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Abstract Background We aimed to determine the time interval between alfentanil and rocuronium administration, at a 50% probability of preventing pain-induced withdrawal movement from rocuronium injection (TimeAR50). Methods A total of 64 patients scheduled for general anesthesia were enrolled in this study (33 men and 31 women). Anesthesia was induced with target-controlled infusion of propofol, at an effect-site target concentration of 3 μg/mL. Then, alfentanil 15 μg/kg was injected for 30 s. After 60 s, rocuronium 0.6 mg/kg was administered to the first patient. The Dixon’s up-and-down method was used to determine the time interval for each subsequent patient (interval of 5 s). Mean arterial pressure (MAP) and heart rate (HR) were recorded at three time points: T0, pre-induction; T1, before rocuronium injection; and T2, 1 min after rocuronium injection. Results The TimeAR50 ± standard deviation (SD) was 5.6 ± 3.7 s and 21.9 ± 5.6 s in the male and female patients, respectively. Based on the probit regression, the TimeAR50 was 4.7 s (95% confidence interval [CI], 1.2–7.6 s) and 20.3 s (95% CI, 7.7–26.1 s) in the male and female patients, respectively. The TimeAR95 was 10.6 s (95% CI, 7.7–25.3 s) and 35.0 s (95% CI, 28.1–95.5 s) in the male and female patients, respectively, with significantly higher values in females than in males (P |