高效液相色谱-同位素稀释质谱法定量分析人生长激素

摘要

A method was developed for the quantification of human growth hormone ( hGH ) by protein purification and isotope dilution-high performance liquid chromatography mass spectrometry ( HPLC-IDMS ) . The hGH was purified and fractionated by fast protein liquid chromatography ( FPLC ) , then hGH molecular weight was accurately determined by Fourier transform ion cyclotron resonance mass spectrometer ( FTICR-MS). The purified hGH was hydrolyzed and the separation was performed on an KINETEX C18 column (150 mmí2 mm I. D. , 2. 6 μm) with water ( containing 0. 1% TFA) and acetonitrile isocratic solution as the mobile phase at a flow rate of 0 . 2 mL/min and 40℃. The electrospray source was operated in the positive ion mode, and monitored in the multiple reaction monitoring ( MRM) mode. The measured hGH molecular weight by FTICR-MS was only 0. 31 Da difference from theoretical value. Three amino ( proline, valine and phenylalanine) were clearly separated by isocratic elution within 5 min. Under the optimized conditions, the content of hGH was 186 . 80 μg/g with a RSD of 0 . 5%. The detection results of hGH in international comparison by this method were consistent with the reference value, which validated the feasibility of the established method. The developed method is simple, practical, accurate, reliable and reproducible, and can be used for the hGH quantitation of pure hGH CRM to provide reference for the routine detection of hGH.%建立了人生长激素( hGH)纯化分析和高效液相色谱( HPLC)与同位素稀释质谱( IDMS)联用高准确度绝对定量方法。采用快速蛋白液相色谱来分离纯化hGH,以傅里叶变换离子回旋共振质谱仪( FTICR-MS)准确地测定蛋白质的分子质量。纯化的hGH经酸水解后,采用KINETEX C18色谱柱为分析柱,以水(含0.1%三氟乙酸)和乙腈为流动相,等梯度分离,流速0.2 mL/min,温度40℃,采用电喷雾正离子模式进行电离,选择多反应监测模式进行检测,内标法定量分析。结果表明,FTICR-MS所测得的分子量实测值与理论值仅相差0.31 Da,脯氨酸、缬氨酸和苯丙氨酸在液相条件下5 min内达到基线分离,在最优条件下,hGH含量测定结果为186.80 mg/g,相对标准偏差为0.52%。利用本方法参加国际比对,比对结果与参考值等效一致。本方法具有简易、实用的特点,并且准确可靠,可作为hGH纯品标准物质的定值方法,为hGH的日常检测提供参考。