Abstract
A simple analytical workflow is described for gas chromatographic-mass spectrometry (GC-MS)-based metabolomic profiling of protic metabolites, particularly amino-carboxylic species in biological matrices. The sample preparation is carried out directly in aqueous samples and uses simultaneous in situ heptafluorobutyl chloroformate (HFBCF) derivatization and dispersive liquid-liquid microextraction (DLLME), followed by GC-MS analysis in single-ion monitoring (SIM) mode. The protocol involves ten simple pipetting steps and provides quantitative analysis of 132 metabolites by using two internal standards. A comment on each analytical step and explaining notes are provided with particular attention to the GC-MS analysis of 112 physiological metabolites in human urine.
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References
Andrews MA (1989) Capillary gas-chromatographic analysis of monosaccharides – improvements and comparisons using trifluoroacetylation and trimethylsilylation of sugar o-benzyl-oximes and o-methyl-oximes. Carbohydr Res 194:1–19. https://doi.org/10.1016/0008-6215(89)85001-3
Kostal V, Zahradnickova H, Simek P et al (2007) Multiple component system of sugars and polyols in the overwintering spruce bark beetle, Ips typographus. J Insect Physiol 53(6):580–586. https://doi.org/10.1016/j.jinphys.2007.02.009
Hill M, Parizek A, Kancheva R et al (2010) Steroid metabolome in plasma from the umbilical artery, umbilical vein, maternal cubital vein and in amniotic fluid in normal and preterm labor. J Steroid Biochem Mol Biol 121(3–5):594–610. https://doi.org/10.1016/j.jsbmb.2009.10.012
Rimnacova L, Husek P, Simek P (2014) A new method for immediate derivatization of hydroxyl groups by fluoroalkyl chloroformates and its application for the determination of sterols and tocopherols in human serum and amniotic fluid by gas chromatography-mass spectrometry. J Chromatogr A 1339:154–167. https://doi.org/10.1016/j.chroma.2014.03.007
Simek P, Heydova A, Jegorov A (1994) High-resolution capillary gas-chromatography and gas-chromatography mass-spectrometry of protein and nonprotein amino-acids, amino-alcohols, and hydroxycarboxylic acids as their tert-butyldimethylsilyl derivatives. J High Resoult Chromatogr 17(3):145–152
Kanani HH, Klapa MI (2007) Data correction strategy for metabolomics analysis using gas chromatography-mass spectrometry. Metab Eng 9(1):39–51. https://doi.org/10.1016/j.ymben.2006.08.001
Villas-Boas SG, Smart KF, Sivakumaran S, Lane GA (2011) Alkylation or silylation for analysis of amino and non-amino organic acid by GC-MS? Meta 1(1):3–20. https://doi.org/10.3390/metabo1010003
Husek P (1997) Urine organic acid profiling by capillary gas chromatography after a simple sample pretreatment. Clin Chem 43(10):1999–2001
Husek P, Simek P (2006) Alkyl chloroformates in sample derivatization strategies for GC analysis. Review on a decade use of the reagents as esterifying agents. Curr Pharm Anal 2(1):23–43. https://doi.org/10.2174/157341206775474007
Smart KF, Aggio RBM, Van Houtte JR et al (2010) Analytical platform for metabolome analysis of microbial cells using methyl chloroformate derivatization followed by gas chromatography-mass spectrometry. Nat Protoc 5(10):1709–1729. https://doi.org/10.1038/nprot.2010.108
Wachsmuth CJ, Hahn TA, Oefner PJ et al (2015) Enhanced metabolite profiling using a redesigned atmospheric pressure chemical ionization source for gas chromatography coupled to high-resolution time-of-flight mass spectrometry. Anal Bioanal Chem 407(22):6669–6680. https://doi.org/10.1007/s00216-015-8824-x
Husek P, Svagera Z, Hanzlikova D et al (2016) Profiling of urinary amino-carboxylic metabolites by in-situ heptafluorobutyl chloroformate mediated sample preparation and gas chromatography-mass spectrometry. J Chromatogr A 1443:211–232. https://doi.org/10.1016/j.chroma.2016.03.019
Simek P, Husek P, Zahradnickova H (2008) Gas chromatographic-mass spectrometric analysis of biomarkers related to folate and cobalamin status in human serum after dimercaptopropanesulfonate reduction and heptafluorobutyl chloroformate derivatization. Anal Chem 80(15):5776–5782. https://doi.org/10.1021/ac8003506
Simek P, Husek P, Zahradnickova H (2012) Heptafluorobutyl chloroformate-based sample preparation protocol for chiral and nonchiral amino acid analysis by gas chromatography. In: Alterman MA, Hunziker P (eds) Amino acid analysis: methods and protocols, Methods in molecular biology, vol 828, pp 137–152. https://doi.org/10.1007/978-1-61779-445-2_13
Husek P, Svagera Z, Hanzlikova D et al (2012) Survey of several methods deproteinizing human plasma before and within the chloroformate-mediated treatment of amino/carboxylic acids quantitated by gas chromatography. J Pharm Biomed Anal 67–68:159–162. https://doi.org/10.1016/j.jpba.2012.04.027
Svagera Z, Hanzlikova D, Simek P et al (2012) Study of disulfide reduction and alkyl chloroformate derivatization of plasma sulfur amino acids using gas chromatography-mass spectrometry. Anal Bioanal Chem 402(9):2953–2963. https://doi.org/10.1007/s00216-012-5727-y
Wu YM, Li L (2016) Sample normalization methods in quantitative metabolomics. J Chromatogr A 1430:80–95. https://doi.org/10.1016/j.chroma.2015.12.007
Bouatra S, Aziat F, Mandal R et al (2013) The human urine metabolome. PLoS One 8(9). https://doi.org/10.1371/journal.pone.0073076
Cimlova J, Kruzberska P, Svagera Z et al (2012) In situ derivatization-liquid liquid extraction as a sample preparation strategy for the determination of urinary biomarker prolyl-4-hydroxyproline by liquid chromatography-tandem mass spectrometry. J Mass Spectrom 47(3):294–302. https://doi.org/10.1002/jms.2952
Dettmer K, Stevens AP, Fagerer SR et al (2012) Amino acid analysis in physiological samples by GC-MS with propyl chloroformate derivatization and iTRAQ-LC-MS/MS. In: Alterman MA, Hunziker P (eds) Amino acid analysis: methods and protocols, Methods in molecular biology, vol 828, pp 165–181. https://doi.org/10.1007/978-1-61779-445-2_15
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This work was supported by the Czech Science Foundation, project No. 17-22276S.
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Hušek, P. et al. (2018). GC-MS Metabolomic Profiling of Protic Metabolites Following Heptafluorobutyl Chloroformate Mediated Dispersive Liquid Microextraction Sample Preparation Protocol. In: Theodoridis, G., Gika, H., Wilson, I. (eds) Metabolic Profiling. Methods in Molecular Biology, vol 1738. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7643-0_11
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DOI: https://doi.org/10.1007/978-1-4939-7643-0_11
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