Quantification of Daridorexant in Human Plasma Using RPHPLC: A Bioanalytical Method Development and Validation

Abstract

A novel analytical approach for quantifying Daridorexant (DXT) in plasma has been devised using suvorexant as an internal standard (IS), To separate the DXT from the plasma, liquid-liquid extraction (LLE) was utilized, followed by RP-HPLC analysis using a PDA. DXT recovery was tested with several extraction solvents (n-butyl chloride, ether / toluene (1: 1), and methyl t-butyl ether (MTBE)). Analytes were separated on an Inertsil ODS column (250 x 4.6 mm, 5µ) using a 0.5 mM sodium acetate buffer (pH-3.5): methanol (78:22 v/v) as mobile phase pumped at 0.5 mL/min. The temperature in the column was kept constant at 25 °C and drug was monitored at 225 nm. The proposed technique was validated in accordance with the USFDA guidelines. Linearity was established in a range of 0.25 to 10.00 µg/mL. The LOQ was determined to be 0.13 µg/mL. The mean accuracy (% CV) was found to be less than 15% of ranging from 92 to 115% of the nominal concentration. In HQC 8.50 µg/mL and LOQ 0.750 µg/mL, the matrix impact was 2.24 and 4.51%, respectively. Plasma Quality Control (QC) samples were kept at -20° C. After three freeze-thaw cycles, first intraday quality control analysis revealed an accuracy of 4% of nominal with an accuracy (% CV) of 3.8%. QC plasma samples have been demonstrated to stay stable at -20 °C for up to 10 months. The method can be used to support clinical studies and/or bioavailability and bioequivalence studies.