The Study On Separation Of Lomefloxacin Hydrochloride And Propranolol Enantiomers By RP-HPLC Combining With Pre-column Derivatization

When the enantiomers of chiral drugs enter chiral environment of biosystem, they will be identified as different molecules. So there is enantioselective effect in pharmacodynamics, pharmacokinetics and toxicology. For instance, the two enantiomers of propoxyphene have different pharmacological activity, dextropropoxyphene is a pain-killer but levopropoxyphene is a cough remedy and there are many such examples. Therefore, it is necessary to study the enantiomer of chiral drugs and the single enantiomers have advantages of better therapeutic effect, more security, and so on because of their lower dosage to reduce side effects. So the study of chiral drugs has important scientific value and practical importance. In recent years, the growth rate of single-enantiomer drugs'market is more than 20 % per year which thoroughly illustrates that the study of chiral drugs is very important no matter for widely practical prospect or huge market demanding.Both propranolol and lomefloxacin hydrochloride are drugs with one chiral center. So they have two enantiomers respectively. Besides that, both of them are secondary amines of more reaction activity. Propranolol is widely used in clinic as an importantβ-blocker drug used to treat the diseases of hypertension, arrhythmia, angina. Lomefloxacin hydrochloride is the third generation of quinolones which has been extensively noticed by pharmaceutical industry for the advantages of long half-life, broad- spectrum, high efficiency and low toxicity. However, the study of these drugs is almost just on racemic drugs instead of the single enantiomer till now. Accordingly, the separation of their racemic to carry out the research of the single enantiomer would have important practical significance. So this experiment researched the chiral separation methods of these two secondary amine drugs in vitro and in plasma.The NEIC and GITC are isothiocyanates and widely used to analyze amino acids and amino or alcoholic hydroxyl drugs as chiral derivatization reagent. Therefore, this experiment developed a method which applied GITC as a pre-column derivatization reagent to react with LMFX, then a pair of diastereoisomers produced and separated by RP-HPLC finally. Meanwhile, this experiment also separated the enantiomers of propranolol with GITC as a pre-column derivatization reagent.The work developed in this dissertation can be summarized as following four aspects:1. A pre-column derivatization RP-HPLC method is established to separate the enantiomers of lomefloxacin hydrochloride. GITC, which is a pre-column derivatization reagent reacts with LMFX to generate a pair of diastereoisomers, and then separate these diastereoisomers by RP-HPLC. Chromatographic conditions of RP-HPLC analysis are that mobile phase is V(methanol):V(3mmol·L-1 tetrabutyl ammonium bromide aqueous solution: 5mmol·L-1 Na2HPO4 aqueous solution=1:2)=25:75, the flow rate is 1 mL·min-1 and the detection wavelength is 284nm. Under these chromatographic conditions, a baseline separation (the resolution is 1.52) of LMFX-GITC diastereoisomers was achieved. The diastereoisomers of LMFX-GITC appeared a good linearity relationship between chromatogra- phic peak area and mass concentration over the range of 1.0～27.5μg·mL-1. The linear regression equations of non-enantiomer 1 and 2 were: A= 39.021ρ+10.79(r=0.9993) and A=42.466ρ+1.5748(r=0.9996) respectively. The RSD of within-day and between-day precision were all less than 3 %.2. A pre-column derivatization RP-HPLC method is established to separate the enantiomers of propranolol. GITC is selected as a pre-column derivatization reagent. Let PL react with GITC to generate a pair of diastereoisomers, then separate the diastereoisomers by RP-HPLC. Chromatographic conditions of RP-HPLC analysis are as follows, mobile phase is V(methanol):V(20mmol ? L-1 KH2PO4 aqueous solution)=75:25, the flow rate is 1 mL·min-1 and the detection wavelength is 220nm. Under these chromatographic conditions, a perfect separation of diastereoisomers generated by PL and GITC was achieved and Rs=3.03. The HPLC can be completed within 20 minutes, therefore, it's very fast. The diastereoisomers of PL-GITC appeared a good linearity relationship between chromato- graphic peak area and mass concentration over the range of 1.0~277.78μg·mL-1. The linear regression equations of diastereoisomers S and R were A=42367ρ-2159.5(r=0.9999) and A=57156ρ+59009(r=0.9999) respectively. The RSD of within-day and between-day precision were all less than 3 %.3. An analytical method is established to separate enantiomers of propranolol in plasma. The diastereoisomers of PL-GITC appeared a good linearity relationship within the range of 1.0～277.78μg·mL-1 between chromatographic peak area and mass concentration. The linear regression equations of non-enantiomer S and R were A=886.43ρ-1409.2(r=0.9992) and A=3099.1ρ+2523.7(r=0.9991) respectively. The RSD of within-day and between-day precision were all less than 5 %. Recoveries were 98.95 % and 105.76 % and the RSD were all less than 7.13 %.4. The separation and analytical methods on lomefloxacin enantiomers in plasma have been preliminary studied. The results showed that there were a lot of works left to optimize the method to accomplish the PL enantiomer analysis in vivo.