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Simultaneously characterizing and quantifying chloramphenicol and its metabolite using LC/MS/MS

he mass spectrometer was operated in negative ion mode. The acquisition method was set up using a total ion scan (from 120 to 600 amu/z) as a survey scan followed by two product ion scans under the aid of information dependent data acquisition (IDA) function, the last step is a multiple reaction monitoring (MRM) mode (321.03 to 152.03) to quantify chloramphenicol. The mass to charge ratio range for the two product ion scans was from 120 to 600 amu/z. Separation was carried out on a Phenomenex Synergi Hydro-RP (2.0 X 50) column using a mobile phase consisting of a mixture of 0.05% formic acid in water (solvent A) and 0.05% formic acid in acetonitrile (solvent B) at a constant flow rate of 0.15 mL/min. The initial condition consisted of a mixture of 95% of solvent A and 5% of solvent B. The percentage of acetonitrile was increased from 5 to 60% in 4 minutes, and then was increased from 60 to 90% in one minute before changing back to the initial condition. The column was reequilibrated for one minute before the next injection was made.

Results

The incubation samples were run using the method mentioned above. Within one run total ion chromatogram of single mass spectrum as a survey scan (Figure 1), single MS scan of the peak 1 at elution time t = 3.75 minutes. (Figure 2) from the survey scan and its product ion spectrum (Figure 3), single MS scan of the peak 2 at t = 4.31 minutes. (Figure 4) from the survey scan and its product ion spectrum (Figure 5), and one MRM chromatogram (Figure 6) information were collected.

Qualitative Confirmation: The qualitative confirmation of chloramphenicol (CAP) is based on unique mass spectrum characteristics of this compound as evaluated by other authors [1,2,3] using different instruments. One unique aspect of this compound is the fact that it contains two chlorine atoms, thus giving rise to unique isotopic patterns. From this experiment settings it is clea
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