Quantification of human growth hormone by amino acid composition analysis using isotope dilution liquid-chromatography tandem mass spectrometry

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• 作者：Ji-Seon Jeong ; Hyuk-Min Lim ; Sook-Kyung Kim ; Hyung-Keun Ku ; Kyung-Hwa Oh ; Sang-Ryoul Park  ; ;   ; ^{srpark@kriss.re.kr}
• 关键词：Amino acid (AA) analysis ; HPLC&ndash ; MS/MS ; Isotope dilution mass spectrometry (IDMS) ; Acid hydrolysis ; Protein quantification ; Human growth hormone
• 刊名：Journal of Chromatography A
• 出版年：2011
• 出版时间：23 September 2011
• 年：2011
• 卷：1218
• 期：38
• 页码：6596-6602
• 全文大小：467 K

文摘

We describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution¨CHPLC¨Cmass spectrometry (ID¨CHPLC¨CMS) method. Sample purity was confirmed using capillary zone electrophoresis, HPLC and MS. The analyte protein, human growth hormone (hGH), was effectively hydrolyzed by incubation with 8 M hydrochloric acid at 130 ¡ãC for 48 h, where at least 1 ¦ÌM of hGH was treated to avoid possible degradation of released amino acids during hydrolysis. Using a reversed-phase column, the analytes (isoleucine, phenylalanine, proline and valine) were separated within 5 min using an isocratic eluent comprising 10 % acetonitrile containing 0.1 % trifluoroacetic acid. The detection limit (signal to noise ratio of 3) of amino acids was 5.5?.2 fmol per injection. The quantification precision (RSD) of amino acids for intra- and inter-day assays was less than 0.98 % and 0.39 % , respectively. Comparison with other biochemical and instrumental methods revealed substantially higher accuracy and reproducibility of the ID¨CHPLC¨CMS/MS method as expected. The optimized hydrolysis and analytical conditions in our study were suitable for accurate quantification of hGH.