Institute of Toxicology, Core Unit Proteomics, Hannover Medical School, 30625 Hannover, Germany.
Molecules. 2021 May 27;26(11):3206. doi: 10.3390/molecules26113206.
In consideration of its relatively constant urinary excretion rate, creatinine (2-amino-1-methyl-5-imidazol-4-one, MW 113.1) in urine is a useful endogenous biochemical parameter to correct the urinary excretion rate of numerous endogenous and exogenous substances. Reliable measurement of creatinine by gas chromatography (GC)-based methods requires derivatization of its amine and keto groups. Creatinine exists in equilibrium with its open form creatine (methylguanidoacetic acid, MW 131.1), which has a guanidine and a carboxylic group. Trimethylsilylation and trifluoroacetylation of creatinine and creatine are the oldest reported derivatization methods for their GC-mass spectrometry (MS) analysis in human serum using flame- or electron-ionization. We performed GC-MS studies on the derivatization of creatinine (d-creatinine), [-H]creatinine (d-creatinine, internal standard) and creatine (d-creatine) with ,-(trimethylsilyl)trifluoroacetamide (BSTFA) using standard derivatization conditions (60 min, 60 °C), yet in the absence of any base. Reaction products were characterized both in the negative-ion chemical ionization (NICI) and in the positive-ion chemical ionization (PICI) mode. Creatinine and creatine reacted with BSTFA to form several derivatives. Their early eluting ,,-(trimethylsilyl) derivatives (8.9 min) were found to be useful for the precise and accurate measurement of the sum of creatinine and creatine in human urine (10 µL, up to 20 mM) by selected-ion monitoring (SIM) of / 271 (d-creatinine/d-creatine) and / 274 (d-creatinine) in the NICI mode. In the PICI mode, SIM of / 256, / 259, / 272 and / 275 was performed. BSTFA derivatization of d-creatine from a freshly prepared solution in distilled water resulted in formation of two lMate-eluting derivatives (14.08 min, 14.72 min), presumably creatinyl-creatinine, with the creatininyl residue existing in its enol form (14.08 min) and keto form (14.72 min). Our results suggest that BSTFA derivatization does not allow specific analysis of creatine and creatinine by GC-MS. Preliminary analyses suggest that pentafluoropropionic anhydride (PFPA) is also not useful for the measurement of creatinine in the presence of creatine. Both BSTFA and PFPA facilitate the conversion of creatine to creatinine. Specific measurement of creatinine in urine is possible by using pentafluorobenzyl bromide in aqueous acetone.
肌酸酐（2-氨基-1-甲基-5-咪唑-4-酮，MW 113.1）在尿液中的排泄率相对稳定，因此是校正许多内源性和外源性物质尿排泄率的有用内源性生化参数。通过基于气相色谱（GC）的方法可靠地测量肌酸酐需要衍生其胺和酮基。肌酸酐与肌酸（甲基胍基乙酸，MW 131.1）处于平衡状态，肌酸具有胍基和羧酸基。三甲硅烷基化和三氟乙酰化是最早报道的用于火焰或电子电离的人血清中肌酸酐和肌酸的 GC-质谱（MS）分析的衍生化方法。我们使用标准衍生化条件（60 分钟，60°C）对肌酸酐（d-肌酸酐）、[-H]肌酸酐（d-肌酸酐，内标）和肌酸（d-肌酸）与,-（三甲基硅基）三氟乙酰胺（BSTFA）进行 GC-MS 研究，但没有使用任何碱。在负离子化学电离（NICI）和正离子化学电离（PICI）模式下对反应产物进行了表征。肌酸酐和肌酸与 BSTFA 反应生成几种衍生物。它们早期洗脱的,-（三甲基硅基）衍生物（8.9 分钟）被发现可用于通过选择离子监测（SIM）在 NICI 模式下精确和准确地测量人尿中肌酸酐和肌酸的总和（10 µL，高达 20 mM），/ 271（d-肌酸酐/d-肌酸）和 / 274（d-肌酸酐）。在 PICI 模式下，进行了 / 256、/ 259、/ 272 和 / 275 的 SIM。在蒸馏水中新鲜制备的溶液中，BSTFA 衍生化 d-肌酸生成两种洗脱时间较长的衍生物（14.08 分钟，14.72 分钟），可能是肌酸酰肌酸，肌酸酰基残基以烯醇形式（14.08 分钟）和酮形式（14.72 分钟）存在。我们的结果表明，BSTFA 衍生化不允许通过 GC-MS 对肌酸和肌酸酐进行特异性分析。初步分析表明，五氟丙酸酐（PFPA）也不适用于在肌酸存在下测量肌酸酐。BSTFA 和 PFPA 都促进肌酸转化为肌酸酐。在水丙酮中使用五氟苯甲溴可实现对尿中肌酸酐的特异性测量。
