( With the advent of liquid chr...)Simultaneously characterizing and quantifying chloramphenicol and its metabolite using LC/MS/MS

With the advent of liquid chromatography/ mass spectrometry and information dependent data acquisition function in mass spectrometry, it has become possible to characterise metabolites while to quantifying their parents during metabolic stability studies. This was once impossible to be analysed in one injection.

Abstract

The Q TRAP instrument is based on a triple quadrupole ion path and is capable of doing conventional multiple reaction monitoring (MRM) mode quantitation as a tandem mass spectrometry. On top of that it also has several high sensitivity ion trap MS scans using the final quadrupole as a linear ion trap. MS3 spectrum can be obtained from this instrument. Usually MS3 is sufficient to establish the fragmentation cascade because all fragments are generated abundantly without lower mass information cut off comparing to 3D ion traps. The application of this new hybrid RF/DC quadruple-linear ion trap mass spectrometer to the quantitation and characterization of chloramphenicol in its metabolic stability study using rat liver S9 is described. The limit of quantitation is around the parts per billion (ppb) level. In vitro half life has been calculated. Chloramphenicol is mainly metabolized by conjugation with glucuronic acid, which is in a good agreement with USP in vivo data. This technique could be applied widely in drug discovery, such as pharmacokinetic screening, metabolic stability study, etc., as well as food analysis. The quadrupole-linear ion trap proved to be a sensitive and reliable HPLC detection system that provided important chemical structure and quantitation information simultaneously.

Introduction

Chloramphenicol (CAP) is a wide range antibiotic which interferes with protein synthesis of many gramnegative and gram-positive bacteria, and has toxic effects on humans. The main potential human toxicity is depression of red blood cell production in bone marrow leading to a plastic aneamia and leukemia in children especially. It is important to identify chloramphenicol and monitor its exposure levels in different subjects.

Quantification of chloramphenicol and its metabolites in its metabolic stability study using rat liver S9 is described. With the advent of liquid chromatography/mass spectrometry and information dependent data acquisition function in mass spectrometry, it has become possible to quantify chloramphenicol and its metabolites and characterize them at the same time. This was once impossible to be analyzed in a sample within a single chromatographic run.

Materials and methods

Biological Methods: Chloramphenicol was incubated with rat liver S9 fractions at a substrate concentration of 50 micro molar and a protein concentration of 1 mg/ml in 100 mM potassium phosphate buffer at pH 7.4. Reactions were initiated by the addition of nicotinamide adenine dinucleotide phosphate reduced form (NADPH), uridine diphosphoglucuronic acid (UDPGA), and adenosine phosphate phosphosulfate (APPS) to give the final cofactor concentrations of 2 mM, 4 mM, and 4 mM, respectively. Incubation mixtures (2 mL total volume) were shaken in a water bath kept at 37C. An aliquot of 200 micro liter incubation solutions was sampled at t=0, 10, 20, 30, 40, 50, and 60 minutes. The reactions were terminated by adding two volumes of ice-cold acetonitrile. The samples were then vortex mixed and centrifuged. The supernatants were dried and the residues reconstituted in a 100 micro liter of 0.1% formic acid in water and acetonitrile (95:5, v/v). Ten micro liters was injected onto HPLC.

HPLC/MS: LC/MS analyses of chloramphenicol in vitro samples were carried out by coupling a Shimadzu 10AD system to a SCIEX Q TRAP^TM LC/MS/MS System. The HPLC eluent was introduced to the mass spectrometer using a TurboIonSpray. The 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 clear that the peak one is CAP-glucuronide, and peak two is the parent CAP in Figure 1. Single mass spectrum of CAP shows [M-H]- pair m/z 321 and 323 (Figure 4). Product ion spectrum (Figure 5) shows two predominant ions m/z 121 and 152, which correspond to [C6H4NO2-H]- and [C6H4NO2+CH2O]-. Ions m/z 194 [m/z 321-C2H2NOCl2]-, 176 [m/z 194-(H2O)]-, 219 [m/z 321-(HCOCl2)-2H]-, 249 [m/z 321-HCl2]-, and 257/259 [m/z 321-(HCOCl)]- also present in the spectrum (Figure 5). The product ion spectrum shows the isotopic ion pair of m/z 257 and 259, which gives approximately equal abundance, indicating the loss of one chlorine atom (either 35Cl or 37Cl) from the 35Cl 37Cl parent ion. The single MS spectrum (Figure 2.) of CAP metabolite and its product ion spectrum (Figure 3.) stated that the metabolite is glucuronidation of CAP. Figure 4. Single MS spectrum of peak 2 at t = 4.31 minutes from Figure 1. Figure 5. Product ion spectrum of peak 2 (M/z 323) at t = 4.31 minutes from Figure 1. Figure 6. MRM quantitation chromatogram of the same analysis run shown in Figure 1. Quantitative Confirmation: The MRM mode quantitation of CAP has been obtained within the same run with the qualitative information collections. The results show in Table 1 with an example chromatogram (t=60 minutes) shows in Figure 6. It is a fast run (CAP elution time t = 4.31minutes). T1/2 was calculated to be 46 minutes and its plot shows in Figure 7.

Conclusions

As stated previously, the advantages for this method are fast run time (about 5 minutes for a entire run) and simultaneously doing qualitative and quantitative work together. The instrument is based on a quadrupole ion path and is capable of doing conventional multiple reaction monitoring (MRM) mode quantitation as a tandem mass spectrometer. On top of that it also has several high sensitivity ion trap MS scans using the final quadrupole as a linear ion trap. MS3 spectrum can be obtained from this instrument. Usually MS3 is sufficient to establish the fragmentation cascade because all fragments are generated abundantly without lower mass information cut off comparing with 3D ion traps.

Chloramphenicol eluted from the LC at t = 4.31 minutes. During the runs, one major metabolite -glucuronide of CAP was identified as well as CAP itself. This metabolite was well confirmed with both single scan and product of 497 (321 parent + 176 Glucuronic acid portion) scan in one chromatographic run along with quantitation using MRM mode. The metabolism result is in a good agreement with USP in vivo data. The quadrupole-linear ion trap proved to be a sensitive and reliable HPLC detection system that provided important chemical structure and quantitation information simultaneously.

References

1. J. Assoc. Off. Anal. Chem. 61, 1247 (1978). 2. Center for Veterinary Medicine (2001), Guidance for Industry: Mass Spectrometry for Confirmation of the Identity of Animal Drug Residues. 3. Al Pfenning, Sherri Turnipseed, Jose Roybal, Cathy Burns, Mark Madson, Joseph Storey, Rebecca Lee, U.S. Food And Drug Administration Laboratory Information Bulletin No. 4289, August 2002, Confirmation of Chloramphenicol Residues In Crawfish by Electrospray LC/MS.