Background: The accurate detection of remifentanil and sufentanil in biological samples is challenged by their rapid metabolism and instability, complicating clinical and forensic toxicology analysis. This study aimed to evaluate the stability of remifentanil, sufentanil, and their primary metabolites—remifentanil acid and norsufentanil—in human whole blood and urine. Methods: A high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for simultaneous quantification, demonstrating satisfactory linearity (0.10–200 ng/mL, r2 > 0.99), detection limits (0.01–0.20 ng/mL), and recovery rates (85.06–119.42%). Stability was assessed under varying temperatures (4 ◦C, −20 ◦C, −80 ◦C) and anticoagulant conditions (EDTA-K2, sodium heparin, sodium citrate) over 35 days. Results: Remifentanil exhibited significant instability in whole blood, degrading over 50% within 6 h at 4 ◦C, whereas stability was markedly improved at −80 ◦C and in sodium citrate-containing samples. Remifentanil acid remained stable for up to 35 days at −80 ◦C. Sufentanil was generally more stable, particularly at −80 ◦C in both blood and urine, while norsufentanil remained stable for 7 days at −20 ◦C in citrate-anticoagulated blood but degraded rapidly at 4 ◦C. These findings support specific recommendations for sample preservation, including storage at −80 ◦C and the use of sodium citrate as an anticoagulant, to enhance detection reliability in toxicological and pharmacokinetic studies.
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