Determination of volatile alkylpyrazines in microbial samples using gas chromatography-mass spectrometry coupled with head space-solid phase microextraction

So Hyeon Jeon,Yun Gyong Ahn,Jeoung Hwa Shin,Yong Pyo Kim

doi:10.1186/s40543-016-0096-3

Abstract

Determination of volatile alkylpyrazines (VAPs) in microbial samples is needed for the understanding of microbial metabolism. An analytical method for screening and quantification of six volatile alkylpyrazines was developed and successfully applied to the real microbial samples. Headspace-solid phase microextraction (HS-SPME) which is a simple extraction simultaneously carrying out clean-up and pre-concentration of VAPs in microbial samples was adopted. GC-TOF/MS with CombiPal SPME autosampler was used for fast screening of microbial samples and the optimization of HS-SPME condition. Separation and quantification of six VAPs that have the potential to be detected in microbial samples using quadrupole GC/MS in chemical ionization (CI) mode were carried out. The optimal extraction conditions for HS-SPME at 50 °C and 50 min were investigated. To eliminate matrix effects, DB-WAX column was more appropriate to separate each alkylpyrazines from the interferences than DB-5MS column. Chemical ionization provided enhanced confirmation from adduct ions ([M + 29]+ and [M + 41]+) of methane reagent gas as well as increasing signal intensity of molecular ions for quantitative analysis. Linear calibration curves of six alkylpyrazines were obtained within a range between 1 and 50 ng/g. This proposed method was successfully applied to the microbial samples. 2-Methylpyrazine and 2,5-dimethylpyrazine were determined in the level of 6.0~34.0 ng/g.

Highlights

Pyrazines are heterocyclic aromatic organic compounds containing nitrogen atoms in the 1- and 4-position of the aromatic ring
We report a suitable analytical method for screening and quantification of six volatile alkylpyrazines (VAPs) in microbial samples
Chemicals and reagents Six VAPs (2-methylpyrazine (MP), 2,5-dimethylpyrazine (DMP), 2-ethylpyrazine (EP), 2-ethyl-3,5-dimethylpyrazine (EDMP), 2-isopropyl-3-methoxy pyrazine (IPMP), and 2-ethoxy-3-ethylpyrazine (EEP); see Table 1 for their full chemical names and information) for Gas chromatography/mass spectrometry (GC/MS) analysis were purchased from Sigma-Aldrich

Summary

Methods

Chemicals and reagents Six VAPs (2-methylpyrazine (MP), 2,5-dimethylpyrazine (DMP), 2-ethylpyrazine (EP), 2-ethyl-3,5-dimethylpyrazine (EDMP), 2-isopropyl-3-methoxy pyrazine (IPMP), and 2-ethoxy-3-ethylpyrazine (EEP); see Table 1 for their full chemical names and information) for GC/MS analysis were purchased from Sigma-Aldrich Homogenized sample of 1 mL was spiked with 100 ng of the internal standard in the 4 mL headspace vial for quantitative analysis. Instrumentation To optimize extraction time and temperature, HS-SPME sampling was performed using an Agilent GC (Wilmington, Delaware, USA)—Pegasus 4D TOFMS (St. Joseph, Michigan, USA) equipped with a combiPAL SPME autosampler (CTC Analytics, Zurich, Switzerland). A 75 μm carboxen/polydimethylsiloxane (CAR/PDMS) fiber which has been demonstrated as an optimal fiber for the alkylpyrazines analysis in the previous studies (de Oliveira et al 2004) was purchased from Supelco (Bellefonte, PA, USA) and used. GCCI/MS was performed by an Agilent 7890B gas chromatograph, equipped with a 7693 mass selective detector single quadrupole mass spectrometer system (Palo Alto, CA, USA). Acquisition was carried out in both scan and SIM mode

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