| Drusentan (LU135252, (+)-2(S)-(4,6-Dimethoxypyrimidin-2-yloxy)-3- methoxy-3,3-diphenylpropionic acid) is an ET receptor antagonist that selectively blocks the ET type A (ETA) receptor. It is currently undergoing Phase 3 investigation for use in resistant hypertention. Several lines of evidence support the notion that the endothelin system (ETS) is involved in the pathogenesis of hypertension and chronic heart failure. Endothelin (ET) receptor antagonists interrupt the ETS and may be useful for treating cardiovascular disease. At present, the methods for enantiomeric separation of racemic darusentan have not been reported. In the present study, an ES-OVM column was used to develop a method to separate darusentan enantiomers and determine their contents.The aim of this study is to investigate metabolites of darusentan from in vivo and in vitro biotransformation. Structure characterization of metabolites is a key to drug metabolism studies. Recently, high-performance liquid chromatography coupled with mass spectrometry has become a powerful technique for isolating and identifying structures of unknown metabolites present in biological samples. On this basis, HPLC-MS/MS was applied to analyze the metabolites of darusentan in rat urine, feces, plasma, bile and rat tissue samples for the first time. We aimed to characterize metabolites of darusentan and to establish possible metabolic pathways in rat. Part one Enantiomeric separation and determination of racemic darusentan by HPLCObjective: To establish an HPLC method for the enantiomeric separation and determination of racemic darusentan.Methods: An ES-OVM column(4.6 mm×150 mm, 5μm) was used with the mobile phase of 30 mmol/L phosphate buffer (pH 3.35)-acetonitrile (60:40) at the detection wavelength of 248 nm. The flow rate was 0.8 ml/min and the column temperature was fixed on 20 ?C.Results: The calibration curves of darusentan and its (R)-isomer were linear in the concentration range of 0.01~0.10 mg/ml. The recoveries were 99.6% and 99.5%, with RSDs of 0.75% and 0.82%. Both of the limits of detection were 1ng.Conclusion: The methods we established are proved to be accurate and sensitive, and can be used for the chiral separation and determination of racemic darusentan.Part two Study on metabolites of darusentan from in vitro biotransformationObjective: To find metabolites of darusentan from in vitro biotransformations by multiple means, such as microbial transformation, incubation of liver microsome, anaerobic culture of intestinal tract bacteria, and prepare pure metabolite as far as possible.Methods: (1)Microbial transformation experiment: We selected a robust strain for the transformation of darusentan by screening twelve kinds of strain. Chromatographic conditions: Reverse-phase silica gel symmetry Column with the column temperature set at 25 ?C. The mobile phase of a gradient mixture of A (methanol) and B (0.025% formic acid in water) programmed according to the following protocol: 0-25 min, 10%A~70%A; 25-55 min, 70%A~86%A; 55-60 min, 86%A~98%A; 60-70 min, 98%A and finally 10% A maintained for 8 min. The flow rate was 1 ml/min. We studied the mass fragmentation patterns of the metabolite in the positive and negative ion mode by full scan mass spectra, product ion scan (PI) and elucidated its structure. (2)Culture solution of intestinal bacteria were produced from rat feces and then incubated with darusentan to find whether there were metabolites. (3)Liver microsome experiment: Darusentan was added to the incubation liquid which was prepared according to the pertinent literature, the incubation culture was treated and analyzed by HPLC-DAD and HPLC-MS/MS. The metabolite was characterized by MRM-IDA-EPI modes on a hybrid triple quadrupole-linear ion trap mass spectrometer. Chromatographic conditions: Reverse-phase silica gel symmetry Column with the column temperature set at 30 ?C. The mobile phase of a gradient mixture of A (0.05% formic acid in methanol) and B (0.05% formic acid in water) programmed according to the following protocol: 0-5 min, 20%A~60%A; 5-20 min, 60%A~95%A; 20-34 min, 95%A; 34-35 min, 95%A~20%A and finally 20%A maintained for 8 min. Mass conditions: ion spray voltage 5.5 kV; turbo spray temperature 650 ?C; nebulizer gas (gas 1), 60 psi; heater gas (gas 2), 65 psi; curtain gas 25 psi.Results: (1)Microbial transformation experiment: After screening, Cunninghamella elegans, AS 3.970 was selected as exclusive to transform darusentan. One metabolite of darusentan was characterized by full scan mass spectra and product ion scan (PI). (2)We fonud no metabolites in culture solution of intestinal bacteria. (3)Liver microsome experiment: we found one metabolite in the rat liver microsome for both SD and Wistar rat, and there was no difference between the two. The metabolite of darusentan was characterized as (4,6-Dimethoxypyrimidin-2-yloxy)-3-hydroxyl-3,3- diphenyl- propionic acid by MRM-IDA-EPI modes on a hybrid triple quadrupole- linear ion trap mass spectrometer.Conclusion: From microbial transformation and rat liver microsome experiment we found two metabolites of darusentan and elucidated their structure. But we did not get a pure product from in vitro biotransformation. Part three Study on metabolites of darusentan from in vivo biotransformationObjective: To summarize fragmentation rules of darusentan and develop a high sensitive and efficient liquid chromatography-mass spectrometry (LC-MS) method for detection and characterization of the metabolites of darusentan in rat.Methods: First, we studied the mass fragmentation patterns of darusentan in the positive and negative ion mode by full scan mass spectra, product ion scan (PI) and precursor ion scan(PREC). On the basis of the summarized new rules, metabolites were characterized by the combined use of the PREC-IDA-EPI and MRM-IDA-EPI modes on a hybrid triple quadrupole- linear ion trap mass spectrometer, for the first time. Chromatographic conditions: Reverse-phase silica gel symmetry Column (C18, 150 mm×4.6 mm, 5.0μm) with the column temperature set at 30 ?C. The mobile phase of a gradient mixture of A (0.05% formic acid in methanol) and B (0.05% formic acid in water) programmed according to the following protocol: initial 20% A increased to 60% in 5 min, increased to 95% in 15 min; maintained at 95% for 14 min; decreased to 20% A in 1 min and finally 20% A maintained for 8 min .The total run time per sample was 43 min. The flow rate was 0.8 ml/min with an injection volume of 10μl. Mass conditions: ion spray voltage 5.5 kV; turbo spray temperature 650 ?C; nebulizer gas (gas 1), 60 psi; heater gas (gas 2), 65 psi; curtain gas 25 psi.Results: According to the summarized fragmentation rules, retention times, accurate molecular weights and characteristic fragment ions, ten metabolites of darusentan were characterized by combination use of the two modes. Except spleen and heart, metablites were found in urine, fence, bile, blood, liver, gaster, pulmo, nephros and small intestine.Conclusion: The main metabolism site was liver and the principal excretion pathways for metabolites of darusentan were bile and urine. In this study, a high sensitive and effective LC-MS method for on-line qualitative analysis of the metobolites has been developed. |
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