目的: 用HPLC-MS/MS结合超滤法测定人血清中丙戊酸钠和奥氮平相互作用后的游离药物浓度,考察丙戊酸钠和奥氮平蛋白结合率的相互影响,指导临床合理用药。方法: 用超滤法得到丙戊酸钠和奥氮平相互作用后游离血清样品,以同位素丙戊酸-d6和奥氮平-d8为内标,血清样品加甲醇涡旋后离心取上清液,测定血清中丙戊酸钠和奥氮平浓度,最后计算蛋白结合率。丙戊酸采用KinetexXB-C18（50 mm×3 mm,3.5 μm）色谱柱,以水（A）-甲醇（0.5 mmol · L-1氟化铵）（B）为流动相,等度洗脱,流速0.35 mL · min-1,柱温40 ℃,进样量5 µL,质谱采用电喷雾离子源（ESI源）,单离子监测负离子模式进行检测。奥氮平采用KinetexPolarC18（100 mm×2.1 mm,2.6 µm）色谱柱,以水（0.2%甲酸）（A）-甲醇（B）为流动相,梯度洗脱,流速0.40 mL · min-1,柱温50 ℃,进样量5 µL,质谱采用电喷雾离子源（ESI源）,多反应监测正离子模式进行检测。结果: 奥氮平对丙戊酸钠血浆蛋白结合率有显著性影响（P<0.05）,随着奥氮平药物浓度升高,丙戊酸钠体外血浆蛋白结合率逐渐下降,当丙戊酸钠药物浓度增加后,其蛋白结合率则降低。而低、中、高浓度（25、50、100 μg · mL-1）的丙戊酸钠对奥氮平蛋白结合率均没有显著影响。结论: 丙戊酸钠与奥氮平联用存在血浆蛋白竞争性相互作用,且奥氮平血浆蛋白结合能力强于丙戊酸钠,导致联用后丙戊酸游离药物浓度增加,药理效应及毒副作用增强,建议临床连用丙戊酸钠和奥氮时监测丙戊酸钠游离药物浓度,减少不良反应的发生,提高丙戊酸用药的有效性和安全性。
Objective: To determine the concentration of free drugs in human serum after the interaction between sodium valproate and olanzapine using HPLC-MS/MS combined with ultrafiltration, and to investigate the mutual influence of their protein binding ratesto provide guidance for rational clinical drug use. Methods: Free serum samples after the interaction between sodium valproate and olanzapine were obtained by ultrafiltration method. Isotopes valproate-d6 and olanzapine d8 were used as the internal standard. The serum samples were vortexed with methanol, centrifuged, and the supernatantwas collected to determine the concentration of sodium valproate and olanzapine in the serum. Finally, the protein binding rate was calculated. Valproic acid was analyzed using a Kinetex XB-C18(50 mm×3 mm, 3.5 μm) column with water (A) -methanol (0.5 mmol · L-1 ammonium fluoride)(B) as the mobile phase under isocratic elution at a flow rate of 0.35 mL · min-1. The column temperature was set to 40 ℃, and the injection volume was 5 µL. Mass spectrometry detection was performed using an electrospray ionization source (ESI source) in single ion monitoring negative ion mode. Olanzapine was determined using a KinetexPolarC18 (100 mm×2.1 mm, 2.6 µm) column with water (0.2% faformic acid)(A) -methanol (B) as the mobile phase by gradient elution at the flow rate of 0.40 mL · min-1. The column temperature was 50 ℃, and the injection volume was 5 µL. Mass spectrometry detection was performed using an electrospray ionization source (ESI source) in multiple reaction monitoring positive ion mode. Results: Olanzapine had a significant effect on the plasma protein binding rate of sodium valproate (P<0.05). With the increase of olanzapine concentration, the plasma protein binding rate of sodium valproate gradually decreased in vitro, and with the increase of sodium valproate concentration, its protein binding rate also decreased. However, sodium valproate at low, medium and high concentrations (25, 50, 100 μg · mL-1) had no significant effect on the protein binding rate of olanzapine. Conclusion: The combination of sodium valproate and olanzapine results in a competitive plasma protein-binding interaction, with olanzapine exhibiting a stronger binding affinity than sodium valproate. This leads to an increased concentration of free valproate after co-administration, and enhances its pharmacological effects and potential side effects. It is recommended to monitor the concentration of free sodium valproate when combined with olanzapine, to reduce the risk of adverse reactions and improve the efficacy and safety of valproic acid treatment.
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