A simple and specific, rapid resolution liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and validated for determination of CHA in human plasma using neochlorogenic acid as the internal standard. Plasma samples were precipitated with methanol and separated on a Zorbax C18 column (50×2.1 mm, i.d. 1.8 m) at a flow rate of 0.4 mL/min using a gradient mobile phase of methanol-water containing 0.1% formic acid (v/v). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring in negative ESI mode. The method was fully validated over the concentration range of 10–2000 ng/mL. The indicators of interand intra-day precision (RSD%) were all within 10.7%, and the accuracy (RE%) was ranged from -3.0% to 10.6%. Moreover, we evaluated this bioanalytical method by re-analysis of incurred samples as an additional measure of assay reproducibility. This method was successfully applied to pharmacokinetic study of CHA in Chinese subjects with advanced solid tumor after intramuscular injection administration of CHA for injection (CAFI).

This study presented evidence about the immunomodulation function of CHA in patients with recurrent high-grade glioma. In CHA responsive patients, CHA may change the gene expression and upregulate the immune system activity to inhibit tumor development, thereby making it a potential antitumor drug.

Our study provides an insight into the metabolism of CHA injection in humans and supports the clinical human mass balance study. The results showed that M3 and M4 had various interactions against malignant glioma-related targets.

Overall these findings indicated CHA as a potential therapeutic approach to reduce glioma growth through promoting M1-polarized macrophage and inhibiting M2 phenotypic macrophage.