration–versus–time plot of ST-246 (tecovirimat) as the main immediate metabolite of NIOCH-14 after single oral administration of 600 mg of NIOCH-14 to healthy human volunteers (n = 4).