HPLC/MS/MS for quantification of two types of neurotransmitters in rat brain and application: Myocardial ischemia and protection of Sheng-Mai-San

Sheng-Mai-San (SMS), a traditional Chinese multiherbal formula, is widely used in clinic for the treatment of myocardial ischemia (MI) in China. Recently, it has been shown that the change of neurotransmitters in central nervous system is closely related to the pathogenesis of MI, whether SMS might affect the neurotransmitters at central nervous system in MI patients has not been studied yet. In this study, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of two types of neurotransmitters (neuropeptides and monoaminergic neurotransmitters) in rat brain tissue was developed. The instrument was operated under the multiple reaction monitoring (MRM) mode using electrospray ionization (ESI) in the positive ion mode. A good linear relationship with coefficients ≥0.99 was achieved over the concentration ranges of 10–1000 ng mL−1 for 5-hydroxytryptamine (5-HT) and norepinephrine (NE); 2–250 ng mL−1 for methionine-enkephalin (M-ENK) and leucine-enkephalin (L-ENK). Quantification limit for 5-HT and NE was 4.0 ng mL−1; and 2.0 ng mL−1 for M-ENK and L-ENK. The intra- and inter-day precision was less than 15% and accuracy was within ±15%. The analysis revealed significant reductions at the levels of 5-HT (p < 0.01), NE (p < 0.01), M-ENK (p < 0.05) and L-ENK (p < 0.01) in the MI group compared to the control group. These findings demonstrate that myocardial ischemia reduces the concentrations of 5-HT, NE, M-ENK and L-ENK in rat brains, while SMS shows protective effects on MI associated with reversing these four neurotransmitters to sham levels.