Suh Jung H, Kim Robert, Yavuz Burcu, Lee Daniel, Lal Ashutosh, Ames Bruce N, Shigenaga Mark K
Nutrition and Metabolism Center, Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Oct 15;877(28):3418-27. doi: 10.1016/j.jchromb.2009.06.041. Epub 2009 Jul 2.
The total concentrations of four sulfur amino acid (SAA) metabolite redox couples (reduced and oxidized forms of homocysteine, cysteine, glutathione, and cysteinylglycine) in human blood are assayed with a simple and sensitive method by liquid chromatography-electrospray positive ionization-tandem mass spectrometry. To prevent ex vivo thiol oxidation, iodoacetamide (IAM) is used immediately following the blood draw. To selectively enrich for S-carboxyamidomethylated SAA, and other cationic amino acids metabolites, proprietary strong cation-exchange solid phase extraction tips are used. Analytes are further derivatized with isopropylchloroformate (IPCF) to esterify the amino and the carboxylic groups. Double derivatization with IAM and IPCF improves the reverse phase liquid chromatography separation of SAA metabolites. The use of detection mode of multiple-reaction monitoring (MRM) allows sensitive and specific simultaneous detection of SAA. The internal standards used to account for the matrix effects of human plasma and erythrocytes were plant glutathione analogue, homoglutathione, and stable isotopes of cystine and homocystine. The method was validated for its linearity, accuracy, and precision. Excellent linearity of detection (r(2)>0.98) was observed over relevant ranges for plasma and erythrocyte samples, and the limits of detection were established to be between 5 and 20nM. Relative standard deviations were <9% for within-day variations and <15% for between-day variations. The method was used to assess thiol redox states in plasma and erythrocytes isolated from healthy subjects and thalassemia patients.
采用液相色谱 - 电喷雾正离子串联质谱法，通过一种简单且灵敏的方法测定人血液中四种含硫氨基酸（SAA）代谢物氧化还原对（同型半胱氨酸、半胱氨酸、谷胱甘肽和半胱氨酰甘氨酸的还原型和氧化型）的总浓度。为防止离体硫醇氧化，采血后立即使用碘乙酰胺（IAM）。为选择性富集S - 羧酰胺甲基化的SAA和其他阳离子氨基酸代谢物，使用了专有的强阳离子交换固相萃取柱。分析物用异丙基氯甲酸酯（IPCF）进一步衍生化，以酯化氨基和羧基。IAM和IPCF的双重衍生化改善了SAA代谢物的反相液相色谱分离。采用多反应监测（MRM）检测模式可实现对SAA的灵敏且特异的同时检测。用于校正人血浆和红细胞基质效应的内标物为植物谷胱甘肽类似物、高谷胱甘肽以及胱氨酸和同型胱氨酸的稳定同位素。该方法在线性、准确性和精密度方面得到了验证。在血浆和红细胞样品的相关范围内观察到了出色的检测线性（r(2)>0.98），检测限确定为5至20nM。日内变异的相对标准偏差<9%，日间变异的相对标准偏差<15%。该方法用于评估从健康受试者和地中海贫血患者分离的血浆和红细胞中的硫醇氧化还原状态。
