Metabolomics is a comprehensive analysis of metabolites existing in biological systems. As one of the important “omics” tools, the approach has been widely employed in various fields in helping to better understand the complex cellular metabolic states and changes. Bacterial metabolomics has gained a significant interest as bacteria serve to provide a better subject or model at systems level. The approach in metabolomics is categorized into untargeted and targeted which serves different paradigms of interest. Nevertheless, the bottleneck in metabolomics has been the sample or metabolite preparation method. A custom-made method and design for a particular species or strain of bacteria might be necessary as most studies generally refer to other bacteria or even yeast and fungi that may lead to unreliable analysis. The paramount aspect of metabolomics design comprises sample harvesting, quenching, and metabolite extraction procedures. Depending on the type of samples and research objective, each step must be at optimal conditions which are significantly important in determining the final output. To date, there are no standardized nor single designated protocols that have been established for a specific bacteria strain for untargeted and targeted approaches. In this paper, the existing and current developments of sample preparation methods of bacterial metabolomics used in both approaches are reviewed. The review also highlights previous literature of optimized conditions used to propose the most ideal methods for metabolite preparation, particularly for bacterial cells. Advantages and limitations of methods are discussed for future improvement of bacterial metabolomics.

The pentose phosphate pathway (PPP) plays a key role in many metabolic functions, including the generation of NADPH, biosynthesis of nucleotides, and carbon homeostasis. In particular, the intermediates of PPP have been found to be significantly perturbed in bacterial metabolomic studies. Nonetheless, detailed analysis to gain mechanistic information of PPP metabolism remains limited as most studies are unable to report on the absolute levels of the metabolites. Absolute quantification of metabolites is a prerequisite to study the details of fluxes and its regulations. Isotope tracer or labeling studies are conducted in vivo and in vitro and have significantly improved the analysis and understanding of PPP. Due to the laborious procedure and limitations in the in vivo method, an in vitro approach known as Group Specific Internal Standard Technology (GSIST) has been successfully developed to measure the absolute levels of central carbon metabolism, including PPP. The technique adopts derivatization of an experimental sample and a corresponding internal standard with isotope-coded reagents to provide better precision for accurate identification and absolute quantification. In this review, we highlight bacterial studies that employed isotopic tracers as the tagging agents used for the absolute quantification analysis of PPP metabolites.