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

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 µL 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°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 (r² > 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, R²Y = 0.977 and Q² (cumulative) = 0.877]. Copyright © 2009 John Wiley & Sons, Ltd.