The Fiehn Protocol in Metabolomics

Martin Perkins

10th May 2016

Agilent 7200 GC/Q-TOF, Conference, Fiehn Extraction, GC/Q-TOF, HRAM GC-MS, John Innes Centre, Max Planck Institute, Metabolomics, MOX-TMS, Nathan Hawkins,

The Early History of Metabolomics

Back in 1997, reliable, modern LC-MS systems were science fiction: ion traps were the only affordable solution for most labs, the first TOF-MS systems were being developed, and triple quadrupoles cost (almost) as much as an NMR.

At the cutting edge of Bioanalytical Chemistry, some interesting stuff was happening at the Max Planck Institute in Golm, at UMIST and Aberystwyth Universities in the UK and at Imperial College in London where several groups were working on metabolite profiling in plants, humans and microbes.

Between 1998 and 2001 a flurry of papers had been published in this emerging field. The weapons of choice were 1H-NMR and GC-MS and it had a catchy new name too: metabolomics had arrived.

GC-MS in Metabolomics

On the GC-MS platform the analysis of polar metabolites, as their MOX-TMS derivatives, was widely adopted having been developed and popularized by the team at MPI Golm (whose alumni include Professors Ute Roessner and Oliver Fiehn).

Whilst this is the most widely used protocol for GC-MS based untargeted metabolite fingerprinting, there are some significant issues with derivative stability for some metabolites (up to 50% reduction in peak area over 24h) that adversely affected data quality when samples are prepared in batches.

Anatune’s Contribution

Starting in 2004, Anatune was approached by several groups looking for a solution to this problem.

With GERSTEL’s newly released Maestro software and the dual-rail GERSTEL MultiPurpose Sampler, we were able to provide several UK based metabolomics groups with systems that could automate the two step derivatization on top of the GC instrument.

What’s more, Maestro had a great new feature called Prep-ahead which meant the preparation of samples could be overlapped to allow a freshly prepared sample to be delivered to the instrument: just-in-time for the start of the next GC run; neatly resolving the derivative stability issue.

Automating GC-MS sample preparation solves two key problems. Firstly, it takes the slog out of prepping large numbers of samples. Secondly, by eliminating sources of human error the data quality is improved.

It has to be said that if GC-MS sample preparation can be automated to good effect, the same will be true for LC-MS.


With the renaissance of GC-MS in field of metabolomics, we’ve had renewed interest in complete metabolomics solutions, incorporating MOX-TMS derivatization solutions, both with the Agilent 5977 MSD and Agilent 7200 GC/Q-TOF.

Last year, we worked in partnership with the John Innes Centre in Norwich to deliver a system to their metabolomics core facility. This incorporated the MOX-TMS derivatization but, as we now had new tools for, and more space on the MPS, we could extend its capabilities…

We therefore developed and built a MOX-TMS Solution incorporating:

  • biphasic solvent extraction of fresh or finely ground freeze dried tissue
  • liquid-liquid extraction using the GERSTEL Multi-Position Vortexer (mVORX)
  • clarification of the phases using the Anatune CF-200 Robotic Centrifuge
  • separation of the organic and aqueous phases
  • evaporation of the separate phases using the GERSTEL Multi-Position Evaporation Station (mVAP)
  • MOX-TMS derivatization of polar and non-polar fractions

This system is affectionately known as ‘The Beast of Norwich’ and we are now working with several other customers on similar solutions.


Anatune have historically worked with research groups doing metabolomics to automate online, just-in-time MOX-TMS derivatization.

With the availability of new modules in the GERSTEL MPS family and the Agilent 7200 GC/Q-TOF providing next generation of HRAM GC-MS systems, we are working with both new and existing customers, to automate their existing manual sample extraction and derivatization workflows, and provide fully integrated platforms for GC-MS based metabolomics.

Anatune can build metabolomics solutions for online (GC-MS, LC-MS) and offline sample preparation.

With the modularity and flexibility of the GERSTEL MPS PrepStation, we can automate what you currently do manually, and provide complete solutions using instrumentation from Agilent Technologies and GERSTEL.

Anatune will be sponsoring Metabolomics 2016 in the Conference Centre, Dublin and will be there with our partners GERSTEL and Agilent.

If you’re there too, come to our booth to find out how we can work together to automate your MOX-TMS workflows.

If you can’t meet us there and would like to know more call us on +44 (0)1223 279210 or email