摘要
目的结合氘代盐酸水解和柱前衍生化,建立超高效液相色谱-四极杆串联飞行时间质谱法(UPLC-Q-TOF-MS)同时测定胸腺法新原料药中9种痕量D-氨基酸含量的方法。方法采用氘代盐酸水解,选择Nα-(2,4-二硝基-5-氟苯基)-L-丙氨酰胺[Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide,FDAA]进行柱前衍生化;选择ACCQ-TAGTM ULTRA C18色谱柱(2.1 mm×100 mm,1.7μm),以0.1%(V/V)甲酸/水溶液-乙腈为流动相,流速为0.3 mL·min^-1,线性梯度洗脱;Xevo G2-S Q-TOF采用电喷雾离子(ESI)源和正离子监测模式,通过标准曲线法对胸腺法新原料药中9种D-氨基酸含量进行准确测定。结果9种D-氨基酸衍生产物在一定范围内进样量与峰响应值呈良好的线性关系(r>0.9912);定量限低至0.05~0.30 pmol;精密度和平均加标回收率等均符合2015年版《中国药典》方法学要求。5家企业的胸腺法新原料药中D-氨基酸含量测定结果为25.16~1322.01μg·g^-1,其中D-Glu、D-Asp、D-Lys和D-Ser的含量较高,分别为1105.45~1322.01、614.15~740.50、358.06~388.76和138.78~291.60μg·g^-1。结论所建立的方法灵敏、高效、可靠,可同时测定胸腺法新中9种痕量D-氨基酸的含量,为该合成多肽药物全面控制消旋肽杂质提供新的方法参考。
OBJECTIVE To develop an ultra-high performance liquid chromatography coupled with quadrupole tandem time-of-flight mass spectrometry method for simultaneous determination of nine trace D-amino acids in thymalfasin,combined with deuterated acid hydrolysis and precolumn-derivatization.METHODS The sample was hydrolyzed by deuterated acid,followed by precolumn-derivatization with Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide(FDAA).The analysis was then performed on an ACCQ-TAGTM ULTRA C18 column(2.1 mm×100 mm,1.7μm),with mobile phase comprising water containing 0.1%formic acid-acetonitrile gradiently eluted at a flow rate of 0.3 mL·min^-1.Nine D-amino acids in thymalfasin were correctly quantified using standard curves by Xevo G2-S Q-TOF coupled with electrospray ion source in positive ion mode.RESULTS The standard curves of the nine D-amino acids derivatives had good linearity in the ranges of the tested concentrations(r>0.9912).The limits of quantitation of all D-amino acids derivatives were as low as 0.05-0.30 pmol.The precision and recoveries met the requirements of Chinese Pharmacopoeia(2015).The contents of D-amino acids in the samples from five companies were determined to be 25.16-1322.01μg·g^-1.Among them,D-glutamate,D-aspartate,D-lysine and D-serine had higher contents,which were 1105.45-1322.01,614.15-740.50,358.06-388.76 and 138.78-291.60μg·g^-1,respectively.CONCLUSION The method is sensitive,efficient and reliable,working well for simultaneous determination of nine trace D-amino acids in thymalfasin,which provides a reference for the comprehensive control of racemic peptide impurities in this synthetic polypeptide drug.
作者
林洁虹
陈钢
邵泓
汪泓
林梅
LIN Jie-hong;CHEN Gang;SHAO Hong;WANG Hong;LIN Mei(Shanghai Institute of Pharmaceutical Industry,China State Institute of Pharmaceutical Industry,Shanghai 201203,China;Shanghai Institute for Food and Drug Control,Shanghai 201203,China)
出处
《中国药学杂志》
CAS
CSCD
北大核心
2020年第11期933-938,共6页
Chinese Pharmaceutical Journal
基金
国家自然科学青年基金项目资助(81803422)。
关键词
胸腺法新
消旋肽
氘代酸水解
柱前衍生化
超高效液相色谱-四极杆串联飞行时间质谱法
thymalfasin
racemic peptide
deuterated acid hydrolysis
pre-column derivatization
ultra-high performance liquid chromatography coupled with quadrupole tandem time-of-flight mass spectrometry
