ANALYSIS OF MYCOLIC ACIDS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND FLUOROMETRIC DETECTION - IMPLICATIONS FOR THE IDENTIFICATION OF MYCOBACTERIA IN CLINICAL-SAMPLES

Mycolic acids from Mycobacterium phlei and M, bovis cell wall skeleton s (CWSs) were analyzed by HPLC. After saponifying lyophilized CWSs in methanolic KOH the mycolic acids were quantitatively extracted into ch loroform. Aliquots of the CWS mycolic acid extracts were then derivati zed prior to HPLC analysis with a UV reagent, p-bromophenacylbromide ( PB PB), and three fluorescent reagents, 4-bromomethhyl-6,7-dimethoxyco umarin, 4-bromomethyl-7-acetoxycoumarin and 3-bromomethyl-7-methoxy-1, 4-benzoxazin-2-one. A synthetic alpha-branched carboxylic acid was der ivatized with the same reagents and used as an internal standard along with the mycolic acids. The derivatized samples were analyzed by reve rsed-phase HPLC on a Waters Novapak C-18, 4 mu m particle size, 150 mm x 3.9 mm stainless-steel column. Two solvent systems were used: (1) m ethanol and methylene chloride with the column at 30 degrees C, and (2 ) methanol and isopropanol with the column at 50 degrees C. Detection sensitivity with the fluorescent reagents was 16-50 times greater than the sensitivity observed with PBPB-derivatized samples. Unique mycoli c acid elution profiles for the two mycobacterial species could be ach ieved with each of the solvent systems and derivatization reagents tes ted. Thus, the HPLC analysis of pre-column derivatized mycolic acids w as useful as a means of rapidly identifying mycobacterial species. Rep lacement of methylene chloride with isopropanol and PBPB with a fluore scent derivatizing reagent could increase the safety and sensitivity o f the assay, and make it more useful for the clinical identification o f mycobacterial infections.