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Optimizing Solvents and Derivatizing Agents for Metabolomic Profiling of Human Plasma Using GC–MS

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Abstract

Metabolites provide a rich source of information regarding individual physiological states influenced by differences in diet, disease, health, and lifestyle. Effective sample preparation is a crucial step for the development of a sensitive, effective, and robust analytical method. In the present study, the metabolite extraction and identification protocol was optimized to achieve a reliable sample preparation technique for human plasma metabolites using gas chromatography–mass spectrometry (GC–MS). We compared the response of different sample preparation techniques with several extraction solvents [chloroform (CHCl3), methanol (MeOH), acetonitrile (ACN), MeOH and ACN (50:50), MeOH/MeOH/water (MMW)] coupled with N, O-Bis (trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane (BSTFA + 1% TMCS), and N-Methyl-N-(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane (MSTFA + 1% TMCS) derivatizing reagents. The suitability of the extraction solvent and derivatizing reagent was assessed for human plasma metabolite identification. We conclude that extraction of metabolites using methanol/methanol/water and 50 min silylation at 70 °C with N-methyl-N-(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane is efficient combination for proper derivatization and identification of metabolites. This protocol resulted in identifying a total of 87 metabolites including various amino acids, carboxylic acids, fatty acids, and polysaccharides. The optimized method provides an efficient, robust, and reproducible analytical method for metabolomic analysis in human plasma.

Graphical Abstract

Schematic representation of metabolite extraction and analysis using gas chromatography mass spectrometry

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The detailed protocols, data, and material are available upon request.

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Funding

The laboratory of Dr. Ashutosh Shrivastava is supported by the King George’s Medical University Intramural Grant Award and a research grant from CCRH, Ministry of AYUSH, Government of India (Z.28015/01/2018-HPC-EMR-AYUSH-D). Alok Mishra is the recipient of senior research fellowship from the University Grant Commission, New Delhi, India.

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Authors and Affiliations

  1. Center for Advance Research, King George’s Medical University, Lucknow, Uttar Pradesh, 226003, India

    Anshuman Srivastava, Alok Mishra & Ashutosh Shrivastava

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  1. Anshuman Srivastava
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  2. Alok Mishra
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  3. Ashutosh Shrivastava
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Contributions

AS and AS conceptualized and planned the study. AS and AM conducted the experiments. AS, AS, and AM analyzed the data and wrote the manuscript. All authors agree with the final version of the manuscript.

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Correspondence to Ashutosh Shrivastava.

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The authors have No competing interests to declare.

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The study was approved by the KGMU institutional ethics committee (Ref. Code: 107. ECM II B-Ph.D./P2). Informed consent was obtained from the subjects before collection of the samples.

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Srivastava, A., Mishra, A. & Shrivastava, A. Optimizing Solvents and Derivatizing Agents for Metabolomic Profiling of Human Plasma Using GC–MS. Chromatographia 86, 523–534 (2023). https://doi.org/10.1007/s10337-023-04266-z

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