Liquid chromatography/isotope ratio mass spectrometry analysis of halogenated benzoates for characterization of the underlying degradation reaction in Thauera chlorobenzoica CB-1T.

Abstract

Halogenated benzoic acids occur in the environment due to their widespread agricultural and pharmaceutical use. Compound-specific stable isotope analysis (CSIA) has developed over the last decades for investigation of in situ transformation and reaction mechanisms of environmental pollutants amenable by gas chromatography (GC). As polar compounds are unsuitable for GC analysis we developed a method to perform liquid chromatography (LC)/CSIA for halogenated benzoates. LC/isotope ratio mass spectrometry (IRMS) utilizing a LC-Surveyor pump coupled to a MAT 253 isotope ratio mass spectrometer via a LC-Isolink interface was applied. For chromatographic separation a YMC-Triart C18 column and a potassium hydrogen phosphate buffer (150mM, pH7.0, 40°C, 200μL mL-1 ) were used, followed by wet oxidation deploying 1.5molL-1 ortho-phosphoric acid and 200g L-1 sodium peroxodisulfate at 75μL mL-1 . Separation of benzoate and halogenated benzoates could be achieved in less than 40min over a concentration range of 2 orders of magnitude. Under these conditions the dehalogenation reaction of Thauera chlorobenzoica 3CB-1T using 3-chloro-, 3-bromo- and 4-chlorobenzoic acid was investigated resulting in inverse carbon isotope fractionation for meta-substituted benzoic acids and minor normal fractionation for para-substituted benzoic acids. Together with the respective growth rates this led to the assumption that dehalogenation of para-halobenzoic acids follows a different mechanism from that of meta-halobenzoic acids. A new LC/IRMS method for the quantitative determination of halogenated benzoates was developed and used to investigate the in vivo transformation pathways of these compounds, providing some insights into degradation and removal of these widespread compounds by T. chlorobenzoica 3CB-1T .