Pre-column liquid chromatographic determination of dipicolinic acid from bacterial endospores

Bente Aa. Lomstein, Bo Barker Jørgensen

Limnol. Oceanogr. Methods 10:227-233 (2012) | DOI: 10.4319/lom.2012.10.227

ABSTRACT: A high performance liquid chromatographic (HPLC) method for fluorescence detection of dipicolinic acid (DPA), a unique component of bacterial endospores, was developed for rapid and highly sensitive quantification of endospores. DPA was pre-column complexed to the lanthanide metal terbium (Tb3+). Surplus free Tb3+ and the Tb3+-DPA complex were separated by reverse-phase gradient chromatography and the fluorescence emission was read at 545 nm after exitation at 270 nm. The limits of quantification (LOQ) and detection (LOD) were 0.26 and 0.08 nM, respectively. The method was used to detect DPA extracted from Bacillus subtilis endospores, DPA extracted from B. subtilis endospores added to more complex substrates such as marine sediment and canned tuna and DPA extracted from indigenous endospores in a coastal marine sediment. Endospores represented 4% of the microbial community (bacterial cells + endospores) in active surface sediment from the Aarhus Bay.