Analysis of mycolic acids by high-performance liquid chromatography and fluorimetric detection implications for the identification of mycobacteria in clinical samples

Abstract

Mycolic acids from Mycobacterium phlei and M. bovis cell wall skeletons (CWSs) were analyzed by HPLC. After saponifying lyophilized CWSs in methanolic KOH, the mycolic acids were quantitatively extracted into chloroform. Aliquots of the CWS mycolic acid extracts were then derivatized prior to HPLC analysis with a UV reagent, p-bromophenacylbromide (PBPB), and three fluorescent reagents, 4-bromomethyl-6,7-dimethoxycoumarin, 4-bromomethyl-7-acetoxycoumarin and 3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one. A synthetic α-branched carboxylic acid was derivatized with the same reagents and used as an internal standard along with the mycolic acids. The derivatized samples were analyzed by reversed-phase HPLC on a Waters Novapak C 18, 4 μm particle size, 150 mm × 3.9 mm stainless-steel column. Two solvent systems were used: (1) methanol and methylene chloride with the column at 30°C, and (2) methanol and isopropanol with the column at 50°C. Detection sensitivity with the fluorescent reagents was 16–50 times greater than the sensitivity observed with PBPB-derivatized samples. Unique mycolic acid elution profiles for the two mycobacterial species could be achieved with each of the solvent systems and derivatization reagents tested. Thus, the HPLC analysis of pre-column derivatized mycolic acids was useful as a means of rapidly identifying mycobacterial species. Replacement of methylene chloride with isopropanol and PBPB with a fluorescent derivatizing reagent could increase the safety and sensitivity of the assay, and make it more useful for the clinical identification of mycobacterial infections.