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The use of trimethylsilyl cyanide derivatization for robust and broad-spectrum high-throughput gas chromatography–mass spectrometry based metabolomics

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Abstract

Reproducible and quantitative gas chromatography–mass spectrometry (GC-MS)-based metabolomics analysis of complex biological mixtures requires robust and broad-spectrum derivatization. We have evaluated derivatization of complex metabolite mixtures using trimethylsilyl cyanide (TMSCN) and the most commonly used silylation reagent N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). For the comparative analysis, two metabolite mixtures, a standard complex mixture of 35 metabolites covering a range of amino acids, carbohydrates, small organic acids, phenolic acids, flavonoids and triterpenoids, and a phenolic extract of blueberry fruits were used. Four different derivatization methods, (1) direct silylation using TMSCN, (2) methoximation followed by TMSCN (M-TMSCN), (3) direct silylation using MSTFA, and (4) methoximation followed by MSTFA (M-MSTFA) were compared in terms of method sensitivity, repeatability, and derivatization reaction time. The derivatization methods were observed at 13 different derivatization times, 5 min to 60 h, for both metabolite mixtures. Fully automated sample derivatization and injection enabled excellent repeatability and precise method comparisons. At the optimal silylation times, peak intensities of 34 out of 35 metabolites of the standard mixture were up to five times higher using M-TMSCN compared with M-MSTFA. For direct silylation of the complex standard mixture, the TMSCN method was up to 54 times more sensitive than MSTFA. Similarly, all the metabolites detected from the blueberry extract showed up to 8.8 times higher intensities when derivatized using TMSCN than with MSTFA. Moreover, TMSCN-based silylation showed fewer artifact peaks, robust profiles, and higher reaction speed as compared with MSTFA. A method repeatability test revealed the following robustness of the four methods: TMSCN > M-TMSCN > M-MSTFA > MSTFA.

Improved GC-MS profiling of Complex Biological Mixtures by TMSCN based Derivatization

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Abbreviations

BSA:

Bis(trimethylsilyl)acetamide

BSTFA:

N,O-Bis(trimethylsilyl)trifluoroacetamide

EI:

Electron impact

HCN:

Hydrogen cyanide

MEOX:

Methoxiamine

M-MSTFA:

Methoximation followed by MSTFA-based silylation

M-TMSCN:

Methoximation followed by TMSCN-based silylation

MPS:

Multi-purpose sampler

MSTFA:

N-methyl-N-(trimethylsilyl)trifluoroacetamide

PARAFAC2:

Parallel Factor Analysis 2

PCA:

Principal component analysis

RI:

Retention index

TMCS:

Trimethylchlorosilane

TMS:

Trimethylsilyl

TMSCN:

Trimethylsilyl cyanide

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Acknowledgments

The authors thank the Faculty of Science for support to the elite-research area “Metabolomics and bioactive compounds” with a PhD stipendium to B. Khakimov and The Ministry of Science and Technology for a grant to University of Copenhagen (S.B. Engelsen) with the title “Metabolomics infrastructure” under which the GC-MS was acquired.

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Authors and Affiliations

  1. Quality and Technology, Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 30, Frederiksberg C, 1958, Copenhagen, Denmark

    Bekzod Khakimov & Søren Balling Engelsen

  2. Plant Biochemistry, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, 1871, Copenhagen, Denmark

    Bekzod Khakimov, Mohammed Saddik Motawia & Søren Bak

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  1. Bekzod Khakimov
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  2. Mohammed Saddik Motawia
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  3. Søren Bak
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  4. Søren Balling Engelsen
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Correspondence to Bekzod Khakimov.

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Khakimov, B., Motawia, M.S., Bak, S. et al. The use of trimethylsilyl cyanide derivatization for robust and broad-spectrum high-throughput gas chromatography–mass spectrometry based metabolomics. Anal Bioanal Chem 405, 9193–9205 (2013). https://doi.org/10.1007/s00216-013-7341-z

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