Simultaneous Determination of Four Monoamine Neurotransmitters and Seven Effective Components of Zaoren Anshen Prescription in Rat Tissue using UPLC-Ms/Ms

Background: Zaoren Anshen Prescription (ZAP) is widely used as a classic Chinese Traditional Medicine (TCM) prescription for the treatment of palpitations and insomnia in China. Some studies have identified the main active components for its anti-insomnia effect and observed changes of some endogenous components that are closely related to its anti-insomnia effect. However, simultaneous determination of four monoamine neurotransmitters and seven effective components of ZAP and the investigation of their distribution in tissues by using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) have not been reported. Methods: An ultra-performance liquid chromatography with tandem mass spectrometry method was developed and validated for simultaneous quantification of four monoamine neurotransmitters (norepinephrine, dopamine, 5-hydroxy tryptamine and 5-hydroxyindoleacetic acid) and seven prescription components (danshensu, protocatechualdehyde, spinosin, 6``` -feruylspinosin, salviaolic acid B, schisandrin and deoxyschisandrin) in rats’ tissues. Tissue samples were prepared by protein precipitation with acetonitrile. Chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of acetonitrile and 0.01% formic acid water. An electrospray ionization triple quadrupole concatenation mass spectrometer was set to switch between positive and negative modes in single run time. All the components were quantitated by multiple-reaction monitoring scanning. Results: The lower limits of quantitation for all analytical components were 0.78 ng/mL-1.99 ng/mL in the heart, liver, spleen, lung, kidney and brain. All the calibration curves displayed good linearity (r> 0.99544). The precision was evaluated by intra-day and inter-day assays, and the relative standard deviation (RSD) values were all within 12.67%. The relative errors of the accuracy were all within ± 19.88%. The recovery ranged from 76.00% to 98.78% and the matrix effects of eleven components were found to be between 85.10% and 96.40%. Conclusion: This method was successfully applied to study the distribution of seven components from ZAP and the concentration changes of four monoamine neurotransmitters after oral ZAP in six tissues.