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Optimization and Validation of a HS-SPME/GC–MS Method for the Analysis of Gluten-Free Bread Volatile Flavor Compounds


Flavor is one of the most important, but also the least researched parameter of bread quality. Numerous volatile compounds that contribute to the flavor profile of bread are usually extracted by the headspace solid-phase microextraction (HS-SPME) method. The development of a reliable and sensitive HS-SPME method requires the selection and optimization of all key extraction parameters as well as performance of a validation procedure. The aim of this study was to optimize the temperature and time of HS-SPME of volatile flavor compounds of gluten-free bread and to validate an optimized HS-SPME/gas chromatography-mass spectrometry (GC-MS) method for their quantification. This study included 62 volatile flavor compounds from the chemical classes of aldehydes, ketones, alcohols, acids, esters, aromatic hydrocarbons, phenols, sulfur compounds, pyrazines, pyrroles, and furans. The determined optimal conditions for extraction with carboxen/divinylbenzene/polydimethylsiloxane fiber were temperature 60 °C and time 60 min. Calibration was performed using a standard addition method. Linearity and precision were achieved within the examined method range for all compounds analyzed, with correlation coefficients r ≥ 0.999. The relative SDs of the determined method repeatability and intermediate precision ranged from 0.3 to 18.9% and from 2.4 to 31.2%, respectively. The obtained limits of detection and quantification differed significantly among the analyzed compounds and ranged from 49.0 pg/kg to 94.5 μg/kg and 163 pg/kg to 315 μg/kg, respectively. Based on the obtained results, the proposed optimized method can be successfully applied for the quantification of all the analyzed bread volatile flavor compounds.

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The authors wish to thank The Dow Chemical Company for supplying hydrocolloids. This work is based upon the work from COST Action 18101 SOURDOMICS—Sourdough biotechnology network towards novel, healthier and sustainable food and bioprocesses (;|name:overview), where the author D.N. is Management Committee member, and N.Č.M. and B.V. are members of a Working Group 7. SOURDOMICS is supported by COST (European Cooperation in Science and Technology). COST is a funding agency for research and innovation networks. COST Actions help connect research initiatives across Europe and enable scientists to grow their ideas by sharing them with their peers—thus boosting their research, career, and innovation.


This study was funded by Croatian Science Foundation (projects VH/VT 09/01/279 and GbP-FFood IP-06–2016 3789).

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Conceptualization: S.D., N.Č.M., D.N., B.V. Methodology: S.D., N.Č.M. Formal analysis and investigation: S.D. Writing—original draft preparation: S.D. Writing—review and editing: S.D., N.Č.M., D.N., B.V., M.K., M.H., D.Ć. Funding acquisition: D.Ć. Resources: D.Ć., M.H., M.K. Supervision: D.Ć., M.H.

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Correspondence to Nikolina Čukelj Mustač.

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Saša Drakula, Nikolina Čukelj Mustač, Dubravka Novotni, Bojana Voučko, Marina Krpan, Mirjana Hruškar, and Duška Ćurić declare that they have no conflict of interest.

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Drakula, S., Mustač, N.Č., Novotni, D. et al. Optimization and Validation of a HS-SPME/GC–MS Method for the Analysis of Gluten-Free Bread Volatile Flavor Compounds. Food Anal. Methods 15, 1155–1170 (2022).

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  • Gluten-free
  • Bread crumb and crust
  • Volatile flavor compounds
  • HS-SPME time and temperature
  • GC–MS
  • Method development