Simultaneous enantioseparation of a basic active pharmaceutical ingredient compound and its neutral intermediate using reversed phase and normal phase liquid chromatography with a new type of polysacc

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• 作者：Lili Zhou ; Vincent Antonucci ; Mirlinda Biba ; Xiaoyi Gong ; Zhihong Ge
• 关键词：Simultaneous enantioseparations ; Chiral liquid chromatographic method ; Active pharmaceutical ingredient ; Polysaccharide stationary phase
• 刊名：Journal of Pharmaceutical and Biomedical Analysis
• 出版年：2010
• 出版时间：5 January 2010
• 年：2010
• 卷：51
• 期：1
• 页码：153-157
• 全文大小：551 K

文摘

Simultaneous enantioseparation of a basic API compound, (R)-2-Amino-N-[2-[1,2-dihydro-1-(methylsulfonyl) spiro [3H-indole-3,4′-piperidin]-1′-yl]-2-oxo-1-[(phenylmethyloxy) ethyl]-2-methylpropanamide monomethanesulfonate (compound-A) and its neutral penultimate intermediate, (R)-2-BOC-Amino-N-[2-[1,2-dihydro-1-(methylsulfonyl) spiro [3H-indole-3,4′-piperidin]-1′-yl]-2-oxo-1-[(phenylmethyloxy) ethyl]-2-methylpropanamide monomethanesulfonate (compound-B) was investigated using reversed phase (RPLC) and normal phase liquid chromatography (NPLC). After an initial screening, a Sepapak-4 column, a new type of polysaccharide chiral stationary phase (CSP), was selected for further method development based on hits on separation selectivity for both compounds under RPLC and NPLC. After comparing the pros and cons, a method utilizing the Sepapak-4 chiral column (150 mm × 4.6 mm, 3 μm particle size) in RPLC mode was finally developed. Separations were performed in gradient elution mode starting at 50 % A (10 mM, NH₄COOH at pH 6.5)/50 % B (50/50 EtOH/MeCN) to 25 % A (10 mM, NH₄COOH at pH 6.5)/75 % B (50/50 EtOH/MeCN). The flow rate was 1.0 mL/min; the column temperature was 50 °C; the UV wavelength was 220 nm and the mass spectrometric detection was APCI in the positive ionization mode. The reaction mixture sample was directly diluted in ethanol. Baseline enantioseparation were achieved for both compound-A and its intermediate simultaneously with resolution greater than 2.0. The method was validated in terms of injection precision, linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, and ruggedness. The specificity of the method was further evaluated by spiking a mixture of enantiomers of compound-A and its intermediate into a reaction matrix containing all of the synthetic reagents. No matrix interference was observed across the elution windows of compound-A and its intermediate. Additionally, the peak purity of each enantiomer was evaluated by mass spectra, indicating the specificity of the method.