title: A novel approach for HPLC determination of 2-cynaoacetamide using derivatization procedure with 2-hydroxyacetophenone as a new useful derivatization reagent.
abstract: A novel and sensitive derivatization procedure for the determination of 2-cynaoacetamide in pharmaceutical samples using liquid chromatography with the fluorescence detection was discovered. The method is based on derivatization of 2-cynaoacetamide using 2-hydroxyacetophenone as a new derivatization reagent. The product of derivatization reaction was isolated and characterized using spectroscopic techniques namely LC-MS, NMR and IR. The structure of 2-cyanoacetamide derivative was unambiguously assigned as a 2-amino-4-phenylfuran-3-carboxamide. Two derivatization systems were optimized in terms of reaction temperature, reaction time, pH and concentration of 2-hydroxyacetophenone, and a new pre- and post-derivatization HPLC methods were developed. The separations on HPLC with pre-column derivatization were accomplished using stationary phase based on a XBridge C18 column (100×4.6, 3.5μm) and isocratic elution using the mobile phase acetonitrile - 0.1% formic acid (30:70, v/v). The separations on the HPLC with post-column derivatization were performed on stationary phase on a TSKgel Amide-80 column (150×4.6mm, 3μm). The mobile phase was a mixture of acetonitrile, methanol and 10mM sodium formate buffer at pH=4.5 in ratio 3:2:95 (v/v). Both HPLC methods were fully validated in terms of linearity, sensitivity (limit of detection and limit of quantification), accuracy and precision according to ICH guidelines. The pre-column derivatization method was linear in the range 1.1-2000μg/l with method accuracy≥98.2% and method precision RSD≤4.8%. The post-column derivatization method was linear in the range 12-2000μg/l. Method accuracy≥96.3% and method precision RSD≤3.5%. Proposed new methods were proved to be highly sensitive, simple and rapid, and were successfully applied to the determinations of 2-cynaoacetamide in pregabalin.
authors.affiliation: Zentiva, k.s. Praha, a Sanofi Company, U Kabelovny 130, 102 37, Praha 10, Czech Republic. Electronic address: michal.dousa@seznam.cz.
authors.forename: Michal
authors.initials: M
authors.lastname: Douša
authors.affiliation: Zentiva, k.s. Praha, a Sanofi Company, U Kabelovny 130, 102 37, Praha 10, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Břicháč
authors.affiliation: Zentiva, k.s. Praha, a Sanofi Company, U Kabelovny 130, 102 37, Praha 10, Czech Republic.
authors.forename: Marcela
authors.initials: M
authors.lastname: Tkadlecová
authors.affiliation: Zentiva, k.s. Praha, a Sanofi Company, U Kabelovny 130, 102 37, Praha 10, Czech Republic.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Man
authors.affiliation: Zentiva, k.s. Praha, a Sanofi Company, U Kabelovny 130, 102 37, Praha 10, Czech Republic.
authors.forename: Josef
authors.initials: J
authors.lastname: Zezula
authors.affiliation: Zentiva, k.s. Praha, a Sanofi Company, U Kabelovny 130, 102 37, Praha 10, Czech Republic.
authors.forename: Josef
authors.initials: J
authors.lastname: Hájíček
authors.affiliation: Zentiva, k.s. Praha, a Sanofi Company, U Kabelovny 130, 102 37, Praha 10, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Pekárek
mesh_terms: D000098:Acetophenones / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009570:Nitriles / Q000032:analysis*; D000069583:Pregabalin / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D000098:Acetophenones; D007202:Indicators and Reagents; D009570:Nitriles; C087908:2'-hydroxyacetophenone; D000069583:Pregabalin; C031594:2-cyanoacetamide
keywords: 2-cyanoacetamide; 2-hydroxyacetophenone; Derivatization; Fluorescence detection; Pregabalin