Abstract：
A gas chromatography-mass spectrometry (GC-MS) method was developed and validated in relevant concentration ranges for the simultaneous measurement of L-lysine (Lys, L) and its Nε- and Nα-methylated (M), Nε- and Nα-acetylated (Ac), Nε-carboxymethylated (CM) and Nε-carboxyethylated (CE) metabolites in human urine. Analyzed Lys metabolites were the post-translational modification (PTM) products Nε-mono-, di- and trimethyllsine, Nε-MML, Nε-DML, Nε-TML, respectively, Nα-ML, Nε-AcL, Nα-AcL, and its advanced glycation end-products (AGEs) Nε-CML, Nε-CM-[2,4,4-2H3]Lys (d3-CML), Nε-CEL and furosine. AGEs of arginine (Arg) and cysteine (Cys) were also analyzed. De novo synthesized trideutero-methyl esters (R-COOCD3) from unlabelled amino acids and derivatives were used as internal standards. Native urine samples (10 µL aliquots) were evaporated to dryness under a stream of nitrogen. Analytes were esterified using 2 M HCl in methanol (60 min, 80 °C) and subsequently amidated by pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C). The generated methyl ester-pentafluoropropionyl (Me-PFP) derivatives were reconstituted in borate buffer and extracted immediately with toluene. GC-MS analyses were performed by split-less injection of 1-µL aliquots, oven-programmed separation and negative-ion chemical ionization (NICI). Mass spectra were generated in the scan mode (range, m/z 50-1000). Quantification was performed in the selected-ion monitoring (SIM) mode using a dwell time of 50 or 100 ms for each ion. The GC-MS method was suitable for the measurement of Lys and all of its metabolites, except for the quaternary ammonium cation Nε-TML. The Me-PFP derivatives of Lys, Arg and Cys and its metabolites eluted in the retention time window of 9 to 14 min. The derivatization of Nε-CML, d3-CML and Nε-CEL was accompanied by partial Nε-decarboxylation and formation of the Me-PFP Lys derivative. The lowest derivatization yield was observed for Nε-DML, indicating a major role of the Nε-DML group in Lys derivatization. The GC-MS method enables precise (relative standard deviation, RSD < 20%) and accurate (bias, < ± 20%) simultaneous measurement of 33 analytes in human urine in relevant concentration ranges. We used the method to measure the urinary excretion rates of Lys and its PTM metabolites and AGEs in healthy black (n = 39) and white (n = 41) boys of the Arterial Stiffness in Offspring Study (ASOS). No remarkable differences were found indicating no ethnic-related differences in PTM metabolites and AGEs except for Nε-monomethyllysine and S-(2-carboxymethylcysteine).
摘要：
开发了气相色谱-质谱（GC-MS）方法，并在相关浓度范围内进行了验证，以同时测量L-赖氨酸（Lys，L)及其Nε-和Nα-甲基化(M)，Nε-和Nα-乙酰化（Ac），人尿液中的Nε-羧甲基化（CM）和Nε-羧乙基化（CE）代谢物。分析的Lys代谢物是翻译后修饰（PTM）产物Nε-单-，二-和三甲基吡啶，Nε-MML,Nε-DML,Nε-TML,分别,Nα-ML,Nε-AcL,Nα-AcL,及其晚期糖基化终产物(AGEs)Nε-CML，Nε-CM-[2,4,4-2H3]Lys(d3-CML)，Nε-CEL和糠氨酸。还分析了精氨酸(Arg)和半胱氨酸(Cys)的AGEs。从未标记的氨基酸和衍生物从头合成三氘甲酯(R-COOCD3)用作内标。将天然尿液样品(10μL等分试样)在氮气流下蒸发至干燥。使用2MHCl在甲醇中酯化分析物（60分钟，80°C），然后在乙酸乙酯中通过五氟丙酸酐酰胺化（30分钟，65°C)。将产生的甲酯-五氟丙酰基(Me-PFP)衍生物在硼酸盐缓冲液中重构,并立即用甲苯萃取。通过1微升等分试样的无分割进样进行GC-MS分析,烘箱程序分离和负离子化学电离（NICI）。在扫描模式下产生质谱(范围，m/z50-1000)。在选择离子监测(SIM)模式下进行定量,每个离子的停留时间为50或100ms。GC-MS方法适用于Lys及其所有代谢物的测定,除了季铵阳离子Nε-TML。Lys的Me-PFP衍生物，Arg和Cys及其代谢物在9至14分钟的保留时间窗口中洗脱。Nε-CML的衍生化，d3-CML和Nε-CEL伴随着部分Nε-脱羧和Me-PFPLys衍生物的形成。对于Nε-DML观察到最低的衍生产率，表明Nε-DML基团在Lys衍生中的主要作用。GC-MS方法可实现精确(相对标准偏差，RSD<20%)和准确(偏差，<±20％）在相关浓度范围内同时测量人体尿液中的33种分析物。我们使用该方法测量了后代研究（ASOS）的健康黑人（n=39）和白人（n=41）男孩的Lys及其PTM代谢物和AGEs的尿排泄率。没有发现显着差异，表明除Nε-单甲基赖氨酸和S-（2-羧甲基半胱氨酸）外，PTM代谢物和AGEs没有种族相关差异。
