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HPLC–UV method for simultaneous determination of adenosine triphosphate and its metabolites in Mycobacterium smegmatis

Akhova Anna V., Tkachenko Alexander G.

Acta Chromatographica

2019

Vol. 31, no. 1

45--48

Method for the analysis of intracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in Mycobacterium smegmatis that involves rapid extraction procedure based on sonication of cells in perchloric acid, as well as separation of nucleotides by ion-pair reversed-phase high-performance liquid chromatography and ultraviolet (UV) detection at 254 nm, is developed. The analytes were separated with mobile phase consisted of acetonitrile and 50 mM monobasic potassium phosphate (pH 4.6) with 25 mM tetrabutylammonium hydrogensulfate in a ratio of 0.5:99.5% within 30 min. The calibration curves were linear in the range of 20–1000 pmol of ATP and 10–1000 pmol of ADP and AMP with correlation coefficient (r2) of ≥0.9998. The proposed method is applicable for mycobacterium cultures taken over a wide range of optical density and physiological states. Concentrations of ATP, ADP, and AMP in mycobacterial extracts varied from 2.61 ± 0.27 to 9.60 ± 0.19 nmol/mg dry weight, from 1.75 ± 0.12 to 5.86 ± 0.09 nmol/mg dry weight, and from 0.55 ± 0.08 to 4.40 ± 0.07 nmol/mg dry weight, respectively, depending on the physiological state.

Computerized "mycobacterium" type recognition using numerical data base of mycolic acids elution profiles

Glapiński J., Walkiewicz R., Safianowska A., Grubek-Jaworska H.

Biocybernetics and Biomedical Engineering

2004

Vol. 24, no. 1

25-38

The aim of this study was to create a standarized numerical data base of high pressure liquid chromatography elution (HPLC) profiles of mycolic acids extracted from the "Mycobacterium" cell wall, with 28 mycobacterial American Type Culture Collection (ATCC) strains taken as a reference for the study. The final result of our work is a notebook of standarized elution profiles and a computer program (MYCAM) for highly effective and specific automatic "Mycobacterium" species identification.