Dicussions Of Evaluation Strategies On HPLC Method For Impurity Profile Analysis Of Complex Samples

Control of impurities in drugs is the key to ensure people’s medication safety. Inrecent years, the concept of “impurity profile control”, which emphasizes that everyimpurity in the drug be under reasonable control according to its characteristics, isbecoming mature. As the first part of impurity profile control, the separation ofimpurities in complex samples is particularly important. Related substances control isthe main content of impurity profile control, and RPLC is the main method for relatedsubstances analysis throughout all national pharmacopoeias. For the development andevaluation of HPLC method, how to understand impurity profile of analysis object,how to find the key factors affecting the separation of critical peak pairs and how todetermine the retention time of every impurity have been the focus problems thatdraw drug control agencies, drug factory, researchers’attention.Based on the Rumsfeld Quadrants theory in risk analysis, we introduced a newstrategy, which is called “four quadrants evaluation strategy”, to evaluate HPLCmethod for complex samples. By answering the following two questions, the impurityseparation situations could be divided into four quadrants. The first question iswhether the impurity is known or unknown, and the second one is whether theimpurity could be detected by the HPLC method. The four quadrants representdifferent situations. The fist, second, third, and fourth one mean detected knownimpurities, detected unknown impurities, undetected unknown impurities andundetected known impurities respectively. The first quadrant evaluates the HPLCmethod’s detection ability of known impurities, and all known impurities should belocated in the first quadrant for a good HPLC method. If a known impurity is notdetected by the HPLC method, then it would be located in the fourth quadrant, whichrepresents the shortcomings of the method. The second quadrant evaluates the HPLCmethod’s detection ability of unknown impurities, and for a good HPLC method newemerging impurities should be detected effectively by the method. If the newemerging impurity, which is also unknown, is not detected by the method, then itwould be located in the third quadrant.Four quadrants evaluation strategy includes the following five parts. First,understanding the complexity of impurity profile actively by summarizing the rules of biosynthetic pathway, production process and degradation mechanism. Second,optimizing HPLC method to improve its detection ability for complex samples,namely to increase the impurities located in the first and second quadrants and reducethe risk deriving from the third quadrant. The optimization process was achieved byusing DOE method on the base of full exploration of key factors affecting theseparation of critical peak pairs. Third, indentifying known impurities and suggestingchemical structures of unknown impurities by combining column switching LC-MStechnology with MS fragmentation rules of known impurities. Fourth, predicting theretention time of the fourth quadrant impurity in the chromatographic system byexploring the QSRR of impurity profile. Fifth, developing a new separation methodwith totally different selectivity to validate the separation ability of the new HPLCmethod. And this part is not involved in the dissertation.In this dissertation, by choosing the macrolides and penicillin antibiotics whichcontains rich impurities as the analysis objects, four quadrants evaluation strategy wasfirstly introduced to evaluate the HPLC method for complex impurity profile.Through the above study, the HPLC methods of the two types of antibiotics wereimproved and the understanding of their impurity profiles were perfected.