An HPLC-DAD method combined with second-order calibration based on the alternating trilinear decomposition (ATLD) algorithm with the aid of region selection was developed to simultaneously and quantitatively characterize the synergistic relationships and cumulative excretion of the four bioactive ingredients of Radix Gentianae Macrophyllae in vivo. Although the analytes spectra substantially overlapped with that of the biological matrix, the overlapping profiles between analytes and co-eluting interferences can be successfully separated and accurately quantified by the ATLD method on the basis of the strength of region selection. The proposed approach not only determined the content change but also revealed the synergistic relationships and the cumulative excretion in vivo of the four ingredients in urine and feces samples collected at different excretion time intervals. In addition, several statistical parameters were employed to evaluate the accuracy and precision of the method. Quantitative results were confirmed by HPLC-mass spectrometry. Satisfactory results indicated that the proposed approach can be utilized to investigate the pharmacokinetics of Radix Gentianae Macrophyllae excretion in vivo.
Keywords: HPLC-DAD; Pharmacokinetic analysis; Radix Gentianae Macrophyllae; Second-order calibration.