Peters R, Hellenbrand J, Mengerink Y, Van der Wal Sj
DSM Research, Competence Center Molecular Identification & Quantification, P.O. Box 18, MD 6160, Geleen, The Netherlands.
J Chromatogr A. 2004 Mar 26;1031(1-2):35-50. doi: 10.1016/j.chroma.2003.10.100.
2-Nitrophenylhydrazine (2-NPH) is widely used for the derivatization of carboxylic acids, aldehydes and ketones, in industrial and biological samples. These compounds react with 2-NPH to form derivatives, which are separated by high-performance liquid chromatography (HPLC) and detected with diode array detection (DAD). The UV spectra give information about the functionality of the compounds: carboxylic acid or ketone/aldehyde. Most of the eluting compounds in "known" samples are well characterised by the retention time (comparison with those of standards) of the 2-NPH derivative and their UV spectrum. The identification of different unknown 2-NPH derivatives of carboxylic acids, ketones and/or aldehydes, in industrial or biological samples, based on retention time and/or UV spectrum is not sufficient. These unknown 2-NPH compounds can be identified with on-line atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) based on the molecular mass or/and the fragmentation of the derivative. A novel and specific on-line HPLC-DAD-APCI(-)-MS method is described for the determination of carboxylic acids, ketones and aldehydes, after on-line pre-column derivatization with 2-NHP. The fragmentation of different 2-NPH derivatives were investigated and the possibilities of APCI(-)-MS detection were demonstrated by the on-line identification of an unknown derivative, which turned out to be a side product between 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2-NPH in the presence of high concentrations of a cyclic amide in the sample solution.
2-硝基苯肼（2-NPH）广泛用于工业和生物样品中羧酸、醛和酮的衍生化。这些化合物与2-NPH反应形成衍生物，通过高效液相色谱（HPLC）分离，并用二极管阵列检测（DAD）进行检测。紫外光谱提供了有关化合物官能团的信息：羧酸或酮/醛。“已知”样品中的大多数洗脱化合物通过2-NPH衍生物的保留时间（与标准品比较）及其紫外光谱得到很好的表征。基于保留时间和/或紫外光谱来鉴定工业或生物样品中羧酸、酮和/或醛的不同未知2-NPH衍生物是不够的。这些未知的2-NPH化合物可以基于衍生物的分子量或/和碎片通过在线大气压化学电离质谱（APCI-MS）进行鉴定。本文描述了一种新颖且特异的在线HPLC-DAD-APCI(-)-MS方法，用于在2-NHP在线柱前衍生化后测定羧酸、酮和醛。研究了不同2-NPH衍生物的碎片情况，并通过在线鉴定一种未知衍生物证明了APCI(-)-MS检测的可能性，该未知衍生物原来是样品溶液中高浓度环状酰胺存在下1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐与2-NPH之间的副产物。
