title: 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 polysaccharide stationary phase.
abstract: 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 x 4.6 mm, 3 microm particle size) in RPLC mode was finally developed. Separations were performed in gradient elution mode starting at 50% A (10 mM, NH(4)COOH at pH 6.5)/50% B (50/50 EtOH/MeCN) to 25% A (10 mM, NH(4)COOH at pH 6.5)/75% B (50/50 EtOH/MeCN). The flow rate was 1.0 mL/min; the column temperature was 50 degrees C; the UV wavelength was 220nm 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.
authors.affiliation: Merck Research Laboratories, Merck and Co. Inc., Rahway, NJ 07065, USA. lili_zhou@merck.com
authors.forename: Lili
authors.initials: L
authors.lastname: Zhou
authors.forename: Vincent
authors.initials: V
authors.lastname: Antonucci
authors.forename: Mirlinda
authors.initials: M
authors.lastname: Biba
authors.forename: Xiaoyi
authors.initials: X
authors.lastname: Gong
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Ge
mesh_terms: D000621:Aminoisobutyric Acids / Q000032:analysis / Q000737:chemistry*; D002219:Carbamates / Q000032:analysis / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D013237:Stereoisomerism; D013449:Sulfonamides / Q000032:analysis / Q000737:chemistry*
chemical_list: C546724:2-amino-N-(2-(1,2-dihydro-1-(methylsulfonyl)spiro(3H-indole-3,4'-piperidin)-1'-yl)-2-oxo-1-((phenylmethyloxy)ethyl))-2-methylpropanamide monomethanesulfonate; C546725:2-tert-butyloxycarbonylamino-N-(2-(1,2-dihydro-1-(methylsulfonyl)spiro(3H-indole-3,4'-piperidin)-1'-yl)-2-oxo-1-((phenylmethyloxy) ethyl))-2-methylpropanamide monomethanesulfonate; D000621:Aminoisobutyric Acids; D002219:Carbamates; D013449:Sulfonamides