Skip to main content

A simple and fast method for metabolomic analysis by gas liquid chromatography—mass spectrometry



The metabolomic profile is an essential tool for understanding the physiological processes of biological samples and their changes. In addition, it makes it possible to find new substances with industrial applications or use as drugs. As GC-MS is a very common tool for obtaining the metabolomic profile, a simple and fast method for sample preparation is required.


The aim of this research was to develop a direct derivatization method for GC-MS to simplify the sample preparation process and apply it to a wide range of samples for non-targeted metabolomic analysis purposes.


One pot combined esterification of carboxylic acids with methanol and silylation of the hydroxyl groups was achieved using a molar excess of chlorotrimethylsilane with respect to methanol in the presence of pyridine.


The metabolome profile obtained from different samples, such as bilberry and cherry cuticles, olive leaves, P. aeruginosa and E. coli bacteria, A. niger fungi and human sebum from the ceruminous gland, shows that the procedure allows the identification of a wide variety of metabolites. Aliphatic fatty acids, hydroxyfatty acids, phenolic and other aromatic compounds, fatty alcohols, fatty aldehydes dimethylacetals, hydrocarbons, terpenoids, sterols and carbohydrates were identified at different MSI levels using their mass spectra.


The metabolomic profile of different biological samples can be easily obtained by GC-MS using an efficient simultaneous esterification-silylation reaction. The derivatization method can be carried out in a short time in the same injection vial with a small amount of reagents.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3


  1. Al-Mutlaq, K. F., Standley, L. J., & Simoneit, B. R. T. (2008). Composition and sources of extractable organic matter from a sediment core in Lake Kivu, East African rift valley. Applied Geochemistry, 23(5), 1023–1040.

    CAS  Article  Google Scholar 

  2. Barakat, A. O., Peakman, T. M., & Rullkötter, J. (1994). Isolation and structural characterization of 10-oxo-octadecanoic acid in some lacustrine sediments from the Nördlinger Ries (southern Germany). Organic Geochemistry, 21(8–9), 841–847.

    CAS  Article  Google Scholar 

  3. Bassindale, A. R., & Stout, T. (1985). The interaction of electrophilic silanes (Me3SiX, X = ClO4, I, CF3SO3, Br, Cl) with nucleophiles. The nature of silylation mixtures in solution. Tetrahedron Letters, 26(28), 3403–3406.

    CAS  Article  Google Scholar 

  4. Belge, B., Comabella, E., Graell, J., Gatius, F., Guillén, P., Llovera, M., & Lara, I. (2017). Cuticular wax composition of “Celeste” and “Somerset” cherry fruit. In Acta Horticulturae (pp. 39–646). Leuven: International Society for Horticultural Science (ISHS).

    Chapter  Google Scholar 

  5. Belge, B., Llovera, M., Comabella, E., Graell, J., & Lara, I. (2014). Fruit cuticle composition of a melting and a nonmelting peach cultivar. Journal of Agricultural and Food Chemistry, 62(15), 3488–3495.

    CAS  Article  PubMed  Google Scholar 

  6. Beloborodova, N. V., Khodakova, A. S., & Olenin, A. J. (2009). Are phenylcarboxylic acids really markers in severe sepsis? Critical Care, 13(4), P41.

    Article  PubMed Central  Google Scholar 

  7. Bianchi, G., Vlahov, G., Anglani, C., & Murelli, C. (1992). Epicuticular wax of olive leaves. Phytochemistry, 32(1), 49–52.

    CAS  Article  Google Scholar 

  8. Birkofer, L., Konkol, W., & Ritter, A. (1961). Di- und Tripeptide aus silylierten Aminosäuren; der Trimethylsilylrest als Schutzgruppe für SH- und OH-Funktionen. Chemische Berichte, 94(5), 1263–1267.

    CAS  Article  Google Scholar 

  9. Blomquist, G., Andersson, B., Andersson, K., & Brondz, I. (1992). Analysis of fatty acids. A new method for characterization of moulds. Journal of Microbiological Methods, 16(1), 59–68.

    CAS  Article  Google Scholar 

  10. Brokl, M., Soria, A. C., Martínez-Castro, I., Sanz, M. L., & Ruiz-Matute, A. I. (2009). Characterization of O-trimethylsilyl oximes of trisaccharides by gas chromatography-mass spectrometry. Journal of Chromatography A, 1216(22), 4689–4692.

    CAS  Article  PubMed  Google Scholar 

  11. Carrapiso, A. I., & García, C. (2000). Development in lipid analysis: Some new extraction techniques and in situ transesterification. Lipids, 35(11), 1167–1177.

    CAS  Article  PubMed  Google Scholar 

  12. Chance, D. L., Gerhardt, K. O., & Mawhinney, T. P. (1998). Gas-liquid chromatography-mass spectrometry of hydroxy fatty acids as their methyl esters tert.-butyldimethylsilyl ethers. Journal of Chromatography A, 793(1), 91–98.

    CAS  Article  PubMed  Google Scholar 

  13. Curran, D. P., Scanga, S. A., & Fenk, C. J. (1984). Reduction of substituted.DELTA.2-isoxazolines. Synthesis of .beta.-hydroxy acid derivatives. The Journal of Organic Chemistry, 49(19), 3474–3478.

    CAS  Article  Google Scholar 

  14. Dembitsky, V. M., Řezanka, T., & Shubinat, E. E. (1993). Unusual Hydroxy fatty acids from some higher fungi. Phytochemistry, 34(4), 1057–1059.

    CAS  Article  Google Scholar 

  15. du Preez, I., & Loots, D. T. (2013). New sputum metabolite markers implicating adaptations of the host to Mycobacterium tuberculosis, and vice versa. Tuberculosis, 93(3), 330–337.

    CAS  Article  PubMed  Google Scholar 

  16. Eras, J., Ferran, J., Perpiña, B., & Canela, R. (2004). Chlorotrimethylsilane, a reagent for the direct quantitative analysis of fats and oils present in vegetable and meat samples. Journal of Chromatography A, 1047(1), 157–161.

    CAS  Article  PubMed  Google Scholar 

  17. Eras, J., Montañes, F., Ferran, J., & Canela, R. (2001). Chlorotrimethylsilane as a reagent for gas chromatographic analysis of fats and oils. Journal of Chromatography A, 918(1), 227–232.

    CAS  Article  PubMed  Google Scholar 

  18. Eras, J., Oró, R., Torres, M., & Canela, R. (2008). Direct quantitation of fatty acids present in bacteria and fungi: Stability of the cyclopropane ring to chlorotrimethylsilane. Journal of Agricultural and Food Chemistry, 56(13), 4923–4927.

    CAS  Article  PubMed  Google Scholar 

  19. Felde, R., & Spiteller, G. (1994). Search for plasmalogens in plants. Chemistry and Physics of Lipids, 71(1), 109–113.

    CAS  Article  Google Scholar 

  20. Franke, R., Briesen, I., Wojciechowski, T., Faust, A., Yephremov, A., Nawrath, C., & Schreiber, L. (2005). Apoplastic polyesters in Arabidopsis surface tissues—A typical suberin and a particular cutin. Phytochemistry, 66(22), 2643–2658.

    CAS  Article  PubMed  Google Scholar 

  21. Gu, Q., David, F., Lynen, F., Rumpel, K., Xu, G., De Vos, P., & Sandra, P. (2010). Analysis of bacterial fatty acids by flow modulated comprehensive two-dimensional gas chromatography with parallel flame ionization detector/mass spectrometry. Journal of Chromatography A, 1217(26), 4448–4453.

    CAS  Article  PubMed  Google Scholar 

  22. Huang, H., Burghardt, M., Schuster, A.-C., Leide, J., Lara, I., & Riederer, M. (2017). Chemical composition and water permeability of fruit and leaf cuticles of Olea europaea L. Journal of Agricultural and Food Chemistry, 65(40), 8790–8797.

    CAS  Article  PubMed  Google Scholar 

  23. Ivanova, A., Delcheva, I., Tsvetkova, I., & Kujumgiev, A. (2002). GC-MS analysis and anti-microbial activity of acidic fractions obtained from Paeonia peregrina and Paeonia tenuifolia Roots. Zeitschrift für Naturforschung C, 57, 624–628.

    CAS  Article  Google Scholar 

  24. Kunst, L., & Samuels, A. L. (2003). Biosynthesis and secretion of plant cuticular wax. Progress in Lipid Research, 42(1), 51–80.

    CAS  Article  PubMed  Google Scholar 

  25. Lee, M., Cho, J. Y., Lee, Y. G., Lee, H. J., Lim, S., Il, Lee, S. Y., et al. (2016). Furan, phenolic, and heptelidic acid derivatives produced by Aspergillus oryzae. Food Science and Biotechnology, 25(5), 1259–1264.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Lehtonen, L., Peltonen, R., & Eerola, E. (1996). Computerised gas-liquid chromatography of bacterial cellular fatty acids in analysis of bacterial mixtures. Journal of Microbiological Methods, 25(3), 317–327.

    CAS  Article  Google Scholar 

  27. Macherius, A., Kuschk, P., Haertig, C., Moeder, M., Shtemenko, N. I., Bayona, A. H., et al. (2011). Composition changes in the cuticular surface lipids of the helophytes Phragmites australis and Juncus effusus as result of pollutant exposure. Environmental Science and Pollution Research, 18(5), 727–733.

    CAS  Article  PubMed  Google Scholar 

  28. Moldovan, Z., Jover, E., & Bayona, J. M. (2002). Gas chromatographic and mass spectrometric methods for the characterisation of long-chain fatty acids: Application to wool wax extracts. Analytica Chimica Acta, 465(1–2), 359–378.

    CAS  Article  Google Scholar 

  29. Nakao, R., Oka, K., & Fukumoto, T. (1981). A simple method for the esterification of carboxylic acids using chlorosilanes. Bulletin of the Chemical Society of Japan, 54(4), 1267–1268.

    CAS  Article  Google Scholar 

  30. Otera, J. (2003). Esterification. Weinheim: Wiley-VCH Verlag.

    Book  Google Scholar 

  31. Peschel, S., Franke, R., Schreiber, L., & Knoche, M. (2007). Composition of the cuticle of developing sweet cherry fruit. Phytochemistry, 68(7), 1017–1025.

    CAS  Article  PubMed  Google Scholar 

  32. Pierce, A. E. (1968). Silylation of organic compounds. Rockford, Illinois: Pierce Chemical Company.

    Google Scholar 

  33. Ruiz-Matute, A. I., Hernández-Hernández, O., Rodríguez-Sánchez, S., Sanz, M. L., & Martínez-Castro, I. (2011). Derivatization of carbohydrates for GC and GC-MS analyses. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 879(17–18), 1226–1240.

    CAS  Article  PubMed  Google Scholar 

  34. Sauer, R. O. (1944). Derivatives of the methylchlorosilanes. I. Trimethylsilanol and its simple ethers. Journal of the American Chemical Society, 66(10), 1707–1710.

    CAS  Article  Google Scholar 

  35. Shantha, N. C., & Napolitano, G. E. (1992). Gas chromatography of fatty acids. Journal of Chromatography A, 624(1–2), 37–51.

    CAS  Article  Google Scholar 

  36. Söderholm, S. L., Damm, M., & Kappe, C. O. (2010). Microwave-assisted derivatization procedures for gas chromatography/mass spectrometry analysis. Molecular Diversity, 14(4), 869–888.

    CAS  Article  PubMed  Google Scholar 

  37. Sonntag, N. O. V. (1953). The reaction of aliphatic chlorides. Chemical Reviews, 52(2), 237–416.

    CAS  Article  Google Scholar 

  38. Tomàs, A., Tor, M., Villorbina, G., Canela, R., Balcells, M., & Eras, J. (2009). A rapid and reliable direct method for quantifying meat acylglycerides with monomode microwave irradiation. Journal of Chromatography A, 1216(15), 3290–3295.

    CAS  Article  PubMed  Google Scholar 

  39. van Look, G., & Simchen, J. . H. . (1995). Silylating agents. Fluka Chemica AG. Sigma-Aldrich: Buchs, Switzerland.

    Google Scholar 

  40. Wanikawa, A., Shoji, H., Hosoi, K., & Nakagawa, K.-I. (2002). Stereospecificity of 10-hydroxystearic acid and formation of 10-ketostearic acid by lactic acid bacteria. Journal of the American Society of Brewing Chemists, 60(1), 14–20.

    CAS  Article  Google Scholar 

  41. Yunoki, K., Ishikawa, H., Fukui, Y., & Ohnishi, M. (2008). Chemical properties of epidermal lipids, especially sphingolipids, of the antarctic minke whale. Lipids, 43(2), 151–159.

    CAS  Article  PubMed  Google Scholar 

Download references


The DBA centre is a certified agent TECNIO in the category of technology developers from the Government of Catalonia. The authors would like to thank the Catalan Government for the quality accreditation given to its research groups (EMO/1787/2015). The authors would like to thank the University of Lleida for the grant “Ajuts per a personal predoctoral de la UdL en formació i ajuts Jade Plus” awarded to Diana Cosovanu.

Author information




JE conceived and designed the research. DC, ML and JE conducted the experiments. DC, GV and JE analysed the data, and RC and JE wrote the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Jordi Eras.

Ethics declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

The study was approved by the Research Ethics Committee of the Hospital Universitari Arnau de Vilanova de Lleida de la Gerència Territorial ICS Lleida, Alt Pirineu i Aran—GSS (82/2014; 745/2018). The investigation was conducted according to the Declaration of Helsinki and the BCP (CHM9/ICH/135/95).

Informed consent

An informed consent to provide sebum for research purposes was given by the donor and a written consent was obtained from the participant.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 2422 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Cosovanu, D., Llovera, M., Villorbina, G. et al. A simple and fast method for metabolomic analysis by gas liquid chromatography—mass spectrometry. Metabolomics 17, 22 (2021).

Download citation


  • Metabolomics
  • Non-targeted analysis
  • Derivatization
  • GC/MS
  • Hydroxy fatty acids