Development and validation of a gas chromatography/mass spectrometry method for the metabolic profiling of human colon tissue

Abstract

In this study, a gas chromatography/mass spectrometry (GC/MS) method was developed and validated for the metabolic profiling of human colon tissue. Each colon tissue sample (20 mg) was ultra-sonicated with 1 mL of a mixture of chloroform/methanol/water in the ratio of 20:50:20 (v/v/v), followed by centrifugation, collection of supernatant, drying, removal of moisture using anhydrous toluene and finally derivatization using N-methyl-N-trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS). A volume of 1 microL of the derivatized mixture was injected into the GC/MS system. A total of 53 endogenous metabolites were separated and identified in the GC/MS chromatogram, all of which were selected to evaluate the sample stability and precision of the method. Of the identified endogenous metabolites 19 belonging to diverse chemical classes and covering a wide range of the GC retention times (Rt) were selected to investigate the quantitative linearity of the method. The developed GC/MS method demonstrated good reproducibility with intra- and inter-day precision within relative standard deviation (RSD) of +/-15%. The metabolic profiles of the intact tissue were determined to be stable (100 +/- 15%) for up to 90 days at -80 degrees C. Satisfactory results were also obtained in the case of other stability-indicating studies such as freeze/thaw cycle stability, bench-top stability and autosampler stability. The developed method showed a good linear response for each of the 19 analytes tested (r(2) > 0.99). Our GC/MS metabolic profiling method was successfully applied to discriminate biopsied colorectal cancer (CRC) tissue from their matched normal tissue obtained from six CRC patients using orthogonal partial least-squares discriminant analysis [two latent variables, R(2)Y = 0.977 and Q(2) (cumulative) = 0.877].

Tissue metabolomic fingerprinting reveals metabolic disorders associated with human gastric cancer morbidity

“…The use of GC/MS in the metabolic profiling of esophageal cancer (19), colorectal cancer (10,26,27,35), liver cancer (16), bladder cancer (30) and ovarian cancer (32) has been reported, while few studies have investigated the metabolic profiles of GC tissue. This study explored the metabolic fingerprinting of GC tumor specimens and their matched normal mucosa.…”

A Plasma Metabolomic Investigation of Colorectal Cancer Patients by Liquid Chromatography-Mass Spectrometry

“…Recently, metabolite profiling between tumoral tissue and normal mucosa from CRC patients found significant variations [5][6][7][8]. These reports suggest that cancerous tissues up-regulate both the energy and amino acid metabolisms.…”

Metabonomics

“…Different extraction strategies such as homogenization alone, or homogenization followed by ultrasonication, or ultrasonication 24.4. GC-MS-BASED TISSUE METABONOMICS alone (usually applicable for small amount of tissue, 20 mg) can be adopted [13,61]. The choice of extraction solvent depends on the nature of the metabonomic study.…”