LC-MS/MS combined with in vivo microdialysis sampling from conscious rat striatum for simultaneous determination of active constituents of Yuanhu- Baizhi herb pair and endogenous neurotransmitters: Application to pharmacokinetic and pharmacodynamic study.

The compatibility of Rhizoma Corydalis(Yuanhu,YH) and Radix Angelicae dahuricae (Baizhi,BZ) as a herb pair Yuanhu-Baizhi(YB) can produce synergistic analgesic effect. However, the underlying mechanism of this herb pair compatibility is not elucidated yet. A LC-MS/MS method combined with in vivo microdialysis sampling from awaken rats' striatum was developed to simultaneously quantitate three endogenous neurotransmitters, dopamine(DA), glutamate (Glu), and 5-hydroxytryptamine (5-HT), and nine components of Yuanhu-Baizhi herb pair in rats. The microdialysates were derivatizated with benzoyl chloride and analyzed on Agilent Poroshell 120EC-C18 column. The mobile phase consisted of aqueous solution with 0.05% formic acid and 2.0 mM ammonium formate (A), and acetonitrile (B) delivered at gradient elution. All the twelve analytes were quantified using an electrospray ionization tandem mass spectrometry in the selective reaction monitoring mode. The method was validated following FDA's guidance on bioanalytical validation. The standard curves were linear ( r2 > 0.991) over the corresponding concentration ranges. For three neurotransmitters, the intra-day and inter-day precision (RSD) were not greater than 14.9%, and the intra-day and inter-day accuracy (RE) ranged from -14.2∼ 7.2%. The LLOQ of 5-HT, DA and Glu were 0.50 nM, 0.50 nM and 20.00 nM, respectively. For nine components in Yuanhu-Baizhi herb pair, the intra-day and inter-day precision (RSD) were less than 15.0%, and the intra-day and inter-day accuracy (RE) ranged from -12.5 ∼14.8%. The novel developed method was applied to pharmacokinetic (PK) and pharmacodynamic (PD) study of Yuanhu-Baizhi herb pair in rats. The results showed that the pharmacokinetic behavior of nine components in the herb pair was significantly different with that of single herb, mainly referring to the bioavailability of five alkaloids from Yuanhu increased whereas that of four coumarins decreased in rat striatum. Pharmacodynamic study based on Glu, DA, and 5-HT revealed that the content of neurotransmitters varied when rats were administered Yuanhu-Baizhi herb pair or single herb. PD analysis results demonstrated the compatibility of Yuanhu and Baizhi has the synergistic effect and the effect of attenuation from the point of the neurotransmitters' view. In conclusion, the pharmacokinetic and pharmacodynamic study based on in vivo microdialysis sampling from awaken animals and LC-MS/MS quantitation is in favor of revealing the compatibility mechanism of herb pairs.