Reaserch Of New Method To Detect CATR, ATR And Transformation Between Them During Decoction

Objective:?1?A new method of reversed phase ion pair chromatography for the determination of carboxyatractyloside and atractyloside in Xanthii Fructus was being established.This method will solve the problems of large impurity interference and lacking in baseline separation.?2?Establishment of enriching and purifing method for the detection of atractyloside in patent medicines with reversed phase ion pair chromatography.?3?Establishment of UPLC-MS/MS method to determine carboxyatractyloside and atractyloside in Xanthii Fructus.Apply this method to the safety evaluation and quality control of patent medicines containing Xanthii Fructus.?4?This study will explore dynamic variation of the main toxicants in Fructus Xanthii during decocting and provide reference for the production and process control of patent medicines containing Xanthii Fructus.Methods:?1?The HPLC method was used with Agilent Eclipse XDB-C₁₈?250 mm × 4.6 mm,5.0?m?column;the mobile phase consisted of acetonitrile and 0.15%sodium dihydrogen phosphate?0.12% tetrabutyl ammonium hydroxide,pH3.5 was adjusted by phosphoric acid??39:61?,the UV detection wavelength was 203 nm.?2?Atractyloside in ultrasonic extraction of patent medicine was collected and purified by D101 macroporous resinand WAX SPE column in turn.?3?Agilent 1290 UPLC coupled with Agilent 6460 three quadrupole mass spectrometer was used.ESI negative mode was performed in multiple reaction monitoring?MRM?.The quantitative ions for carboxyatractyloside,atractyloside and chloramphenicol are m/z 769.1 ? 688.8,725.0 ? 644.9,320.8 ? 151.7,respectively.?4?Analyzing the contents of carboxyatractyloside and atractyloside during decocting with reversed phase ion pair chromatography and find out the changes among them.Results:?1?Under the above conditions,the calibration curves of carboxyatractyloside and atractyloside were good linear in the ranges of0.15-3.7 ?g?r = 0.9999?and 0.10-2.5 ?g?r = 1.000?.The average recoveries were 101.4%?RSD 1.7%?and 101.6%?RSD 1.2%?,respectively.?2?Atractyloside was collected and purified by this sample treating method.The average recovery was 95.7% with the RSD being1.2%.The results of 5 patent medicines were 226.3,464.6,386.1,66.0,1044.7 ?g·g-1.?3?Carboxyatractyloside and atractyloside had good linear relationship in the range of 9.73-973 ng·mL^-1 and 50.5-4040 ng·mL^-1,respectively.The average recovery were 98.9%?RSD4.3%?and 102.4%?RSD3.8%?.The contents of carboxyatractyloside and atractyloside in 8samples from different origins and varies of formulations could be detected successfully.?4?Samples of water decoction: As the reflux time going on,in rude Xanthii Fructus,the contents of both two toxicantsincrease in 2 hours attributing to dissolution,then atractyloside continued to rise but carboxyatractyloside decreased sharply duing to transformation;in fried Xanthii Fructus,there was no carboxyatractyloside and the content of atractyloside increased quickly in 2 hours,then came to a plateau,which implies that atractyloside was stable.Conclusion:?1?Reversed phase ion pair chromatography is simple and applicable for the good separation effect of carboxyatractyloside and atractyloside.?2?Atractyloside could be collected and purified by D101 macroporous resin and WAX solid phase extraction column.This method could be applied to pretreatment of complex samples.?3?This established UPLC-MS/MS method could be applied to determine carboxyatractyloside and atractyloside for safety evaluation and quality control of patent medicines contain Xanthii Fructus.?4?Carboxyatractyloside would transform into atractyloside to reduce the toxicity by extending decoction time.