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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