To establish an ultra-high performance liquid chromatography tandem high resolution mass spectrometry (UHPLC-HRMS) method for the determination of serum demthylzeylasteral concentration in diabetic nephropathy patients, which treated by tripterygium wilfordii polyglycosides tablets (TGTs), and to apply this method in the exploratory study of clinical pharmacodynamic marker.

Serum samples and ¹⁸O-demethylzeylasteral which was used as the internal standard were processed by solid phase extraction method and took the supernatant for the determination. Chromatographic separation of the analytes was accomplished using a BEH C₁₈ column (2.1 mm×50.0 mm, 1.7 μm) under gradient elution, where the mobile phase consisted of a mixture of 0.1% formic acid aqueous solution and acetonitrile, as well as 0.1% formic acid aqueous solution. The flow rate was set at 0.3 mL·min^-1 and the column temperature at 30 ℃. The mass spectrometer was equipped with an electrospray ionization source with positive multiple reaction monitoring mode. The specificity, standard curve and lower limit of quantification, precision and recovery rate, matrix effect and stability were investigated. This method was applied to determine the serum concentration of demethylzeylasteral from 15 patients with oral TGTs. Receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were utilized to explore the correlation between serum concentration of demethylzeylasteral and efficacy in treating diabetic nephropathy.

The calibration curve of demethylezylasteral was linear over the range of 1-200 ng·mL^-1, and the lower limit of quantification was 1 ng·mL^-1. The precision and accuracy relative standard deviation of intra-day and inter-day was less than 10% and 15%, respectively. The average absolute recovery was more than 89%, no matrix effect has been affected and all stability tests met the acceptance criteria. The AUC of the efficacy of TGTs predicted by serum demethylzeylasteral quantitation was 0.66 (95% confidence interval was 0.53-0.78), the cut-off value was 61.73 ng·mL^-1, the sensitivity was 83.3% and the specificity was 46.7% (P<0.05).

The UHPLC-HRMS method established in this study has the advantages of simple operation, high sensitivity, accurate and reliable results, which can be suitable for determining serum concentration of demethylzsylasteral and studying clinical drug efficacy markers of TGTs.