Application of Parallel Reaction Monitoring to the Development and Validation of a Quantitative Assay for ST-246 in Human Plasma

In this work, we developed and validated a robust and sensitive method of liquid chromatography with high-resolution mass spectrometry in parallel reaction monitoring (PRM) mode for ST-246 (tecovirimat) quantification in human blood plasma. The method was compared with the multiple reaction monitoring (MRM) technique and showed better selectivity and similar sensitivity in a wider concentration range (10–5000 ng/mL). Within this range, intra- and interday variability of precision and accuracy were within acceptable ranges in accordance with the European Medicines Agency guidelines, and recovery was 87.9–100.6%. Samples were stable at 4 °C within 48 h and at −20 °C up to 3 months. The recovery and matrix effects in the proposed HRMS method were about 5% higher than those reported for the MRM method, but the PRM method showed better accuracy with comparable precision. It was found that the ST-246 concentration shown by the PRM method is approximately 24% higher than the output of the MRM one. Nonetheless, the high selectivity with similar sensitivity, as compared with traditional MRM methods, makes the proposed approach attractive for research and clinical use.