Increasing Throughput in Metabolite Identification Studies with Ultra Performance Liquid Chromatography / Tandem Mass Spectrometry

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Erin Chambers;Mary E. Lame*;Marian Twohig;Diane M. Diehl;Amit S. Kalgutkar*
AAPS 2006; October 30; San Antonio TX
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Indomethacin; Indomethacin amide; Buspirone
Human Liver Microsomes incubation extract
ACQUITY UPLC™ BEH C18 1.7 µm N/A 2.1 mm 50 mm ACQUITY UPLC™ BEH Shield RP18 1.7 µm N/A 2.1 mm 50 mm
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To evaluate the utility of UPLC ™/tandem MS for the rapid metabolite identification for drugs with diverse physiochemical properties.


Metabolic profiles of test compounds in NAPDH-supplemented human liver microsomes were then analyzed with a UPLC ™ system coupled to a triple quadrupole mass spectrometer operating in the electrospray ionization positive mode. Precursor and product ion scanning modes were used to evaluate metabolite formation and characterization. The LC system consisted of either an embedded polar group column or a C 18column. The dimensions of both columns were 2.1 x 50 mm, with 1.7 µm particles.


Utility of the UPLC ™/tandem MS approach in the rapid detection and characterization of metabolites of structurally diverse drugs with a broad spectrum of physiochemical attributes (acidic, basic and neutral compounds) was examined. Compared with traditional LC-MS/MS, UPLC ™/MS/MS demonstrates significant advantages such as faster separations, sharper peaks, and increased sensitivity with minimal sample preparation and without changing the detection system. The increased speed and resolution of the UPLC ! methods allowed us to decrease our analysis time from 45 minutes to 5-6 minutes, resulting in approximately a 7-9-fold increase in productivity. The increased sensitivity of the UPLC ! method has enabled us to reduce our injection volume by 4-fold, from 20 µL to 5 µL of sample. An extensive metabolite ID profile can be generated in 2 analysis runs or less with the afore-described methods. It is often necessary to perform a minimum of six scan functions per run in precursor or product ion mode to generate the data necessary for preliminary metabolite identification. The high-speed MRM capabilities of the MS coupled to a UPLC ™ system are ideal for this purpose. This combination facilitates the acquisition of a large number of scan functions from highly resolved peaks in one run while maintaining adequate peak characterization.


UPLC/MS/MS for metabolite identification results in a 7- to 9-fold increase in productivity, a 4-fold improvement in sensitivity and the ability to determine an extensive metabolite ID profile.

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