Analysis of Isoprenoid Pathway Metabolites by LC-MS

By: Rudolf Köhling, Roland Meier, Bernhard Schönenberger and Roland Wohlgemuth

Sigma-Aldrich®, Buchs, Switzerland
, Biofiles, Vol. 8, No. 17

Isoprenoid pathway metabolites such isoprenoidphosphates and-pyrophosphates are central biological intermediates leading to sterols, dolichols, ubiquinones, prenylated natural products and proteins. The development of new methodologies for their analytical characterization and quantitation is key for detailed investigations of those pathways on a molecular level. It is therefore of much interest to develop new high-performance separation methods which are able to determine a wide range of isoprenoid-(pyro)phosphates in parallel.

Single unit isoprenoid(pyro)phosphates

The separation of these phosphorylated compounds depends largely on stereoselective and ionic interactions. This required the rather unusual choice of a Cyclodextrin column operated in the HILIC mode and controlled through pH and ionic strength of the aqueous mobile phase.

The performance of the cyclodextrine-based Astec Cyclobond 2000 stationary phase greatly surpassed the original method1 and for the first time enabled baseline separation of IP/DMAP and IPP/DMAPP respectively. Another published method2 was shown to distinguish pyrophosphates of different numbers of isoprene units, but not isomeric isoprenoid(pyro)phosphates such as IPP/DMAPP or GP/NP.

Eluent pH 4 through 8 were tested, but a successful separation occurred at a pH < 7 only. The flowrate turned out to be another critical parameter with the optimum value of 0.5ml/min., while a flow of 1ml/min. resulted in loss of baseline separation.

Multiple units isoprenoid(pyro)phosphates

A general approach for the separation of unknown metabolites is the usage of two different stationary phases with opposite polarities, such as C18 and HILIC. Although HILIC is well-suited to the LC/MS detection mode, as the buffers and solvents are compatible with MS ion sources such as ESI, HILIC separations of highly polar analytes are influenced strongly by the character of buffer and stationary phase.

In case of the higher isoprenoid(pyro)phosphates, HILIC resulted in a poor chromatography and could not be applied. However, Ion-Pair Chromatography3,4 using MS-compatible reagents and Fused Core® RP-columns turned out to be a powerful alternative. Dihexylamine Acetate, (DHAA, 92467) functioning as an IPC reagent, had several positive effects on the separation and detection of isoprenoid phosphates:

1. It is invisible in ESI(-) mode
2. Enhances the detection of [M-H]- ions and
3. Results in a high efficiency on Ascentis Express C8/C18 columns
(Fig 3).

This work was presented as a poster at the 2013 Metabolomic
Society Conference hosted in Glasglow, Scotland

sh6636 Biofiles 8.17.indd

Figure 1.

HILIC separation of the ismeres IP/DMAP and IPP/DMAPP using the cyclodextrine-based stationary phase Astec Cyclobond I 2000

Figure 2. HILIC separation of the ismeres IP/DMAP and IPP/DMAPP using the cyclodextrine-based stationary phase Astec Cyclobond I 2000.

Separation of homologous series of isoprenoid phosphates and pyrophosphates

Figure 3. Separation of homologous series of isoprenoid phosphates and pyrophosphates. Adjusting flow and gradient allows the chromatography of the longest homologue (geranylgeranyl(pyro)phosphate, GGP/GGPP) with the same setup.




  1. M. Lange, et al. Isoprenoid Biosynthesis. Metabolite Profiling of Peppermint Olil Gland Secretory Cells and Application to Herbicide Target Analysis. Plant Physiol. 127, 305-14 (2001).
  2. J.M. Buescher, et al. Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Fast and Robust Quantification of Anoinic and Aromatic Metabolites. Anal.Chem. 82, 4403-12 (2010).
  3. L.R. Snyder, et al. Introduction to Modern Liquid Chromatography, 3rd ed., Wiley (2010).
  4. M. Holcapek, et al. High performance liquid chromatography-mass spectrometric ananlysis of sulfonated dyes and internediates. J.Chromatogr. A  926(1), 175-86 (2001).


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