title: Quantification of human growth hormone by amino acid composition analysis using isotope dilution liquid-chromatography tandem mass spectrometry.
abstract: We describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-HPLC-mass spectrometry (ID-HPLC-MS) 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 °C for 48 h, where at least 1 μM 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 5.5-6.2 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-HPLC-MS/MS method as expected. The optimized hydrolysis and analytical conditions in our study were suitable for accurate quantification of hGH.
authors.affiliation: Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, 1 Doryong-dong, Yuseong-gu, Daejeon 305-340, South Korea.
authors.forename: Ji-Seon
authors.initials: JS
authors.lastname: Jeong
authors.forename: Hyuk-Min
authors.initials: HM
authors.lastname: Lim
authors.forename: Sook-Kyung
authors.initials: SK
authors.lastname: Kim
authors.forename: Hyung-Keun
authors.initials: HK
authors.lastname: Ku
authors.forename: Kyung-Hwa
authors.initials: KH
authors.lastname: Oh
authors.forename: Sang-Ryoul
authors.initials: SR
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019382:Human Growth Hormone / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D007201:Indicator Dilution Techniques; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D019382:Human Growth Hormone