RAPID ANALYSIS OF ANTIBIOTIC-CONTAINING MIXTURES FROM FERMENTATION BROTHS BY USING LIQUID-CHROMATOGRAPHY ELECTROSPRAY IONIZATION-MASS SPECTROMETRY AND MATRIX-ASSISTED LASER-DESORPTION IONIZATION-TIME-OF-FLIGHT-MASS SPECTROMETRY
Bl. Ackermann et al., RAPID ANALYSIS OF ANTIBIOTIC-CONTAINING MIXTURES FROM FERMENTATION BROTHS BY USING LIQUID-CHROMATOGRAPHY ELECTROSPRAY IONIZATION-MASS SPECTROMETRY AND MATRIX-ASSISTED LASER-DESORPTION IONIZATION-TIME-OF-FLIGHT-MASS SPECTROMETRY, Journal of the American Society for Mass Spectrometry, 7(12), 1996, pp. 1227-1237
A crucial step in the isolation of antibiotic substances is establishi
ng whether or not the isolated material represents a new chemical enti
ty. Because of the importance of molecular weight to this process-know
n as dereplication-mass spectrometry has traditionally played an activ
e role. In this communication a strategy for utilizing liquid chromato
graphy-mass spectrometry (LC/MS) for novelty assessment is described.
Crude extracts (20-50 mu g) are chromatographed by conventional bore h
igh-performance liquid chromatography (1 mL/min) after which a postcol
umn split to divert roughly one-tenth of the sample to the mass spectr
ometer for molecular weight determination by electrospray ionization (
ESI) mass spectrometry. The majority of the effluent is sent to a UV d
etector and ultimately collected as 1-min fractions for biological tes
ting. As a secondary confirmation of molecular weight, an aliquot of e
ach fraction (< 5%) is taken for analysis by matrix-assisted laser des
orption ionization (MALDI). The improved efficiency of this approach o
ver more traditional schemes utilizing off-line fraction collection an
d conventional ionization methods can be explained by several factors.
First, the superior sensitivity of ESI and MALDI means that less mate
rial is required for successful analysis. Second, on-line LC/MS optimi
zes the efficiency of sample transfer and saves both time and labor. F
urthermore, the concentration dependence of ESI allows a majority of t
he material injected for LC/MS to be recovered for biological testing
without compromising the signal available for molecular weight determi
nation. As a validation of the above method, crude extracts containing
two well-characterized antibiotics-teicoplanin and phenelfamycin-were
examined. Results from these analyses are presented along with data f
rom the analysis of a potent unknown antifungal sample. (C) 1996 Ameri
can Society for Mass Spectrometry.