Quantification of daridorexant, lemborexant and suvorexant in whole blood using liquid chromatography-tandem mass spectrometry

Abstract Suvorexant, lemborexant, and daridorexant are dual orexin receptor antagonists (DORAs) used to treat insomnia, offering a lower abuse potential due to their lack of gamma-aminobutyric acid activity compared to traditional sleep medications. Still, DORAs remain drugs of forensic interest due to their accessibility, long half-lives, and potential risk for next-day residual drowsiness, impaired motor coordination, and decreased alertness, which may feature prominently in cases of driving impairment or drug-facilitated sexual assault. Thus, developing analytical methods to detect these compounds, particularly the more novel lemborexant and daridorexant, is crucial for forensic toxicological testing. This study aimed to develop and validate an LC-MS/MS method for quantifying daridorexant, lemborexant, and suvorexant in blood. An acidic/neutral liquid-liquid extraction (LLE) using n-butyl chloride was optimized to isolate the three DORAs and the internal standard, suvorexant-d6, from bovine blood. All accuracy, precision and key validation parameters met acceptability requirements per ANSI/ASB 036. LLE recovery was >94%, with the calibration range between 0.25-500 ng/mL for all analytes. The LLOQ was 0.25 ng/mL. Matrix effects were -54.2% to 75.7%. Bias ranged between -10.9% to 8.8%, while %CV was <17.7%. Two-fold dilution integrity studies yielded a bias <-7.6%, with %CV <7.6%. Exogenous/endogenous interferences were negligible. Re-injection of the blank following the highest calibrator was free from carryover. Extracts were stable beyond 48 hours when stored at 4°C. A proof-of-concept study using authentic blood samples containing suvorexant, compared to previously reported concentrations, showed no consistent decrease over time, highlighting the need for further studies to determine optimal storage conditions for long-term stability. Although further studies are needed with authentic samples containing the more novel DORAs lemborexant or daridorexant, this validated method supports broader adoption in forensic toxicology, enhancing the detection and monitoring of these emerging sedative-hypnotics in forensic investigations.