Konieczna Lucyna, Roszkowska Anna, Niedźwiecki Maciej, Bączek Tomasz
Department of Pharmaceutical Chemistry, Medical University of Gdańsk, Gen. J. Hallera 107, 80-416 Gdańsk, Poland.
Department of Pediatrics, Hematology and Oncology, Medical University of Gdańsk, Dębinki 7, 80-211 Gdańsk, Poland.
J Chromatogr A. 2016 Jan 29;1431:111-121. doi: 10.1016/j.chroma.2015.12.062. Epub 2015 Dec 23.
A simple and sensitive method using dispersive liquid-liquid microextraction (DLLME) followed by liquid chromatography coupled to mass spectrometry (LC-MS) with a hydrophilic interaction chromatography (HILIC) column was developed for the simultaneous determination of 13 compounds of different polarities, comprising monoamine neurotransmitters (dopamine, norepinephrine, epinephrine and serotonin) along with their respective precursors and metabolites, in human urine samples. The microextraction procedure was based on the fast injection of a mixture of ethanol (disperser solvent) and dichloromethane (extraction solvent) into a human urine sample, forming a cloudy solution in the Eppendorf tube. After centrifugation, the sedimented phase was collected and subsequently analyzed by LC-HILIC-MS in about 12min without a derivatization step. The separation was performed on an XBridge Amide™ BEH column 3.0×100mm, 3.5mm and the mobile phase consisted of phase A: 10mM ammonium formate buffer in water pH 3.0 and phase B: 10 mM ammonium formate buffer in acetonitrile, under gradient program elution. Tyrosine, tryptophan, 5-hydroxytryptophan, dopamine, epinephrine, norepinephrine, serotonin, 3-methoxytyramine, 5-hydroxyindole-3-acetic acid, 3,4-dihydroxy-l-phenylalanine and norvaline (internal standard) were detected in the positive ionization mode. While vanillylmandelic acid, homovanillic acid, 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxybenzylamine (internal standard) were detected in the negative ionization mode. Parameters influencing DLLME and LC-HILIC-MS were investigated. Under the optimum conditions, the proposed method exhibited a low detection limit (5-10ngmL(-1)), and good linearity with R between 0.9991 and 0.9998. The recoveries in human urine samples were 99.0%±3.6%. for the 13 studied biogenic amines with intra- and inter-day RSDs of 0.24-9.55% and 0.31-10.0%, respectively. The developed DLLME-LC-MS method could be successfully applied for the determination of trace amounts of polar endogenous compounds, such as neurotransmitters, in human urine samples, including samples with a reduced volume obtained from pediatric patients.
建立了一种简单灵敏的方法，采用分散液液微萃取（DLLME），随后使用亲水作用色谱（HILIC）柱的液相色谱-质谱联用（LC-MS），用于同时测定人尿液样本中13种不同极性的化合物，包括单胺类神经递质（多巴胺、去甲肾上腺素、肾上腺素和5-羟色胺）及其各自的前体和代谢物。微萃取过程基于将乙醇（分散剂溶剂）和二氯甲烷（萃取溶剂）的混合物快速注入人尿液样本中，在Eppendorf管中形成浑浊溶液。离心后，收集沉淀相，随后在约12分钟内通过LC-HILIC-MS进行分析，无需衍生化步骤。分离在XBridge Amide™ BEH柱3.0×100mm，3.5μm上进行，流动相由A相：pH 3.0的10mM甲酸铵水溶液和B相：乙腈中的10mM甲酸铵缓冲液组成，采用梯度洗脱程序。酪氨酸、色氨酸、5-羟色氨酸、多巴胺、肾上腺素、去甲肾上腺素、5-羟色胺、3-甲氧基酪胺、5-羟吲哚-3-乙酸、3,4-二羟基-L-苯丙氨酸和正缬氨酸（内标）在正离子模式下检测。而香草扁桃酸、高香草酸、3,4-二羟基苯乙酸和3,4-二羟基苄胺（内标）在负离子模式下检测。研究了影响DLLME和LC-HILIC-MS的参数。在最佳条件下，所提出的方法具有低检测限（5-10ngmL⁻¹），良好的线性，R在0.9991至0.9998之间。人尿液样本中13种研究的生物胺的回收率为99.0%±3.6%，日内和日间相对标准偏差分别为0.24-9.55%和0.31-10.0%。所建立的DLLME-LC-MS方法可成功应用于测定人尿液样本中痕量的极性内源性化合物，如神经递质，包括从儿科患者获得的少量样本。
