Quantitative analysis of medium-chain polyhydroxyalkanoates in bacterial cells via gas chromatography-mass spectrometry: classical method revision and optimization

Abstract

A classical method of polyhydroxyalkanoates characterization via gas chromatography-mass spectrometry provides a fast, stable, and reproducible method of identification and quantification with high precision. However, the structure of currently applied internal standards differs from polyhydroxyalkanoates monomers potentially leading to analysis accuracy loss. To optimize the conventional analysis process for medium-chain-length-polyhydroxyalkanoates we suggested salicylic acid (2-hydroxybenzoic acid) use as a much more appropriate internal standard for polyhydroxyalkanoates quantification. A number of organic solvents of different nature and properties were considered as more suitable alternatives to the traditionally used chloroform. Cell culture of Pseudomonas helmanticensis was used as a medium-chain-length-polyhydroxyalkanoates producer for primary experiments, Pseudomonas putida and Cupriavidus necator were used for method verification. We showed that more frequently used benzoic acid as an internal standard is not very suitable for medium-chain-length-polyhydroxyalkanoates analysis. It was found that 2-hydroxybenzoic acid is a much more appropriate internal standard and provides highly accurate polyhydroxyalkanoates estimation. Following optimization of the sample treatment parameters has shown that using dichloromethane as solvent providing better analysis sensitivity, as well as improves its eco-safety and economical aspects. The method developed in this study was successfully verified and can be used to evaluate polyhydroxyalkanoates productivity of bacterial cells of different taxonomic classifications.