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Abstract No.: MoP-159
Session: Metabolomics, Metabonomics
Presentation date: Mon, Aug 28, 2006
Presentation time: 09:50 – 11:20

A Fast and Sensitive Method for Determination of Endogenous ATP Analog ApppI and Mevalonate Pathway Metabolite IPP Using Ion-Pairing High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry

Seppo Auriola1, Marjo Jauhiainen1, Hannu Monkkonen2, Johanna Kuokkanen1, Jukka Monkkonen1

1 University of Kuopio, Kuopio, Finland
2 University of Sheffield, Sheffied, United Kingdom

Correspondence address: Seppo Auriola, University of kuopio, Pharmaceutical Chemistry, Harjulantie 1, PL1627, Kuopio, Fi-70211 Finland.

Keywords: Chromatography, Liquid (LC); Electrospray Ionization (ESI); Metabolism, Metabolites; Nucleotides.

Novel aspect: LC-MS Analysis of a novel endogenous nucleotide metabolite.

 

Mevalonate pathway produces several molecules which are important to cell development and growth, for instance isoprenoid lipids such as isopentenyl (IPP), farnesyl (FPP) and geranylgeraniol pyrophosphates (GGPP). A class of drugs widely used for treatment of many metabolic bone diseases is nitrogen containing bisphoshonates (N-BPs), which act by inhibiting one of the key enzymes of the intracellular mevalonate pathway, FPP synthase. N-BPs can cause the accumulation of intracellular IPP and consequently induce formation of an ATP analog ApppI, which leads to apoptosis in vitro.
We describe here a fast and sensitive method for extraction and determination of endogenous ATP analog ApppI and mevalonate pathway metabolite isopentenyl pyrophosphate (IPP) in cultured cancer cells. A simple extraction procedure of the analytes from the cell samples was performed using cold acetonitrile and water. Reversed-phase high performance liquid chromatography (RP-HPLC) method that is compatible with negative ion electrospray ionization mass spectrometry (ESI-MS) was used for separation and detection of the molecules. For quantitation, selective reaction monitoring (SRM) was used and characteristic fragment ions m/z 159 for IPP and m/z 406 for ApppI were followed. As an ion-pairing reagent, a volatile dimethylhexylamine (DMHA) was added to mobile phase to improve the retention of the highly hydrophilic, anionic molecules in the reversed-phase C18 column. Molecules were eluted with increasing methanol gradient with simultaneous decreasing of DMHA concentration. For method reliability evaluation, some important validation parametres such as linearity, repeatability and accuracy were determined. Since the analytes can degrade in aqueous solution due to enzyme activity, sample stability during analyzing process was also evaluated.
This method is simple, fast and sensitive and it can be applied to analysis of other structurally related molecules. The method was used to analyze the intracellular contents of ApppI and IPP in different bisphosphonate treated cancer cells.

References:
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3. M. Cichna, M. Raab, H. Daxecker, A. Griesmacher, M. M. Muller and P. Markl, J. Chromatogr. B 787, 381 (2003).
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