Quantification of aristolochic acids and their DNA adducts in mice kidney and liver by HPLC-MS/MS / 中国中药杂志

This study aims to establish a quantitative method of 4 aristolochic acids-DNA adducts in mice kidney and liver based on high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) for monitoring the content changes of aristolochic acids-DNA adducts. A Shiseido Capcellpak AQ C_(18) column(3 mm×100 mm, 3 μm) was used, with a mixture of 0.2% acetic acid-5 mmol·L~(-1) ammonium acetate as the aqueous phase and methanol as the organic phase for gradient elution. The multiple reaction monitoring(MRM) scanning method under positive mode by electrospray ionization(ESI) was performed for the detection of the aristolochic acids-DNA adducts which formed by combining aristolochic acid Ⅰ/Ⅱ with deoxyadenosine, deoxyguanosine, and deoxycytidine, respectively. Balb/c mice were given Guanmutong extract by gavage, and the relative content of aristolochic acids-DNA adducts in liver and kidney samples were analyzed within 60 days. It was found that the concentration of 4 aristolochic acids-DNA adducts in the kidney was significantly higher than that in the liver, and there were about 15.87 adducts in per 1×10~6 normal deoxynucleosides, which was 4.5-7.5 times than that of the liver. What's more, some adducts can still be detected on the 30 th day after administration. The concentration of the adducts in the liver was highest on the first day after administration, and a second peak appeared during the 7 th to 14 th days. The results indicated that aristolochic acids-DNA adducts are difficult to eliminate in vivo, and it is of great significance to study the mechanism of liver and kidney injury of aristolochic acid.

HPLC fingerprint of Qishen Granules and its multi-component quantitative analysis / 中草药

Objective: To establish the HPLC fingerprint and determine main components of Qishen Granules (QG), so as to provide a scientific basis for its quality control. Methods: HPLC analysis was performed on an Agilent Eclipse plus C18 column (250 mm × 4.6 mm, 5 μm). The gradient elution was performed by the mobile phase consisting of acetonitrile-0.1% formic acid and 0.1% formic acid aqueous with the flow rate of 1.0 mL/min, the detection wavelength was set at 254 nm, and the column temperature was 30 ℃. Fingerprints of ten batches of QG were determined, and the similarities among fingerprints were evaluated. Attributing analysis of the common peaks was achieved by comparing the retention times with the chromatograms of single constituent drugs, and identifications of common peaks were performed on LC-Q TOF-MS and nine components were further confirmed by the reference substances, the content of the nine compounds was subsequently analyzed by HPLC. Results: The similarities of 10 batches of QG were all greater than 0.991. There were 18 common peaks marked in total, peaks 1, 2, 8, 14 and 18 from Astragalus membranaceus var. mongholicus, peaks 3, 4, 5, 6, 7, 9, 10 and 11 from Lonicera japonica, peaks 12, 13, 15 and 16 from Salvia miltiorrhiza, and peaks 17 and 18 from Glycyrrhiza uralensis. Based on the identification of the common peaks, nine components such as chlorogenic acid (peaks 4), calycosin-7-glucoside (peaks 8), isochlorogenic acid B (peaks 9), isochlorogenic acid A (peaks 10), ononin (peaks 14), salvianolic acid B (peaks 15), salvianolic acid A (peaks 16), glycyrrhizic acid (peaks 17), and formononetin (peaks 18) were identified and quantified. The content of the nine components was determined as 6.676-10.213, 0.628-0.963, 1.018-1.886, 1.082-1.972, 0.477-0.790, 11.327-17.788, 0.519-0.908, 2.000-3.638, and 0.010-0.016 mg/g, respectively. Conclusion: The method established in this study shows good characteristics, specificity, and repeatability, which can provide scientific basis for the quality control of QG.