Day, F., O’Sullivan, J., Ramzan, F., Pook, C. Polar metabolomics using trichloroacetic acid extraction and porous graphitic carbon stationary phase. Metabolomics 20, 77 (2024). https://doi.org/10.1007/s11306-024-02146-7
This paper is the 2nd from Frankie Day’s PhD and one of only three we could find in the literature applying Porous Graphitic Carbon [PGC] stationary phase for LC-MS metabolomics. We worked hard to quantify the performance of this method so that we could be confident it really was as good as it looks. This offers an important and powerful alternative to the limited options currently available to researchers trying to resolve polar metabolites. These include the notorious Hydrophobic Interaction Liquid Chromatography, or HILIC, as well as derivatisation for Gas Chromatography and Mass Spectrometry. These both have some fairly major limitations and so this new method has great potential to open up this area of metabolomics to large-scale, high-throughput analysis.
A second innovation was the use of aqueous trichloroacetic acid to extract the metabolites from human serum and precipitate protein. This extract was directly compatible with liquid chromatography, unlike alternative techniques, resulting in a fast and simple sample extraction protocol. Combined with the short 15 minute LC-MS run time this potentiates analysis of close to a hundred samples per day. The method demonstrated robust quantitation (CV < 0.30) of polar metabolites within a logP range of − 9.1 to 5.6. We used an isotopically labelled internal standard mixtures (QReSS mix, Cambridge Isotope Labs) to quantify reproducibility (CV < 0.16) and effective recovery (70–130%) of metabolites. Quality control dilution series demonstrated that ~ 80% of annotated metabolites could be accurately quantified (Pearson’s correlation coefficient > 0.80) within their concentration range.
Frankie has gone on to apply this method to a set of nearly 700 serum samples from the GutBugs clinical trial of faecal microbiome transplants to try to identify microbial metabolites responsible for host-microbiome interactions.
Fig. 1 Polar metabolite methodology developed in this study: a) Peripheral blood samples were obtained from six healthy individuals, and metabolites extracted from 20 µL of serum (methods). b) Following centrifugation, the supernatant (50 µL) was transferred into an autosampler vial and diluted 1:3 with MQ H2O (150 µL). c) Sample data were acquired by LC–MS/MS using a 150mm porous graphitic column and Q-Exactive MS using the data-dependent analysis mode. d) Raw data was processed, cleaned, annotated and statistically analysed. e) Recovery (%) of spiked internal QReSS standards was calculated using QReSS internal standard spiked blank (MQ) samples as a baseline for comparison. Samples that fall between the solid red lines represent reliably quantifed metabolites. The dotted red line represents an alternative cut of (50% and above) for lowly but linear and repeatably recovered metabolites. f) The CV of each QreSS internal standard, across 3 repeats from each individual. The dashed red line represents the CV threshold of 0.15 specifed for targeted analysis by the United States Food and Drug Agency (FDA, 2018, 2019). Reproduced from original publication.
Frankie’s PhD was funded by a University of Auckland Scholarship. Chris Pook’s Fellowship was funded by a donation from Shundi Group. The authors thank Kalita Prangnell and Eric Thorstensen for their support. The authors acknowledge support from the Mass Spectrometry Hub, a Strategic Research Initiative at the University of Auckland.
Full text freely available at Metabolomics journal website.
Authors and Affiliations
Francesca Day, Justin O’Sullivan, Farha Ramzan & Chris Pook: Liggins Institute, The University of Auckland, Auckland, New Zealand
Justin O’Sullivan: The Maurice Wilkins Centre, The University of Auckland, Auckland, New Zealand
Justin O’Sullivan: MRC Lifecourse Epidemiology Unit, University of Southampton, Southampton, UK
Justin O’Sullivan: Australian Parkinson’s Mission, Garvan Institute of Medical Research, 384 Victoria Street, Sydney, Darlinghurst, NSW, 2010, Australia
Justin O’Sullivan: A*STAR Singapore Institute for Clinical Sciences, Singapore, Singapore
Chris Pook: School of Chemical Sciences, University of Auckland, 23 Symonds St., Auckland, 1010, New Zealand