Pastor-Belda M, Fernández-García A J, Campillo N, Pérez-Cárceles M D, Motas M, Hernández-Córdoba M, Viñas P
Department of Analytical Chemistry, Faculty of Chemistry, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, E-30100 Murcia, Spain.
Department of Legal and Forensic Medicine, Faculty of Medicine, Biomedical Research Institute (IMIB-Arrixaca), University of Murcia, Spain.
J Chromatogr A. 2017 Aug 4;1509:43-49. doi: 10.1016/j.chroma.2017.06.041. Epub 2017 Jun 16.
Glyoxal (GO) and methylglyoxal (MGO) are α-oxoaldehydes that can be used as urinary diabetes markers. In this study, their levels were measured using a sample preparation procedure based on salting-out assisted liquid-liquid extraction (SALLE) and dispersive liquid-liquid microextraction (DLLME) combined with gas chromatography-mass spectrometry (GC-MS). The effect of the derivatization reaction with 2,3-diaminonaphthalene, the addition of acetonitrile and sodium chloride to urine, and the DLLME step using the acetonitrile extract as dispersant solvent and carbon tetrachloride as extractant solvent were carefully optimized. Quantification was performed by the internal standard method, using 5-bromo-2-chloroanisole. The intraday and interday precisions were lower than 6%. Limits of detection were 0.12 and 0.06ngmL, and enrichment factors 140 and 130 for GO and MGO, respectively. The concentrations of these α-oxoaldehydes in urine were between 0.9 and 35.8ngg levels (creatinine adjusted). A statistical comparison of the analyte contents of urine samples from non-diabetic and diabetic patients pointed to significant differences (P=0.046, 24 subjects investigated), particularly regarding MGO, which was higher in diabetic patients. The novelty of this study compared with previous procedures lies in the treatment of the urine sample by SALLE based on the addition of acetonitrile and sodium chloride to the urine. The DLLME procedure is performed with a sedimented drop of the extractant solvent, without a surfactant reagent, and using acetonitrile as dispersant solvent. Separation of the analytes was performed using GC-MS detection, being the analytes unequivocal identified. The proposed procedure is the first microextraction method applied to the analysis of urine samples from diabetic and non-diabetic patients that allows a clear differentiation between both groups using a simple analysis.
乙二醛（GO）和甲基乙二醛（MGO）是α-氧代醛，可作为糖尿病尿液标志物。在本研究中，采用基于盐析辅助液液萃取（SALLE）和分散液液微萃取（DLLME）结合气相色谱-质谱联用（GC-MS）的样品制备方法测定其含量。仔细优化了与2,3-二氨基萘的衍生化反应、向尿液中添加乙腈和氯化钠的效果，以及使用乙腈提取物作为分散溶剂和四氯化碳作为萃取溶剂的DLLME步骤。采用内标法，以5-溴-2-氯苯甲醚进行定量分析。日内和日间精密度均低于6%。GO和MGO的检测限分别为0.12和0.06 ng/mL，富集因子分别为140和130。这些α-氧代醛在尿液中的浓度在0.9至35.8 ng/g水平之间（肌酐校正）。对非糖尿病患者和糖尿病患者尿液样本中分析物含量的统计比较表明存在显著差异（P = 0.046，共调查24名受试者）），尤其是MGO，其在糖尿病患者中含量更高。与以往方法相比，本研究的新颖之处在于基于向尿液中添加乙腈和氯化钠的SALLE处理尿液样本。DLLME步骤是用一滴沉淀的萃取溶剂进行的，不使用表面活性剂试剂，并使用乙腈作为分散溶剂。采用GC-MS检测对分析物进行分离，可明确鉴定分析物。所提出的方法是应用于糖尿病和非糖尿病患者尿液样本分析的第一种微萃取方法，通过简单分析即可清晰区分两组。
