Coumarins are the primary bioactive ingredients in Radix Glehniae, named Beishashen in China, which possesses many pharmacological activities, including anticancer, anti-inflammation and antivirus activities. In the present study, we employed a sensitive and selective high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantification of nine coumarins in rat urine and bile: scopoletin (1), xanthotoxol (2), xanthotoxin (3), psoralen (4), isoimpinellin (5), bergapten (6), oxypeucedanin (7), imperatorin (8) and isoimperatorin (9). Pimpinellin (10) was used as the internal standard (IS). The urine and bile samples were pretreated by liquid-liquid extraction with ethyl acetate (EtOAc). The chromatographic separation was carried out on a C₁₈ column with gradient elution. The detection of analytes was performed on a tandem mass system equipped with a turbo ion spray interface in positive mode using multiple-reaction monitoring (MRM). The specificity, linearity, accuracy, precision, recovery, matrix effect and several stabilities were validated for coumarins in rat urine and bile samples. The results showed that this method is robust, specific and sensitive and it can successfully fulfill the requirements of the excretion study of the nine coumarins in Radix Glehniae.
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