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Application of on‐line nano‐liquid chromatography/mass spectrometry in metabolite identification studies
Author(s) -
Liu Jingzhou,
Zhao Zhiyang,
Teffera Yohannes
Publication year - 2012
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.5332
Subject(s) - chemistry , chromatography , high performance liquid chromatography , metabolite , mass spectrometry , tandem mass spectrometry , liquid chromatography–mass spectrometry , protein precipitation , in vivo , resolution (logic) , biochemistry , microbiology and biotechnology , artificial intelligence , computer science , biology
Metabolite identification is an important part of the drug discovery and development process. High sensitivity is necessary to identify metabolic products in vitro and in vivo . The most common method utilizes standard high‐performance liquid chromatography (4.6 mm i.d. column and 1 mL/min flow rate) coupled to tandem mass spectrometry (HPLC/MS/MS). We have developed a method that utilizes a nano‐LC system coupled to a high‐resolution tandem mass spectrometer to identify metabolites from in vitro and in vivo samples. Using this approach, we were able to increase the sensitivity of analysis by approximately 1000‐fold over HPLC/MS. In vitro samples were analyzed after simple acetonitrile precipitation, centrifugation, and dilution. The significant improvement in sensitivity enabled us to conduct experiments at very low substrate concentrations (0.01 μM), and very low incubation volumes (20 μL). In vivo samples were injected after simple dilution without any pre‐purification. All the metabolites identified by conventional HPLC/MS/MS were also identified using the nano‐LC method. This study demonstrates a very sensitive approach to identifying phase I and II metabolites with throughput and separation equivalent to the standard HPLC/MS/MS method. Copyright © 2012 John Wiley & Sons, Ltd.