Determination of Paeoniflorin in Rat Plasma by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry and its Application to a Pharmacokinetic Study.
In this study, a rapid and highly sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was established for the quantification of paeoniflorin in rat plasma. The analyte and the internal standard (IS), hyperoside, were extracted from rat plasma via liquid-liquid extraction with ethyl acetate. LC separation was carried on a Phenomenex Luna C18 column within 2.5 min. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization and operated in the multiple reaction monitoring and negative ion modes. The precursor to product ion transitions monitored for paeoniflorin and the IS were m/z 524.8→449.0 and 463.1→300.0, respectively. The assay was validated with a linear range of 0.02~25.6 μg/mL for paeoniflorin. The intra- and inter-day precisions (relative standard deviation%) were within 9.7%, and the recoveries were greater than 65.2%. Paeoniflorin was proven to be stable during all sample storage, preparation and analytical procedures. The method was successfully applied to a pharmacokinetic study of the Baixiangdan Capsule in eight female rats.
Simultaneous UPLC-MS/MS determination of four components of Socheongryong-tang tablet in human plasma: Application to pharmacokinetic study.
The purpose of this study was to develop a method for simultaneous analysis of schizandrin, ephedrine, paeoniflorin, and cinnamic acid as constituents of Socheongryong-tang tablet in human plasma using UPLC-MS/MS. These four components were separated using water containing 0.01% formic acid and methanol as a mobile phase by gradient elution at a flow rate of 0.3 mL/min with a HALO-C18 column (2.1 mm × 100 mm, 2.7 μm particle size). Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique operated in multiple reaction monitoring mode. Mass transitions were m/z 432.9 → 384.1 for schizandrin, 165.8 → 148.1 for ephedrine, 525.0 → 449.2 for paeoniflorin, 146.8 → 102.9 for cinnamic acid, and 340.0 → 324.0 for papaverine as internal standard. Liquid-liquid extraction and protein precipitation with ethyl acetate-methanol (1:2, v/v) were used to obtain these four components. Chromatograms showed high resolution, sensitivity, and selectivity without interference by plasma constituents. Calibration curves of schizandrin, ephedrine, paeoniflorin, and cinnamic acid in human plasma ranged from 0.02 to 8 ng/mL, 0.5 to 200 ng/mL, 0.2 to 80 ng/mL, and 1 to 400 ng/mL, respectively. Calibration curves of each analyte displayed excellent linearity, with correlation coefficients > 0.99. For all four components, both intra- and inter-day precisions (CV%) were <5.99%. The accuracy was 99.35-103.30% for schizandrin, 98.48-104.38% for ephedrine, 97.06-103.34% for paeoniflorin, and 99.97-104.36% for cinnamic acid. This analytical method developed in this study satisfied the criteria of international guidance. It could be successfully applied to pharmacokinetic studies of schizandrin, ephedrine, paeoniflorin, and cinnamic acid after oral administration of Socheongryong-tang tablet to humans.
Inhibition of l-type voltage dependent calcium channels causes impairment of long-term potentiation in the hippocampal CA1 region in vivo
Long-term potentiation (LTP), in the hippocampal CA1 region is dependent on postsynaptic calcium influx. It is generally accepted that calcium influx occurs via activation of the NMDA receptor channel complex. However, studies in vitro using a high-frequency stimulus protocol (> or =200 Hz) demonstrated previously an NMDA receptor-independent form of LTP that is dependent upon activation of L-type voltage-dependent calcium channels (VDCCs). Here we have investigated a role for L-type VDCCs in LTP in vivo. Two structurally different, L-type VDCC blockers, verapamil (1, 3 and 10 mg/kg) and diltiazem (1, 10 and 20 mg/kg), depressed the induction of LTP in a dose-dependent manner. Increased activation of L-type VDCCs by Bay K 8644, an L-type agonist, however, did not enhance LTP. The NMDA receptor antagonist D-AP5 (5 and 20 mM injected i.c.v) impaired, but failed to block fully LTP in vivo. A reduced level of LTP could still be recorded following co-administration of verapamil and D-AP5. The level of LTP recorded was similar to that observed in the presence of either verapamil (10 mg/kg) or D-AP5 alone. These results suggest that activation of the NMDA receptor/channel and L-type VDCCs are involved in the induction of LTP in area CA1 in vivo. However, it appears that activation of other receptor/channels may also play a role in this form of LTP.