| m-Nisoldipine is a new 1, 4-dihydropyridine calcium ion antagonist, to be presented as a couple of enantiomers, which were firstly composed in School of Pharmacy, Hebei Medical University. The main effect of dihydropyridine calcium ion antagonist was relaxing blood vessel, and they showed remarkable effect on hepertension, cardiac angina etc. Nowadays, the study of m-nisoldipine is in pre-clinical research.In the present study, the micellar electrokinetic capillary chromatography (MEKC) method was established to determine m-nisoldipine and its five related substances. The methods for the enantioseparation of m-nisoldipine were developed by the cyclodextrin electrokinetic capillary chromatography (CD-EKC). The capillary electrophoresis was an alternative method to quality control of m-nisoldipine. The metabolites of m-nisolipine were studied by microbial transformation method. After studied the fragmentation pathway of m-nisoldipine and its metabolites, the liquid chromatography - mass spectrometry method was established to simultaneous analysis of m-nisoldipine and its three metabolites in rat plasma. This study is useful to assess the pharmacology and toxicology characters of m-nisolipine and its metabolites, to understand the interactions of drugs in metabolic pathways and to alter the chemical structure of m-nisolipine for new drugs.Part one Determination of m-Nisoldipine by micellar electrokinetic capillary chromatographyObjective: To establish a method to determine m-nisoldipine by micellar electrokinetic capillary chromatographyMethods: An uncoated fused silica capillary column was used, with running buffer of 10 mmol/L sodium dihydrogen phosphate-5 mmol/L borax buffer (containing 30 mmol/L SDS, pH 8.1), at the applied voltage of 30 kV and the detection wavelength of 214 nm.Results: The method of determination of m-nisoldipine was established. The calibration curve for m-nisoldipine was linear in the concentration range of 30～120μg/ml with the detection limit of 0.9μg/ml (r=0.9990).Conclusion: The method, which is simple, fast and low cost, is suitable to determination of m-nisolidpine as HPLC method. It is more suitable to control the quality of m-nisoldpine in the process of manufacture and storage.Part two Capillary electrophoretic enantioseparation of m-nisoldipine using cylodextrinsObjective: To enantioseparate m-nisoldipine using two differentβ-cylodextrins by electrokinetic capillary electrophoresisMethods: The methods of m-nisoldipine enantiomers separation were performed using an anionic cyclodextrin—SBE-β-CD or CM-β-CD as chiral selector. The influences of chiral selectors, pH, buffer type and concentration, addition of organic modifier and temperature on the chiral separation of m-nisoldipine were studied. Moreover, in correlation with the molecular structures of nisoldipine and nimodipine, possible chiral recognition mechanisms were discussed.Results: The elaborated methods of m-nisoldipine enantiomers separation were successfully performed using an anionic cyclodextrin—SBE-β-CD or CM-β-CD as chiral selector. However, the results indicated that SBE-β-CD was a better chiral selector for enantioseparation of the neutral m-nisoldipine. Furthermore, comparing the two SBE-β-CDs, the derivative with higher DS (7.0) induced better enantioresolution than the one with low DS (4.0). As chiral recognition mechanisms, the dihydropyridine ring was the main chiral recognition site for DHPs, whereas the o-ntryl (such as nisoldipine) may affect on the chiral recognition.Conclusion: The method was another choice to enantioseparation of m-nisoldipine, and was the basic work for furthermore study of pharmaceutical formulations and plasma concentration. The chiral recognition mechanisms was confirmed the earlier proposals, and was the theory support for enantioseparation of other DHPs.Part three A screening method for m-nisoldipine and its related substances by micellar electrokinetic capillary chromatographyObjective: To develop a method for the determination of m-nisoldipine and its five related substances using micellar electrokinetic capillary chromatography.Methods: An uncoated fused silica capillary column was used, with the running buffer contained 20 mmol/L borax, 20 mmol/L SDS and 15% (v/v) acetonitrile at pH 8.68, at the applied voltage of 25 kV and the detection wavelength of 214 nm. The key factors were systematically investigated and the forced degradation products of m-nisoldipine were also tested.Results: In this work, an MEKC method was first developed for determination of m-nisoldipine in the presence of its five related substances within 9 min. The concentration of acetonitrile and sodium dodecyl sulfate (SDS) was the important factors. In the hydrogen peroxide (30%, v/v) experiment, an unknown peak which also appeared in other degradation was observed. The accelerated tests indicated that the drug was unstable when degradation was forced using hydrogen peroxide, and was stable against photochemical forced degradation.Conclusion: The method, which is fast and low cost, is an alternative method to control the quality of m-nisoldpine, and is the reference to determine other DHPs simultaneously.Part four The study on metabolites of m-nisoldipine by microbial transformation using HPLC-PDAObjective: To establish the microbial transformation method of m-nisoldipine, identify the chemical structures of the metabolites and propose the possibie metabolic pathways of m-nisoldipineMethods: The metabolites were discovered by comparing the HPLC-PDA chromatograms of 14 fungal strains samples with the corresponding blanks. Then they were prepared by preparative liquid chromatography.Results: The chemical structures of two metabolites were identified by 1H-NMR, 13C-NMR and ESI-MS2. Moreover, the possibie metabolic pathways of m-nisoldipine were proposed.Conclusion: The method of microbial transformation may simulate the drug metabolism of m-nisoldipine in vivo, which can be used as effective supplementary tools of drug metabolism research.Part five Simultaneous analysis of m-nisoldipine and its three metabolites in rat plasma by liquid chromatography - mass spectrometryObjective: To develope a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of m-nisoldipine and its three metabolites in rat plasma, and study the tendency of metabolites after m-nisolipine orally administrated.Methods: The prepared suspensions of m-nisolipine were orally administrated to six rats at a dose of 9 mg/kg. Blood samples were obtained from each rat at 15, 30, 60, 90, 120, 180, 300, 480, 720 and 1440 min after the administration and collected in heparinized centrifuge tube, respectively. Then the samples were centrifuged and separated plasma was employed for liquid-liquid extraction (LLE) prior to analysis. The analysis was performed on a Symmetry RP-C18 column (150 mm×4.6 mm i.d., 5μm) with the mobile phase of acetonitrile-water (72: 68, v/v) at a flow rate of 0.8 ml/min. The Qtrap 3200 triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) scan mode using electrospray ionization (ESI) source. Metabolite M and M2 was detected in positive condition, while m-nisoldipine and M1 in negative condition.Results: A selective and sensitive LC-MS-MS method was firstly established using MRM mode for quantification of m-nisoldipine and its three metabolites in plasma samples. All the validation data, such as accuracy, precision, intra-day and inter-day repeatability, were within the required limits. The tendency of metabolites was similar with the parent drug's. The metabolite M1, the only potential active metabolite, presented in plasma at concentrations approximately equal to the parent compound after m-nisolipine administrated. Additionally, an unknown peak (molecular weight 386) was observed when detected the precursor-product ion transitions m/z 387.3→331.3 using MRM in positive mode. This indicated that the drug may be degraded or biotransformed into another compound which may be the isomer of metabolite M. However, the chemical structure of the unknown peak needs further research.Conclusion: The method was sensitive and suitable for simultaneous analysis of m-nisoldipine and its three metabolites in vivo. This study provides necessary evidences for the research and new drug development of m-nisoldipine.
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