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Mass Spec Method for Amphotericin B

Question asked by annlarson47 on Jun 5, 2020
Latest reply on Jun 5, 2020 by annlarson47


I'm trying to develop a method to test amphotericin B using our Agilent G6125B single-quad LC/MSD . We need it to be quantitative for amphotericin B, but are less interested in impurities. My first attempts were based on the following article and I did not get a peak while monitoring ions at 925.1, 905, or 742.3 m/z. . It seems like a solid method so I might need to make a few changes to get it to work on our single-quad.

MP A: 0.1 formic acid in 50:50 acetonitrile:methanol
MP B: 10mM ammonium formate, pH 3 with 1% acetonitrile and 0.2% formic acid
column; agilent Zorbax SB-C18 2.1x50mm 1.8µm

Amphotericin B test concentration was about 5µg/ml, dissolved first in DMSO:MeOH and then diluted in MeOH.
Gradient is 35% A to 90% A over 2 min, returning to 35% A at 2.7. Flow was 0.3 ml/min at start of run, increasing to 0.4 ml/min within 2 min, and returning to 0.3 ml/min at 2.7 min. Total run time is 4 min.
Current MSD settings: gas 10 L/min, 300°C, 50 psig. Capillary 3500 eV. Fragmentation 15 V.
Thanks for any suggestions!