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An analytical method is presented for the quantitative analysis of microbial metabolites, consisting of a derivatization reaction combined with gas chromatographic analysis coupled to mass spectrometry

Microbial metabolomics with gas chromatography/mass spectrometry.

ANALYTICAL CHEMISTRY, no. 4 (2006): 1272-1281

Cited by: 306|Views22
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Abstract

An analytical method was set up suitable for the analysis of microbial metabolomes, consisting of an oximation and silylation derivatization reaction and subsequent analysis by gas chromatography coupled to mass spectrometry. Microbial matrixes contain many compounds that potentially interfere with either the derivatization procedure or a...More

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Introduction
  • An analytical method was set up suitable for the analysis of microbial metabolomes, consisting of an oximation and silylation derivatization reaction and subsequent analysis by gas chromatography coupled to mass spectrometry.
  • The development of a generic analytical method fulfilling these requirements is very challenging, especially in view of the wide range of compound classes and large range of metabolite concentrations present in the samples
  • Another challenge applies especially to the analysis of microbial samples: large amounts and numbers of components derived from the growth medium and the buffer used for quenching may be present, and their concentration may vary significantly from sample to sample, for instance, when comparing microorganisms grown on different growth media or harvested at different time points during growth.
  • An elaborate method validation is needed to check the performance of the method for metabolites from different compound classes
Highlights
  • An analytical method was set up suitable for the analysis of microbial metabolomes, consisting of an oximation and silylation derivatization reaction and subsequent analysis by gas chromatography coupled to mass spectrometry
  • The development of a generic analytical method fulfilling these requirements is very challenging, especially in view of the wide range of compound classes and large range of metabolite concentrations present in the samples. Another challenge applies especially to the analysis of microbial samples: large amounts and numbers of components derived from the growth medium and the buffer used for quenching may be present, and their concentration may vary significantly from sample to sample, for instance, when comparing microorganisms grown on different growth media or harvested at different time points during growth
  • The relative standard deviations (RSDs) of the response and the derivatization efficiencies for most test compounds were satisfactory, i.e., with RSDs below 10% and derivatization efficiencies higher than 50%, respectively
  • In this paper, an analytical method is presented for the quantitative analysis of microbial metabolites, consisting of a derivatization reaction combined with gas chromatographic analysis coupled to mass spectrometry
  • The analytical variation of the method was much smaller than the biological variation in the E. coli samples, proving the suitability of the method to analyze differences in the metabolome of microorganisms
  • The generic method has been applied for the analysis of a large number of microorganisms, e.g. B. subtilis, P. freudenreichii, Trichoderma reesei, and Pseudomonas putida
Methods
  • The derivatization and GC/MS analysis were optimized using a representative set of test compounds with varying physical and chemical properties.
  • For this purpose, metabolites from different chemical classes, i.e., amino acids, organic acids, sugars, and sugar phosphates, were chosen.
  • The combination of pyridine as solvent, ethoxyamine as oximation reagent, and MSTFA as silylation reagent resulted in the most satisfactory results with respect to derivatization efficiencies and application range
Results
  • The relative standard deviations (RSDs) of the response and the derivatization efficiencies for most test compounds were satisfactory, i.e., with RSDs below 10% and derivatization efficiencies higher than 50%, respectively.
  • The calculated derivatization efficiency is an estimate and can, deviate from the actual derivatization efficiency, if, for example, the MS response factor of a compound is lower or higher compared to the reference compounds used for quantification.
  • The derivatization efficiency for glucose 6-phosphate calculated from the GC-FID data was 80% and was comparable with the derivatization efficiency for glucose
Conclusion
  • The aim is to determine the differences in the complete metabolomes of cells, body fluids, or tissue.
  • J. Plant Cell 2002, 14, 1437-1440In this paper, an analytical method is presented for the quantitative analysis of microbial metabolites, consisting of a derivatization reaction combined with gas chromatographic analysis coupled to mass spectrometry.
  • Plant Cell 2002, 14, 1437-1440In this paper, an analytical method is presented for the quantitative analysis of microbial metabolites, consisting of a derivatization reaction combined with gas chromatographic analysis coupled to mass spectrometry
  • This method is part of a metabolomics platform in the laboratory consisting of several GCand LC-based methods to analyze, all metabolites from cells, body fluids, and tissue.
  • The research is aimed at improving the performance of the method for critical metabolites and lowering the detection limits
Tables
  • Table1: Repeatability (RSD) of the Response and Derivatization Efficiency for Several Metabolitesa compound
  • Table2: Linearity and Quantification Limit of Standards Spiked to E. coli Cell Extracts quantification limit compound
  • Table3: Recovery of Standards Spiked to E. coli Cell Extract compounds
  • Table4: Stability of the Analysis of Metabolites in Standards and in P. freudenreichii Cell Extractsa metabolite
  • Table5: Precision of Quantificationa in Standard Solutions and Cell Extracts of B. subtilis metabolite
Download tables as Excel
Funding
  • The authors thank Roelie Bijl, Machtelt Braaksma, Nicole van Luijk, and Karin Overkamp for cultivating the microorganisms and extracting the samples, William van Dongen for reviewing the manuscript, Roche Vitamins (currently DSM Nutritional Products), DSM Food Specialties and Friesland Foods for financial support, and Markus Wyss and Werner Bretzel (currently DSM Nutritional Products) for fruitful discussions. Received for review September 21, 2005
Study subjects and analysis
microbial samples: 15
In addition, a decrease in the response of the cholic acid standard was observed after ∼20 analyses. As a result, the RSD of the cholic acid standard after 15 microbial samples was 6%, after 20 microbial samples 12%, and after 30 microbial samples 26%. The somewhat higher RSDs for phosphoenolpyruvic acid and cholic acid in the standard solutions compared to their RSDs in cell extract can probably be attributed to the presence or increase of active places in the analytical system when samples are injected

samples: 20
In standard solutions, these “protective” compounds from the matrix are not present, causing the RSDs to be higher than in cell extracts. In general, 20 samples could be analyzed using the same injection liner. The performance of a few quality standards added to the microbial samples was checked for each measurement; if the performance of the quality standards deteriorated, the injection liner was changed

microbial samples: 15
12b not presenta a not presenta valine not presenta a not presenta proline not presenta a not presenta glycine not presenta a not presenta succinic acid not presenta a not presenta. 2 (32)c 3 5 (21)c 5 1 1 2 2 8 2 2 4 6 2 a Metabolite present in sample, concentration not known; not present in standard. b RSD of cholic acid in the standards after 15 microbial samples was 6% (n ) 8), after 20 samples 12% (n ) 10), and after 30 samples 26% (n ) 18). c Unstable metabolite. Standard Solutions malic acid fructose glucose 6-phosphate repeatabilityb (i.e., interbatch precision) reproducibility (i.e., interbatch precision) act. conc (μg/L) SD (μg/L) CV (%) df SD (μg/L) CV (%) df

samples: 8
To prove the suitability of the GC/MS method for microbial metabolomics, the method was applied to a set of samples from phenylalanine producing E. coli. A total of eight samples, harvested at different time points during fermentation (Figure 1), were analyzed and the concentrations for the different. Analytical Chemistry, Vol 78, No 4, February 15, 2006 metabolites were compared

samples: 5
Analytical Chemistry, Vol 78, No 4, February 15, 2006 metabolites were compared. The first five samples were harvested in the logarithmic growth phase, and the last three samples were harvested in the stationary growth phase. Glucose was used as the carbon source

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