Separation and identification of Se-methylselenogalactosamine -: a new metabolite in basal human urine -: by HPLC-ICP-MS and CE-nano-ESI-(MS)2
Separation and identification of Se-methylselenogalactosamine -: a new metabolite in basal human urine -: by HPLC-ICP-MS and CE-nano-ESI-(MS)2
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DOI：
10.1039/b406589a
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发表时间：
2004-01-01
影响因子：
3.4
通讯作者：
Gammelgaard, B
中科院分区：
文献类型：
--
作者：
Bendahl, L;Gammelgaard, B
从人尿中分离出三种微量代谢物。其中两种经纳米电喷雾质谱（nESI-MS）鉴定分别为硒甲基硒- n -乙酰氨基葡萄糖胺和硒甲基硒半乳糖胺。在制备反相高效液相色谱分离之前，将人尿池冻干并在甲醇中重组。除了主要的尿液代谢物半甲基硒- n -乙酰半乳糖胺外，该系统还分离出7种以上的次要代谢物，并用ICP-MS进行检测。分离三个代谢物组分，在反相系统中重新色谱，并在不同的分离系统中进一步纯化，然后进行nESI-MS分析。通过CE-nESI-MS分析，其中一个组分在m/z 285附近识别出特征硒模式，（MS） 2片段分别在m/z 267、173和155处产生a片段。根据现有的数据，不可能鉴定出这种硒化合物。第二组分中硒化合物与硒甲基硒- n -乙酰氨基葡萄糖共洗脱。该化合物经分离纯化后，经nESI-MS鉴定。第三部分通过离子对色谱和阴离子交换色谱进一步纯化，重组并进行CE-nESI-MS。该化合物的m/z为258，（MS） 2在m/z为162处产生一个片段，对应于甲基硒的损失。这表明该化合物的结构为硒甲基硒半乳糖胺。为了验证该化合物的性质，将相应的n -乙酰己糖胺水解制备了硒甲基硒半乳糖胺和硒甲基硒葡萄糖胺。这些标准品的质谱与纯化后的尿液化合物的质谱相同。尿硒化合物与硒甲基硒半乳糖胺在反相色谱系统中共洗脱，能够分离硒甲基硒半乳糖胺和硒甲基硒葡萄糖胺。对未补充硒的志愿者的基础尿液样本的分析显示，硒甲基硒半乳糖胺的存在，而硒甲基硒- n -乙酰半乳糖胺的代谢物是硒补充后尿液中的主要代谢物。因此，这种新的代谢物可能是基础尿中的主要代谢物。
Three minor metabolites were isolated from human urine. Two of these were identified by nano electrospray ionisation mass spectrometry (nESI-MS) as Se-methylseleno-N-acetylglucosamine and Se-methylselenogalactosamine, respectively. A human urine pool was lyophilised and reconstituted in methanol prior to fractionation by preparative reversed phase HPLC. In addition to the major urinary metabolite, Semethylseleno-N-acetylgalactosamine, more than seven minor metabolites were separated by this system and detected by ICP-MS. Three of the metabolite fractions were isolated, re-chromatographed in the reversed phase system and further purified in different separation systems before analysis by nESI-MS. By CE-nESI-MS analysis of one of the fractions, the characteristic selenium pattern was recognized around m/z 285 and ( MS) 2 fragmentation resulted in a fragments at m/z 267, 173 and 155, respectively. It was not possible to identify this selenium compound on basis of the available data. The selenium compound in the second fraction showed co-elution with a Se-methylseleno-N-acetylglucosamine standard. The identity of this compound was verified by nESI-MS after further purification by size exclusion chromatography. The third fraction was further purified by ion-pair and anion exchange chromatography, reconstituted and subjected to CE-nESI-MS. The m/z of the compound was 258 and ( MS) 2 resulted in a fragment at m/z 162, corresponding to loss of methylselenium. This indicated that the structure of the compound was Se-methylselenogalactosamine. To verify the identity of the compound, the Se-methylselenogalactosamine and the Se-methylselenoglucosamine were prepared by hydrolysis of the corresponding N-acetylhexosamines. The mass spectra of these standards were identical and also identical to the mass spectra of the purified urine compound. The urine selenium compound co-eluted with Se-methylselenogalactosamine in a reversed phase chromatographic system able to separate Se-methylselenogalactosamine and Se-methylselenoglucosamine. Analysis of basal urine samples from volunteers who had not been supplemented with selenium showed the presence of Se-methylselenogalactosamine when only traces of the metabolite Se-methylseleno-N-acetylgalactosamine, which is the major metabolite in urine after selenium supplementation was present. Hence, this new metabolite may be the main metabolite in basal urine.