Analysis Of The 3S,5S-enantiomer In Atorvastatin Calcium

Atorvastatin calcium is blood lipid lowering agent of statins class, the brand drug of atorvastatin calcium which named LIPITOR（?） was first launched in the US on 17 December 1996, and became the best-selling pharmaceutical by 2003. As a drug for treatment of Primary hypercholesterolemia and mixed hyperlipidemia, atorvastatin calcium has greatly brought the market share growth due to its notable effective and safety. The monograph of Atorvastatin calcium was now listed in current European Pharmacopoeia （EP7.1） and United States Pharmacopoeia （USP34）.Atorvastatin calcium has 2 stereo centers resulting in 4 theoretical stereoisomers including the atorvastatin calcium molecule itself. The current monograph of atorvastatin calcium listed on USP34 and EP7.1 briefly controlled 3S,5R-diastereomer and 3S,5S-enantiomer impurities. 3S,5R-diastereomer could easily separate from atorvastatin by RP-HPLC, which was also listed in the monograph. The key point is separation of 3S,5S-enantiomer. However, we found that the existing literature for the analysis of enantiomers susceptible to interference from other impurities, the better method is needed. On the other hand, if we can control its impurities in the starting material （A9）, the product quality could be control more strictly. This work mainly contains two aspects as follows.The chapter lis a literature review. It introduces the social value of the atorvastatin calcium, and also illustrates the importance for its conduct of the study; additionally introduced several common methods of chiral drugs split; finally introduced the drug atorvastatin calcium isomers analysis of the status quo and the significance of the topic.The chapter 2 of this paper has systematic and deeply study the analysis method of 3S,5S-enantiomer, a determination method of 3S,5S-enantiomer was developed based on screening of the column, detection wavelength and especially the optimization of mobile phase. Firstly, a new normal Phase-HPLC method for determination of atorvastatin calcium 3S,5S-enantiomerbased on monograph of atorvastatin calcium on European Pharmacopoeia（EP） was developed, the author found that by adding an appropriate amount of methanol to the mobile phase resulted in good separating effect after repetition test, and finally the HPLC condition was:the chromatographic separation was performed on CHIRAL PAK AD-H（250x4.6mm,5μm） with the mobile phase consisting of n-Hexane-Methanol-Ethanol-Acetic acid（95:3:2:0.3）, the flow rate was 1.0 mL.min^-1, the detection wavelength was 244nm, the column temperature was 35 ℃, the injection volume was 20μL. Result: Atorvastatin was well separated from 3S,5S-enantiomer, the 3S,5S-enantiomer was also well separated from other impurities; LOQ was 4.77ng, LOD was 0.95ng, Six solutions with LOQ concentration was performed and the RSD of peak area was 5.2%, RSD of retention time was 0.38%, which showed good repeatability; the solution also showed good stability. This method was applied to determine our product, meanwhile the stability experiment was performed, result:3S,5S-enantiomershowed no significant change under stress condition. According to this method, other impurities other than the diastereomers can be better separated, the baseline is relatively stable, and thus can more accurately atorvastatin calcium medicament analyzed enantiomer testing.Chapter 3, A study of isomers in starting material of atorvastatin calcium（A9） was performed due to the demand of CTD regulation filling. According the rule of "Quality control from the source", the 3S,5R-diastereomer and 3S,5S-enantiomer impurities should be controlled in the starting material （A9）. There is no literature reports this by experiment by now. A new Normal Phase-HPLC method was developed for detection of the isomers in A9. Because A9 not absorb in UV, which was a trouble during the development, the author introduced p-Nitro benzoic group to A9 by derivatization, thereby A9 became UV-absorbed, which result in an appropriate analytical method. The photographic condition was mostly the same with Normal Phase-HPLC method for 3S,5S-enantiomer except mobile phase which was consisting of n-Hexane-Ethanol-Diethyl amine （96:4:0.15）. Result: A9 Derivative, A9 enantiomer Derivative and A9 diastereomer Derivative could achieve baseline separation; LOD of A9 Derivative was 0.97ng, LOQ was 1.94ng. LOD of A9 diastereomer Derivative was 1.06ng, LOQ was 2.11ng; and the RSD of A9 diastereomer Derivative detection in parallel experiment （n=6） was 3.5% , which was 3.9% when detected A9 enantiomer derivative; Moreover, a derivatization experiment was designed to confirm the yield when low assay of isomers in A9 （about 0.2%）, and the result consistent with isomers assay before derivatization.the structure of A9 diastereomer derivative and A9 enantiomer derivative was confirmed by various spectra technology such as¹H-NMR and¹H-¹H COSY.Chapter 4, a study of the isomers in telbivudine was performed based on experience above. By adding an appropriate amount of methanol to the mobile phase, the enantiomer and epimer could be well separated from telbivudine, which significant simplified the analysis procedure. An additional method validation including of LOD, LOQ, repeatability and stability of solution was performed.