To establish an ultra-high performance liquid chromatography coupled with triple quadrupole mass (HILIC-UPLC-MS/MS) for determination of sixteen nucleosides and nucleobases in Isatidis Radix. Chromatographic separation was carried out on a Acquity UPLC BEH Amide column with gradient elution of acetonitrile (containing 0.1% acetic acid) and water (containing 0.8% acetic acid and 10 mmol L−1 ammonium acetate) at a flow rate of 0.3 mL min−1, the column temperature was set at 35 °C; Waters Xevo™ TQ worked in multiple reaction monitoring mode. Each component were separating in 11 min. All calibration curves were linear (r2 > 0.997) over the tested ranges. The limits of detection of these compounds were 0.02–42.54 ng mL−1. The limits of quantitation of these compounds were 0.05–98.18 ng mL−1. The average recoveries were in the range of 93.81–105.77% with RSD value less than 7.5%. The result showed that almost all of these Isatidis Radix were rich in nucleosides and nucleobases, but their contents were obviously various. The method was simple and fast with high precision, sensitivity and repeatability, which can be used for determination of this type class of nucleosides and nucleobases in other medicinal herbs.
Amino acids, neurotransmitters, purines, and pyrimidines are bioactive molecules that play fundamental roles in maintaining various physiological functions. Their metabolism is closely related to the health, growth, development, reproduction, and homeostasis of organisms. Most recently, comprehensive measurements of these metabolites have shown their potential as innovative approaches in disease surveillance or drug intervention. However, simultaneous measurement of these metabolites presents great difficulties. Here, we report a novel quantitative method that uses hydrophilic interaction ultra-high-performance liquid chromatography–tandem mass spectrometry (HILIC-UPLC–MS/MS), which is highly selective, high throughput, and exhibits better chromatographic behavior than existing methods. The developed method enabled the rapid quantification of 37 metabolites, spanning amino acids, neurotransmitters, purines, and pyrimidines pathways, within 6.5 min. The compounds were separated on an ACQUITY UPLC® BEH Amide column. Serum and brain homogenate were extracted by protein precipitation. The intra- and interday precision of all of the analytes was less than 11.34 %, and the accuracy was between −11.74 and 11.51 % for all quality control (QC) levels. The extraction recoveries of serum ranged from 84.58 % to 116.43 % and those of brain samples from 80.80 % to 119.39 %, while the RSD was 14.61 % or less for all recoveries. This method was used to successfully characterize alterations in the rat brain and, in particular, their dynamics in serum. The following study was performed to simultaneously test global changes of these metabolites in a serotonin antagonist p -chlorophenylalanine (PCPA)-induced anxiety and insomnia rat model to understand the effect and mechanism of PCPA. Taken together, these results show that the method is able to simultaneously monitor a large panel of metabolites and that this protocol may represent a metabolomic method to diagnose toxicological and pathophysiological states.
Overview of experimental workflow
危险品化学品经营许可证（带存储）