Reference compound for use in analysis of levosimendan batches
A kind of reference compound, compound technology, applied in potential genotoxic impurities as a reference compound, the analytical field of determination of potential genotoxic impurities in levosimendan batch samples
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Embodiment 1
[0018] Example 1. [[4-(2-Azido-3-methyl-5-oxotetrahydrofuran-2-yl)phenyl]hydrazono]propanedinitrile (mixture of diastereoisomers) preparation
[0019] 6-(4-Aminophenyl)-4,5-dihydro-5-methyl-3(2H)-pyridazinone (153 g, 0.75 mol) was dissolved in acetic acid (750 ml). Solid sodium nitrite (210 g, 3.0 mol) was slowly added to the solution at 10-20°C. After the addition, the mixture was stirred at 10-20°C for 90 minutes. The reaction mixture was quickly poured into a 20°C solution of malononitrile (150 g, 2.3 mol) and water (1500 ml). The resulting mixture was stirred at 20-25°C for 60 minutes and then filtered, washing with water. The wet solid was extracted successively with tetrahydrofuran (300ml) and ethyl acetate (1900ml). The combined extracts were dried over sodium sulfate and the solvent was evaporated under vacuum. The residue was triturated with ethyl acetate (200ml), filtered and the filtrate evaporated in vacuo. The residue (8.0 g) was purified by chromatography o...
Embodiment 2
[0020] Example 2.[[4-(2-Azido-3-methyl-5-oxotetrahydrofuran-2-yl)phenyl]hydrazono]propane dinitrile (I) as a reference compound in HPLC chromatography Applications
[0021] The HPLC (High Pressure Liquid Chromatography) method for the analysis of compound (I) in the levosimendan starting material was based on a C-8 reverse phase HPLC column with UV detection at 360 nm. The mobile phase consisted of acetonitrile and phosphate buffer pH 2.1. Reagents: 1. Methanol, HPLC grade 2. Sodium dihydrogen phosphate, NaH 2 PO 4 x H 2 O, Merck 3. Orthophosphoric acid, H 3 PO 4 , Merck 4. Phosphate buffer pH 2.1:
[0022] 1.8g sodium dihydrogen phosphate (NaH 2 PO 4 ) was dissolved in water and 2.0 ml phosphoric acid was added. If necessary, adjust the pH with 2M sodium hydroxide or 1M phosphoric acid and dilute to 1000.0ml with water. 5. Dimethyl sulfoxide, BDH6. Acetonitrile, HPLC grade 7. Solvent: a mixture of 200ml methanol and 800ml water 8. [[4-(2-azido-3-methyl-5-oxotetrahy...
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