盐酸头孢他美酯分散片有关物质的色谱-质谱结构鉴定

Separation and identification of related substances in cefetamet pivoxil hydrochloride dispersible tablets by LC-MS method

目的:采用液相色谱-飞行时间质谱法对盐酸头孢他美酯分散片中有关物质进行结构鉴定。方法:采用HPLC梯度洗脱方法对有关物质进行测定,色谱柱为C_(18)(4.6 mm×250 mm,5μm),流动相A为0.005 mol·L^(-1)四丁基氢氧化铵溶液(用磷酸调节p H至4.5),流动相B为乙腈,梯度洗脱,流速1.0mL·min^(-1),检测波长为232 nm;采用液相色谱-飞行时间质谱技术对未知杂质进行鉴定,色谱柱为C_(18)(2.1 mm×100 mm,2.7μm),流动相A为10 mmol·L^(-1)醋酸铵溶液,流动相B为甲醇-乙腈(50∶50),梯度洗脱,流速0.2mL·min^(-1),采用正离子扫描(ESI+)方式,在m/z 100~1 200范围内进行质谱扫描。结果:头孢他美酯与其有关物质能较好地分离,检测出10个有关物质,Ⅰ:(6R,7R)-3-苯乙烯基-7-[(Z)-2-(2-特戊酰氨基-4-噻唑基)-2-(甲氧亚氨基)乙酰氨基]-8-氧代-5-硫杂^(-1)-氮杂双环[4,2,0]辛-2-稀-2-甲酸新戊酰氧甲酯;Ⅱ:(6R,7R)-3-甲基-7-[(Z)-2-(2-氨基-4-噻唑基)-2-(甲氧亚氨基)乙酰氨基]-8-氧代-5-硫杂^(-1)-氮杂双环[4,2,0]辛-2-稀-2-羧酸;Ⅲ:(6R,7R)-3-甲基-7-[(Z)-2-(2-特戊酰氨基-4-噻唑基)-2-(甲氧亚氨基)乙酰氨基]-8-氧代-5-硫杂^(-1)-氮杂双环[4,2,0]辛-2-稀-2-羧酸;Ⅳ:(6R,7R)-3-甲基-7-[(Z)-2-(2-羟甲基氨基-4-噻唑基)-2-(甲氧亚氨基)乙酰氨基]-8-氧代-5-硫杂^(-1)-氮杂双环[4,2,0]辛-2-稀-2-甲酸新戊酰氧甲酯;Ⅴ:(6R,7S)-3-甲基-7-[(Z)-2-(2-氨基-4-噻唑基)-2-(甲氧亚氨基)乙酰氨基]-8-氧代-5-硫杂^(-1)-氮杂双环[4,2,0]辛-2-稀-2-甲酸新戊酰氧甲酯;Ⅵ:(6R,7R)-3-甲基-7-[(Z)-2-[2-(2-甲氨基-4-噻唑基)-2-(甲氧亚氨基)乙酰氨基]-2-(甲氧亚氨基)乙酰氨基]-8-氧代-5-硫杂^(-1)-氮杂双环[4,2,0]辛-2-稀-2-甲酸新戊酰氧甲酯;Ⅶ:(6R,7R)-3-甲基-7-[(Z)-2-[2-(2-氨基-4-噻唑基)-2-(甲氧亚氨基)乙酰氨基]-2-(甲氧亚氨基)乙酰氨基]-8-氧代-5-硫杂^(-1)-氮杂双环[4,2,0]辛-2-稀-2-甲酸新戊酰氧甲酯;Ⅷ:(6R,7R)-3-甲基-7-[(Z)-2-(2-特戊酰氨基-4-噻唑基)-2-(甲氧亚氨基)乙酰氨基]-8-氧代-5-硫杂^(-1)-氮杂双环[4,2,0]辛-2-稀-2-甲酸新戊酰氧甲酯;Ⅸ、Ⅹ:((6S,7S)-3-甲基-7-((Z)-2-(甲氧基亚氨基)-2-(2-((((4-((Z)^(-1)-(甲氧基亚氨基)-2–(6R,7R)-3-甲基-8-氧代-2-(((新戊酰氧基)甲氧基)羰基)-5-硫杂^(-1)-氮杂双环[4.2.0]辛-2-烯-7-基)氨基)-2-氧代乙基)-2-噻唑基)氨基)甲基)氨基)-4-噻唑基)乙酰氨基)-8-氧代-5-硫杂^(-1)-氮杂双环[4.2.0]辛-2-烯-2-羧酸,它们都具有头孢菌素的基本母核7-氨基去乙酰氧基头孢烷酸(7-ADCA),其中有关物质Ⅰ、Ⅲ、Ⅵ尚未见文献报道。结论:色谱-质谱联用技术能有效地鉴定盐酸头孢他美酯分散片中有关物质,为其生产工艺和质量控制提供参考依据。

Objective：Separation and identification of the related substances in cefetamet pivoxil hydrochloride by high performance liquid chromatography coupled with time-of-flight mass spectrometry（HPLC-TOF/MS）.Methods：To establish an HPLC-gradient elution method for the determination of related substances in cefetamet pivoxil hydrochloride dispersible tablets,use a C_（18）（4.6 mm×250 mm,5 μm）column,with the mobile phase A of 0.005 mol·L（-1） tetrabutyl ammonium hydroxide solution（adjusted with phosphoric acid to a p H of 4.5）and the mobile phase B of acetonitrile by gradient elution programme,and with a flow rate of 1.0mL·min（-1）,at the detection wavelength of 232 nm;An HPLC-TOF/MS system（C_（18）,2.1 mm×100 mm,2.7 μm）was performed by gradient elution programme with the mobile phase A of 10 mmol·L（-1） ammonium acetate and the mobile phase B of methanol-acetonitrile（50∶50）at a flow rate of 0.2mL·min（-1）.The analysis was run with electric spray interface under the positive ion mode within the mass range of m/z 100（-1） 200.Results：Each pair of adjacent peaks due to cefetamet pivoxil and individual impurities can be well separated by using established HPLC-gradient elution method.Ten related substances of cefetamet pivoxil hydrochloride containing with cephalosporin mother nucleus（7-ADCA）were detected,three of them had been reported previously. Ⅰ：（pivaloyloxy）methyl（6R,7R）-7-（Z）-2-（2-aminothiazol-4-yl）-2-（methoxyimino）acetamido）-8-oxo-3-methyl-5-thia（-1）-azabicyclo[4.2.0]oct-2-ene-2-carboxylate;Ⅱ：（6R,7R）-7-（（Z）-2-（methoxyimino）-2-（2-pivalamidothiazol-4-yl）acetamido）-8-oxo-3-（（Z）-styryl）-5-thia（-1）-azabicyclo[4.2.0]oct-2-ene-2-carboxylate;Ⅲ：（6R,7R）-7-（（Z）-2-（methoxyimino）-2-（2-pivalamidothiazol-4-yl）acetamido）-3-methyl-8-oxo-5-thia（-1）-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid;Ⅳ：（6R,7R）-7-（（Z）-2-（2-（（hydroxymethyl）amino）thiazol-4-yl）-2-（methoxyimino）acetamido）-3-methyl-8-oxo-5-thia（-1）-azabicyclo[4.2.0]oct-2-ene-2-carboxylate;Ⅴ：（6R,7S）-7-（（Z）-2-（2-aminothiazol-4-yl）-2-（methoxyimino）acetamido）-3-methyl-8-oxo-5-thia（-1）-azabicyclo[4.2.0]oct-2-ene-2-carboxylate;Ⅵ：（6R,7R）-7-（（Z）-2-（methoxyimino）-2-（2-（（Z）-2-（methoxyimino）-2-（2-（methylamino）thiazol-4-yl）acetamido）thiazol-4-yl）acetamido）-3-methyl-8-oxo-5-thia（-1）-azabicyclo[4.2.0]oct-2-ene-2-carboxylate;Ⅶ：（6R,7R）-7-（（Z）-2-（2-（（E）-2-（2-aminothiazol-5-yl）-2-（methoxyimino）acetamido）thiazol-4-yl）-2-（methoxyimino）acetamido）-3-methyl-8-oxo-5-thia（-1）-azabicyclo[4.2.0]oct-2-ene-2-carboxylate; Ⅷ：（6R,7R）-7-（（Z）-2-（methoxyimino）-2-（2-pivalamidothiazol-4-yl）acetamido）-3-methyl-8-oxo-5-thia（-1）-azabicyclo[4.2.0]oct-2-ene-2-carboxylate; Ⅸ and Ⅹ：（6S,7S）-7-（（Z）-2-（methoxyimino）-2-（2-（（（（4-（（Z）（-1）-（methoxyimino）-2-（（（6R,7R）-3-methyl-8-oxo-2-（（（pivaloyloxy）methoxy）carbonyl）-5-thia（-1）-azabicyclo[4.2.0]oct-2-en-7-yl）amino）-2-oxoethyl）thiazol-2-yl）amino）methyl）amino）thiazol-4-yl）acetamido）-3-methyl-8-oxo-5-thia（-1）-azabicyclo[4.2.0]oct-2-ene-2-carboxylate;Conclusion：The hyphenated LC-MS method is useful for the identification of related substances in cefetamet pivoxil hydrochloride dispersible tablets.The results obtained are referential experiences for the optimization of manufacturing process and quality control.